SCAP重组腺病毒对ATDC5细胞增殖和凋亡的影响

Effects of SCAP recombinant adenoviruses on proliferation and apoptosis of ATDC5 cells

目的构建固醇调节元件结合蛋白裂解激活蛋白(SRREBP cleavage activating protein,SCAP)基因干扰(small interfering RNA,siRNA)和过表达重组腺病毒,观察其对ATDC5软骨干细胞增殖及凋亡的影响。方法应用pAd EasyTM腺病毒载体系统构建重组腺病毒质粒p Ad-SCAPsiRNA和p AdSCAP,在HEK293细胞中分别包装和扩增。RT-PCR和Western blot检测ATDC5细胞中SCAP的表达,流式细胞仪检测SCAP腺病毒对ATDC5细胞周期和凋亡的影响,Western blot检测凋亡相关蛋白Cleaved Caspase-3、p-JNK的表达。结果成功获腺病毒si SCAP(滴度为2.5×10^(10)PFU/m L)和Ad-SCAP(滴度为3.0×10^(11)PFU/m L)。RT-PCR和Western blot结果表明:siSCAP和Ad-SCAP能分别有效抑制和增强ATDC5细胞中SCAP的表达。流式细胞仪检测结果表明:与正常组和空病毒组比较,si SCAP腺病毒处理组S期细胞比例升高15.45%和16.07%(P<0.05),Ad-SCAP组比例降低14.27%和13.45%(P<0.05);siSCAP组细胞凋亡率降低13.10%和11.50%(P<0.05),Ad-SCAP组升高10.51%和10.17%(P<0.05)。Western blot检测结果与流式细胞仪检测结果一致。结论成功构建SCAP腺病毒;敲低SCAP能促进ATDC5细胞增殖并抑制凋亡;过表达SCAP则抑制增殖、促进凋亡。

Objective To construct recombinant adenoviral vectors containing small interfering RNA （siRNA） targeting human SREBP cleavage activating protein （SCAP） gene and recombinant adenoviral vector containing SCAP gene, and investigate their effects on proliferation and apoptosis of ATDC5 stem cells. Methods By using pAdEasyTM adenovirus vector system, recombinant shuttle vector containing siRNA sequence targeting SCAP gene （pSES-HUS-SCAP siRNA） and recombinant shuttle vector containing SCAP gene （pAdTrack-SCAP） were constructed, and then transferred with pAdEasy-1 to generate recombinant adenovirus plasmids pAd-SCAPsiRNA and pAd-SCAP with an electroporation method. Subsequently, the plasmids were transferred into HEK-293 cells by lipofectamine-mediated transfection to package and amplify the recombinant adenoviruses Ad-siSCAP and Ad-SCAP. The ATDC5 cells were infected with the recombinant adenoviruses Ad-siSCAP and Ad-SCAP, respectively, and the expressions of SCAP at mRNA and protein levels were detected by RT-PCR and Western blotting. The effects of Ad-siSCAP and Ad-SCAP adenoviruses on the proliferation and apoptosis of ATDC5 cells were detected by flow cytometry. The expressions of Cleaved Caspase-3 and p-JNK were detected by Western blotting. Results Recombinant adenoviruses Ad-SCAPsiRNA with high titer of 2.5 × 10^10 PFU/mL and Ad-SCAP with 3.0 × 10^11 PFU/mL were successfully obtained. Both the SCAP mRNA and protein levels were significantly decreased in the ATDC5 cells after infection with Ad-siSCAP, and increased after infection with Ad-SCAP. Flow cytometry showed that the ATDC5 cells infected with Ad-siSCAP had more cells at S phase than the normal control and Ad-RFP （increased by 15.45% and 16. 07%, respectively, P 〈 0. 05）, and the apoptotic rate of the Ad-siSCAP group was decreased by 13.10% and 11.50% （ P 〈 0.05 ） when compared with the 2 control groups. However, the ATDC5 cells infected with Ad-SCAP had less cells at S phase than the normal control and Ad-GFP （ decreased by 14.27% and 13.45%, respectively, P 〈 0. 05 ）, and the apoptotic rate of the Ad-SCAP group was increased by 10.51% and 10.17% （P 〈 0. 05 ） when compared with the 2 control groups. Western blotting results of Cleaved Caspase-3 and p-JNK were in accordance with the results shown by flow cytometry. Conclusion Infection of ATDC5 cells with Ad-SCAPsiRNA promotes the proliferation and suppresses the apoptosis, while infection of ATDC5 cells with Ad-SCAP suppresses the proliferation and promotes the apoptosis.