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首页> 外文期刊>Oncology reports >A preliminary study on the construction of double suicide gene delivery vectors by mesenchymal stem cells and the in vitro inhibitory effects on SKOV3 cells
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A preliminary study on the construction of double suicide gene delivery vectors by mesenchymal stem cells and the in vitro inhibitory effects on SKOV3 cells

机译：间充质干细胞构建双自杀基因传递载体的初步研究及体外对SKOV3细胞的抑制作用

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The aim of the present study was to investigate the efficacy of using human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) as gene delivery vectors in the treatment of ovarian cancer. Lentivectors overexpressing cytosine deaminase (CD) and herpes simplex virus thymidine kinase (HSV-tk) (pGC-FU-CD-TK) were constructed, and confirmed by enzyme digestion, DNA sequence and western blotting. Quantitative PCR (PCR) was used to verify the overexpression of the fusion gene (CD and HSV-tk). SKOV3 cells were co-cultured with MSCs/tk+CD+ at a 1:1 ratio, and were then treated with the prodrugs (GCV) and/or 5-fluoro-cytosine (5-FC) at different concentrations, and the cytotoxic effects were evaluated using MTT assay and flow cytometry. DNA sequencing demonstrated that the sequence of HSV-tk and CD genes were consistent with the objective sequence and western blotting verified that the constructed lentivector could produce the HSV-tk/CD gene. The packed titer was 2.00e+8 TU/ml. The pGC-FU-CD-TK could be stably transferred to hUCB-MSCs, and the infection efficiency was almost 80%. RT-PCR demonstrated that the expression levels of the HSV-tk/CD fusion gene in MSCs/tk+CD+ group was 75 times that in the negative control (P0.05). Compared with GCV or 5-FC alone, the growth inhibition rate (GIR) was significantly higher in the combined treatment (F=85.35, P0.05). The reconstructed MSCs/tk+ CD + vectors were capable of slowing down the growth of human SKOV3 cells in the presence of prodrugs in vitro. The use of combination chemotherapy exhibited a more significant inhibitory effect than using a single prodrug.
机译：本研究的目的是研究使用人脐血来源的间充质干细胞（hUCB-MSC）作为基因传递载体治疗卵巢癌的功效。构建了过量表达胞嘧啶脱氨酶（CD）和单纯疱疹病毒胸苷激酶（HSV-tk）（pGC-FU-CD-TK）的慢病毒载体，并通过酶消化，DNA序列和蛋白质印迹进行了证实。定量PCR（PCR）用于验证融合基因（CD和HSV-tk）的过表达。将SKOV3细胞与MSCs / tk + CD +以1：1比例共培养，然后用不同浓度的前药（GCV）和/或5-氟胞嘧啶（5-FC）处理，并具有细胞毒性作用使用MTT测定法和流式细胞术评估。 DNA测序表明，HSV-tk和CD基因序列与目的序列一致，Western blotting证实所构建的慢病毒载体可以产生HSV-tk / CD基因。堆积效价为2.00e + 8TU / ml。可以将pGC-FU-CD-TK稳定转移到hUCB-MSCs中，感染效率几乎达到80％。 RT-PCR显示，MSCs / tk + CD +组中HSV-tk / CD融合基因的表达水平是阴性对照组的75倍（P <0.05）。与单独使用GCV或5-FC相比，联合处理的生长抑制率（GIR）显着更高（F = 85.35，P <0.05）。在体外存在前药的情况下，重建的MSCs / tk + CD +载体能够减慢人类SKOV3细胞的生长。与使用单一前药相比，联合化疗显示出更显着的抑制作用。

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