电穿孔法转染hTERT基因至脂肪来源干细胞增强其增殖能力的研究

发布时间：2018-03-25 13:12

  本文选题：穿孔　切入点：法转　出处：《复旦大学》2009年硕士论文

【摘要】： 1.目的脂肪干细胞(adipose derived stromal cells,ADSCs)是一类有增殖分化潜能的间充质细胞,但其传代次数往往有限,实验中无法有一个标准用细胞系,造成偏差,利用电穿孔技术,将人端粒酶催化亚基(human telomerase reverse transcriptase,hTERT)转染至SD大鼠脂肪来源干细胞(adipose derived stromal cells,ADSCs),观察及检测外源性hTERT在ADSCs内表达及对ADSCs寿命及增殖能力的影响。 2.方法 (1)参照GenBank的hTERT基因序列设计引物,按照Qiagen RNA试剂盒提取程序从人食管癌组织中提取hTERT RNA,逆转录为cDNA,PCR扩增目的基因,hindⅢ酶切pcDNA3.1质粒,将目的基因与载体pcDNA3.1连接,构建pcDNA3.1-hTERT质粒。 (2)提取SD大鼠腹股沟脂肪组织,反复冲洗,组织剪剪碎至1mm大小,Ⅰ型胶原酶消化30min,200目微孔过滤后离心收集沉淀,种植并且培养细胞。 (3)利用电穿孔技术,将hTERT-pcDNA3.1转染ADSCs,PCR法检测hTERT表达,G418筛选1月,回收ADSCs,按照TRAP-PCR银染法端粒酶活性检测试剂盒检说明书测端粒酶活性,运用Pl-annexin V染色,流式细胞仪测第15代转染及未转染ADSCs细胞凋亡比例。观察转染前后细胞形态变化以及第15代转染与未转染细胞生长活力区别。 3.结果:成功提取人食管癌组织中hTERT片段,PAGE及DNA测序证实符合目标基因,成功构建hTERT-pcDNA3.1质粒,成功利用电穿孔技术转染,ADSCs细胞内hTERT RNA表达,TRAP法检测端粒酶活性增强,细胞形态无明显变化,15代细胞Pl-annexin V染色检验细胞凋亡比例证实转染后15代细胞凋亡比例明显减少。细胞生长活力明显增强。 4.结论:成功构建hTER-ADSCs细胞,为构建ADSCs永生化细胞系打下基础。
[Abstract]:1. the purpose of adipose derived stem cells (adipose derived stromal cells, ADSCs) is a kind of proliferation and differentiation potential of mesenchymal cells, but its passage is often limited, the experiment cannot have a standard deviation, with cells by electroporation, the human telomerase catalytic subunit (human telomerase reverse transcriptase, hTERT) was transfected into SD rat adipose derived stem cells (adipose derived stromal cells, ADSCs), to observe the expression and detection of exogenous hTERT in ADSCs and the impact on the life of ADSCs and proliferation.
2. method
(1) hTERT primers were designed according to the gene sequence of GenBank, extraction program to extract the hTERT RNA from human esophageal carcinoma Qiagen RNA kit according to reverse transcription cDNA, PCR gene, hind restriction enzyme pcDNA3.1 plasmid, the target gene connected with pcDNA3.1 vector to construct pcDNA3.1-hTERT plasmid.
(2) extract the fat tissue of groin in SD rats, wash it repeatedly, cut the tissue to 1mm size, digest 30min with type I collagenase, centrifuge, collect and precipitate after 200 mesh micropore filtration, plant and culture cells.
(3) by electroporation, transfection of hTERT-pcDNA3.1 ADSCs, PCR method to detect the expression of hTERT, G418 screening in January, according to the recovery of ADSCs TRAP-PCR silver staining telomerase activity detection kit inspection manual measurement of telomerase activity by Pl-annexin V staining, measured fifteenth transfected and untransfected ADSCs cells apoptosis rate was observed by flow cytometry. Before and after the changes of cell morphology and the fifteenth generation of transfected and untransfected cell growth vigor difference.
3. results: the successful extraction of human esophageal carcinoma hTERT fragments, PAGE and DNA sequencing with target gene, hTERT-pcDNA3.1 plasmid was successfully constructed and successfully transfected by electroporation, the expression of hTERT RNA in ADSCs cells, enhance the telomerase activity by TRAP assay, cells had no significant morphological changes, cells of the 15 generation Pl-annexin V staining test confirmed the apoptosis ratio after transfection 15 cells apoptosis was significantly reduced. The cell growth activity was significantly enhanced.
4. conclusion: hTER-ADSCs cells were successfully constructed to lay a foundation for the construction of ADSCs immortalized cell lines.

【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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