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目的通过基因工程修饰法建立肿瘤坏死因子相关凋亡诱导配体(TRAIL)稳定过表达的人脐带间充质干细胞亚系(h UC-MSC_TRAIL)。方法人TRAIL全长蛋白编码序列(CDS)由PCR法扩增而得,PCR产物经NotⅠ和MluⅠ双酶切并纯化后,亚克隆至经同样双酶切的慢病毒表达载体p LEX-MCS。重组载体经PCR法和限制性内切酶酶切法鉴定,再行DNA直接测序验证后命名为人TRAIL表达慢病毒载体pLEX-h TRAIL。p LEXh TRAIL与相应包装质粒ps PAX2和p MD2.G经聚乙烯亚胺介导共转染HEK293T细胞以包装慢病毒。P4代h UC-MSC经慢病毒感染24 h,再行嘌呤霉素筛选2周后,抽提细胞基因组DNA,行PCR法鉴定h TRAIL c DNA在h UC-MSC基因组中的整合情况;同时抽提细胞总RNA,并行RT-PCR法检测外源h TRAIL在h UC-MSC中的m RNA表达水平,以及实时定量RT-PCR法检测周期调控相关蛋白Cyclin D1、Cyclin E1、p21~(WAF1/CIP1和p27的表达,采用方差分析和t检验进行统计学分析。结果 PCR法和限制性内切酶酶切法鉴定结果表明,本研究已成功构建人TRAIL慢病毒表达载体p LEX-h TRAIL,直接DNA测序结果证实克隆得到的人TRAIL蛋白编码序列准确无误;病毒包装及细胞感染的鉴定结果说明,慢病毒感染法可成功介导外源人TRAIL在h UC-MSC的稳定整合和高表达;实时定量RT-PCR法检测结果则显示,与对照慢病毒感染后的细胞相比,h TRAIL表达慢病毒感染后其细胞周期调控相关蛋白Cyclin D1、Cyclin E1、p21~(WAF1/CIP1)和p27的m RNA表达水平分别是对照组的1.19倍(P=0.141)、0.94倍(P=0.745)、0.95倍(P=0.047)和1.01倍(P=0.567),表明外源TRAIL高表达对体外培养的h UC-MSC生长增殖等表型无显著影响。结论本研究经基因工程修饰法成功构建了具外源TRAIL稳定高表达的h UC-MSC亚细胞系,该亚细胞系的建立为后续靶向攻击TRAIL敏感肿瘤细胞的细胞治疗的探索奠定了基础。
Objective To establish a human umbilical cord mesenchymal stem cell line (h UC-MSC_TRAIL) stably overexpressed by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by genetic engineering. Methods Human TRAIL full-length protein coding sequence (CDS) was amplified by PCR. The PCR product was digested with NotⅠand MluⅠ and then subcloned into lentiviral vector p LEX-MCS. The recombinant vector was identified by PCR and restriction endonuclease digestion, and then verified by DNA sequencing and named as human TRAIL lentiviral vector pLEX-h TRAIL. p LEXh TRAIL and corresponding packaging plasmid ps PAX2 and p MD2.G were co-transfected into HEK293T cells via polyethylenimine to package lentivirus. P4 generation of h UC-MSCs was infected with lentivirus for 24 h. After puromycin selection for 2 weeks, cell genomic DNA was extracted and the integration of h TRAIL c DNA in h UC-MSC genome was identified by PCR. The expression of m RNA in h UC-MSCs was detected by RT-PCR and real-time quantitative RT-PCR to detect the expression of cyclin D1, Cyclin E1, p21 WAF1 / CIP1 and p27 were analyzed by ANOVA and t-test.Results The results of PCR and restriction enzyme digestion showed that the human TRAIL lentiviral vector pLEX-h TRAIL was constructed successfully in this study, Direct DNA sequencing confirmed that the cloned human TRAIL protein coding sequence was correct; virus packaging and cell infection identification results show that lentiviral infection can successfully mediated foreign human TRAIL h UC-MSC stable integration and high expression; The result of real-time quantitative RT-PCR showed that hTRAIL expression of cyclin D1, Cyclin E1, p21 WAF1 / CIP1 and p27 after lentivirus infection were significantly higher than that of control lentivirus The mRNA expression level of m RNA in control group was 1. (P = 0.141), 0.94 times (P = 0.745), 0.95 times (P = 0.047) and 1.01 times (P = 0.567), respectively, indicating that the high exogenous TRAIL expression on proliferation and proliferation of h UC- Type.Conclusion This study successfully constructed a hUCC-MSC sub-cell line with stable and high expression of exogenous TRAIL genetically modified, which was established for the subsequent cell-targeted therapy targeting TRAIL-sensitive tumor cells The exploration laid the foundation.