發(fā)布時(shí)間：2018-06-01 10:50

  本文選題：血管生成素基因 + T細(xì)胞　； 參考：《山東大學(xué)學(xué)報(bào)(醫(yī)學(xué)版)》2016年12期

【摘要】：目的通過基因重組技術(shù)構(gòu)建人血管生成素-1(Ang-1)基因慢病毒表達(dá)載體,并檢測其在人臍帶間充質(zhì)干細(xì)胞(hUC-MSCs)的表達(dá)及對(duì)hUC-MSCs免疫抑制能力的影響。方法應(yīng)用Trizol法從hUC-MSCs提取總RNA,反轉(zhuǎn)錄獲取c DNA,PCR擴(kuò)增獲得編碼Ang-1的序列克隆到GV287載體中。將重組GV287-Ang-1載體質(zhì)粒和慢病毒包裝質(zhì)粒pHelper 1.0和p Helper 2.0共轉(zhuǎn)染293T細(xì)胞,收集病毒上清,純化濃縮測定病毒滴度。采用熒光顯微鏡觀察轉(zhuǎn)染效率,Western blotting法檢測Ang-1蛋白表達(dá),并通過CCK8試劑盒檢測T淋巴細(xì)胞增殖活性。結(jié)果 Ang-1基因擴(kuò)增PCR產(chǎn)物與預(yù)期大小一致。重組慢病毒GV287-Ang-1質(zhì)粒經(jīng)PCR和DNA測序分析顯示,所得結(jié)果與目的基因序列一致且插入方向正確。包裝慢病毒濃縮懸液滴度為2×108TU/m L,最佳感染復(fù)數(shù)為8。GV287-Ang-1轉(zhuǎn)染組細(xì)胞Ang-1表達(dá)顯著高于未轉(zhuǎn)染組和GV287轉(zhuǎn)染組。過表達(dá)Ang-1的hUC-MSCs對(duì)T淋巴細(xì)胞的增殖抑制顯著高于單純的hUC-MSCs。結(jié)論成功構(gòu)建攜帶Ang-1基因的慢病毒載體GV287-Ang-1,并可有效轉(zhuǎn)染hUC-MSCs過表達(dá)Ang-1蛋白,且能顯著提高h(yuǎn)UC-MSCs的免疫抑制能力。
[Abstract]:Objective to construct the lentivirus expression vector of human angiopoietin-1 (Ang-1) gene and to detect its expression in human umbilical cord mesenchymal stem cells (hUC-MSCs) and its effect on the immunosuppressive ability of human angiopoietin-1 (Ang-1) gene in human umbilical cord mesenchymal stem cells (hUC-MSCs). Methods Trizol was used to extract total RNAs from hUC-MSCs and reverse transcription-PCR was used to amplify the coding Ang-1 sequence into GV287 vector. The recombinant GV287-Ang-1 vector plasmid and lentivirus packaging plasmid pHelper 1.0 and p Helper 2.0 were co-transfected into 293T cells to collect the virus supernatant and purify and concentrate to determine the titer of the virus. The transfection efficiency was observed by fluorescence microscope and the expression of Ang-1 protein was detected by Western blotting. The proliferative activity of T lymphocytes was detected by CCK8 kit. Results the PCR products amplified by Ang-1 gene were consistent with the expected size. The recombinant lentivirus GV287-Ang-1 plasmid was sequenced by PCR and DNA. The results were consistent with the target gene sequence and the insertion direction was correct. The concentration and suspension titer of lentivirus was 2 脳 108TU/m L, and the best number of infection was that the Ang-1 expression in the 8.GV287-Ang-1 transfected group was significantly higher than that in the untransfected group and GV287 transfection group. The inhibition of T lymphocyte proliferation by hUC-MSCs with overexpression of Ang-1 was significantly higher than that of hUC-MSCs. Conclusion the lentivirus vector GV287-Ang-1 carrying Ang-1 gene can be successfully constructed, and can effectively transfect hUC-MSCs overexpression of Ang-1 protein, and can significantly improve the immunosuppressive ability of hUC-MSCs.
【作者單位】： 山東大學(xué)齊魯兒童醫(yī)院兒科醫(yī)學(xué)研究所;山東大學(xué)齊魯醫(yī)院低溫醫(yī)學(xué)研究室;山東大學(xué)齊魯兒童醫(yī)院血液腫瘤科;
【基金】：山東省自然科學(xué)基金(ZR2013HM001)
【分類號(hào)】：R457.7

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