本文選題：Insulin/EGFP基因 + 轉(zhuǎn)染　； 參考：《蘭州大學(xué)》2010年碩士論文

【摘要】： 目的：1,探討人臍帶間充質(zhì)干細(xì)胞(human umbilical cord mesenchymal stem cells hUCMSCs)體外分離、培養(yǎng)、純化條件,并對(duì)hUCMSCs的生物學(xué)特性進(jìn)行研究；2,構(gòu)建含人重組胰島素(insulin)與增強(qiáng)型綠色熒光蛋白(EGFP)基因慢病毒表達(dá)載體pLenti6.3-insulin-IRES-EGFP,并進(jìn)行慢病毒顆粒的包裝,純化,濃縮及滴度測(cè)定；3,以GFP作為報(bào)告基因,篩選慢病毒轉(zhuǎn)染人臍帶間充質(zhì)干細(xì)胞最適MOI值,為進(jìn)一步進(jìn)行攜帶外源基因的轉(zhuǎn)染探索條件；4,研究攜帶重組人胰島素慢病毒顆粒轉(zhuǎn)染人臍帶間充質(zhì)干細(xì)胞后,基因及蛋白水平的表達(dá)情況,為進(jìn)一步進(jìn)行體內(nèi)回植提供研究基礎(chǔ)。 方法：1,將剔除動(dòng)脈和靜脈的新鮮人臍帶組織剪成小塊培養(yǎng)(1mm×1mm×1mm),培養(yǎng)1-2周后得到貼壁細(xì)胞,倒置相差顯微鏡下觀察細(xì)胞形態(tài),細(xì)胞計(jì)數(shù)繪制細(xì)胞生長(zhǎng)曲線；應(yīng)用流式細(xì)胞儀鑒定細(xì)胞表面標(biāo)志；化學(xué)染色檢測(cè)體外誘導(dǎo)成脂和成骨分化的能力；2,應(yīng)用PCR方法擴(kuò)增獲得insulin基因序列,在目的基因上游加上BamHI,下游加上AscI兩個(gè)酶切位點(diǎn)；將PCR產(chǎn)物進(jìn)行T載體克隆,轉(zhuǎn)化入感受態(tài)DH5α細(xì)胞中,通過篩選獲得重組質(zhì)粒pMD18T-insulin,測(cè)序分析選定序列正確的克隆提取質(zhì)粒；用限制性內(nèi)切酶分別酶切pMD18T-insulin和pLenti6.3-IRES-EGFP質(zhì)粒,將insulin基因片段和pLenti6.3-IRES-EGFP載體連接,轉(zhuǎn)化入感受態(tài)DH5α細(xì)胞中,通過篩選獲得慢病毒表達(dá)載體pLenti6.3-insulin-IRES-EGFP,并進(jìn)行測(cè)序。中量抽提慢病毒載體,將慢病毒表達(dá)載體轉(zhuǎn)染293T細(xì)胞,包裝病毒,純化及濃縮并測(cè)定病毒滴度。3,以EGFP作為報(bào)告基因,分別以MOI值0,1,3,5,7,10,15,20轉(zhuǎn)染細(xì)胞,觀察48小時(shí),72小時(shí),96小時(shí),熒光表達(dá)情況,篩選慢病毒轉(zhuǎn)染人臍帶間充質(zhì)干細(xì)胞最適轉(zhuǎn)染時(shí)間及MOI值；4,轉(zhuǎn)染人臍帶間充質(zhì)干細(xì)胞,應(yīng)用qPCR及RT-PCR方法檢測(cè)重組人胰島素基因表達(dá)情況,應(yīng)用westen bloting方法檢測(cè)胰島素蛋白表達(dá)情況。 結(jié)果：1,體外培養(yǎng)一周,細(xì)胞從組織塊中爬出；細(xì)胞傳代培養(yǎng)至第10代,無(wú)明顯形態(tài)及增值能力改變；細(xì)胞陽(yáng)性表達(dá)CD44, CD106,而CD34, HLA-DR表達(dá)陰性。體外誘導(dǎo)實(shí)驗(yàn)證實(shí),hUCMSCs具有成脂及成骨分化的能力。2,聚合酶鏈反應(yīng)獲得長(zhǎng)度347bp帶有BamHI和AscI兩個(gè)酶切位點(diǎn)序列的目的基因,經(jīng)與克隆載體pMD18-T載體和慢病毒表達(dá)載體pLenti6.3-IRES-EGFP連接,構(gòu)建慢病毒表達(dá)載體pLenti6.3-insulin-IRES-EGFP。成功包裝病毒,純化及濃縮并測(cè)定病毒滴度。3,重組慢病毒載體具有較高的轉(zhuǎn)染效率,當(dāng)轉(zhuǎn)染復(fù)數(shù)(MOI)值是10時(shí),轉(zhuǎn)染效率最高可達(dá)到90%。應(yīng)用qPCR及RT-PCR方法檢測(cè)重組人胰島素基因表達(dá)情況,證實(shí)基因水平的表達(dá)量,轉(zhuǎn)染組為陽(yáng)性,未轉(zhuǎn)染組為陰性。應(yīng)用westen bloting方法檢測(cè)胰島素蛋白表達(dá)情況,證實(shí)轉(zhuǎn)染組細(xì)胞及上清表達(dá)陽(yáng)性；未轉(zhuǎn)染組表達(dá)均為陰性。 結(jié)論：1,hUCMSCs能在體外培養(yǎng)、擴(kuò)增并具骨髓間充質(zhì)干細(xì)胞相似的生物學(xué)特性；2,成功構(gòu)建慢病毒表達(dá)載體pLenti6.3-insulin-IRES-EGFP并包裝病毒顆粒純化及濃縮并測(cè)定病毒滴度；3,篩選出最適MOI值為10,攜帶重組人胰島素基因慢病毒顆粒轉(zhuǎn)染hUCMSCs后,其在基因及蛋白水平均陽(yáng)性表達(dá)胰島素。
[Abstract]:Objective: 1, to study the isolation, culture and purification of human umbilical cord mesenchymal stem cells (human umbilical cord mesenchymal stem cells hUCMSCs) in vitro, and to study the biological characteristics of hUCMSCs; 2, to construct a lentivirus expression vector containing recombinant human recombinant insulin (insulin) and enhanced green fluorescein (EGFP) gene. -EGFP, and carry out the packaging, purification, concentration and titer determination of lentivirus particles; 3, using GFP as the reporter gene, screening the optimal MOI value of lentivirus transfected human umbilical cord mesenchymal stem cells to further carry out transfection conditions with exogenous gene, and 4, study the transfection of human umbilical cord mesenchymal stem cells with recombinant human insulin lentivirus particles. The expression level of gene and protein will provide a basis for further research on in vivo replantation.
Methods: 1, the fresh human umbilical cord tissues removed from the arteries and veins were cut into small mass culture (1mm x 1mm x 1mm). After 1-2 weeks of culture, the adherent cells were obtained. The cell morphology was observed under the inverted phase contrast microscope and the cell growth curve was plotted by the cell count; the cell surface markers were identified by the flow cytometry; the chemical staining was used to detect the formation of lipid and formation in vitro. The ability of bone differentiation: 2, the insulin gene sequence was amplified by PCR method, the upstream of the target gene was added with BamHI, and the downstream was added with the AscI two enzyme cutting sites, and the PCR product was cloned by T vector and transformed into the receptor DH5 alpha cells. The recombinant plasmid pMD18T-insulin was obtained by screening, and the correct cloning extract was sequenced and analyzed. PMD18T-insulin and pLenti6.3-IRES-EGFP plasmids were cut by restriction endonuclease, the insulin gene fragment and pLenti6.3-IRES-EGFP vector were linked and transformed into the receptive DH5 alpha cells. The Lentivirus Expression Vector pLenti6.3-insulin-IRES-EGFP was screened and sequenced. The lentivirus vector was extracted from the lentivirus vector, and the lentivirus vector was extracted. The vector transfected 293T cells, packed the virus, purified and condensed the virus titer.3, and transfected EGFP as the reporter gene, transfected cells with MOI value 0,1,3,5,7,10,15,20 respectively, observed 48 hours, 72 hours, 96 hours, fluorescence expression, screening the optimal transfection time and MOI value of human umbilical cord mesenchymal stem cells transfected by lentivirus; 4, transfection of human umbilical cord The expression of recombinant human insulin gene was detected by qPCR and RT-PCR, and the expression of Insulin protein was detected by westen bloting.
1, 1, in vitro culture, the cells crawled out of the tissue block, and the cells were cultured to tenth generations without obvious morphologic and value-added changes. The positive expression of CD44, CD106, and CD34, HLA-DR expression was negative. In vitro induction experiments confirmed that hUCMSCs has the ability of lipid and osteogenic differentiation.2, and the polymerase chain reaction obtained the length 347bp The target genes of the two enzyme cutting sites of BamHI and AscI were linked with the cloned carrier pMD18-T vector and the Lentivirus Expression Vector pLenti6.3-IRES-EGFP to construct the Lentivirus Expression Vector pLenti6.3-insulin-IRES-EGFP. to successfully package the virus, purify and concentrate and determine the virus titer.3. The recombinant lentivirus carrier has high transfection efficiency. The transfection complex (MOI) value was 10, the highest transfection efficiency could reach the 90%. application qPCR and RT-PCR method to detect the recombinant human insulin gene expression. The expression of gene level was confirmed, the transfection group was positive and the untransfected group was negative. The expression of Insulin protein was detected by westen bloting method, and the expression of cell and supernatant in the transfected group was confirmed. The expression of untransfected group was negative.
Conclusion: 1, hUCMSCs can be cultured in vitro, amplification and similar biological characteristics of bone marrow mesenchymal stem cells. 2, successful construction of Lentivirus Expression Vector pLenti6.3-insulin-IRES-EGFP and packaging virus particles to purify and concentrate and determine virus titer; 3, the optimum MOI value is 10, carrying recombinant human insulin gene lentivirus particles turn. After hUCMSCs staining, they expressed insulin at both gene and protein levels.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329

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