hMTH1缺陷细胞株建立及生物学特性研究

Construction of hMTH1 gene deficient cell strain and observation of its biologic characters

目的 建立hMTH1缺陷细胞株 ,观察该缺陷所引起的生物学效应。方法 用hMTH1基因反义RNA绿色荧光蛋白真核表达载体 (pEGFP C1 T)转染人胚肺成纤维细胞 (HLF) ,半定量RT PCR鉴定其对hMTH1基因mRNA表达的抑制效果。同时观察转染细胞生长形态 ,绘制生长曲线 ,用流式细胞术观察细胞周期 ,软琼脂培养法鉴定恶性程度。结果 pEGFP C1 T载体在转染细胞内可较稳定表达 ,缺陷细胞株hMTH1mRNA表达水平比HLF细胞下降了 47%。hMTH1缺陷细胞生长形态无明显变化 ,细胞倍增时间为 0 89d ,与HLF细胞 0 93d接近 ,细胞周期各时相未受影响 ,软琼脂培养未见细胞集落。结论 hMTH1缺陷细胞株成功建立 ,该缺陷不足以单独引起可观察的生物学效应。

Objective To construct hMTH1 deficient cell strain and observe the effects induced by hMTH1 deficiency.Methods HLF was transfected with the eukaryotic expression plasmids of hMTH1 gene antisense RNA(pEGFP-C1-T).The efficacy of antisense inhibition was verified by RT-PCR.Morphology,growth character,cell cycle and growth status in soft agar of transfected HLF were observed.Results pEGFP-C1-T vector was successfully expressed in HLF.The mRNA expression level of hMTH1 gene in the deficient cell was decreased by 47% as compared with that in HLF.No obvious changes of biologic character were observed. Conclusion The hMTH1 deficient cell strain was successfully constructed.When transfected HLF growed in normal environment biologic characters didn't change obviously.