中华心血管病杂志
中華心血管病雜誌
중화심혈관병잡지
Chinese Journal of Cardiology
2013年
1期
54-59
,共6页
戴晓春%周希%黄晓燕%王良国%林素%杨德业
戴曉春%週希%黃曉燕%王良國%林素%楊德業
대효춘%주희%황효연%왕량국%림소%양덕업
肌细胞,心脏%细胞凋亡%线粒体%转录因子
肌細胞,心髒%細胞凋亡%線粒體%轉錄因子
기세포,심장%세포조망%선립체%전록인자
Myocytes,cardiac%Apoptosis%Mitochondria%Transcription factors
目的 采用RNA干扰技术选择性下调大鼠心肌细胞中转录因子Pax-8的表达,引起线粒体功能的变化,借此探讨线粒体对心肌细胞凋亡的影响.方法 将H9C2(2-1)大鼠胚胎心肌细胞分为3组:实验组[针对Pax-8基因的小干扰RNA(Pax-8 siRNA)组],阴性对照组[非特异性siRNA(NC siRNA)组]和空白对照组(BC siRNA组).荧光分光光度法检测半胱天冬酶(caspase-3)活性以反映细胞凋亡,RT-PCR测定B细胞淋巴瘤/白血病-2(B cell lymphoma/lewkmia-2,Bcl2)和其同源类似物Bax的mRNA表达,Western blot测定Bcl2、Bax和胞浆细胞色素C(Cyto)的蛋白表达,流式细胞仪检测细胞线粒体膜电位(△ψm,用JC-1单体/聚合物比值来衡量线粒体去极化的比例,比值越大,△ψm越低).结果 Pax-8 siRNA组心肌细胞caspase-3活性(0.167 ±0.012)明显高于NC siRNA组(0.075 ±0.021)和BC siRNA组(0.072 ±0.019),P均＜0.05.Pax-8 siRNA组Bcl2 mRNA和蛋白的表达水平(分别为0.61 ±0.06和0.94±0.11)明显低于NC siRNA组(分别为0.90±0.070和1.39±0.15)和BC siRNA组(分别为0.94±0.09和1.49 ±0.20),P均＜0.05.Pax-8 siRNA组Bax mRNA和蛋白表达水平(分别为1.05±0.10和1.25±0.12)明显高于NC siRNA组(分别为0.72 ±0.03和0.99 ±0.12)和BC siRNA组(分别为0.64±0.03和0.92±0.06),P均＜0.05.Pax-8 siRNA组心肌细胞线粒体释放Cyto至胞浆的量(0.75±0.14)明显高于NC siRNA组(0.51±0.06)和BC siRNA组(0.48±0.07),P均＜0.05.Pax-8 siRNA组JC-1单体/聚合物比值(0.163±0.011)明显高于NCsiRNA组(0.092 ±0.015)和BC siRNA组(0.072±0.025),P均＜0.05,提示△ψm明显低于NCsiRNA组和BC siRNA组(P均＜0.05).NC siRNA组与BC siRNA组上述指标比较,差异均无统计学意义.结论 Pax-8基因干扰心肌细胞,促进了心肌细胞的凋亡,线粒体参与了心肌细胞凋亡,推测心肌细胞的凋亡可能是通过线粒体途径实现的.
目的 採用RNA榦擾技術選擇性下調大鼠心肌細胞中轉錄因子Pax-8的錶達,引起線粒體功能的變化,藉此探討線粒體對心肌細胞凋亡的影響.方法 將H9C2(2-1)大鼠胚胎心肌細胞分為3組:實驗組[針對Pax-8基因的小榦擾RNA(Pax-8 siRNA)組],陰性對照組[非特異性siRNA(NC siRNA)組]和空白對照組(BC siRNA組).熒光分光光度法檢測半胱天鼕酶(caspase-3)活性以反映細胞凋亡,RT-PCR測定B細胞淋巴瘤/白血病-2(B cell lymphoma/lewkmia-2,Bcl2)和其同源類似物Bax的mRNA錶達,Western blot測定Bcl2、Bax和胞漿細胞色素C(Cyto)的蛋白錶達,流式細胞儀檢測細胞線粒體膜電位(△ψm,用JC-1單體/聚閤物比值來衡量線粒體去極化的比例,比值越大,△ψm越低).結果 Pax-8 siRNA組心肌細胞caspase-3活性(0.167 ±0.012)明顯高于NC siRNA組(0.075 ±0.021)和BC siRNA組(0.072 ±0.019),P均＜0.05.Pax-8 siRNA組Bcl2 mRNA和蛋白的錶達水平(分彆為0.61 ±0.06和0.94±0.11)明顯低于NC siRNA組(分彆為0.90±0.070和1.39±0.15)和BC siRNA組(分彆為0.94±0.09和1.49 ±0.20),P均＜0.05.Pax-8 siRNA組Bax mRNA和蛋白錶達水平(分彆為1.05±0.10和1.25±0.12)明顯高于NC siRNA組(分彆為0.72 ±0.03和0.99 ±0.12)和BC siRNA組(分彆為0.64±0.03和0.92±0.06),P均＜0.05.Pax-8 siRNA組心肌細胞線粒體釋放Cyto至胞漿的量(0.75±0.14)明顯高于NC siRNA組(0.51±0.06)和BC siRNA組(0.48±0.07),P均＜0.05.Pax-8 siRNA組JC-1單體/聚閤物比值(0.163±0.011)明顯高于NCsiRNA組(0.092 ±0.015)和BC siRNA組(0.072±0.025),P均＜0.05,提示△ψm明顯低于NCsiRNA組和BC siRNA組(P均＜0.05).NC siRNA組與BC siRNA組上述指標比較,差異均無統計學意義.結論 Pax-8基因榦擾心肌細胞,促進瞭心肌細胞的凋亡,線粒體參與瞭心肌細胞凋亡,推測心肌細胞的凋亡可能是通過線粒體途徑實現的.
목적 채용RNA간우기술선택성하조대서심기세포중전록인자Pax-8적표체,인기선립체공능적변화,차차탐토선립체대심기세포조망적영향.방법 장H9C2(2-1)대서배태심기세포분위3조:실험조[침대Pax-8기인적소간우RNA(Pax-8 siRNA)조],음성대조조[비특이성siRNA(NC siRNA)조]화공백대조조(BC siRNA조).형광분광광도법검측반광천동매(caspase-3)활성이반영세포조망,RT-PCR측정B세포림파류/백혈병-2(B cell lymphoma/lewkmia-2,Bcl2)화기동원유사물Bax적mRNA표체,Western blot측정Bcl2、Bax화포장세포색소C(Cyto)적단백표체,류식세포의검측세포선립체막전위(△ψm,용JC-1단체/취합물비치래형량선립체거겁화적비례,비치월대,△ψm월저).결과 Pax-8 siRNA조심기세포caspase-3활성(0.167 ±0.012)명현고우NC siRNA조(0.075 ±0.021)화BC siRNA조(0.072 ±0.019),P균＜0.05.Pax-8 siRNA조Bcl2 mRNA화단백적표체수평(분별위0.61 ±0.06화0.94±0.11)명현저우NC siRNA조(분별위0.90±0.070화1.39±0.15)화BC siRNA조(분별위0.94±0.09화1.49 ±0.20),P균＜0.05.Pax-8 siRNA조Bax mRNA화단백표체수평(분별위1.05±0.10화1.25±0.12)명현고우NC siRNA조(분별위0.72 ±0.03화0.99 ±0.12)화BC siRNA조(분별위0.64±0.03화0.92±0.06),P균＜0.05.Pax-8 siRNA조심기세포선립체석방Cyto지포장적량(0.75±0.14)명현고우NC siRNA조(0.51±0.06)화BC siRNA조(0.48±0.07),P균＜0.05.Pax-8 siRNA조JC-1단체/취합물비치(0.163±0.011)명현고우NCsiRNA조(0.092 ±0.015)화BC siRNA조(0.072±0.025),P균＜0.05,제시△ψm명현저우NCsiRNA조화BC siRNA조(P균＜0.05).NC siRNA조여BC siRNA조상술지표비교,차이균무통계학의의.결론 Pax-8기인간우심기세포,촉진료심기세포적조망,선립체삼여료심기세포조망,추측심기세포적조망가능시통과선립체도경실현적.
Objective To observe the effects of paired box gene 8 (Pax-8) silencing by RNA interference on mitochondrial function and cardiomyocytes apoptosis.Methods The cultured H9C2 (2-1) myocytes were divided into 3 groups:short interference RNA targeting Pax-8 (Pax-8 siRNA) group,nonspecific siRNA group as the negative control (NC siRNA),and blank control group (BC siRNA).Fluo rescence spectrophotometry was used to detect the activity of caspase-3.RT-PCR was performed to detect mRNA expression of Bcl2 and Bax.The protein expression of Bcl2、Bax and cytoplasm of Cytothrome was examined by Western blot.Changes of △ψm were detected by flow cytometry.△ψm with JC-1 monomer/polymer ratio was calculated for measuring mitochondrial depolarization proportion.Results Compared to NC siRNA and BC siRNA group(0.075 ±0.021,0.072 ±0.019),the activity of caspase-3 in Pax-8 siRNA group(0.167 ±0.012) was significantly increased (P ＜0.05) ； Bcl2 mRNA and protein expression in Pax-8siRNA group (0.61 ±0.06,0.94 ±0.11) were significantly downregulated compared with NC siRNA group (0.90±0.070,1.39±0.15) and BC siRNA group (0.94 ±0.087,1.49±0.20) (P＜0.05)； Bax mRNA and protein expression in Pax-8 siRNA group (1.05 ± 0.10,1.25 ± 0.12) were markedly upregulated compared with NC siRNA group (0.72 ± 0.03,0.99 ± 0.12) and BC siRNA group (0.64 ±0.03,0.92 ± 0.06),P ＜ 0.05 ； cytosolic cytochrome expression in Pax-8 siRN A group (0.75 ± 0.14) was significantly upregulated compared with NC siRNA group (0.51 ± 0.06) and BC siRNA group (0.48 ±0.07) (P ＜ 0.05) ； JC-1 monomer/polymer ratio in Pax-8 siRNA group (0.163 ± 0.011) was significantly increased compared with NC siRNA group(0.092 ±0.015) and BC siRNA group (0.072 ± 0.025) (P ＜0.05) indicating mitochondrial membrane potential was significantly reduced in Pax-8 siRNA group.Above parameters were similar between NC siRNA group and BC siRNA group (P ＞ 0.05).Conclusion Inhibiting Pax-8 results in enhanced cardiomyocytes apoptosis through the mitochondrial pathway.
