目的 探讨蛋白激酶C(PKC)与钙敏感受体(CaR),在心肌缺血预适应(IPC)中的保护作用.方法 采用细胞培养方法 ,体外培养大鼠乳鼠心肌细胞,模拟缺血预适应模型,实验分为7组:①正常对照组(C),②缺血再灌注组(1/R),③IPC组,④IPC+PKC抑制剂组(IPC+PKCI),⑤IPC+PKCI+CaR激动剂组(IPC+PKCI+CARS),⑥IPC+CaRS组,⑦IPC+CaR抑制剂组(IPC+CaRI).分别用TUNEL,Hoechst 33342染色法检测细胞凋亡,四甲基偶氮唑比色法(MTT)观察细胞存活率,Western Blot法检测胞浆内caspase-12,CaR,钙蛋白酶(calpain)表达.结果 光镜下,I/R组细胞核缩小,呈强蓝色荧光,染色质浓缩,出现凋亡小体,其他各实验组有不同程度的荧光增强,尤以IPC+PKCI+CaRS组多见强蓝色荧光细胞核.心肌细胞存活率和凋亡率,I/R组[(62.99±0.65)%,(19.13±0.87)%],IPC组[(78.67±0.37)%,(14.21±0.74)%],IPC+PKCI组[(71.09±0.52)%.(20.46±0.81)%],IPC+PKCI+CaRS组[(66.10±0.75)%,(24.89±1.43)%],IPC+CaRS组[(69.56±0.44)%,(21.64±0.77)%],IPC+CaRI组[(85.81±0.60)%,(13.12±0.69)%]明显低于或高于对照组[(100.00)%,(6.02±0.31)%],除IPC+CaRI组心肌细胞存活率外,其他各组与对照组比较差异有统计学意义(P＜0.05或＜0.01).Western Blot测定,胞浆白CaR蛋白在IPC+PKCI,IPC+CaRS,IPC+PKCI+CaRS组表达较IPC组高,IPC+CaRI组较IPC组低,caspase-12蛋白在I/R,IPC+CaRS,IPC+PKCI+CaRS 组,相对分子质量(胁)为60×103的活性片段表达均较高,calpain在I/R,IPC,IPC+PKCI,IPC+PKCI+CaRS,IPC+CaRS组表达均高于对照组和IPC+CaRI组,其中I/R组最高,对照组最低,IPC+CaRI组次之.结论 PKC与CaR受体之间的相互作用在IPC中可以通过减少肌浆网钙释放而起到对心肌的保护作用.Protective mechanism of the interaction between protein kinase C and calcium sensing receptor in jschemiapreconditioning DU Li-juan,WANG Yah-li,SUN Zhi-rui,ZHAO Ya-jun,LI Quan-feng,WANG Li-na,ZHANG Wei-hua Departmem of Pothophysioloty,Harbin Medical University,Harbin 150081,China
目的 探討蛋白激酶C(PKC)與鈣敏感受體(CaR),在心肌缺血預適應(IPC)中的保護作用.方法 採用細胞培養方法 ,體外培養大鼠乳鼠心肌細胞,模擬缺血預適應模型,實驗分為7組:①正常對照組(C),②缺血再灌註組(1/R),③IPC組,④IPC+PKC抑製劑組(IPC+PKCI),⑤IPC+PKCI+CaR激動劑組(IPC+PKCI+CARS),⑥IPC+CaRS組,⑦IPC+CaR抑製劑組(IPC+CaRI).分彆用TUNEL,Hoechst 33342染色法檢測細胞凋亡,四甲基偶氮唑比色法(MTT)觀察細胞存活率,Western Blot法檢測胞漿內caspase-12,CaR,鈣蛋白酶(calpain)錶達.結果 光鏡下,I/R組細胞覈縮小,呈彊藍色熒光,染色質濃縮,齣現凋亡小體,其他各實驗組有不同程度的熒光增彊,尤以IPC+PKCI+CaRS組多見彊藍色熒光細胞覈.心肌細胞存活率和凋亡率,I/R組[(62.99±0.65)%,(19.13±0.87)%],IPC組[(78.67±0.37)%,(14.21±0.74)%],IPC+PKCI組[(71.09±0.52)%.(20.46±0.81)%],IPC+PKCI+CaRS組[(66.10±0.75)%,(24.89±1.43)%],IPC+CaRS組[(69.56±0.44)%,(21.64±0.77)%],IPC+CaRI組[(85.81±0.60)%,(13.12±0.69)%]明顯低于或高于對照組[(100.00)%,(6.02±0.31)%],除IPC+CaRI組心肌細胞存活率外,其他各組與對照組比較差異有統計學意義(P＜0.05或＜0.01).Western Blot測定,胞漿白CaR蛋白在IPC+PKCI,IPC+CaRS,IPC+PKCI+CaRS組錶達較IPC組高,IPC+CaRI組較IPC組低,caspase-12蛋白在I/R,IPC+CaRS,IPC+PKCI+CaRS 組,相對分子質量(脅)為60×103的活性片段錶達均較高,calpain在I/R,IPC,IPC+PKCI,IPC+PKCI+CaRS,IPC+CaRS組錶達均高于對照組和IPC+CaRI組,其中I/R組最高,對照組最低,IPC+CaRI組次之.結論 PKC與CaR受體之間的相互作用在IPC中可以通過減少肌漿網鈣釋放而起到對心肌的保護作用.Protective mechanism of the interaction between protein kinase C and calcium sensing receptor in jschemiapreconditioning DU Li-juan,WANG Yah-li,SUN Zhi-rui,ZHAO Ya-jun,LI Quan-feng,WANG Li-na,ZHANG Wei-hua Departmem of Pothophysioloty,Harbin Medical University,Harbin 150081,China
목적 탐토단백격매C(PKC)여개민감수체(CaR),재심기결혈예괄응(IPC)중적보호작용.방법 채용세포배양방법 ,체외배양대서유서심기세포,모의결혈예괄응모형,실험분위7조:①정상대조조(C),②결혈재관주조(1/R),③IPC조,④IPC+PKC억제제조(IPC+PKCI),⑤IPC+PKCI+CaR격동제조(IPC+PKCI+CARS),⑥IPC+CaRS조,⑦IPC+CaR억제제조(IPC+CaRI).분별용TUNEL,Hoechst 33342염색법검측세포조망,사갑기우담서비색법(MTT)관찰세포존활솔,Western Blot법검측포장내caspase-12,CaR,개단백매(calpain)표체.결과 광경하,I/R조세포핵축소,정강람색형광,염색질농축,출현조망소체,기타각실험조유불동정도적형광증강,우이IPC+PKCI+CaRS조다견강람색형광세포핵.심기세포존활솔화조망솔,I/R조[(62.99±0.65)%,(19.13±0.87)%],IPC조[(78.67±0.37)%,(14.21±0.74)%],IPC+PKCI조[(71.09±0.52)%.(20.46±0.81)%],IPC+PKCI+CaRS조[(66.10±0.75)%,(24.89±1.43)%],IPC+CaRS조[(69.56±0.44)%,(21.64±0.77)%],IPC+CaRI조[(85.81±0.60)%,(13.12±0.69)%]명현저우혹고우대조조[(100.00)%,(6.02±0.31)%],제IPC+CaRI조심기세포존활솔외,기타각조여대조조비교차이유통계학의의(P＜0.05혹＜0.01).Western Blot측정,포장백CaR단백재IPC+PKCI,IPC+CaRS,IPC+PKCI+CaRS조표체교IPC조고,IPC+CaRI조교IPC조저,caspase-12단백재I/R,IPC+CaRS,IPC+PKCI+CaRS 조,상대분자질량(협)위60×103적활성편단표체균교고,calpain재I/R,IPC,IPC+PKCI,IPC+PKCI+CaRS,IPC+CaRS조표체균고우대조조화IPC+CaRI조,기중I/R조최고,대조조최저,IPC+CaRI조차지.결론 PKC여CaR수체지간적상호작용재IPC중가이통과감소기장망개석방이기도대심기적보호작용.Protective mechanism of the interaction between protein kinase C and calcium sensing receptor in jschemiapreconditioning DU Li-juan,WANG Yah-li,SUN Zhi-rui,ZHAO Ya-jun,LI Quan-feng,WANG Li-na,ZHANG Wei-hua Departmem of Pothophysioloty,Harbin Medical University,Harbin 150081,China
Objective To investigate the protective mechanism of protein kinase C(PKC)and calcium sensing receptor(CaR)in ischemia preconditioned rat hearts.Methods Using cell culture method,in vitro cultured inhibitor(IPC+CaRI).Apoptosis was detected using TUNEL and Hoechst33342 cell viability was detected by MTT,the protein expression of easpase-12,calpain and CaR in endochylema were detected using Wedtetm blot.ResultsIn I/R group nucleus was shrank,big blue,chromatin concentrated,apoptotle body appeared.Other groups haddifferent fluorescence intensity varying degree,IPC+PKCI+CaRS group had more big blue nucleus.Myocardialcell viability and apoptotic rate,I/R group[(62.99±0.65)%,(19.13±0.87)%],IPC group[(78.67±0.37)%,(14.21±0.74)%],IPC+PKCI group[(71.09±0.52)%,(20.46±0.81)%],IPC+PKCI+CaRS group(66.10±0.75)%,(24.89±1.43)%],IPC+CaRS group[(69.56±0.44)%,(21.64±0.77)%],IPC+CaRI group(85.81±0.60)%,(13.12±0.69)%],all had a difference(P＜0.05 or＜0.01)compared with C group[(100.00)%,(6.02±0. 31)%].Western blot identified that CaR expression in IPC+PKCI and IPC+CaRS,IPC+PKCI+CaRS groupswas more than that in IPC and IPC+CaRI groups;easpase-12 had more active fragment(60×103)in I/R,IPC+CaRS,IPC+PKCI+CaRS groups;ealpain expressions in I/R,IPC,IPC+PKCI,IPC+PKCI+CaRS,IPC+CaRSgroups were higher than those in C and IPC+CaRI,I/R group was the highest one,C group the second,IPC+CaRI the third.Conclusion The interaction of PKC and CaR can reduce the intracellular Ca2+ from sarcoplasmicreticulum thus provide a protection.
