靶向性Fas基因转导卵巢癌细胞系SKOV_3的实验研究

Research on cancer-specific transfection of fas gene in ovarian epithelial carcinoma cell line SKOV_3

目的为了解决Fas基因转导中靶向性差的问题,在基因上游插入人端粒酶逆转录酶(human telomerase reverse tran-scriptase,hTERT)启动子,构建真核表达质粒,了解其对Fas表达的提高程度。方法将Fas基因自质粒pcDNA3/Fas中用限制性内切酶KpnⅠ和XbaⅠ切出,插入到KpnⅠ和XbaⅠ双酶切后的载体pAd/TERT中hTERT启动子的下游,构建pAd/TERT-Fas,并进行酶切鉴定和测序鉴定。然后转染pAd/TERT-Fas、pAd/TERT和pcDNA3/Fas到低表达Fas的SKOV3,并命名为T-F、T和F。对转染克隆细胞进行逆转录PCR、实时PCR、Western免疫印迹和流式细胞分析,了解Fas基因在mRNA和蛋白水平的表达情况。结果质粒pAd/TERT-Fas经酶切及测序鉴定构建成功。转染SKOV3鉴定结果如下:实时聚合酶链反应中T-F的循环阈值(cycle threshold,CT)值为29,F的CT值为31.4,T和未转染SKOV3不表达Fas;Western免疫印迹结果显示在F和T-F组Fas蛋白有表达,T-F组表达明显强于F组;流式细胞分析Fas在未转染SKOV3、T、F、T-F组分别为1.1%,4.9%,10.9%,15.1%。结论本研究成功构建真核表达质粒pAd/TERT-Fas,并转染SKOV3,结果显示hTERT启动子可提高Fas表达。

Objective To explore an approach of targeting and elevating expression of fas gene driven by human telomerase reverse transcriptase（hTERT） promoter in ovarian epithelial carcinoma. Methods Fas gene was isolated from pcDNA3/Fas vector with Kpn I and Xba I , and then inserted into the pAd/TERT vector under the control of hTERT promoter through the same restriction sites. The plasmid pAd/TERT-Fas （ T-F group ） was transfected into SKOV3 , while pcDNA3/Fas （ F group ） and pAd/TERT （ T group ） were trans- fected into SKOV3 as controls. Fas mRNA and protein were measured with RT-PCR,real-time PCR,Western blot and flow cytometry, respectively. Results The construction of pAd/TERT-Fas was confirmed by sequencing and restriction endonuclease analysis. The values of cycle threshold（CT） by real-time PCR in T-F group, F group, T group, SKOV3 group were 29,31. g, 0 and 0, respectively. Western blot showed that fas protein expressed in F and T-F groups, especially in T-F group. Flow cytometry showed that expression level of fas protein in SKOV3 ,T,F and T-F groups were 1.1% ,4.9% ,10.9% and 15.1% ,respectively. Conclusion The results indi- cate that hTERT promoter could up-regulate the expression of fas gene in ovarian epithelial carcinoma.