本文選題：穿孔　切入點(diǎn)：法轉(zhuǎn)　出處：《復(fù)旦大學(xué)》2009年碩士論文

【摘要】： 1.目的脂肪干細(xì)胞(adipose derived stromal cells,ADSCs)是一類有增殖分化潛能的間充質(zhì)細(xì)胞,但其傳代次數(shù)往往有限,實(shí)驗(yàn)中無法有一個(gè)標(biāo)準(zhǔn)用細(xì)胞系,造成偏差,利用電穿孔技術(shù),將人端粒酶催化亞基(human telomerase reverse transcriptase,hTERT)轉(zhuǎn)染至SD大鼠脂肪來源干細(xì)胞(adipose derived stromal cells,ADSCs),觀察及檢測外源性hTERT在ADSCs內(nèi)表達(dá)及對ADSCs壽命及增殖能力的影響。 2.方法 (1)參照GenBank的hTERT基因序列設(shè)計(jì)引物,按照Qiagen RNA試劑盒提取程序從人食管癌組織中提取hTERT RNA,逆轉(zhuǎn)錄為cDNA,PCR擴(kuò)增目的基因,hindⅢ酶切pcDNA3.1質(zhì)粒,將目的基因與載體pcDNA3.1連接,構(gòu)建pcDNA3.1-hTERT質(zhì)粒。 (2)提取SD大鼠腹股溝脂肪組織,反復(fù)沖洗,組織剪剪碎至1mm大小,Ⅰ型膠原酶消化30min,200目微孔過濾后離心收集沉淀,種植并且培養(yǎng)細(xì)胞。 (3)利用電穿孔技術(shù),將hTERT-pcDNA3.1轉(zhuǎn)染ADSCs,PCR法檢測hTERT表達(dá),G418篩選1月,回收ADSCs,按照TRAP-PCR銀染法端粒酶活性檢測試劑盒檢說明書測端粒酶活性,運(yùn)用Pl-annexin V染色,流式細(xì)胞儀測第15代轉(zhuǎn)染及未轉(zhuǎn)染ADSCs細(xì)胞凋亡比例。觀察轉(zhuǎn)染前后細(xì)胞形態(tài)變化以及第15代轉(zhuǎn)染與未轉(zhuǎn)染細(xì)胞生長活力區(qū)別。 3.結(jié)果:成功提取人食管癌組織中hTERT片段,PAGE及DNA測序證實(shí)符合目標(biāo)基因,成功構(gòu)建hTERT-pcDNA3.1質(zhì)粒,成功利用電穿孔技術(shù)轉(zhuǎn)染,ADSCs細(xì)胞內(nèi)hTERT RNA表達(dá),TRAP法檢測端粒酶活性增強(qiáng),細(xì)胞形態(tài)無明顯變化,15代細(xì)胞Pl-annexin V染色檢驗(yàn)細(xì)胞凋亡比例證實(shí)轉(zhuǎn)染后15代細(xì)胞凋亡比例明顯減少。細(xì)胞生長活力明顯增強(qiáng)。 4.結(jié)論:成功構(gòu)建hTER-ADSCs細(xì)胞,為構(gòu)建ADSCs永生化細(xì)胞系打下基礎(chǔ)。
[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.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R329

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