CN109394784A - The screening model of anti-head-neck carcinoma tumour medicine - Google Patents
The screening model of anti-head-neck carcinoma tumour medicine Download PDFInfo
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Abstract
The invention discloses a kind of screening model of anti-head-neck carcinoma tumour medicine, the specific steps of building include: 1) to be restructured hollow fiber reactor surface using acetic acid plasma-based, sterilization treatment；2) separation incidence tumor is unicellular；3) by the unicellular implantation hollow fiber reactor of head-neck carcinoma tumour, cell attachment culture is carried out；4) it determines drug target, using healthy mice as administration object, takes hollow fiber reactor to be implanted into back of mice, establish mouse head-neck carcinoma cancer pathology model afterwards for 24 hours；5) drug target is applied to mouse head-neck carcinoma cancer pathology model, be administered once every 2 days, construct the screening model of anti-head-neck carcinoma tumour medicine.The present invention can quickly carry out anti-head-neck carcinoma tumour medicine evaluating drug effect, and the time needed for highly shortened the effect experiment of traditional mice xenograft model is no more than 20 days from experiment to result, and can reduce clinical trial cost, easy to spread.
Description
Technical field
The present invention relates to medicine effects to screen field, in particular to a kind of screening model of anti-head-neck carcinoma tumour medicine.
Background technique
Head and neck neoplasm includes tumor colli, ear-nose-throat department tumour and oral and maxillofacial tumor three parts.Neck is swollen
Tumor belongs to general surgery in general hospital, and relatively common is exactly thyroid tumors；Ear-nose-throat department tumour common are laryngocarcinoma,
Paranasal sinus cancer etc.；Oral and maxillofacial tumor is common for various carcinoma of mouth, such as tongue cancer, gingival carcinoma, cheek cancer.Therefore, incidence
The tumour occurred, as many as original site and histological type occupy first of general tumour.And head and neck neoplasm recurrence or transfer are suffered from
The median survival interval of person only about 6 months.Patient survival dramatic decrease after first-line treatment failure, it is dead usually in 3 months.
Chemotherapy is a kind of primary treatment regimen to advanced metastatic cancer patient at present, but since chemotherapeutics lacks tumour cell
Specificity also kills a large amount of marrow and other molecular marker for increased proliferation normal cells while killing tumor cell, have it is more serious not
Good reaction.Embolic chemotherapy traditional simultaneously is easy to produce drug resistance after carrying out multiple chemotherapy, this will affect chemotherapy effect.Thus
It is more more and more urgent for the accurate medical demand of individual.Precisely on the one hand medical treatment improves the accuracy treated to individuation, separately
On the one hand it can also be effectively reduced the randomness and blindness of patient medication, to mitigate the injury of patient medication.In recent years,
It is patient source tumour transplatation object model (patient- with the high animal model of clinical patient medication curative effect correlation
Derivedxenograft model, PDX model) it is widely used in precisely medical treatment, but traditional mouse xenografts at present
Model has construction complexity, overlong time, both expensive and there is a problem of clinical application deficiency.
Summary of the invention
For above-mentioned existing anti-head-neck carcinoma tumour screening drug model construction complexity, overlong time, somewhat expensive with
And the problem of clinical application deficiency, the present invention provide a kind of screening model of anti-head-neck carcinoma tumour medicine.
In order to solve the above-mentioned technical problem, the technical solution adopted by the present invention is that:
A kind of screening model of anti-head-neck carcinoma tumour medicine, wherein its construct specific steps include:
1) hollow fiber reactor surface is restructured by 5~15min, 10~30min of sterilization treatment using acetic acid plasma-based；
2) head-neck carcinoma cell strain or head-neck carcinoma sufferer tumor tissues are subjected to digestion process, obtain head-neck carcinoma tumour
It is unicellular；
3) it by the unicellular implantation hollow fiber reactor of head-neck carcinoma tumour, seals, and hollow fiber reactor is put
Enter in culture dish, RPMI-1640 culture medium be added, carries out cell attachment culture, take out hollow fiber reactor after 1~3 day,
For use；
4) drug target is determined, using healthy mice as administration object, the hollow fiber reactor for taking step 3) to obtain is implanted into
Mouse head-neck carcinoma cancer pathology model is established at healthy mice back afterwards for 24 hours；
5) drug target is applied to mouse head-neck carcinoma cancer pathology model, be administered once every 1~2 day, in administration 2
It, 4 days, 8 days, 10 days taking-up hollow fiber reactors, incidence tumor single cell active is detected, and with head-neck carcinoma tumour
Thus single cell active is completed as the index for evaluating anti-head-neck carcinoma tumour medicine to the anti-head-neck carcinoma tumour medicine
The building process of screening model.
Preferably, the screening model of the anti-head-neck carcinoma tumour medicine, wherein the hollow fiber reactor packet
Include more hollow fiber conduits arranged in parallel；Each doughnut pipe surface is distributed with multiple holes.
Preferably, the screening model of the anti-head-neck carcinoma tumour medicine, wherein the diameter of the hollow fiber conduit
It is 70~100 μm, wall thickness is 2~4 μm, and length is 3~5cm.
Preferably, the screening model of the anti-head-neck carcinoma tumour medicine, wherein the hollow fiber reactor
Having a size of 3cm × 0.5cm~5cm × 0.5cm；The diameter of described hole is 100~300nm.
Preferably, the screening model of the anti-head-neck carcinoma tumour medicine, wherein be implanted into hollow fiber reactor
Before healthy mice back, pretreated step is also carried out；The pre-treatment step specifically: extract anesthetic note with insulin needle
Enter mouse peritoneal, and cuts the notch of 0.5cm wide at back.
Preferably, the screening model of the anti-head-neck carcinoma tumour medicine, wherein the head-neck carcinoma tumour is slender
The implant density of born of the same parents is 2 × 105Cell/5 μ l.
Preferably, the screening model of the anti-head-neck carcinoma tumour medicine, wherein the administration route is abdominal cavity note
It penetrates.
Preferably, the screening model of the anti-head-neck carcinoma tumour medicine, wherein the head-neck carcinoma tumour is slender
Cytoactive detection specifically:
1) hollow fiber reactor is soaked in the first pipettor of 2ml, 200 μ l 0.5% is contained in the first pipettor
Thiazolyl blue solution and 1000 μ l phosphate buffer solutions, hollow fiber reactor are kept in dark place 30min, are centrifugated out incidence
Tumor is unicellular；
2) hollow fiber reactor is placed in the second pipettor of 2ml containing 500 μ l dimethyl sulfoxides, be kept in dark place for 24 hours；
3) the 100 μ l of cell solution of step 2) of learning from else's experience processing measures 570nm absorbance value, as 96 porose discs to judge head
The single celled survival rate of neck tumor.
Preferably, the screening model of the anti-head-neck carcinoma tumour medicine, wherein the drug target is anti-neck
Portion's cancerous swelling tumor medicine.
The utility model has the advantages that
(1) present invention provide it is a kind of using hollow fiber reactor carry out cell culture method belong to 3D cell culture,
Cell is grown in caliber has enough contact points that can provide rise signal, compared to not utilizing hollow fiber reactor
The growth of mode cell is very fast, and cell physiological state also cultivates growing state intracorporal close to biology than 2D.
(2) hollow fiber reactor includes more hollow fiber conduit compositions arranged in parallel, each doughnut pipe surface
Multiple holes are equipped with, hole is conducive to nutrient transmission and product secretion when cell culture, improves cell survival rate and growth
Speed, by hollow fiber reactor implantation mouse it is subcutaneous after due to tube wall is thin and have exchange nutrition and drug hole, carry out
Cell culture educates that the tumor mode speed of growth is fast, and success rate is higher than tradition, is conducive to clinical patient tumour cell heteroplastic transplantation,
Do not influence drug permeation.
(3) present invention can quickly carry out anti-head-neck carcinoma tumour medicine evaluating drug effect, and it is different to highly shortened traditional mouse
Time needed for the effect experiment of kind transplantation model is no more than 20 days from experiment to result, and can reduce clinical trial cost, easily
In popularization.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of hollow fiber reactor in the present invention；
Fig. 2 is general head-neck carcinoma tumor cell survival datagram in the present invention；
Fig. 3 is patient source neck cancerous swelling viability datagram in the present invention.
Specific embodiment
Specific embodiments of the present invention will be further explained with reference to the accompanying drawing.It should be noted that for
The explanation of these embodiments is used to help understand the present invention, but and does not constitute a limitation of the invention.In addition, disclosed below
The each embodiment of the present invention involved in technical characteristic can be combined with each other as long as they do not conflict with each other.
Embodiment 1: the foundation of mouse head-neck carcinoma cancer pathology model
1, the preparation of hollow fiber reactor
With reference to Fig. 1, hollow fiber reactor includes more doughnuts 1 arranged in parallel, each doughnut pipe surface
It is distributed with multiple holes 2, hollow fiber reactor is polypropylene or polychloroethylene with the material of hollow fibre filament on the way, hollow
The diameter of fibre pipe is 70~100 μm, and wall thickness is 2~4 μm, and length is 3~5cm, and the diameter of hole is 100~300nm, experiment
140 hollow fiber reactors are selected, with acetic acid plasma-based by hollow fiber reactor surfaction 10min, and are irradiated with UV light
Sterilizing 30 minutes, to ensure that cell is all in germ-free condition.
2, mouse head-neck carcinoma tumor model is established
2.1 establish general head-neck carcinoma tumor models
Head-neck carcinoma cell strain is subjected to digestion process, implantation incidence tumor is slender in 40 hollow fiber reactors
Born of the same parents SAS, by the unicellular SAS (2 × 10 of head-neck carcinoma tumour5Cells/5 μ l) using siphonage implantation cut into 4.5cm ×
In the lumen of 0.5cm hollow fiber reactor, then both ends open is folded into 0.5cm inside and is sealed, to ensure that cell does not flow out
Hollow fiber reactor is finally incubated in 10 centimeters of culture dish by hollow fiber reactor, and culture medium RPMI- is added
1640, after the attaching of 36 hour cells, determine drug target, using healthy mice as administration object, experiment is selected 7 weeks big
BALB/c mouse 10, the biological cell reactor of 10 mouse implantation unicellular SAS of incidence tumor, by 30g insulin
Needle draws 200L arcotic (900L PBS+50L Zoletil 50+50L Rompun 2%), injects abdominal cavity, is carrying on the back after anesthesia
Portion cuts the notch of the about 0.5cm wide of a width, gos deep into notch using scissors, scratches the mucous membrane between skin and muscle, will be hollow
Fiber reactor is placed on micro medicine spoon, and is implanted into back of mice, using sutures by wound suture 2-3 needle, Mei Ge little
Mouse back is implanted into 4 hollow fiber reactors, establishes the general head-neck carcinoma cancer pathology model of mouse afterwards for 24 hours.
2.2 establish mouse pathogeny head-neck carcinoma tumor model
Head-neck carcinoma sufferer tumor tissues are subjected to digestion process, are implanted into head-neck carcinoma in 100 hollow fiber reactors
Sufferer tumour is unicellular, by head-neck carcinoma sufferer tumour unicellular (2 × 105Cells/5 μ l) it is cut using siphonage implantation
In lumen at 2.5cm × 0.5cm hollow fiber reactor, then both ends open is folded into 0.5cm inside and is sealed, it is thin to ensure
Born of the same parents do not flow out hollow fiber reactor, are finally incubated at GNTA in 10 centimeters of culture dish, and culture medium RPMI-1640 is added,
After the attaching of 36 hour cells, drug target is determined, using healthy mice as administration object, experiment selects BALB/c 7 weeks big small
Mouse 25,30g insulin needle is drawn into 200L arcotic, arcotic is 900L PBS+50L Zoletil 50+50L Rompun
2%, PBS are phosphate buffered saline solution, and Zoletil 50 is hydrochloride for injection Tiletamine, for anesthesia injection, contain sequestered
Anesthetic ingredient Tiletamine and the azoles for having tranquillizer and myorelaxant effects concurrently draw the West, have that induction period is short, side reaction is small
With the advantage of safety；Rompun is xylazine hydrochloride, has calm, analgesia and central myorelaxant effects, passes through
The reasonable compatibility of PBS, Zoletil 50 and Rompun makes anaesthetic effect in experimentation reach best.Arcotic injects mouse
Abdominal cavity, cuts the notch of the about 0.5cm wide of a width at back after anesthesia, go deep into notch using scissors, scratch skin and muscle
Between mucous membrane, hollow fiber reactor is placed on micro medicine spoon, and is implanted into back of mice, is stitched wound using sutures
2-3 needle is closed, each back of mice is implanted into 4 hollow fiber reactors, establishes mouse pathogeny head-neck carcinoma cancer pathology mould afterwards for 24 hours
Type.
Embodiment 2: the screening model of anti-head-neck carcinoma tumour medicine is established
1, experimental group
10 mouse of general head-neck carcinoma tumor model, set up SAS blank group, SAS application group separately, and every group each 5.Wherein
MCF naive mice does not apply anticancer drug, and SAS application group mouse applies Doxorubicin (DOX) drug, and dosage is
Doxorubicin 20mg/kg。
25 mouse of pathogeny head-neck carcinoma tumor model, set up control group, cis group, Carbo group, Doce group and 5- separately
Fu group, every group each 5, cis group injection Cisplatin drug, Carbo group injection carboplatin drug, Doce group injection
Docetaxel drug, 5-Fu group injection 5-Fu drug, formulation rate are Cisplatin 20mg/kg, carboplatin 20mg/
Kg, Docetaxel 20mg/kg, 5-Fu 20mg/kg.
2, the screening model of anti-head-neck carcinoma tumour medicine
10 mouse of general head-neck carcinoma tumor model, SAS naive mice do not apply anticancer drug, SAS application group
Mouse applies Doxorubicin (DOX) drug, is administered once every 2 days, and dosage is Doxorubicin 100mg/kg, applies
Hollow fiber reactor is taken out from experimental mice back respectively within medicine 10 days, it is living to detect the unicellular SAS of general head-neck carcinoma tumour
Property, and using SAS cell activity as the index for evaluating anti-head-neck carcinoma tumour medicine, it thus completes to general anti-incidence cancerous swelling
The building process of the screening model of tumor medicine.
25 mouse of pathogeny head-neck carcinoma tumor model, control group do not apply drug, cis group injection Cisplatin
Drug, Carbo group injection carboplatin drug, Doce group injection Docetaxel drug, 5-Fu group injection 5-Fu drug are given
Dose be Cisplatin 20mg/kg, carboplatin 20mg/kg, Docetaxel 20mg/kg, 5-Fu 20mg/kg, often
It was administered once every 1 day, took out hollow fiber reactor from experimental mice back respectively in administration 10 days, detect pathogeny incidence
Tumor single cell active, and using pathogeny head-neck carcinoma tumour single cell active as the finger for evaluating anti-head-neck carcinoma tumour medicine
Mark, thus completes the building process of the screening model to the anti-head-neck carcinoma tumour medicine of pathogeny.
3, cell activity is tested
1) hollow fiber reactor is soaked in the pipettor of 2ml, it is molten containing 200 μ l, 0.5% thiazolyl blue in pipettor
Liquid and 1000 μ l phosphate buffer solutions, hollow fiber reactor are kept in dark place 30min, are centrifugated out cell；
2) hollow fiber reactor is placed in the second pipettor of 2ml containing 500 μ l dimethyl sulfoxides, be kept in dark place for 24 hours；
3) the 100 μ l of cell solution of step 2) of learning from else's experience processing measures 570nm absorbance value, as 96 porose discs to judge head
The survival rate of neck tumor cell culture, and using head-neck carcinoma tumour single cell active as the anti-head-neck carcinoma tumour medicine of evaluation
The index of object.
The selection result of general head-neck carcinoma tumour as shown in Fig. 2, general anti-head-neck carcinoma tumour medicine screening model
Set up SAS blank group, SAS application group separately, for SAS application group compared with SAS blank group, the SAS cell survival rate of SAS application group is obvious
Higher than SAS blank group, illustrate that SAS head-neck carcinoma tumour cell is more sensitive to Doxorubicin drug.
The selection result of pathogeny head-neck carcinoma drug as shown in figure 3, the anti-head-neck carcinoma tumour medicine of pathogeny screening model
Set up control group, cis group, Carbo group, Doce group and 5-Fu group separately, every group each 5, is administered 10 days progress cell activity
Test, compared with control group, cell survival rate is below control group for cis group, Carbo group, Doce group and 5-Fu group, and
Control group is not because apply anti-head-neck carcinoma tumour medicine, cell survival rate highest, and cis group cell survival in application group
Rate is minimum, illustrates that patient is most sensitive to Cisplatin drug.
In conjunction with attached drawing, the embodiments of the present invention are described in detail above, but the present invention is not limited to described implementations
Mode.For a person skilled in the art, in the case where not departing from the principle of the invention and spirit, to these embodiments
A variety of change, modification, replacement and modification are carried out, are still fallen in protection scope of the present invention.
Claims (9)
1. a kind of screening model of anti-head-neck carcinoma tumour medicine, which is characterized in that its construct specific steps include:
1) hollow fiber reactor surface is restructured by 5~15min, 10~30min of sterilization treatment using acetic acid plasma-based；
2) head-neck carcinoma cell strain or head-neck carcinoma sufferer tumor tissues are subjected to digestion process, it is slender obtains head-neck carcinoma tumour
Born of the same parents；
3) it by the unicellular implantation hollow fiber reactor of head-neck carcinoma tumour, seals, and hollow fiber reactor is put into training
It supports in ware, RPMI-1640 culture medium is added, carry out cell attachment culture, take out hollow fiber reactor after 1~3 day, to
With；
4) drug target is determined, using healthy mice as administration object, the hollow fiber reactor implantation for taking step 3) to obtain is healthy
Back of mice establishes mouse head-neck carcinoma cancer pathology model afterwards for 24 hours；
5) drug target is applied to mouse head-neck carcinoma cancer pathology model, be administered once every 1~2 day, in administration 2 days, 4
It, 8 days, 10 days taking-up hollow fiber reactors, detect incidence tumor single cell active, and it is slender with head-neck carcinoma tumour
Thus cytoactive is completed as the index for evaluating anti-head-neck carcinoma tumour medicine to the anti-head-neck carcinoma tumor drug screening mould
The building process of type.
2. the screening model of anti-head-neck carcinoma tumour medicine according to claim 1, which is characterized in that the doughnut
Reactor includes more hollow fiber conduits arranged in parallel；Each doughnut pipe surface is distributed with multiple holes.
3. the screening model of anti-head-neck carcinoma tumour medicine according to claim 2, which is characterized in that the doughnut
The diameter of pipe is 70~100 μm, and wall thickness is 2~4 μm, and length is 3~5cm.
4. the screening model of anti-head-neck carcinoma tumour medicine according to claim 2, which is characterized in that the doughnut
The size of reactor is 3cm × 0.5cm~5cm × 0.5cm；The diameter of described hole is 100~300nm.
5. the screening model of anti-head-neck carcinoma tumour medicine according to claim 1, which is characterized in that doughnut is anti-
Before answering device to be implanted into healthy mice back, pretreated step is also carried out；The pre-treatment step specifically: extracted with insulin needle
Anesthetic injects mouse peritoneal, and cuts the notch of 0.5cm wide at back.
6. the screening model of anti-head-neck carcinoma tumour medicine according to claim 1, which is characterized in that the head-neck carcinoma
The single celled implant density of tumour is 2 × 105Cell/5 μ l.
7. the screening model of anti-head-neck carcinoma tumour medicine according to claim 1, which is characterized in that the administration route
For intraperitoneal injection.
8. the screening model of anti-head-neck carcinoma tumour medicine according to claim 1, which is characterized in that the head-neck carcinoma
The detection of tumour single cell active specifically:
1) hollow fiber reactor is soaked in the first pipettor of 2ml, 200 μ l, 0.5% thiazole is contained in the first pipettor
Blue solution and 1000 μ l phosphate buffer solutions, hollow fiber reactor are kept in dark place 30min, are centrifugated out incidence cancerous swelling
Tumor is unicellular；
2) hollow fiber reactor is placed in the second pipettor of 2ml containing 500 μ l dimethyl sulfoxides, be kept in dark place for 24 hours；
3) the 100 μ l of cell solution of step 2) of learning from else's experience processing measures 570nm absorbance value, as 96 porose discs to judge incidence
The single celled survival rate of tumor.
9. the screening model of anti-head-neck carcinoma tumour medicine according to claim 1, which is characterized in that the drug target
For anti-head-neck carcinoma tumour medicine.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110663648A (en) * | 2019-09-26 | 2020-01-10 | 上海联鑫生物科技有限公司 | A method for establishing a mouse model of human cancer tissue transplanted tumor hollow fiber test method |
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2018
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110663648A (en) * | 2019-09-26 | 2020-01-10 | 上海联鑫生物科技有限公司 | A method for establishing a mouse model of human cancer tissue transplanted tumor hollow fiber test method |
| CN113832106A (en) * | 2019-09-26 | 2021-12-24 | 东部细胞治疗(山东)有限公司 | Hollow fiber tube for human cancer tissue transplantable tumor and application thereof |
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