人canstatin基因稳定转染CHO-K1细胞及其表达产物的生物学作用研究

发布时间：2018-04-05 08:34

本文选题：稳定转染　切入点：脂质体　出处：《南华大学》2006年硕士论文

【摘要】：[目的]将人canstatin cDNA分泌型真核表达载体pSecTag2B／canstatin稳定转染中国仓鼠卵巢(CHO-K1)细胞并获得稳定表达canstatin蛋白产物的细胞株。再用含canstatin的上清液作用人脐静脉内皮细胞(HUVEC-12)以进一步研究canstatin的生物学作用。 [方法]将人canstatin cDNA的重组质粒pSecTag2B／canstatin稳定转染中国仓鼠卵巢(CHO-K1)细胞并获得稳定表达canstatin蛋白产物的细胞株，应用RT-PCR检测细胞中canstatin基因的表达。收集细胞培养上清液中的蛋白，采用western-blotting法鉴定表达产物，用鸡胚绒毛尿囊膜(CAM)实验初步鉴定其生物学活性。通过绘制细胞生长曲线和流式细胞学技术进一步研究canstatin对血管内皮细胞的生物学作用。 [结果]成功的将人canstatin cDNA的重组质粒pSecTag2B／canstatin稳定转染CHO-K1细胞，RT-PCR法证实质粒pSecTag2B／canstatin已成功转染进入CHO-K1细胞。离心收集细胞培养上清液，western-blotting法鉴定上清液中有目的蛋白的表达。该细胞培养上清液在体内能抑制鸡胚绒毛尿囊膜中微血管的生成，可见CHO-K1，，pSecTag2B／canstatin组给药区及其周围血管明显减少甚至未见血管纹理，小血管分支少，而其余四组鸡胚尿囊膜血管生成没有明显影响。通过鸡胚处理前后血管生成记数，CHO-K1，pSecTag2B／canstatin组与其余四组之间经统计学处理差异有显著性(P＜0.05)。通过绘制细胞生长曲线显示
[Abstract]:[objective] to stably transfect human canstatin cDNA secretory eukaryotic expression vector pSecTag2B/canstatin into Chinese hamster ovary cell line CHO-K1 and to obtain a cell line with stable expression of canstatin protein product.Human umbilical vein endothelial cells (HUVEC-12) were treated with supernatant containing canstatin to further study the biological effects of canstatin.[methods] the recombinant plasmid pSecTag2B/canstatin of human canstatin cDNA was stably transfected into Chinese hamster ovary (CHO-K1) cells and the cell lines expressing canstatin protein products stably were obtained. RT-PCR was used to detect the expression of canstatin gene in the cells.The protein in the supernatant of cell culture was collected and the expression product was identified by western-blotting method. The biological activity of the protein was preliminarily identified by chorioallantoic membrane assay.The biological effects of canstatin on vascular endothelial cells were further studied by cell growth curve and flow cytometry.[results] Recombinant plasmid pSecTag2B/canstatin of human canstatin cDNA was successfully transfected into CHO-K1 cells by RT-PCR and confirmed that the plasmid pSecTag2B/canstatin had been successfully transfected into CHO-K1 cells.The expression of the target protein in the supernatant was identified by Western-blotting method.The supernatant of cell culture could inhibit the formation of microvessels in chorioallantoic membrane of chicken embryo in vivo.The other four groups had no significant effect on angiogenesis of allantoic membrane.There was a significant difference between CHO-K1PSecTag2B / canstatin group and the other four groups in the number of angiogenesis before and after embryo treatment (P < 0.05).By drawing the cell growth curve.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R363

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