血管平滑肌细胞增殖与转化生长因子β/smads的表达  

Proliferation of vascular smooth muscle cell and the expression of transforming growth factor beta/smads

作　　者：闫承慧[1] 申景岭[2] 金焰[2] 王柏秋[2] 王哲[2] 张贵寅[2] 李璞[2] 傅松滨[2] 

机构地区：[1]解放军沈阳军区总医院全军心血管病研究所心内科,辽宁省沈阳市110016 [2]哈尔滨医科大学医学遗传学研究室,黑龙江省哈尔滨市150086

出　　处：《中国临床康复》2005年第19期50-52,i001,共4页Chinese Journal of Clinical Rehabilitation

摘　　要：目的:观察增殖和分化两种血管平滑肌细胞表型改变与转化生长因子β/smads信号传递的关系。方法:实验于2003-09/10哈尔滨医科大学医学遗传学研究室完成。取体质量200g左右的健康雄性SD大鼠,常规胶原酶消化法原代培养大鼠血管平滑肌细胞。用体积分数为0.2和0.05(去血清培养)胎牛血清和的Dulbecco改良的Eagle培养基,37℃,体积分数0.05CO2进行培养,2.5g/L胰酶消化传代。血管平滑肌细胞经锥虫蓝染色,存活细胞数在98%以上。形态学上出现典型的“峰和谷”样生长状态,抗平滑肌特异α-肌动蛋白免疫组织化学染色阳性。取3～8代细胞用于实验。流式细胞分析检测不同表型血管平滑肌细胞增殖能力,蛋白质印迹分析方法检测血管平滑肌细胞分化相关基因平滑肌特异性肌动蛋白表达变化,反转录聚合酶链反应和细胞免疫荧光方法检测转化生长因子βI型受体,smad2,smad3,smad4和smad7的表达变化。结果:①流式细胞分析及蛋白印迹分析方法检测发现,去血清培养后,原代培养大鼠血管平滑肌细胞增殖能力下降;DNA合成前期细胞明显增加犤(52.42±2.35)%犦,细胞分化基因平滑肌特异性α-肌动蛋白明显表达。体积分数为0.2胎牛血清培养后,大鼠血管平滑肌细胞增殖旺盛,DNA合成前期细胞数相对较少犤(17.23±1.58)%犦,细胞分化相关基因平滑肌特异性肌动蛋白表达明显下调。②反转录聚合酶链反应和细胞免疫荧光方法检测发现,去血清培养后的分化表型平滑肌细胞中,转化生长因子βI型受体表达增加,smad2和smad3表达无明显变化,smad4表达增加,smad7表达下降。而高血清培养诱导平滑肌细胞转化为增殖表型后,转化生长因子βI型受体表达下降,smad4表达下降,抑制型smad7表达增加,smad2和smad3表达无明显变化。结论:转化生长因子β/smads表达与血管平滑肌细胞表型状态密切相关。血管平滑肌细胞分化时,可能AIM:To observe the phenothpe changes of proliferation and differentiation of vascular smooth muscle cells associated with the signal pathway of transforming growth factor beta/smads.METHODS:The study was finished in the Research Room of Genetics of Harbin Medical University between September and October 2003.Healthy male SD rats,weighing about 200 g,were used.The model of rat vascular smooth muscle cells was constructed by primary culture,and then cultured in the fetal bovine serum with the volume fraction of 0.2 and 0.05 (serum deprivation culture) and Dulbecco modified Eaglethe medium at 37 ℃ ,the volume fraction of carbon dioxide was 0.05,and passaged with 2.5 g/L pancreatin digestion.After trypan blue staining,more than 98% of the vascular smooth muscle cells survived. Morphologically,classic' peak' and ' valley' shape growth states were observed,anti smooth muscle specific alpha actin protein was positive detected with immunohistochemical staining.Cells of the 3rd and 8th passages were used in the study.The proliferation of vascular smooth muscle cells of different phenotype was detected with flow cytometry;The changes of differentiation related gene smooth muscle specific alpha actin protein expression of smooth muscle cells were determined with western blot analysis;The changes of the expressions of transforming growth factor beta type I receptor,smad2,smad3,smad4 and smad7 were detected with reverse transcription polymerase chain reaction and immunofluorescence method.RESULTS:① Flow cytometry analysis and western blot analysis found that after serum deprivation culture,the proliferation of primarily cultured vascular smooth cells was decreased,the cells at DNA G1 phase was obviously increased[(52.42± 2.35)% ],and expression of differentiated gene smooth muscle specific actin was obvious;After culture in the fetal bovine serum with the volume fraction of 0.2,the proliferation of primarily cultured vascular smooth cells was productive,the number of cells at DNA G1 phase was fewer relatively[(17.23± 1.

关 键 词：肌 平滑 血管/病理学 细胞 培养的 受体 转化生长因子β 

分 类 号：R543[医药卫生—心血管疾病]