CN107007578A - 莱菔硫烷在制备治疗白血病药物中的应用 - Google Patents
莱菔硫烷在制备治疗白血病药物中的应用 Download PDFInfo
- Publication number
- CN107007578A CN107007578A CN201710109473.9A CN201710109473A CN107007578A CN 107007578 A CN107007578 A CN 107007578A CN 201710109473 A CN201710109473 A CN 201710109473A CN 107007578 A CN107007578 A CN 107007578A
- Authority
- CN
- China
- Prior art keywords
- cells
- sulforaphane
- leukemia
- gene
- apoptosis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- SUVMJBTUFCVSAD-UHFFFAOYSA-N sulforaphane Chemical compound CS(=O)CCCCN=C=S SUVMJBTUFCVSAD-UHFFFAOYSA-N 0.000 title claims abstract description 116
- SUVMJBTUFCVSAD-JTQLQIEISA-N 4-Methylsulfinylbutyl isothiocyanate Natural products C[S@](=O)CCCCN=C=S SUVMJBTUFCVSAD-JTQLQIEISA-N 0.000 title claims abstract description 56
- 229960005559 sulforaphane Drugs 0.000 title claims abstract description 56
- 235000015487 sulforaphane Nutrition 0.000 title claims abstract description 56
- 208000032839 leukemia Diseases 0.000 title claims abstract description 38
- 239000003814 drug Substances 0.000 title claims abstract description 15
- 230000006907 apoptotic process Effects 0.000 claims abstract description 36
- 108090000397 Caspase 3 Proteins 0.000 claims abstract description 17
- 229940079593 drug Drugs 0.000 claims abstract description 13
- 238000002360 preparation method Methods 0.000 claims abstract description 12
- 230000035755 proliferation Effects 0.000 claims abstract description 12
- 101150024147 bax gene Proteins 0.000 claims abstract description 11
- 108700041737 bcl-2 Genes Proteins 0.000 claims abstract description 11
- 230000001105 regulatory effect Effects 0.000 claims abstract description 6
- 230000002222 downregulating effect Effects 0.000 claims abstract description 5
- 230000030833 cell death Effects 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 8
- 206010000830 Acute leukaemia Diseases 0.000 abstract description 5
- 230000002401 inhibitory effect Effects 0.000 abstract description 5
- 210000004027 cell Anatomy 0.000 description 62
- 108700000707 bcl-2-Associated X Proteins 0.000 description 12
- 102000055102 bcl-2-Associated X Human genes 0.000 description 11
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 10
- 102100021569 Apoptosis regulator Bcl-2 Human genes 0.000 description 10
- 101000971171 Homo sapiens Apoptosis regulator Bcl-2 Proteins 0.000 description 10
- 230000014509 gene expression Effects 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 102000003952 Caspase 3 Human genes 0.000 description 7
- 235000018102 proteins Nutrition 0.000 description 6
- 230000004663 cell proliferation Effects 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 208000031261 Acute myeloid leukaemia Diseases 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- 102000011727 Caspases Human genes 0.000 description 3
- 108010076667 Caspases Proteins 0.000 description 3
- 208000033776 Myeloid Acute Leukemia Diseases 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 238000010839 reverse transcription Methods 0.000 description 3
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 108010087230 Sincalide Proteins 0.000 description 2
- 239000006180 TBST buffer Substances 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- 230000001640 apoptogenic effect Effects 0.000 description 2
- 239000012148 binding buffer Substances 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 238000010609 cell counting kit-8 assay Methods 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 230000003211 malignant effect Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 230000002438 mitochondrial effect Effects 0.000 description 2
- 230000025915 regulation of apoptotic process Effects 0.000 description 2
- 238000003757 reverse transcription PCR Methods 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 108090000672 Annexin A5 Proteins 0.000 description 1
- 102000004121 Annexin A5 Human genes 0.000 description 1
- 238000012406 Annexin V-FITC/PI double staining Methods 0.000 description 1
- 102000051485 Bcl-2 family Human genes 0.000 description 1
- 108700038897 Bcl-2 family Proteins 0.000 description 1
- 235000011299 Brassica oleracea var botrytis Nutrition 0.000 description 1
- 235000017647 Brassica oleracea var italica Nutrition 0.000 description 1
- 240000003259 Brassica oleracea var. botrytis Species 0.000 description 1
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 1
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 1
- 102100030497 Cytochrome c Human genes 0.000 description 1
- 108010075031 Cytochromes c Proteins 0.000 description 1
- 108010040476 FITC-annexin A5 Proteins 0.000 description 1
- 101001066129 Homo sapiens Glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 239000012807 PCR reagent Substances 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102000013535 Proto-Oncogene Proteins c-bcl-2 Human genes 0.000 description 1
- 108010090931 Proto-Oncogene Proteins c-bcl-2 Proteins 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 239000012721 SDS lysis buffer Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 208000024207 chronic leukemia Diseases 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 230000000040 effect on leukemia Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000003394 haemopoietic effect Effects 0.000 description 1
- 208000019691 hematopoietic and lymphoid cell neoplasm Diseases 0.000 description 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 1
- 230000011132 hemopoiesis Effects 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 102000047486 human GAPDH Human genes 0.000 description 1
- 238000010191 image analysis Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 150000002540 isothiocyanates Chemical class 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000006654 negative regulation of apoptotic process Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/13—Amines
- A61K31/145—Amines having sulfur, e.g. thiurams (>N—C(S)—S—C(S)—N< and >N—C(S)—S—S—C(S)—N<), Sulfinylamines (—N=SO), Sulfonylamines (—N=SO2)
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明公开了莱菔硫烷在制备治疗白血病药物中的应用，属于莱菔硫烷的新用途技术领域。本发明的技术方案要点为：莱菔硫烷在制备治疗白血病药物中的应用，即莱菔硫烷通过上调Bax基因和Caspase‑3基因及下调Bcl‑2基因促进白血病细胞凋亡进而抑制白血病细胞的增殖。本发明首次系统探讨了SFN对急性白血病细胞KG1a和K562细胞凋亡的影响，结果证明SFN可以通过调控Bax基因、Bcl‑2基因和caspase‑3基因促进急性白血病细胞凋亡，这不仅是理论上的突破，而且具有潜在的临床意义，为治疗白血病提供了一种全新的思路和潜在的治疗路径。
Description
技术领域
本发明涉及莱菔硫烷的新用途技术领域，具体涉及莱菔硫烷在治疗白血病药物中的应用。
背景技术
莱菔硫烷（Sulforaphane，SFN）是一种存在于西兰花和其它十字花科蔬菜中的异硫氰酸盐物质，具有抗增殖能力，目前研究发现SFN对卵巢癌、膀胱癌、前列腺癌等多种恶性肿瘤细胞的生长具有抑制作用，是一种新型的抗癌成分。但是，SFN对造血肿瘤细胞的作用及机制则报道较少。
细胞周期失控是引起恶性增殖性疾病的重要原因之一。急性白血病是一类造血干细胞恶性克隆性疾病，白血病细胞因为增殖失控、分化障碍、凋亡受阻等机制在骨髓和其它造血组织中大量增殖，并浸润其它组织和器官，同时正常造血受抑制。Bcl-2基因家族是与细胞凋亡紧密相关的家族，其中Bcl-2和Bax分属Bcl-2家族中的抑制凋亡和促进凋亡的两部分，Bcl-2发挥保护细胞的作用，减少细胞凋亡，而Bax则发挥诱导细胞凋亡的作用，两者在细胞内的表达维持在平衡状态，形成一个凋亡调节系统，共同调控细胞增殖和凋亡。当Bax过度表达时形成Bax-Bax同源二聚体，促进细胞凋亡；随着Bcl-2表达量上升，Bax同源二聚体便分开，Bcl-2与Bax可以形成比Bax-Bax更稳定的Bax-Bcl-2二聚体，则抑制细胞的凋亡。因此，Bax/Bcl-2两蛋白之间的比例关系是决定对细胞凋亡抑制作用强弱以及恶性肿瘤预后的关键因素。含半胱氨酸的天冬氨酸蛋白水解（Caspase），Caspase是一组存在于细胞质中具有类似结构的蛋白酶。Caspase与真核细胞凋亡密切相关，并参与细胞的生长、分化与凋亡调节，也是细胞凋亡通路的重要组成部分。其中，Caspase-3被认为是一个涉及在不同的细胞系凋亡执行最常见的成员，这些蛋白质控制线粒体外膜通透性（MOMP）和线粒体膜间隙蛋白如细胞色素C的释放。
在各类成人白血病中，以急性髓系白血病（Acute myeloid leukaemia，AML）发病率最高，且化疗缓解率较低。KGla细胞和K562细胞是分别来源于急性髓系白血病耐药的细胞株和慢性白血病急性变的患者中分离建立的细胞株，是目前研究急性髓系白血病的理想模型，本发明以不同浓度的SFN作用KG1a细胞和K562细胞，观察其对白血病细胞增殖、凋亡及相关基因表达的影响，分析SFN抑制白血病细胞增殖的作用和机制。
发明内容
本发明的目的在于提供莱菔硫烷的新用途，即莱菔硫烷在制备治疗白血病药物中的新应用。
实际上，本发明涉及莱菔硫烷在制备治疗白血病药物中的新应用，即莱菔硫烷通过调控细胞凋亡抑制白血病细胞的增殖。
具体的，本发明涉及莱菔硫烷在制备治疗白血病药物中的新应用，即莱菔硫烷通过调控Bax基因、Bcl-2基因和Caspase-3基因诱导白血病细胞凋亡进而抑制白血病细胞的增殖。
本发明所涉及莱菔硫烷在制备治疗白血病药物中的新应用，即莱菔硫烷通过上调Bax基因和Caspase-3基因及下调Bcl-2基因促进白血病细胞凋亡进而抑制白血病细胞的增殖。
进一步限定，所述的白血病细胞为KG1a细胞和K562细胞。
本发明首次系统探讨了SFN对急性白血病细胞KG1a和K562细胞凋亡的影响，结果证明SFN可以通过调控Bax基因、Bcl-2基因和caspase-3基因促进急性白血病细胞凋亡，这不仅是理论上的突破，而且具有潜在的临床意义，为治疗白血病提供了一种全新的思路和潜在的治疗路径。
附图说明
图1是SFN对KGla细胞和K562细胞增殖活力的影响图，CCK8检测和显微镜观察显示不同浓度的SFN（0、4、8、12μmol/L）作用于KGla细胞和K562细胞后，细胞增殖活力随着细胞浓度和作用时间延长而降低；
图2是流式细胞仪检测SFN对KGla细胞和K562细胞凋亡的影响图，结果显示不同浓度的SFN（0、4、8、12μmol/L）作用KGla细胞48h的早期凋亡率分别为0、3.12%、13.33%、28.18%，不同浓度的SFN（0、4、8、12μmol/L）作用K562细胞48h的早期凋亡率分别为0、2.81%、10.68%、30.27%；
图3是Western blotting检测SFN对白血病细胞中Bax基因、Bcl-2基因和Caspase-3基因蛋白表达的影响图；
图4是RT-PCR检测SFN在白血病细胞中对凋亡基因Bax、Bcl-2和Caspase-3的影响图。
具体实施方式
以下通过实施例对本发明的上述内容做进一步详细说明，但不应该将此理解为本发明上述主题的范围仅限于以下的实施例，凡基于本发明上述内容实现的技术均属于本发明的范围。
实施例
1、实验材料
莱菔硫烷（批号28911701）购自美国LKT Laboratories公司，纯度98%。1640培养液和胎牛血清为美国Gibco公司产品。CCK-8试剂盒购自日本Dojindo公司，Annexin-V/PI细胞凋亡检测试剂（美国BD公司，批号5121706），RNA提取试剂盒、逆转录试剂盒、PCR试剂2×Taq PCRMaster Mix、DNA Marker均购自上海天根，琼脂糖（西班牙Biowest），引物由上海生物工程公司合成，兔抗人Bax mAb、兔抗人Bcl-2 mAb、兔抗人Caspase-3 mAb为美国Abcam公司产品，抗人GAPDH pAb及HRP-羊抗兔pAb为世纪康为公司提供。流式细胞仪（FACSCaibur型）为美国BD公司产品，MK3酶标仪和ND2000超微量分光光度计系美国 Thermo公司产品，凝胶图像分析系统由英国Genegenius公司生产，流式细胞仪为美国BD公司，Eclipse Ti-s倒置显微镜购自日本Nikon公司。
2、细胞培养
人急性髓系白血病细胞KGla和K562细胞株由本实验室保存，以含10%胎牛血清RPMI1640培养基培养，培养基中预先加入青霉素l00U/mL、链霉素100μg/mL、谷氨酰胺100μmol/L，置体积分数为5%的CO2培养箱于37℃培养，根据细胞生长状态及时换液传代，取对数生长期细胞做后续试验。
3、细胞增殖实验
将传代培养的对数生长期细胞，计数，按照每孔细胞密度为1×105/L接种于96孔培养板内，分别加入终浓度分别为0、2、4、6、8、10、12μmol/L的SFN（0.9% NS溶解稀释为2×103μmol/L），37℃培养24h、48h、72h后，每孔加入CCK-8试剂10uL继续孵育4h，在酶联免疫检测仪OD值为450nm处测量各孔的OD值，实验重复3次，取平均值。细胞相对存活率=（实验组-空白组）/（对照组-空白组）×100%。根据KG1a细胞存活率，绘制细胞生长曲线，选取合适SFN浓度做后续实验。
4、流式分析细胞凋亡
收集0、4、8、12μmol/L SFN处理后的KG1a细胞1×106/L，用预冷PBS洗涤细胞2次，加入100μL的1×Binding Buffer悬浮细胞；加5μL的Annexin V-FITC混匀后，再加入5μL的PI染色，4℃避光孵育15min后，加入400μL的1×Binding Buffer悬浮细胞，上流式细胞仪检测。每管获取10000个细胞，用CellQuest分析软件分析凋亡率。
5、western bloting分析Bax、Bcl-2和Caspase-3的蛋白
收集0、4、8、12μmol/L SFN作用48h后的KG1a细胞，用冰预冷的SDS裂解缓冲液溶解20min，12000×g离心15min，抽提蛋白，BCA蛋白浓度检测试剂盒测定蛋白含量，蛋白上样量为30μg，10wt% SDS-PAGE电泳；电转到PVDF膜，5wt%的脱脂奶粉封闭，1:1000一抗4℃过夜，TBST洗膜3次之后，1:5000二抗室温2h，再用TBST洗涤3次，ECL化学发光液显影拍照。用Image J对实验结果进行分析。
6、RT-PCR检测Bax、Bcl-2和Caspase-3的mRNA表达
收集0、4、8、12μmol/L SFN处理48h后的KG1a细胞，用Trizol法提取总RNA，用核酸蛋白定量仪检测RNA纯度和浓度，纯度在1.8-2.0之间，浓度在1-3μg/μL。用逆转录试剂盒进行逆转录合成cDNA，转录体系为20uL。Bax引物序列上游引物：5’-TTTGCTTCAGGGTTTCATCC-3’，下游引物5’-CAGTTGAAGTTGCCGTCAGA-3’，扩增产物246bp；Bcl-2引物序列上游引物：5’-GGATGCCTTTGTGGAACTGT-3’，下游引物：5’-AGCCTGCAGCTTTGTTTCAT-3’，扩增产物236bp；Caspase-3：5’-TGTTTGTGTGCTTCTGAGCC-3’，5’-CACGCCATGTCATCATCAAC-3’，扩增产物817bp；内参β-actin引物序列上游引物：5’- AGAGCTACGAGCTGCCTGAC-3’，下游引物5’-AGCACTGTGTTGGCGTACAG-3’，扩增产物183bp。所用引物由上海生工生物技术有限公司合成。PCR反应体系为20μL，PCR扩增试剂盒 Taq PCR Mastermix 10μL，上下游引物各0.4μL，cDNA1μL，95℃预变性3min，94℃ 30s，扩增参数55℃ 30s，72℃ 60s，30个循环，72℃延伸5min。取5μL PCR产物上样于1wt%的琼脂糖凝胶中，于80V电泳30min。取出凝胶置于凝胶成像分析系统中观察，Image J软件进行扫描灰度值并分析结果。
7、实验结果
1、从图1可以清楚看到SFN对KG1a细胞和k562细胞的生长增殖具有明显的抑制现象，并且随着SFN作用时间延长和剂量增大其增殖呈明显下降趋势。
2、从图2 可以看出SFN可以诱导白血病细胞的凋亡，在SFN作用KG1a细胞和k562细胞48h后，经过Annexin V-FITC/PI双染，流式细胞仪检测发现随着SFN浓度的增加，KG1a细胞的早期凋亡率逐渐增加，0、4、8、12μmol/L SFN作用KG1a细胞和K562细胞48h后，早期凋亡率分别为0、3.12%、13.33%、28.18%和0、2.81%、28.18%和30.27%。由此可知SFN可以诱导白血病细胞凋亡，并且随着SFN浓度的增加，KG1a细胞和K562细胞的早期凋亡率呈逐渐增加趋势。
3、从图3可以看出用0、4、8、12μmol/L的莱菔硫烷干预KG1a细胞、K562细胞48h后，Bax和Caspase-3蛋白表达显著增高，各浓度组与0μmol/L比较差异有统计学意义（P﹤0.05），而Bcl-2表达有所下降，12μmol/L与0μmol/L比较差异有统计学意义（P﹤0.05）。结果表明莱菔硫烷可能通过上调Bax基因，下调Bc-l2基因表达促进细胞凋亡，并且这种表达调控存在于蛋白表达水平。
4、图4采用了PT-PCR检测方法，结果进一步验证了莱菔硫烷具有上调Bax基因和Caspase-3基因及下调Bcl-2基因的功能，莱菔硫烷可以通过上调Bax基因和Caspase-3基因及下调Bcl-2基因促进白血病细胞凋亡，进而抑制白血病细胞的增殖。
以上实施例描述了本发明的基本原理、主要特征及优点，本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明原理的范围下，本发明还会有各种变化和改进，这些变化和改进均落入本发明保护的范围内。
SEQUENCE LISTING
<110> 新乡医学院
<120> 莱菔硫烷在制备治疗白血病药物中的应用
<130> 2017
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> RNA
<213> 人工序列
<400> 1
TTTGCTTCAGGGTTTCATCC 20
<210> 2
<211> 20
<212> RNA
<213> 人工序列
<400> 1
CAGTTGAAGTTGCCGTCAGA 20
<210> 3
<211> 20
<212> RNA
<213> 人工序列
<400> 1
GGATGCCTTTGTGGAACTGT 20
<210> 4
<211> 20
<212> RNA
<213> 人工序列
<400> 1
AGCCTGCAGCTTTGTTTCAT 20
<210> 5
<211> 20
<212> RNA
<213> 人工序列
<400> 1
TGTTTGTGTGCTTCTGAGCC 20
<210> 6
<211> 20
<212> RNA
<213> 人工序列
<400> 1
CACGCCATGTCATCATCAAC 20
<210> 7
<211> 20
<212> RNA
<213> 人工序列
<400> 1
AGAGCTACGAGCTGCCTGAC 20
<210> 8
<211> 20
<212> RNA
<213> 人工序列
<400> 1
AGCACTGTGTTGGCGTACAG 20
Claims (5)
1.莱菔硫烷在制备治疗白血病药物中的应用。
2.根据权利要求1所述的莱菔硫烷在制备治疗白血病药物中的应用，其特征在于：所述的莱菔硫烷通过调控细胞凋亡抑制白血病细胞的增殖。
3.根据权利要求2所述的莱菔硫烷在制备治疗白血病药物中的应用，其特征在于：所述的莱菔硫烷通过调控Bax基因、Bcl-2基因和Caspase-3基因诱导白血病细胞凋亡进而抑制白血病细胞的增殖。
4.根据权利要求3所述的莱菔硫烷在制备治疗白血病药物中的应用，其特征在于：所述的莱菔硫烷通过上调Bax基因和Caspase-3基因及下调Bcl-2基因促进白血病细胞凋亡进而抑制白血病细胞的增殖。
5.根据权利要求2-4中任意一项所述的莱菔硫烷在制备治疗白血病药物中的应用，其特征在于：所述的白血病细胞为KG1a细胞和K562细胞。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710109473.9A CN107007578A (zh) | 2017-02-27 | 2017-02-27 | 莱菔硫烷在制备治疗白血病药物中的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710109473.9A CN107007578A (zh) | 2017-02-27 | 2017-02-27 | 莱菔硫烷在制备治疗白血病药物中的应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107007578A true CN107007578A (zh) | 2017-08-04 |
Family
ID=59440546
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710109473.9A Pending CN107007578A (zh) | 2017-02-27 | 2017-02-27 | 莱菔硫烷在制备治疗白血病药物中的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107007578A (zh) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115515686A (zh) * | 2020-03-18 | 2022-12-23 | 纽约州州立大学研究基金会 | 修饰的短干扰rna组合物及其在癌症治疗中的用途 |
| CN115844869A (zh) * | 2022-12-19 | 2023-03-28 | 新乡医学院 | 莱菔硫烷在逆转耐阿糖胞苷白血病细胞耐药性中的应用和药物组合物 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101120948A (zh) * | 2006-08-08 | 2008-02-13 | 普文英 | 一种具有化学保护、癌症预防和治疗用途的化合物组合物 |
| CN101474170A (zh) * | 2008-12-04 | 2009-07-08 | 中国人民解放军第三军医大学 | 异硫氰酸酯类化合物在制备治疗白血病的药物中的应用 |
| WO2010065329A2 (en) * | 2008-11-25 | 2010-06-10 | The Board Of Regents Of The University Of Texas System | Nanoparticles for cancer treatment |
-
2017
- 2017-02-27 CN CN201710109473.9A patent/CN107007578A/zh active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101120948A (zh) * | 2006-08-08 | 2008-02-13 | 普文英 | 一种具有化学保护、癌症预防和治疗用途的化合物组合物 |
| WO2010065329A2 (en) * | 2008-11-25 | 2010-06-10 | The Board Of Regents Of The University Of Texas System | Nanoparticles for cancer treatment |
| CN101474170A (zh) * | 2008-12-04 | 2009-07-08 | 中国人民解放军第三军医大学 | 异硫氰酸酯类化合物在制备治疗白血病的药物中的应用 |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115515686A (zh) * | 2020-03-18 | 2022-12-23 | 纽约州州立大学研究基金会 | 修饰的短干扰rna组合物及其在癌症治疗中的用途 |
| CN115844869A (zh) * | 2022-12-19 | 2023-03-28 | 新乡医学院 | 莱菔硫烷在逆转耐阿糖胞苷白血病细胞耐药性中的应用和药物组合物 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Meng et al. | Crosstalk between integrin αvβ3 and estrogen receptor-α is involved in thyroid hormone-induced proliferation in human lung carcinoma cells | |
| Han et al. | Triptonide potently suppresses pancreatic cancer cell-mediated vasculogenic mimicry by inhibiting expression of VE-cadherin and chemokine ligand 2 genes | |
| Wang et al. | Methionine enkephalin (MENK) inhibits human gastric cancer through regulating tumor associated macrophages (TAMs) and PI3K/AKT/mTOR signaling pathway inside cancer cells | |
| Xiang et al. | Deregulation of miR-520d-3p promotes hepatocellular carcinoma development via lncRNA MIAT regulation and EPHA2 signaling activation | |
| Yang et al. | Transcriptomics and proteomics analyses of anti-cancer mechanisms of TR35–An active fraction from Xinjiang Bactrian camel milk in esophageal carcinoma cell | |
| Huang et al. | Antiproliferation effect of evodiamine in human colon cancer cells is associated with IGF-1/HIF-1α downregulation | |
| Guo et al. | Salvianolic acid B reverses multidrug resistance in nude mice bearing human colon cancer stem cells | |
| Shi et al. | Anticancer effect of 20 (S)-ginsenoside Rh2 on HepG2 liver carcinoma cells: Activating GSK-3β and degrading β-catenin | |
| Zhang et al. | Kaempferol suppresses androgen-dependent and androgen-independent prostate cancer by regulating Ki67 expression | |
| Wang et al. | Aspirin ameliorates cerebral infarction through regulation of TLR4/NF‑κB‑mediated endoplasmic reticulum stress in mouse model | |
| Zhu et al. | Sennoside A alleviates inflammatory responses by inhibiting the hypermethylation of SOCS1 in CCl4-induced liver fibrosis | |
| Xu et al. | Astemizole promotes the anti-tumor effect of vitamin D through inhibiting miR-125a-5p-meidated regulation of VDR in HCC | |
| Fan et al. | Intervention effects of QRZSLXF, a Chinese medicinal herb recipe, on the DOR-β-arrestin1-Bcl2 signal transduction pathway in a rat model of ulcerative colitis | |
| Zhu et al. | Melittin inhibits lung metastasis of human osteosarcoma: Evidence of wnt/β-catenin signaling pathway participation | |
| Liu et al. | Anti-tumor effects of Skp2 inhibitor AAA-237 on NSCLC by arresting cell cycle at G0/G1 phase and inducing senescence | |
| Yang et al. | Circ DENND4C inhibits pyroptosis and alleviates ischemia-reperfusion acute kidney injury by exosomes secreted from human urine-derived stem cells | |
| Li et al. | Synergism of ursolic acid and cisplatin promotes apoptosis and enhances growth inhibition of cervical cancer cells via suppressing NF-κB p65 | |
| Ebrahim et al. | Prophylactic evidence of MSCs-derived exosomes in doxorubicin/trastuzumab-induced cardiotoxicity: beyond mechanistic target of NRG-1/Erb signaling pathway | |
| Xue et al. | Overexpression of FYN suppresses the epithelial-to-mesenchymal transition through down-regulating PI3K/AKT pathway in lung adenocarcinoma | |
| Liang et al. | Preserved egg digests promote the apoptosis of HT29 and HepG2 cells | |
| Qin et al. | Soluble CD40 ligands sensitize the epithelial ovarian cancer cells to cisplatin treatment | |
| CN107007578A (zh) | 莱菔硫烷在制备治疗白血病药物中的应用 | |
| Wei et al. | Knockdown of Rhotekin 2 expression suppresses proliferation and invasion and induces apoptosis in hepatocellular carcinoma cells | |
| Chen et al. | Flavokawain B inhibits NF-κB inflammatory signaling pathway activation in inflammatory bowel disease by targeting TLR2 | |
| Chang et al. | Antcin K ameliorates cardiotoxin-induced skeletal muscle injury and inflammation via IL-10 regulation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170804 |
|
| RJ01 | Rejection of invention patent application after publication |