CN120400168A - Construction method and application of an animal model of dilated cardiomyopathy - Google Patents
Construction method and application of an animal model of dilated cardiomyopathyInfo
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Abstract
The invention provides a construction method and application of an dilated cardiomyopathy animal model, belongs to the technical field of biological medicine and animal genetic model construction, provides a novel dilated cardiomyopathy zebra fish model, is constructed by specifically over-expressing a truncated dnajb b gene long isomer fragment in zebra fish myocardial cells, has a nucleotide sequence shown as SEQ ID NO.1, and further discovers the application of 4-phenylbutyric acid in preparing drugs for treating dilated cardiomyopathy.
Description
Technical Field
The invention belongs to the technical field of biological medicine and animal genetic model construction, and particularly relates to a construction method and application of an dilated cardiomyopathy animal model.
Background
Dilated cardiomyopathy (Dilated cardiomyopathy), abbreviated as dilated cardiomyopathy, is a common non-ischemic cardiomyopathy. The main clinical symptoms are ventricular enlargement and cardiac contraction dysfunction, and serious heart disease can cause heart failure and sudden cardiac death. It is counted that nearly 50% of sudden death or patients in need of heart transplantation in the adolescent population are due to expanded heart disease. In adults, heart disease is the second most common cause of heart failure next to coronary heart disease, accounting for about one third of all cases of heart failure. It is estimated that the incidence of the heart disease in the general population is 0.04-0.4%, which can affect over 250 ten thousand people worldwide, and thus the medical and socioeconomic burden is extremely heavy. At present, clinically, the treatment aiming at the heart disease is mainly symptomatic treatment for resisting heart failure, and no specific intervention means exists. Therefore, the pathogenesis and action mechanism of the hereditary heart disease can be explored, the early diagnosis and prevention of the disease and the prediction and evaluation of the occurrence and development processes of the disease can be realized, and experimental basis is provided for further developing specific genes aiming at the heart disease and personalized treatment strategies of drug targeted intervention.
The pathogenesis of the heart disease is relatively complex, and mainly comprises primary and acquired factors. Among them, the primary heart disease has strong family history and genetic susceptibility, and the gene mutation is an important cause of the hereditary heart disease. The prevalence of hereditary heart disease accounts for about 30% -50% of all cases of heart disease. To date, more than 50 genetic heart disease-causing genes have been discovered successively. The human DNAJB gene encodes a B6 member of the DnaJ heat shock protein (Hsp) 40 family, functioning as a chaperone protein. It binds to HSP70 and plays a key role in maintaining protein homeostasis by preventing protein misfolding and aggregation. The role of DNAJB protein in neurodegenerative diseases such as huntington's disease and skeletal muscle health, particularly in limb-girdle muscular dystrophy type 1D (LGMD 1D), has been recognized. However, although DNAJB gene is expressed in heart tissue, its function and mechanism of action in heart disease has been under-studied.
Due to the limitation of difficult availability and ethical aspects of clinical samples, animal disease models play an important role in researching the pathological mechanism of human diseases and drug screening tests. Although mice are always the first animal model for researching human diseases, the zebra fish model has the advantages of small size, large number, transparent embryo and the like on the basis that the zebra fish is a vertebrate, and the gene similarity of the zebra fish and human can reach 84 percent, and the zebra fish model is widely developed and applied in the research fields of researching human diseases, such as inheritance, drug screening test and the like. In particular, in the aspect of researching complex cardiovascular diseases such as heart disease, the basic heart rate of zebra fish is about 100 times per minute and is approximately equal to the heart rate level of human being 60-90 times per minute, compared with the heart rate of mice which is about 500 times per minute and is relatively large compared with human being, the action potential of the heart contraction of the zebra fish is relatively close to the action potential of the human being, and the zebra fish also has similar signal paths and drug metabolism paths on drug metabolism. Therefore, in recent years, the zebra fish model has unique feasibility advantages in the fields of research on inheritance of cardiovascular diseases such as heart disease and the like and drug screening development.
At present, the disease model of the heart disease established by taking the zebra fish as an animal model is mainly realized by the technical means of gene editing and drug induction. Gene editing simulates pathological characteristics of hereditary heart disease by mainly knocking out or mutating pathogenic genes related to human heart disease by CRISPR/Cas9 or TALEN and other gene editing technologies, such as myoglobin (TTN), beta-myosin heavy chain (MYH 7), BAG3 molecular chaperone regulatory genes and the like. The drug-induced model mainly uses chemical substances such as Doxorubicin (Doxorubicin), and the chemical substances are exposed to Doxorubicin (chemotherapeutic drugs) in embryo stage or juvenile stage to cause myocardial cell injury, so that ventricular dilatation and contractility are reduced, and the secondary pathological process of the non-hereditary dilated heart disease is simulated. The application of the gene editing technology in the disease model of the zebra fish heart-expanding disease is mostly focused on early embryo stages, the research on adult fish is less, the disease is usually a progressive heart function contraction dysfunction disease based on the heart-expanding disease, the disease is usually aggravated with the age, and finally heart failure is caused. Therefore, there is a need to construct a disease model of the heart disease in adult zebra fish animal models. The drug-induced heart disease model is generally nonspecific, and has certain side effects on other organs besides myocardial injury.
Disclosure of Invention
Aiming at the characteristics of the prior art, such as early embryo or insufficient specificity, the invention provides a construction method and application of an dilated cardiomyopathy adult animal model with cardiomyocyte specificity.
According to the research, through the functional analysis research of zebra fish dnajb b homologous genes in heart disease expansion, the specific overexpression of a section of truncated dnajb b gene long isomer fragment in cardiac muscle cells can cause the typical heart disease expansion phenotype of 3-month-old adult zebra fish such as obvious expansion of ventricles, myocardial damage and heart contraction dysfunction, so that a new genetic heart disease expansion adult zebra fish disease model is established. The research further applies the disease model, and discovers that the specific inhibition drug 4-phenylbutyric acid (4-PBA) of the sarcoplasmic reticulum stress response has a certain therapeutic effect.
The technical scheme of the invention is as follows:
an dilated cardiomyopathy zebra fish model is constructed by specifically over-expressing a truncated dnajb b gene long-form isomer fragment in zebra fish cardiomyocytes;
The nucleotide sequence of the gene fragment is shown as SEQ ID NO. 1.
SEQ ID NO.1 sequence is as follows:
ATGGAGGAGGATTATTACCACATCCTTGGTGTCACGAAAAGCGCATCT
CCTGACGATATAAAGAAAGCGTACAGAAAACTTGCACTAAAATGGCATCCA
GACAAAAACCCCAATGACAAAGAGGAGGCGGAGAAAAGGTTCAAAGAAA
TCTCAGAAGCATATGAAGTCCTGTCAGATGAAAACAAACGGAGAGATTATG
ATAGATATGGTAAACAAGGCCTCTCTAATAGAGGTGGCCATTACGATGATGA
ATATATGGGTGGATTCACATTCCGTAACCCAGAAGACGTCTTCAGGGAATTT
TTTGGAGGTCATGATCCATTTGCAGATTTCTTTGCTGATGACACATTTGAAG
GTTTCTTTGGTGGGCGCCGTCACAGAGGTATGAGCAGGAGCAGGACAGCA
GGTCCATTCTTTCCTGGATTTTCTCCATTTGGTCCGTCCTTTTCTGGATTTGA
CACAGGGTTTTCTCCGTTCGGCCCAATGGGTGGAGGAAGCTTCAGCTCATT
TTCATCGTCTCCATTTGGTGGTGGAGGTGGGATGAGAAACTTCACCTCCAT
TTCCACATCCACCAAATTCATCAACGGAAAGAGGATCACCACTAAACG。
The construction method of the dilated cardiomyopathy zebra fish model comprises the following steps:
(1) A section of truncated dnajb b gene long isomer fragment is obtained by a reverse transcription and PCR method, and the nucleotide sequence of the gene fragment is shown as SEQ ID NO. 1;
(2) Constructing a transgenic overexpression vector plasmid for fusing the gene fragment in the specific overexpression step (1) with a green fluorescent protein coding gene in zebra fish myocardial cells by a molecular cloning technology;
(3) Transferring the transgenic over-expression vector plasmid constructed in the step (2) into zebra fish by an embryo microinjection method, and obtaining the transgenic strain zebra fish of the gene fusion fragment in the step (2) through stable over-expression in a zebra fish living animal mode, namely the dilated cardiomyopathy zebra fish model.
According to a preferred embodiment of the present invention, in step (2), the method for constructing the transgenic overexpressing vector plasmid comprises the steps of:
① The total RNA extracted from the heart tissue of the zebra fish strain Wik is subjected to Reverse Transcription (RT) by using random primers to obtain a total cDNA library, and then amplified by PCR by using primers dnajb b (delta L) -BamHI-F as shown in SEQ ID NO.2 and dnajb b (delta L) -Xhol-R as shown in SEQ ID NO.3 to obtain dnajb b (delta L) cDNA as shown in SEQ ID NO. 1;
The primer sequences were as follows:
dnajb6b(ΔL)-BamHI-F SEQ ID NO.2:
5'-TAGCGGATCCGCCACCATGGAGGAGGATTATTACCACATCCTTGGTG-3';
dnajb6b(ΔL)-Xhol-R SEQ ID NO.3:
5'-AGCTCTCGAGTTGACTGTGAGAGATTTGAGCTGACCGT-3'
② The resulting PCR amplified fragment product of dnajb b (. DELTA.L) cDNA was cloned into pENTRI A plasmid by BamHI and XhoI restriction sites to give pENTRI A-dnajb b (. DELTA.L) plasmid, and then the pENTRI A-dnajb b (. DELTA.L) plasmid was recombined with p5E-cmlc2, p3E-EGFP-polyA and pDest-Tol2pA plasmid by GATEWAY LR cloning enzyme IIPlus to give pDest-cmlc2: dnajb b (. DELTA.L) transgenic overexpressing plasmid.
According to the invention, in the step (3), the construction method of the transgenic strain zebra fish comprises the following steps:
i, injecting the mixture of the transgenic overexpression vector plasmid constructed in the step (2) and the transposase mRNA into embryos of the wild zebra fish 1 cell stage by using an embryo injector, and then culturing;
ii screening injection embryo expressing green fluorescent marker protein (EGFP) signal, and culturing to adult fish as candidate F0 generation;
And (iii) hybridizing the F0 adult fish with the wild zebra fish to obtain an F1 embryo, screening the embryo positive to the green fluorescent marker protein to obtain a stable transgenic strain, and backcrossing the zebra fish with the wild zebra fish, which has EGFP positive signals in the hearts of the F1 individual, and screening the embryo positive to the green fluorescent marker protein to obtain the stable F2 transgenic strain.
Further preferred, the wild type zebra fish is a Wik strain.
According to a preferred embodiment of the invention, in step (3), the transgenic strain zebra fish of 3 months of age is selected as a model of dilated cardiomyopathy zebra fish.
The dilated cardiomyopathy zebra fish model constructed by the method is applied to screening of drugs for treating dilated cardiomyopathy.
Application of 4-phenylbutyric acid in preparing medicine for treating dilated cardiomyopathy.
According to a preferred embodiment of the invention, the dilated cardiomyopathy is a dilated cardiomyopathy resulting from the specific overexpression of a truncated dnajb b gene long isoform fragment in a cardiomyocyte.
Preferably, according to the present invention, the medicament comprises one or more pharmaceutically acceptable carriers or adjuvants.
Further preferably, the auxiliary agent is at least one of a slow release agent, a filler, a binder, a wetting agent, a disintegrating agent, a surfactant or a lubricant.
According to the invention, the medicament is preferably in the form of capsules, pills, tablets, oral liquids, granules, tinctures or injections.
The beneficial effects of the invention at least comprise the following:
1. The invention discovers for the first time that the specific overexpression of a truncated dnajb b gene long isomer fragment in myocardial cells can lead to dilated cardiomyopathy. The invention provides a new hereditary adult zebra fish heart disease expanding model, which has important clinical guiding significance for gene diagnosis, early prevention, prediction and evaluation of disease development process and design of personalized treatment scheme of the heart disease expanding model, and can be used for screening medicines for treating cardiomyopathy.
2. The invention not only establishes a new animal model of the heart disease, but also deeply discusses the pathological mechanism of the heart disease caused by over-expression of a section of truncated dnajb b gene long isomer fragment, in particular to two key pathological events of the increase of the myoplasmatic reticulum stress level and the aggregation of misfolded proteins, and provides new insight for future treatment and prevention strategies.
3. The zebra fish dilated cardiomyopathy model provided by the invention contains a plurality of indexes of heart functions, and can better illustrate successful construction of the model.
4. The invention discovers that 4-phenylbutyric acid can improve the heart phenotype of dilated cardiomyopathy caused by over-expression of a section of truncated dnajb b gene long isomer fragment, and provides a new drug target for preventing and treating dilated heart disease.
Drawings
FIG. 1 is a schematic diagram of a truncated Dnajb b long-form isoform protein fragment-encoding cDNA sequence, abbreviated dnajb b (ΔL), and a wild-type full-length Dnajb b long-form isoform protein-encoding cDNA sequence, abbreviated dnajb b (L), under the drive of a specific overexpressed promoter in cardiomyocytes constructed in accordance with the present invention;
In the figure, A is a schematic diagram of expression plasmid construction, and B is a diagram of the result of detecting over-expression of a truncated Dnajb B (delta L) and wild type full-length Dnajb B (L) control protein by western blotting experiment.
FIG. 2 is a graphical representation of the results of the inventive design of a split mirror of Tg (cmlc 2: dnajb6b [ delta ] L-EGFP), a transgenic zebra fish adult fish of the type Tg (delta L), and a transgenic control zebra fish adult fish of the type Tg (cmlc 2: dnajb6b [ L-EGFP);
In the figure, the heart phenotypes of wild type zebra fish (WT), tg (delta L) transgenic zebra fish and Tg (L) transgenic control zebra fish at 3 months of age are respectively shown, and the arrow indicates that the heart of the Tg (delta L) transgenic zebra fish is enlarged and highlighted visually.
FIG. 3 is a graph showing the results of contrast of cardiac ultrasound testing of wild type zebra fish (WT), tg (ΔL) transgenic zebra fish and Tg (L) transgenic control zebra fish according to the present invention;
in the figure, A is the Ejection Fraction (EF), and B is the shortening Fraction (FS).
Fig. 4 is a representative image of isolated hearts of 3 month old wild type zebra fish (WT), tg (Δl) transgenic zebra fish, and Tg (L) transgenic control zebra fish according to the present invention and quantification of Ventricular Surface Area (VSA) normalization and body weight (BW/g), n=8 fish per group;
In the figure, A is a heart image, and B is the quantification of Ventricular Surface Area (VSA) normalization and body weight (BW/g).
FIG. 5 is a schematic diagram showing the results of the heart histological examination of wild type zebra fish (WT), tg (ΔL) transgenic zebra fish and Tg (L) transgenic control zebra fish of 3 months of age according to the present invention;
In the figures, A-B are representative images of H & E staining and myocardial density quantification, and in the figure, C are Transmission Electron Microscope (TEM) results, the Tg (delta L) transgenic zebra fish is proved to have sarcomere disorder and mitochondrial abnormal swelling.
FIG. 6 is a graph showing survival curves of wild type zebra fish (WT), tg (ΔL) transgenic zebra fish and Tg (L) transgenic control zebra fish for 0-12 months according to the present invention.
FIG. 7 is a graph A showing the detection of the expression of protein folding related proteins in cardiac tissue of dilated cardiomyopathy zebra fish Tg (DeltaL) and Tg (L) control group zebra fish according to the present invention, and a graph B showing the analysis of quantitative analysis of the protein folding related proteins by specifically overexpressing a truncated dnajb B long isomer fragment transgene in cardiomyocytes.
FIG. 8 shows the transcription levels of the gene xbp1-s and the chop gene related to the stress response of the sarcoplasmic reticulum in the heart tissue of the dilated cardiomyopathy zebra fish Tg (delta L) and the control group of Tg (L) caused by the specific overexpression of a truncated dnajb B long isomer fragment transgene in the cardiac muscle cells, wherein A is an electrophoresis chart for detecting the xbp1-s, and B is a result for detecting the chop gene transcription level by quantitative PCR.
FIG. 9 is a schematic representation of the treatment of dilated cardiomyopathy zebra fish Tg (ΔL) caused by specific overexpression of a truncated dnajb b long isomer fragment in cardiomyocytes in accordance with the present invention.
FIG. 10 is a graphical representation of the results of a comparison of cardiac ultrasound measurements of transgenic and wild-type zebra fish according to the present invention after one month of treatment with 4-phenylbutyrate, where A is the Ejection Fraction (EF) and B is the shortening Fraction (FS).
FIG. 11 representative images of isolated hearts and quantification of Ventricular Surface Area (VSA) normalization and body weight (BW/g) of control zebra fish transgenic for Tg (ΔL) and wild-type zebra fish, with n=8 fish per group, following one month treatment with 4-phenylbutyrate;
in the figure, A is a heart image, and B is a quantification of Ventricular Surface Area (VSA) normalization and body weight (BW/g).
FIG. 12 is a graphical representation of the results of a comparison of cardiac histological examination of wild-type control zebra fish and Tg (ΔL) transgenic zebra fish according to the present invention after one month of treatment with 4-phenylbutyrate, wherein A-B is representative image and myocardial density quantification of H & E staining of 3 month old wild-type zebra fish (WT), tg (ΔL) transgenic zebra fish without drug addition (PBS) control treatment, and Tg (ΔL) transgenic zebra fish after treatment with 4-phenylbutyrate, and C is Transmission Electron Microscope (TEM) results.
FIG. 13 is a graph showing survival of wild-type control zebra fish and Tg (ΔL) transgenic zebra fish according to the present invention after one month of treatment with 4-phenylbutyrate.
FIG. 14 is a schematic A and quantitative B representation of myocardial apoptosis of wild type control zebra fish and Tg (ΔL) transgenic zebra fish according to the present invention after one month of treatment with 4-phenylbutyrate.
Detailed Description
The following describes the technical scheme of the present invention further with reference to examples, but the scope of the present invention is not limited thereto.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Sources of reagent materials:
zebra fish source, national zebra fish resource center, wuhan;
Plasmids pENTRI A, p5E-cmlc2, p3E-EGFP-polyA, and pDest-Tol2pA were from Invitrogen, addgene, respectively.
1. Specific overexpression of a truncated dnajb b long isomer fragment, abbreviated dnajb b (delta L), in cardiomyocytes was found to lead to heart disease and heart failure
To explore the function and mechanism of action of dnajb b gene in heart disease and heart failure, the inventors constructed a transgenic line driven by the cardiac myoglobin light chain 2 (cmlc 2) promoter that specifically overexpresses dnajb b (ΔL) isoform fragment in cardiac myocytes and named Tg (cmlc 2: dnajb6b [ ΔL ] -EGFP), abbreviated as Tg (ΔL). As a control, the inventors also constructed a transgenic line that specifically overexpresses the wild-type full-length dnajb b long isomer, abbreviated dnajb b (L), in cardiac myocytes, and designated Tg (cmlc 2: dnajb6b [ L ] -EGFP), abbreviated Tg (L).
First, western blot results confirm high level expression of Dnajb b (Δl) and Dnajb b (L) proteins in heart tissue of the respective transgenic lines. Second, cardiac phenotyping revealed that transgenic lines with Tg (Δl) already exhibited a significant phenotype of ventricular enlargement and cardiac dysfunction at 3 months of age. As a control, no significant abnormalities were seen in the Tg (L) transgenic line. The results of the histological analysis of H & E staining and the ultrastructural examination by transmission electron microscopy revealed that there was a significant amount of myofibril injury and mitochondrial swelling in the heart tissue of the Tg (Δl) transgenic line, whereas the Tg (L) transgenic control line was not significantly abnormal. Again, at the molecular level, the inventors detected abnormal activation of the heart disease and heart failure molecular marker genes nppa and nppb in heart tissue of Tg (Δl) transgenic lines. As a control, no such abnormalities were detected in the Tg (L) transgenic line control group. Finally, survival curve analysis showed that Tg (Δl) transgenic lines died successively from 4 months of age, with only about 60% of the fish surviving to 12 months of age. In contrast, the survival rate of Tg (L) control line was comparable to wild type group fish.
Taken together, these experimental results demonstrate that specific overexpression dnajb b (Δl) in cardiac myocytes can lead to the development of heart disease and heart failure in adult zebra fish.
2. It was found that Dnajb b protein regulates cardiac contractility, sarcoplasmic reticulum stress response and protein quality control by interacting with Grp78 protein
In order to elucidate in depth the pathogenic mechanisms of heart disease and heart failure caused by specific overexpression dnajb b (ΔL) in cardiomyocytes, the inventors propose to envisage sustained elevation of sarcoplasmic reticulum stress response levels and accumulation of misfolded proteins in heart tissue of the Tg (ΔL) transgenic line as major pathological events. First, the inventors demonstrated the existence of direct interactions between human DNAJB (L) long form isoforms and the sarcoplasmic reticulum stress response molecular marker GRP78 protein, respectively, in HEK 293T cells. Second, the inventors detected that dnajb b (L) transcripts were significantly upregulated by in situ hybridization and quantitative RT-PCR following treatment of sarcoplasmic reticulum stress-stimulated drug with tunicamycin (Tunicamycin). Again, the inventors detected significant increases in the cumulative levels of Grp78, LC3II and ubiquitinated proteins, respectively, in heart tissue of Tg (Δl) transgenic lines. Meanwhile, at the transcriptional level, the inventor detects that the expression of two muscle mass network stress response molecular marker genes including an xbp1-s spliceosome and chop are significantly up-regulated.
The experimental results show that Dnajb b has responsiveness to the sarcoplasmic reticulum stress reaction, and the sustained accumulation of misfolded protein aggregates is likely to be the main pathogenic mechanism of the phenotype of heart disease and heart failure caused by Tg (delta L) transgenesis.
3. It was found that 4-phenylbutyric acid, an inhibitor of the sarcoplasmic reticulum stress response, can partially rescue the symptoms of dilated cardiomyopathy and heart failure caused by cardiomyocyte-specific overexpression dnajb b (ΔL)
To continue to explore whether sustained increases in the level of sarcoplasmic reticulum stress and accumulation of misfolded proteins were sufficient to result in the heart disease and heart failure phenotype in Tg (Δl) transgenic lines, the inventors tested the potential therapeutic effect of the sarcoplasmic reticulum stress inhibitor drug 4-phenylbutyrate (4-PBA). First, the inventors optimized the therapeutic dose of 4-PBA by lavage to ensure that it was effective in reducing sustained activation of the sarcoplasmic reticulum stress level molecular marker protein Grp78 in heart tissue of Tg (Δl) transgenic lines. Subsequently, drug treatment of 4-PBA was started before the appearance of the pronounced heart disease and heart failure phenotype in the Tg (ΔL) transgenic line, i.e. at 2 months of age. The results of the study showed that after 1 month of 4-PBA treatment, the ventricular enlargement phenotype of the Tg (Δl) transgenic line was significantly inhibited and the cardiac function was also significantly improved. At the histological and ultrastructural level, 4-PBA treatment significantly slowed the sarcomere degeneration phenotype in heart tissue of Tg (Δl) transgenic lines. On the cellular level, 4-PBA treatment effectively reduces apoptosis, simultaneously inhibits the loss of myocardial cells, and finally improves the overall survival rate of transgenic fish.
Taken together, these results demonstrate that the sarcoplasmic reticulum stress inhibitor drug 4-phenylbutyric acid (4-PBA) can exert a significant therapeutic effect on the heart disease and heart failure phenotype due to specific overexpression dnajb b (Δl) in cardiomyocytes.
Example 1
The embodiment 1 relates to a new construction method of an dilated cardiomyopathy zebra fish model, which is obtained by over-expressing a truncated dnajb b gene long isomer fragment transgene in a zebra fish living animal model, and comprises the following specific steps:
(1) Total RNA extracted from heart tissue of zebra fish strain Wik is Reverse Transcribed (RT) to obtain total cDNA library by using random primer, then primer dnajb b (delta L) -BamHI-F is used as shown in SEQ ID NO.2 and primer dnajb b (delta L) -Xhol-R is used as shown in SEQ ID NO.3, PCR is performed to obtain dnajb b (delta L) cDNA as shown in SEQ ID NO.1, primer dnajb b (L) -BamHI-F is used as shown in SEQ ID NO.4, and primer dnajb b (L) -Xhol-R is used as shown in SEQ ID NO.5, and PCR is performed to obtain dnajb b (L) full-length wild type control cDNA.
Primer sequence:
dnajb6b(ΔL)-BamHI-F SEQ ID NO.2:
5'-TAGCGGATCCGCCACCATGGAGGAGGATTATTACCACATCCTTGGTG-3';
dnajb6b(ΔL)-Xhol-R SEQ ID NO.3:
5'-AGCTCTCGAGTTGACTGTGAGAGATTTGAGCTGACCGT-3';
dnajb6b(L)-BamHI-F SEQ ID NO.4:
5'-TAGCGGATCCGCCACCATGGAGGAGGATTATTACCACATCCTTGGTG-3';
dnajb6b(L)-Xhol-R SEQ ID NO.5:
5'-AGCTCTCGAGCATTTGCTGTCCAGTCGGAGTAACTGC-3'。
(2) The resulting PCR amplified fragment products of truncated dnajb b (ΔL) and full length wild type dnajb b (L) control cDNAs were then cloned into the pENTRI A plasmid (Invitrogen) by BamHI and XhoI restriction sites, respectively, resulting in pENTRI A-dnajb6b (ΔL) and pENTRI1A-dnajb b (L) plasmids. The pENTRI A-dnajb6b (ΔL) or pENTRI A-dnajb6b (L) plasmid was then recombined with the p5E-cmlc2, p3E-EGFP-polyA and pDest-Tol2pA plasmids by means of GATEWAY LR cloning enzyme IIPlus to give the final pDest-cmlc2: dnajb6b (ΔL) and pDest-cmlc2: dnajb6b (L) transgenic overexpressing plasmids.
(3) Preparing an injection needle, namely preparing a needle capable of carrying out microinjection by using a needle pulling instrument to prepare a 1mm capillary tube before microinjection, taking care when preparing the microinjection needle to prevent the needle tip from breaking, mixing the obtained recombinant plasmid with about 50pg of final pDest-cmlc2: dnajb6b (delta L) or pDest-cmlc2: dnajb6b (L) contrast transgene with 50pg of transposase mRNA, adding a phenol red indicator, and injecting the mixture into the microinjection needle by using an Eppendorf long suction head to avoid bubbles in the injection needle;
(4) Microinjection, namely injecting a mixture of 50ng pDest-cmlc2: dnajb6b (delta L) or pDest-cmlc2: dnajb6b (L) control transgenic overexpressed recombinant plasmid and 50pg transposase mRNA into embryos of wild type zebra fish strain 1 cell stage by using an embryo injector, injecting at least 200 embryos, placing the embryos injected with the mixture of recombinant plasmid and 50pg transposase mRNA in an embryo culture dish, adding embryo culture solution (0.292 g/L NaCl, 0.013g/L KCl, 0.044g/L CaCl 2、0.081g/L MgSO4, pH 7.2), simultaneously retaining some uninjected embryos of the same batch as a wild type control group, and placing all the embryos in a 28.5 ℃ incubator for culture;
(5) It was identified whether transgene overexpression was successful by anesthetizing embryos with 0.16mg/ml of tricaine solution (ARGENT CHEMICAL Laboratories) 2-5 days after fertilization to stop heartbeats. Screening injected embryos expressing green fluorescent marker protein (EGFP) signals using a Zeiss microscope as candidate F0 generation for cultivation to adult fish;
(6) The F0 adult fish is hybridized with wild zebra fish to obtain F1 embryo, and green fluorescent marker protein positive embryo is selected to obtain stable transgenic fish strain Tg (cmlc 2: dnajb6b [ delta ] L ] -EGFP) and Tg (cmlc 2: dnajb6b [ L ] -EGFP). The stable F2 transgenic strain is obtained by backcrossing zebra fish with wild zebra fish, wherein EGFP positive signals exist in about 50% of individual hearts in the F1 generation offspring, and green fluorescent marker protein positive embryos are continuously screened, the size of over-expressed proteins is confirmed to be in accordance with expectations through an immunoWestern blotting experiment, and the identified stable F2 transgenic strain Tg (cmlc: dnajb b [ delta ] L-EGFP) (Tg (delta L) and Tg (cmlc: dnajb b [ L-EGFP) (Tg (L) are used for subsequent experiments, and FIG. 1 is a construction schematic diagram of the transgenic strain and verification results of a western blotting experiment.
Example 2
The present example 2 relates to an assay for the effect of transgene overexpression of a truncated dnajb b gene long-form isoform fragment on cardiac function of zebra fish, comprising the following:
(1) Living heart at 3 months of age was observed by anesthetizing wild-type zebra fish (WT), tg (. DELTA.L) transgenic zebra fish and Tg (L) transgenic control zebra fish with 0.02% of tricaine for 5 minutes, looking sideways up, looking at heart contours 1-2cm under gill using a 2.5-fold magnification Leka microscope, and recording with photographs, the results are shown in FIG. 2.
As can be seen from the white light phenotype of fig. 2, tg (Δl) transgenic zebra fish showed significant heart enlargement and protrusion at 3 months of age, which phenotype was not observed in wild type zebra fish (WT) as well as Tg (L) transgenic control zebra fish.
(2) Living body echocardiography at 3 months of age the cardiac performance index of adult zebra fish was measured and analyzed using a 50MHz (MX 700) Vevo 3100 high frequency imaging system. An acoustic gel is applied to the probe surface to provide adequate coupling to the tissue interface. Adult zebra fish of 3 months of age were anesthetized with 0.02% of tricaine for 5 minutes and placed in a sponge with the abdomen facing upwards. A 50MHz (MX 700) sensor was placed over the zebra fish to provide sagittal imaging of the heart. The B mode image adopts an axial imaging view field of 8.00mm, a transverse imaging view field of 8.73mm, a frame rate of 152Hz, a persistence degree and the like, and a transmission focus is positioned at the center of the heart. Image quantification was performed using data packets in VevoLAB stations, and ventricular size was measured from B-mode images using the following criteria, ejection fraction and foreshortening fraction, with the results shown as a-B in fig. 3.
From fig. 3 a-B, it can be seen that the cardiac ejection fraction, shortening Fraction (FS) of the 3 month old Tg (Δl) transgenic zebra fish was significantly reduced compared to wild type zebra fish (WT) and Tg (L) transgenic control zebra fish.
(3) Ventricular surface area to body weight ratio was measured by anesthetizing 3 months of zebra fish, weighing (BW), dissecting the heart of individual zebra fish, imaging in a millimeter scale on a licarpa microscope, and measuring Ventricular Surface Area (VSA). The results are shown in FIG. 4.
As can be seen from fig. 4, the 3 month old Tg (Δl) transgenic zebra fish exhibited a pronounced ventricular dilatation phenotype, which was not observed in wild type zebra fish (WT) as well as Tg (L) transgenic control zebra fish.
(4) Histological hearts taken immediately after 3 months of euthanasia of zebra fish were fixed with 4% formaldehyde fixative, paraffin embedded, sections were stained with hematoxylin and eosin (H & E), photographed using a Ni-U imaging system (nikon), and trabecular muscle density was quantitatively determined using ImageJ software as shown in fig. 5 a-B.
As can be seen from fig. 5 a-B, the Tg (Δl) transgenic zebra fish at 3 months of age showed significantly reduced myofibril density compared to the wild type zebra fish (WT) and the Tg (L) transgenic control zebra fish heart tissue H & E staining, thereby finding myocardial myofibril injury of the Tg (Δl) transgenic zebra fish.
(5) Transmission Electron Microscopy (TEM) in TEM studies, the zebra fish hearts were immediately fixed in a fixing solution (0.1M phosphate buffer with pH 7.2 containing 4% paraformaldehyde and 1% glutaraldehyde) at room temperature for 1h, and then left at 4℃overnight. The fixed samples were then processed and imaged by the wuhansai biotechnology company using an HT7800 transmission electron microscope. The results are shown in FIG. 5C.
In FIG. 5, C demonstrates that abnormal swelling of mitochondria occurs in 3 month old Tg (ΔL) transgenic zebra fish, and in FIG. 5, A-C results indicate that abnormal myocardial fibers and mitochondrial morphology of the Tg (ΔL) transgenic zebra fish affect normal heart function.
As can be seen from the survival analysis of FIG. 6, tg (ΔL) transgenic zebra fish started to die successively at 4 months of age, with only about 60% surviving to 12 months, in contrast to the survival rate of Tg (L) transgenic control zebra fish, which was comparable to wild-type, peer group fish.
The above results demonstrate that 3 month old Tg (Δl) transgenic zebra fish developed an dilated cardiomyopathy phenotype.
Example 3
The procedure of example 3 is as follows, in which the expression of protein related to protein folding in dilated cardiomyopathy zebra fish is caused by specific overexpression of a truncated dnajb b long isomer fragment in cardiomyocytes:
3 wild zebra fish (WT), tg (DeltaL) transgenic zebra fish and Tg (L) transgenic control zebra fish were each 3 pieces and euthanized by soaking in 0.032% glyceryl triacetate solution for 10 minutes, and heart tissue was dissected. Heart tissue was rinsed with PBS and transferred to RIPA buffer with protease inhibitor cocktail (Roche Diagnostic) added. Tissue homogenization was performed using Bullet Blender tissue homogenizer (Next ADVANCE INC). The supernatant was collected for protein concentration determination (502 nm) by centrifugation at 10000g for 20min at 4 ℃. Each group was adjusted to an equal concentration based on the measured protein concentration, 30mg of protein was loaded, and separation was performed using a 12% SDS-PAGE gel under conditions of 70v,50min;120v 2h to stop electrophoresis until bromophenol blue reached the very bottom of the gel, and the power was turned off. Transferring film, namely firstly soaking PVDF in methanol for more than 1min for activation, transferring into 4 ℃ pre-cooled film transferring liquid for standby, and simultaneously soaking 6 pieces of filter paper in the 4 ℃ pre-cooled film transferring liquid. And then taking out the electrophoresis rubber plate, prying the short glass plate by using a seesaw, and cutting off redundant parts in the concentrated rubber. And (3) transferring the separation gel into the pre-cooled transfer film liquid by using a seesaw. And (3) spreading the black negative electrode of the film transfer clamp in a water tank containing film transfer liquid, and then sequentially spreading a sandwich structure. The sandwich structure is that a glass rod is used for removing bubbles, gel and PVDF film (bubbles are avoided between the gel and the PVDF film), wherein the bubbles are removed from a blackboard (negative electrode), a sponge and 3 layers of filter paper (soaked with film transferring liquid), the film transferring liquid can be dripped on the gel, the PVDF film is taken to be contacted with the edge of the gel at one side, and the film is slowly put down and attached, and the 3 layers of filter paper, the sponge and the whiteboard are all laminated. And finally closing the clamping plate, placing the clamping plate into a film transfer groove (black corresponds to a negative electrode and red corresponds to a positive electrode), placing the sandwich structure into the film transfer groove (positive and negative electrodes are aligned), and placing the sandwich structure into a rotor and an ice bag or placing the sandwich structure into a 4-degree refrigerator, and transferring the sandwich structure into an ice water bath for 50 minutes under the condition of constant current 400 MA. After the film transfer is finished, the power supply is turned off, the filter paper is recovered, the PVDF film is soaked by the film transfer liquid and placed in a plastic package bag which is cut in advance, and a target strip is cut. The molecular weight of Grp78 protein is 78kDa, the molecular weight of Lc3II protein is 13kDa, the molecular weight of the Ubiquitin protein is about 10-250kDa, and the molecular weight of the internal reference Actin protein is 42kDa. The PVDF membrane is blocked by shaking at room temperature for 1 hour after being fully dissolved by 5% skimmed milk, the blocked PVDF membrane is placed in TBST membrane washing liquid for washing 3 times for 5 minutes each time, then the primary antibody anti-Grp78(1:2000,Novus Biologicals LLC,catalog#NBP-06274)、anti-LC3(1:3000,Cell signaling Technology,catalog#12741)、anti-β-Actin(1:5000,Santa Cruz Biotechnology,catalog#sc-1615)、anti-Ubiquitin(1:2000,Thermo Fisher Scientific,catalog#PA5-17067) is diluted by 5% BSA according to the dilution multiple, incubated overnight at 4 ℃, and after the primary antibody is recovered, the membrane is washed by TBST for 4 times for 10 minutes each time. Secondary antibody incubation HRP-labeled secondary antibody was diluted with 5% bsa as described above and incubated with shaking at room temperature for 1h. Washing the membrane, namely discarding the secondary antibody, washing the membrane for 4 times by using TBST for 10min each time, and finally developing. The protein bands were subjected to grey value analysis using Image J, see figure 7.
The invention deeply discusses the pathological mechanism of the heart disease caused by the specific over-expression of a truncated dnajb b gene long isomer fragment in cardiac muscle cells. Given that the mouse endogenous Dnajb protein was previously reported to co-localize with the nuclear membrane protein Lamin a/C and that it was translocatable into the Sarcoplasmic Reticulum (SR) lumen after treatment with the sarcoplasmic reticulum stress inducer tunica, and that part was co-localized with the sarcoplasmic reticulum protein glucose regulatory protein 78 (Grp 78), the inventors focused on the misfolding of the protein and the pathological mechanism of the enlarged heart disease caused by elevated levels of sarcoplasmic reticulum stress for over-expression of a truncated dnajb b gene long isoform fragment. In the expanded heart disease zebra fish model newly established by the inventors, as shown in a-B in fig. 7, a significant increase in the aggregation level of protein Grp78, LC3II and ubiquitinated proteins associated with protein folding was detected at 3 months of age. This suggests that accumulation of misfolded protein aggregates is one of the pathogenesis of Tg (Δl) transgenic zebra fish.
Example 4
The procedure of example 4 is as follows, in which the expression of the gene associated with sarcoplasmic reticulum stress in dilated cardiomyopathy zebra fish is specifically over-expressed in cardiomyocytes as a truncated dnajb b long isomer fragment:
3 pieces of wild-type zebra fish (WT), tg (. DELTA.L) transgenic zebra fish and Tg (L) transgenic control zebra fish were each taken at 3 months of age, total RNA was extracted from hearts of adult fish using 400ul Trizol reagent (ThermoFisher Scientific) according to the manufacturer's instructions, and ground using a Bullet Blender tissue grinder (Next ADVANCE INC). The ground tissues were mixed upside down by adding 2/5 volume of RNase-free ddH20 (160. Mu.L for every 400. Mu.L of RNA-easy). After 5min of standing at room temperature, centrifugation was carried out at 20℃under 12000g for 15min. After centrifugation, the solution was seen to separate into an upper aqueous phase and a dark lower precipitate (impurities such as protein, DNA, polysaccharide, etc.). Collecting the supernatant to another grinding tube, adding 400 μl isopropanol with equal volume, mixing upside down, and standing at room temperature for 10min. Centrifugation at 20℃and 12000g for 10min. After centrifugation in this step, white massive precipitates at the bottom of the centrifuge tube are observed, namely RNA, sometimes the visible precipitates cannot be gathered and dispersed on the tube wall, and the supernatant is carefully sucked along the liquid level. The supernatant was removed, 400. Mu.L of 75% alcohol was added, the RNA was sprung up and turned upside down several times, and then centrifuged at 20℃and 9100g for 3min. Removing alcohol, instantly separating at 20deg.C, sucking water, and air drying for 3min. According to the precipitation adding 10 u L DEPC, against the milky precipitation solution, pipetting gun blow mixing, repeatedly blow until RNA completely dissolved. Genomic DNA removal and reverse transcription were performed, and finally real-time fluorescent quantitative PCR was performed. The analysis of the results is shown in FIG. 8.
FIG. 8 demonstrates that at 3 months of age Tg (. DELTA.L) transgenic zebra fish significantly increased the transcript levels of the two sarcoplasmic reticulum stress-related genes, xbp1-s and chop, compared to wild type zebra fish (WT) and Tg (L) transgenic control zebra fish. This suggests that elevated sarcoplasmic reticulum stress levels are the second pathogenesis of Tg (Δl) transgenic zebra fish.
The results of example 3 and example 4 combined may lead to an increase in the level of sarcoplasmic reticulum stress and accumulation of misfolded protein aggregates as a significant pathological event of cardiac dysfunction in Tg (Δl) transgenic zebra fish.
Example 5
The present embodiment relates to the use of 4-phenylbutyrate for improving symptoms of dilated cardiomyopathy and heart failure caused by specific overexpression dnajb b (Δl) in cardiomyocytes, in particular:
Treatment of Tg (Δl) transgenic zebra fish constructed in example 1 with 4-phenylbutyrate can improve or partially rescue phenotypes such as enlarged ventricles, abnormal cardiac function, reduced survival, etc. of zebra fish.
Wild-type control group, tg (delta L) transgenic zebra fish drug-free control group and Tg (delta L) transgenic zebra fish drug-free treatment group were set, and the wild-type and drug-free control groups were infused with Phosphate Buffered Saline (PBS) and the treatment groups were infused with 4-phenylbutyrate (4-PBA) at a dose of 200 micrograms per gram of body weight. The dose was normalized to 2ul per fish, and the control and treatment groups received five days of oral gavage treatment weekly for four weeks, see figure 9. The ejection fraction, shortening fraction, ventricular size, myotrabecular change, survival rate, myocardial apoptosis, etc. of each group of zebra fish were observed according to the adult fish heart function detection method in example 2. The results are shown in fig. 10, 11, 12, 13 and 14.
The results show that compared with the control group without the drug, the Tg (delta L) transgenic zebra fish has the advantages that the heart function is obviously improved after being treated by the 4-phenylbutyric acid for one month, the Ejection Fraction (EF) and the shortening Fraction (FS) are obviously improved, the phenomenon of ventricular enlargement after being treated by the 4-phenylbutyric acid is also obviously improved, the myogenic degeneration phenotype in heart tissues of the Tg (delta L) transgenic strain is obviously slowed down by the 4-PBA treatment on the aspects of histology and ultrastructure, meanwhile, the swelling condition of mitochondria after being treated is also relieved, and the increase of cell death is obviously inhibited by the 4-PBA treatment on the cellular level, so that the overall survival rate of fish is finally improved as seen from the reduction of TUNEL index. It was shown that 4-phenylbutyrate has a therapeutic effect on dilated cardiomyopathy caused by specific overexpression dnajb b (Δl) in cardiomyocytes.
The invention discovers that a section of truncated dnajb b gene long isomer fragment is specifically over-expressed in myocardial tissues and is a primary factor for causing dilated cardiomyopathy for the first time, and provides a novel zebra fish heart disease dilating model. And further elucidate the molecular mechanism of dnajb b gene in heart disease and heart failure, highlighting its key role in maintaining protein homeostasis in cardiomyocytes, in heart of Tg (Δl) transgenic zebra fish, accumulation of ubiquitinated protein aggregation, elevation of endoplasmic reticulum stress molecular markers, and abnormal activation of autophagy were detected, highlighting the core role of Dnajb b protein in promoting protein folding, preventing aggregation, and promoting misfolded protein degradation through proteasome and autophagy pathways.
The novel heart disease expanding model established by the invention can be used for screening related medicines for preventing/treating heart disease expanding caused by potential DNAJB gene mutation. The data presented by the inventors demonstrate that the heart-expanding phenotype of dnajb b (ΔL) animals, which is specifically overexpressed in cardiomyocytes, can be partially alleviated and their longevity prolonged by 4-PBA treatment. Has important clinical guidance significance for early prevention of the heart disease, prediction and evaluation of the disease development process and design of personalized treatment schemes.
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