重组逆转录病毒载体PLXSN-GDNF转染人脐带间充质干细胞的实验研究

发布时间：2018-06-04 08:13

  本文选题：脐带间充质干细胞 + 胶质细胞源性神经营养因子　； 参考：《郑州大学》2012年硕士论文

【摘要】：目的 构建GDNF基因修饰的脐带间充质干细胞(UCMSCs),为组织工程化神经的构建打下基础。 方法 1.用CaCl2法制备感受态大肠杆菌,重组逆转录病毒载体质粒(第三军医大学阮怀珍教授惠赠)转化入感受态细菌并进行抗性筛选,抽提纯化质粒。酶切、测序检测目的基因的正确性。 2.复苏包装细胞PA317,用贴壁培养法培养、传代扩增,待细胞生长至80%融合时,脂质体转染法将重组逆转录病毒PLXSN-GDNF转染进入PA317包装细胞,G418筛选产毒克隆细胞,收集病毒上清液并测定病毒滴度。 3.取健康剖腹产产妇捐献的脐带,用组织块法培养获得原代细胞、胰酶消化传代,取第2代脐带间充质干细胞(UCMSCs),免疫组织化学法检测细胞表面抗原CD44、CD105、CD106、CD34、CD45的表达情况。 4.病毒上清感染脐带间充质干细胞后筛选转基因细胞,即GDNF-UCMSCs,收集细胞并用反转录聚合酶链反应法(RT-PCR)检测GDNF在mRNA水平的表达。 结果 1.Xhol Ⅰ单酶切重组质粒,出现0.66kb和5.93kb两个片段,与预期结果相符。测序检测结果显示重组逆转录病毒PLXSN-GDNF中大鼠GDNF的编码序列完整。 2.用小鼠成纤维细胞NIH3T3检测逆转录病毒滴度,挑选四个克隆检测,最高病毒滴度为6.56×104cfu/ml,选用该病毒上清液感染细胞。 3.原代培养1周时组织块之间出现贴壁的成纤维样细胞,14天左右集落细胞达到80%-90%融合,成螺旋状或放射状。免疫组化检测细胞表面抗原示：CD44、CD105强阳性,CD106弱阳性,CD34、CD45阴性,与脐带间充质干细胞(UCMSCs)的生物学特性相符。 4. RT-PCR法检测显示,GDNF-UCMSCs组呈阳性,而UCMSCs组呈阴性,转基因组GDNF mRNA水平表达明显强于对照组,P0.01,差异有统计学意义,可知GDNF基因在mRNA水平得以表达。 结论 1.获赠的逆转录病毒载体PLXSN-GDNF中大鼠GDNF基因的编码序列完整。 2.成功培养出人脐带间充质干细胞,免疫组织化学染色鉴定结果与脐带间充质干细胞的生物学特征相符合。 3.成功构建了GDNF基因修饰的脐带间充质干细胞,并在mRNA水平得以表达,为组织化工程神经的构建打下了基础。
[Abstract]:Purpose The construction of GDNF gene modified umbilical cord mesenchymal stem cells (UCM SCS) lays the foundation for the construction of tissue engineered nerve. Method 1. The competent E. coli was prepared by CaCl2 method. The recombinant retroviral vector plasmid (Professor Huai Chin Nguyen Huai-chen, third military Medical University) was transformed into the receptive bacteria and screened for resistance, and the plasmid was extracted and purified. The correctness of the target gene was detected by enzyme digestion and sequencing. 2. The resuscitation packaging cells PA317 were cultured with adherent culture method and amplified by passage. When the cells grew to 80% fusion, the recombinant retrovirus PLXSN-GDNF was transfected into the PA317 packaging cells by liposome transfection, and the toxic clones were screened by G418. Collect the supernatant of the virus and determine the titer of the virus. 3. The umbilical cord donated by healthy caesarean women was cultured with tissue block method. The primary cells were cultured and subcultured by trypsin digestion. The UCM SCS were collected from the second generation of umbilical cord mesenchymal stem cells. The expression of CD44 + CD105 + CD106 + CD34 + CD34 + CD45 was detected by immunohistochemical method. 4. After the virus supernatant was infected with umbilical cord mesenchymal stem cells, GDNF-UCMSCswere selected, and the expression of GDNF at mRNA level was detected by reverse transcriptase polymerase chain reaction (RT-PCR). Result The recombinant plasmid of 1.Xhol 鈪，

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