CAJ | 학술논문

目的探讨合成型血管平滑肌细胞(VSMCs)的分离、培养及鉴定方法.方法采用改良Ⅱ型胶原酶消化法分离、体外培养SD大鼠胸主动脉VSMCs,胰酶消化传代获取第3代细胞,分为空白组与血小板衍生因子-BB(PDGF-BB)处理组.噻唑蓝(MTT)比色法及细胞划痕实验分别检测分析细胞增殖活性与迁移活性;倒置显微镜、结晶紫染色观察形态学改变;免疫细胞化学染色鉴定VSMCs并测定分析收缩型标记蛋白α-平滑肌肌动蛋白(SMA)以及合成型标记蛋白平滑肌胚胎型肌球蛋白重链(SMemb)、原肌球蛋白-4(TPM-4)表达量的变化.结果空白组VSMCs呈典型“谷峰状”生长,SMA细胞免疫荧光染色及免疫细胞化学染色阳性率≥95％;PDGF-BB处理组VSMCs由收缩型长梭状形态向合成型扁平状形态转化(P＜0.05),VSMCs增殖及迁移活性分别增加11.48％、1.92倍(P＜0.05);收缩型标记蛋白SMA表达下降(P＜0.05),合成型标记蛋白SMemb及TPM-4表达均增加(P＜0.05).结论 PDGF-BB处理后,VSMCs由收缩表型向合成表型转化,增殖迁移活性增加,收缩型标记蛋白表达下降、合成型标记蛋白呈现高表达,表现为高合成型VSMCs的特点.
목적 탐토합성형혈관평활기세포(VSMCs)적분리、배양급감정방법.방법 채용개량Ⅱ형효원매소화법분리、체외배양SD대서흉주동맥VSMCs,이매소화전대획취제3대세포,분위공백조여혈소판연생인자-BB(PDGF-BB)처리조.새서람(MTT)비색법급세포화흔실험분별검측분석세포증식활성여천이활성;도치현미경、결정자염색관찰형태학개변;면역세포화학염색감정VSMCs병측정분석수축형표기단백α-평활기기동단백(SMA)이급합성형표기단백평활기배태형기구단백중련(SMemb)、원기구단백-4(TPM-4)표체량적변화.결과 공백조VSMCs정전형“곡봉상”생장,SMA세포면역형광염색급면역세포화학염색양성솔≥95％;PDGF-BB처리조VSMCs유수축형장사상형태향합성형편평상형태전화(P＜0.05),VSMCs증식급천이활성분별증가11.48％、1.92배(P＜0.05);수축형표기단백SMA표체하강(P＜0.05),합성형표기단백SMemb급TPM-4표체균증가(P＜0.05).결론 PDGF-BB처리후,VSMCs유수축표형향합성표형전화,증식천이활성증가,수축형표기단백표체하강、합성형표기단백정현고표체,표현위고합성형VSMCs적특점.
Objective To investigate a method to obtain vascular smooth muscle cells (VSMCs),and isolate,culture and identify the VSMCs with highly synthetic phenotype.Methods The primary and subculture of VSMCs from thoracic aorta of SD rats was done by modified collage Ⅱ enzymatic dispersion and trypsin digestion,respectively.The third passage VSMCs were treated with platelet-derived growth factorBB (PDGF-BB).The cell viability and migration were detected by methyl thiazol tetrazolium (MTT) assay and wound-healing assay,respectively.The VSMCs were observed using inverted microscope and crystal violet staining for morphological observation.Immunofluorescence staining and immunocytochemical staining were used to examine contractile marker α-smooth muscle actin (SMA) and synthetic markers nonmuscle MHC isoform-B (SMemb),Tropomyosin-4 (TPM-4) for VSMCs identification and quantitative analysis.Results The post-confluent cells in the control group exhibited the "hill and valley" growth pattern typical of cultured VSMCs.More than 95％ of cells were positively stained by both stains for SMA.VSMCs under PDGF-BB treatment underwent a significant morphological alteration,from spindle to polygonal shape (P ＜0.05).The viability and mobility of VSMCs was increased by 11.48％ and 1.92 times,respectively (P ＜0.05).The expression of both SMemb and TPM-4 proteins was increased,and that of SMA protein decreased,significantly (P ＜ 0.05).Conclusion PDGF-BB treatment could lead to a significant phenotype modulation (highly synthetic VSMCs) in accompanied with enhancement of viability and mobility,highly increased synthetic markers while decreased contractile marker,significantly.