人角化上皮生长因子基因(hKGF)达载体对大鼠肺泡II型细胞增殖的影响

Effect of the neukaryotic vector expressing human keratinocyte growth factor (hKGF) gene on rat AEC II proliferation

目的:构建人角化上皮生长因子基因真核表达载体pEGFP-C2-hKGF,为以后的hKGF基因功能和基因治疗研究提供实验材料。方法:提取人胚肺成纤维细胞(HLF)总RNA,逆转录酶-多聚酶链式反应(RT-PCR)扩增hKGF基因全长cDNA序列,经与pMD18-T载体连接、筛选、克隆、抽提质粒和双酶切后,将纯化的hKGF基因cDNA全长序列插入绿色荧光蛋白表达载体pEGFP-C2中,筛选、克隆、抽提质粒,从而构建hKGF基因真核表达载体pEGFP-C2-hKGF。载体转染后大鼠肺泡II型细胞进行免疫细胞化学及荧光显微镜鉴定,并进行细胞记数,绘制生长曲线。结果:经RT-PCR获得609bp含限制性内切酶位点的DNA片段,T载体克隆后经双酶切、测序,确定该片段为hKGF基因cDNA,进而构建hKGF真核表达载体pEGFP-C2-hKGF,经双酶切,测序确证。转染载体后,有hKGF的阳性表达并随时间逐渐增加,大鼠肺泡II型细胞也随时间出现明显分裂增殖。结论:成功构建hKGF基因真核表达载体pEGFP-C2-hKGF,为进一步应用于基因治疗研究提供工具。

Objective：To construct the pEGFP-C2-hKGF vector,a neukaryotic expression plasmid of hKGF gene in order to provide experimental material for the studies of hKGF gene function and hKGF gene therapy afterward. Methods：The hKGF gene was amplified by reverse transcriptage polymerase chain reaction（RT-PCR）. Total RNA was extracted from human embryo lung fibroblast （HLF）and then cloned into pMD18-T vector. The recombinant plasmid were screened and extracted. The pEGFP-C2-hKGF vector,an eukaryotic expression plasmid of hKGF gene was constructed after the object hKGF cDNA fragment was inserted into pEGFP-C2 vector and the recombinant were screened,cloned and extracted. The recombinant plasmid vector was transferred into AEC Ⅱ,and expression of hKGF was assayed by immunohistochemistry. The effect of AEC Ⅱ proliferation was analyzed by cell count. Results：609bp DNA fragment containing hKGF gene and restriction sites were obtained by RT-PCR. After cloned by pMD18-T vector and certified by restriction analysis and sequencing, pEGFP-C2-hKGF vector was successfully constructed by means of recombinant DNA technology, pEGFP-C2-hKGF encoding the green fluorescent protein （GFP）,capable of directing the expression of functional hKGF successfully. Furthermore,hKGF had the effect on AEC Ⅱ proliferation. Conclusion：The efficient expression vector of hKGF gene is constructed,which can be used to establish the hKGF gene therapy vector .