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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 small interfering RNA (siRNA) of steroid regulatory element binding protein (SRREBP) and overexpression of recombinant adenovirus and observe its effect on the proliferation and apoptosis of ATDC5 chondrocyte stem cells. Methods The recombinant adenovirus plasmid pAd-SCAPsiRNA and pAdSCAP were constructed by pAd EasyTM adenoviral vector system and packaged and expanded in HEK293 cells respectively. The expression of SCAP in ATDC5 cells was detected by RT-PCR and Western blot. The effect of SCAP adenovirus on the cell cycle and apoptosis of ATDC5 cells was detected by flow cytometry. The expressions of Cleaved Caspase-3 and p-JNK were detected by Western blot. Results The adenovirus si SCAP (titer of 2.5 × 10 ~ (10) PFU / ml) and Ad-SCAP (titer of 3.0 × 10 ~ (11) PFU / ml) were successfully obtained. The results of RT-PCR and Western blot showed that siSCAP and Ad-SCAP can effectively inhibit and enhance the expression of SCAP in ATDC5 cells. The results of flow cytometry showed that the proportion of S phase cells in si SCAP-treated group was increased by 15.45% and 16.07% (P <0.05), and the proportion of Ad-SCAP group was reduced by 14.27% and 13.45 % (P <0.05). The apoptotic rate of siSCAP group was decreased by 13.10% and 11.50% respectively (P <0.05), while the Ad-SCAP group was increased by 10.51% and 10.17% (P <0.05). Western blot test results and flow cytometry results. CONCLUSION: SCAP adenovirus was successfully constructed. SCAP knockdown can promote ATDC5 cell proliferation and inhibit apoptosis. Overexpression of SCAP inhibits proliferation and promotes apoptosis.