天堂国产午夜亚洲专区-少妇人妻综合久久蜜臀-国产成人户外露出视频在线-国产91传媒一区二区三区

電穿孔法轉(zhuǎn)染hTERT基因至脂肪來源干細(xì)胞增強(qiáng)其增殖能力的研究

發(fā)布時(shí)間:2018-03-25 13:12

  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

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 程飚,陳崢嶸,吳志偉;PLGA生物膜和延長的神經(jīng)軸突束構(gòu)建人工神經(jīng)的體外研究[J];中華創(chuàng)傷骨科雜志;2004年07期

,

本文編號:1663270

資料下載
論文發(fā)表

本文鏈接:http://sikaile.net/yixuelunwen/shiyanyixue/1663270.html


Copyright(c)文論論文網(wǎng)All Rights Reserved | 網(wǎng)站地圖 |

版權(quán)申明:資料由用戶b0ec6***提供,本站僅收錄摘要或目錄,作者需要?jiǎng)h除請E-mail郵箱bigeng88@qq.com
91欧美一区二区三区成人| 日本一本在线免费福利| 日本人妻熟女一区二区三区| 久久这里只有精品中文字幕| 午夜小视频成人免费看| 精品久久综合日本欧美| 中文字幕一区二区三区中文| 午夜激情视频一区二区| 毛片在线观看免费日韩| 欧美夫妻性生活一区二区| 久久精品伊人一区二区| 青青草草免费在线视频| 黑人粗大一区二区三区| 99国产一区在线播放| 国产精品免费视频久久| 国产高清一区二区白浆| 三级高清有码在线观看| 国产精品香蕉在线的人| 国产精品一区二区有码| 中文字幕日韩精品人一妻| 国产成人精品在线播放| 国内女人精品一区二区三区| 自拍偷拍一区二区三区| 91天堂免费在线观看| 日韩特级黄片免费观看| 91精品欧美综合在ⅹ| 精品老司机视频在线观看| 免费在线成人午夜视频| 好吊妞视频免费在线观看| 国产免费一区二区三区不卡| 成年人视频日本大香蕉久久| 国产香蕉国产精品偷在线观看| 亚洲国产欧美精品久久| 精品香蕉一区二区在线| 国产欧美一区二区另类精品| 精品欧美一区二区三久久| 国产偷拍盗摄一区二区| 日韩一区二区三区18| 欧美日韩中黄片免费看| 又色又爽又黄的三级视频| 国产一区二区不卡在线播放|