| KATP通道E23K突变对阿霉素诱导的细胞凋亡的影响 |
| 投稿时间：2017-04-25 修订日期：2017-05-31 点此下载全文 |
| 引用本文：刘剑芳,王梦龙,叶晶,徐瑶,王震,姜慧敏,叶迪,赵生娣,万军.KATP通道E23K突变对阿霉素诱导的细胞凋亡的影响[J].医学研究杂志,2018,47(2):28-31 |
| DOI：
10.11969/j.issn.1673-548X.2018.02.008 |
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| 基金项目:国家自然科学基金资助项目（81170208） |
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| 中文摘要:目的 研究ATP敏感性钾通道Kir6.2亚基E23K突变对阿霉素诱导的心肌细胞凋亡的影响。方法 （1）在心肌组织中提取总RNA，RT-PCR法获取cDNA，合成特异性引物，用PCR法获得kir6.2、SUR2A的ORF片段，用T4DNA连接酶将上述片段与线性化的pEGFP-C1连接，重组的pEGFP-C1/kir6.2用PCR法进行E23K定点突变，获得真核表达质粒pEGFP C1/kir6.2，pEGFP C1/kir6.2（E23K）及pEGFP C1/SUR2A。（2）重组质粒采用脂质体法瞬时共转染H9C2细胞，使其在H9C2细胞中表达。（3）生理盐水与1mg/L阿霉素（Adriamycin，ADR）分别处理转染后细胞，分为4组：A组为野生型重组质粒+生理盐水处理组（WT+NS）；B组为含E23K突变重组质粒+生理盐水处理组（E23K+NS）；C组为野生型重组质粒+阿霉素损伤组（WT+ADR）；D组为含E23K突变重组质粒+阿霉素损伤组（E23K+ADR）。（4）采用TUNEL法检测心肌细胞凋亡指数（AI）。（5） Western blot法测定caspase-3、Bcl-2、Bax蛋白表达。AlphaEase FC软件取值读取各样品条带灰度值。结果 （1） TUNEL结果示，应用ADR后凋亡比例升高，与WT+ADR组相比，E23K+ADR组凋亡比例更高（P<0.05）。（2）阿霉素处理后，所有组促凋亡相关蛋白（caspase-3、Bax）表达均增高，抗凋亡蛋白（Bcl-2）表达降低，且E23K+ADR组caspase-3与Bax蛋白表达均大于WT+ADR组，Bcl-2蛋白表达低于WT+ADR组（P<0.05）。结论 KATP通道kir6.2亚基E23K突变加重阿霉素诱导的细胞凋亡。 |
| 中文关键词:KATP通道 E23K突变 细胞凋亡 |
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| Effect of E23K Mutant of KATP Channel on Apoptosis Induced by Adriamyci |
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| Abstract:Objective To investigate the effect of E23K mutant on kir6.2 subunit of KATP channel on myocardial cell apoptosis induced by Adriamycin.Methods (1)Total RNA was obtained in myocardial tissue and cDNA was obtained by RT-PCR method. Using specific primers to get ORF fragments of kir6.2, SUR2A by PCR method, using T4DNA ligase to connected the fragment with linearized pEGFP-C1, restructuring pEGFP-C1/kir6.2(E23K)by point mutation fixed in the PCR method, then get the pEGFP-C1/kir6.2, pEGFP-C1/kir6.2 (E23K) and pEGFP-C1/SUR2A. (2) The plasmid transient transfected the H9C2 cells and expressed the target proteins. (3) Saline and 1mg/L adriamycin (ADR) add to the cells after transfection, divided into four groups.A:wild type and saline treatment group (WT + NS); B:E23K mutation and saline treatment group (E23K + NS);C:Wild type and Adriamycin treatment group (WT + ADR);D:E23K mutation and adriamycin treatment group (E23K + ADR). (4) TUNEL method to detect myocardial apoptosis index (AI).(5) The Western blot method to test caspase-3, Bcl-2, Bax protein expression. Alpha Ease FC software to read grey value.Results (1) TUNEL results showed that with an increase in apoptosis after application of ADR, compared with (WT + ADR) group, apoptosis rate of (E23K + ADR) group was higher.(2) After being treated with Adriamycin, the expression of caspase-3 and Bax proteins of all groups were increased, while the Bcl-2 was decreased,and in (E23K + ADR) group, the expression of caspase-3, Bax protein were increased than in (WT + ADR) group but Bcl-2 decreased (P<0.05).Conclusion E23K mutation aggravated the apoptosis of myocardial cell induced by Adriamycin. |
| keywords:KATP channel E23K mutation Apoptosis |
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