人肿瘤坏死因子相关凋亡诱导配体稳定过表达基因工程修饰人脐带间充质干细胞亚细胞系的建立

Genetically engineered human umbilical cord mesenchymal stem cells with stable overexpression of human tumor necrosis factor-related apoptosis-inducing ligand

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 genetically engineered human umbilical cord mesenchymal stem cells （hUC-MSC） with overexpression of hTRAIL for cancer therapy. Methods The entire coding sequence （CDS） of human tumor necrosis factor （TNF）-related apoptosis-inducing ligand （TRAIL） was amplified from cDNAs of hUC-MSC cells. The amplified fragments were purified and digested with Not I and Mlu I, and then inserted into thelentiviral expression vector LentiORF pLEX-MCS. The recombinant was identified with both PCR and restriction digestion. After verification by DNA sequencing, the recombinant （with accurate sequence of hTRAIL CDS） was nominated as pLEX-hTRAIL. The lentiviral expression vector pLEX-hTRAIL and lentivirus packaging plasmids psPAX2 and pMD2.G were co- transfected into virus packaging cell line HEK293T using PEI to produce lentiviruses. Human umbilical cord mesenchymal stem cells were infected with hTRAIL-expressing lentiviruses and then subjected to selection with puromycin. The integration of exogenous hTRAIL cDNA into the genome of hUC-MSCs was detected with PCR and overexpression of hTRAIL was analyzed with both regular and real-time qRT-PCR. Also, the same method was carried out to analyze the mRNA expression of cell cycle regulators Cyclin D1, Cyclin El, p21WAF1/CIP1 and p27. Analysis of variance and t test was used for statistical analysis. Results The recombinant pLEX-hTRAIL with insert of human TRAIL cDNA was verified by PCR, restriction digestion, as well as direct DNA sequencing. After infection, the integration of exogenous hTRAIL cDNA in the genome of hUC-MSC was shown by PCR and the successful expression of exogenous hTRAIL in hUC-MSC was confirmed by both regular and real-time quantification RT- PCR. In addition, the real-time RT-PCR results indicated that the lentiviral system mediated overexpression of exogenous hTRAIL in hUC-MSC did not cause any significant change of expression of cell cycle regulators Cyclin D1 （1.19 fold, P= 0.141）, Cyclin E1 （0.94 fold, P = 0.745）, p21wAw/cIP1 （0.95 fold, P = 0.047）, and p27 （1.01 fold, P = 0.567） as compared with those of control viral infection. Conclusion We reported here an establishment of genetically engineered human umbilical cord mesenchymal stem cells with overexpression of hTRAIL. Its anti-cancer therapeutic potential in vitro and in vivo is awaiting further investigation.