中华心血管病杂志
中華心血管病雜誌
중화심혈관병잡지
Chinese Journal of Cardiology
2010年
2期
160-165
,共6页
潘大彬%柯永胜%刘文洁%蔚有权%唐军%曹蘅
潘大彬%柯永勝%劉文潔%蔚有權%唐軍%曹蘅
반대빈%가영성%류문길%위유권%당군%조형
血管%肌细胞,平滑肌%血小板源性生长因子%转化生长因子β1%周期素依赖激酶抑制剂p21
血管%肌細胞,平滑肌%血小闆源性生長因子%轉化生長因子β1%週期素依賴激酶抑製劑p21
혈관%기세포,평활기%혈소판원성생장인자%전화생장인자β1%주기소의뢰격매억제제p21
Blood vessels%Myocytes,smooth muscle%Platelet-derived growth factor%Transforming growth factor betal%Cyclin-dependent kinase inhibitor p21
目的 探讨血小板衍生生长因子(PDGF)-BB是否通过诱导转化生长因子(TGF)-β_1,表达及其信号系统调节p21~(WAF1)表达.方法 采取8～9周龄雄性Sprague-Dawley大鼠血管平滑肌细胞(VSMC)进行体外培养,分为对照组、中和性抗TGF-β_1抗体组(20μg/ml,抗体组)、PDGF-BB组(40 ng/ml)、PDGF-BB+中和性抗TGF-β_1抗体组(PDGF-BB+抗体组),实验时间为48 h.逆转录酶-聚合酶链式反应检测TGF-β_1mRNA的表达,ELISA方法检测TGF-β_1蛋白的表达.Western blot检测p21~(WAF1)及TGF-β信号系统下游蛋白表达,后者包括TGF-β_1I类受体(在VSMC内表现为ALK-5)、Smurf2、pSmad2/3、Smad4、Smad7.结果 PDGF-BB组的TGF-β_1 mRNA(24 h)和TGF-β_1(48 h)蛋白分子表达水平分别为(447.716±23.911)和(0.309±0.034)ng/ml,明显高于对照组(250.435±17.869)和(0.104±0.013)ng/ml(P均＜0.01).p21WAF1蛋白表达在对照组0.621±0.046和抗体组0.657±0.058之间差异无统计学意义,PDGF-BB组表达仅为0.204±0.033,与对照组和抗体组比较差异均有统计学意义(P均＜0.01),PDGF-BB+抗体组表达为0.530±0.043,与对照组和抗体组相比差异均有统计学意义(P均＜0.05),与PDGF-BB组比较差异亦有统计学意义(P＜0.01).PDGF-BB组ALK-5 0.634±0.060、pSmad2/31.894±0.184、Smad4 1.129±0.136及Smurf2 0.559±0.047的表达水平分别高于对照组和抗体组0.288±0.049和0.300±0.047、0.972±0.096和0.887±0.059、0.665±0.055和0.709±0.058、0.158±0.040和0.224±0.064(P均＜0.01),对照组与抗体组之间差异无统计学意义,以上蛋白PDGF-BB+抗体组的表达分别比PDGF-BB组低了(85±9.5)%、(79±12)%、(91±17)%和(84±5)%.Smad7蛋白表达在对照组0.461±0.035与抗体组0.458±0.031之间差异无统计学意义,PDGF-BB组0.160±0.028与对照组和抗体组比较差异均有统计学意义(P均＜0.01),PDGF-BB+抗体组0.375±0.039与PDGF-BB组比较差异有统计学意义(P＜0.01).结论 PDGF-BB调节p21~(WAF1)的表达部分通过TGF-β_1及其信号系统.
目的 探討血小闆衍生生長因子(PDGF)-BB是否通過誘導轉化生長因子(TGF)-β_1,錶達及其信號繫統調節p21~(WAF1)錶達.方法 採取8～9週齡雄性Sprague-Dawley大鼠血管平滑肌細胞(VSMC)進行體外培養,分為對照組、中和性抗TGF-β_1抗體組(20μg/ml,抗體組)、PDGF-BB組(40 ng/ml)、PDGF-BB+中和性抗TGF-β_1抗體組(PDGF-BB+抗體組),實驗時間為48 h.逆轉錄酶-聚閤酶鏈式反應檢測TGF-β_1mRNA的錶達,ELISA方法檢測TGF-β_1蛋白的錶達.Western blot檢測p21~(WAF1)及TGF-β信號繫統下遊蛋白錶達,後者包括TGF-β_1I類受體(在VSMC內錶現為ALK-5)、Smurf2、pSmad2/3、Smad4、Smad7.結果 PDGF-BB組的TGF-β_1 mRNA(24 h)和TGF-β_1(48 h)蛋白分子錶達水平分彆為(447.716±23.911)和(0.309±0.034)ng/ml,明顯高于對照組(250.435±17.869)和(0.104±0.013)ng/ml(P均＜0.01).p21WAF1蛋白錶達在對照組0.621±0.046和抗體組0.657±0.058之間差異無統計學意義,PDGF-BB組錶達僅為0.204±0.033,與對照組和抗體組比較差異均有統計學意義(P均＜0.01),PDGF-BB+抗體組錶達為0.530±0.043,與對照組和抗體組相比差異均有統計學意義(P均＜0.05),與PDGF-BB組比較差異亦有統計學意義(P＜0.01).PDGF-BB組ALK-5 0.634±0.060、pSmad2/31.894±0.184、Smad4 1.129±0.136及Smurf2 0.559±0.047的錶達水平分彆高于對照組和抗體組0.288±0.049和0.300±0.047、0.972±0.096和0.887±0.059、0.665±0.055和0.709±0.058、0.158±0.040和0.224±0.064(P均＜0.01),對照組與抗體組之間差異無統計學意義,以上蛋白PDGF-BB+抗體組的錶達分彆比PDGF-BB組低瞭(85±9.5)%、(79±12)%、(91±17)%和(84±5)%.Smad7蛋白錶達在對照組0.461±0.035與抗體組0.458±0.031之間差異無統計學意義,PDGF-BB組0.160±0.028與對照組和抗體組比較差異均有統計學意義(P均＜0.01),PDGF-BB+抗體組0.375±0.039與PDGF-BB組比較差異有統計學意義(P＜0.01).結論 PDGF-BB調節p21~(WAF1)的錶達部分通過TGF-β_1及其信號繫統.
목적 탐토혈소판연생생장인자(PDGF)-BB시부통과유도전화생장인자(TGF)-β_1,표체급기신호계통조절p21~(WAF1)표체.방법 채취8～9주령웅성Sprague-Dawley대서혈관평활기세포(VSMC)진행체외배양,분위대조조、중화성항TGF-β_1항체조(20μg/ml,항체조)、PDGF-BB조(40 ng/ml)、PDGF-BB+중화성항TGF-β_1항체조(PDGF-BB+항체조),실험시간위48 h.역전록매-취합매련식반응검측TGF-β_1mRNA적표체,ELISA방법검측TGF-β_1단백적표체.Western blot검측p21~(WAF1)급TGF-β신호계통하유단백표체,후자포괄TGF-β_1I류수체(재VSMC내표현위ALK-5)、Smurf2、pSmad2/3、Smad4、Smad7.결과 PDGF-BB조적TGF-β_1 mRNA(24 h)화TGF-β_1(48 h)단백분자표체수평분별위(447.716±23.911)화(0.309±0.034)ng/ml,명현고우대조조(250.435±17.869)화(0.104±0.013)ng/ml(P균＜0.01).p21WAF1단백표체재대조조0.621±0.046화항체조0.657±0.058지간차이무통계학의의,PDGF-BB조표체부위0.204±0.033,여대조조화항체조비교차이균유통계학의의(P균＜0.01),PDGF-BB+항체조표체위0.530±0.043,여대조조화항체조상비차이균유통계학의의(P균＜0.05),여PDGF-BB조비교차이역유통계학의의(P＜0.01).PDGF-BB조ALK-5 0.634±0.060、pSmad2/31.894±0.184、Smad4 1.129±0.136급Smurf2 0.559±0.047적표체수평분별고우대조조화항체조0.288±0.049화0.300±0.047、0.972±0.096화0.887±0.059、0.665±0.055화0.709±0.058、0.158±0.040화0.224±0.064(P균＜0.01),대조조여항체조지간차이무통계학의의,이상단백PDGF-BB+항체조적표체분별비PDGF-BB조저료(85±9.5)%、(79±12)%、(91±17)%화(84±5)%.Smad7단백표체재대조조0.461±0.035여항체조0.458±0.031지간차이무통계학의의,PDGF-BB조0.160±0.028여대조조화항체조비교차이균유통계학의의(P균＜0.01),PDGF-BB+항체조0.375±0.039여PDGF-BB조비교차이유통계학의의(P＜0.01).결론 PDGF-BB조절p21~(WAF1)적표체부분통과TGF-β_1급기신호계통.
Objective To assess if the modulating effect of platelet-derived growth factor(PDGF)BB on p21~(WAF1) was mediated by upregulating transforming growth factor(TGF)-β_1 expression in vascular smooth muscle cells(VSMC).Methods TGF-β_1 mRNA and protein expressions were measured by reverse transeription-PCR and ELISA,the protein expressions of p21~(WAF1) and the downstream TGF-βsignalling including TGF-β type I receptor(ALK-5 in VSMC),Smurf2,pSmad2/3,Smad4,Smad7 were detected by Western blot.Results PDGF-BB significantly upregulated the expressions of TGF-β_1 at mRNA(0.79-fold)and protein(1.98-fold)levels in VSMC,significantly inhibited the expression of p21WAF1(-67±12)%,and enhanced the expressions of ALK-5,pSmad2/3,Smad4,Smurf2 protein by 1.21-fold.0.95-fold,0.69.fold and 2.55-fold respectively,inhibited Smad7 expression(-65±9)%,these alterations were partially restored by anti-TGF-β_1 neutralizing antibody.Conclusions These findings suggested that PDGF-BB inhibited p21~(WAF1) expression in VSMC partially through upregulating TGF-β_1 expression via PDGFBB and TGF-β signalling pathways.
