胶原酶消化法、组织块贴壁法培养的大鼠RASMCs生长、增殖及分化对比观察

发布时间：2018-02-03 22:26

本文关键词： 血管平滑肌细胞胸主动脉胶原酶消化法组织块贴壁法细胞生长细胞增殖细胞分化大鼠　出处：《山东医药》2017年28期 　论文类型：期刊论文

【摘要】：目的比较胶原酶消化法、组织块贴壁法培养的大鼠胸主动脉平滑肌细胞(RASMCs)生长、增殖及分化的影响。方法选择6~8周龄雄性SD大鼠6只,取胸主动脉及主动脉弓部,保留中膜备用。分别采用胶原酶消化法、组织块贴壁法培养RASMCs。采用α-肌动蛋白免疫荧光染色法鉴定RASMCs,并比较两种方法培养第0、24、36、48、60、72、84、96 h RASMCs生长情况。采用CCK-8法检测细胞增殖情况,流式细胞术检测细胞周期。采用实时定量PCR法检测细胞表型标志基因表达,包括收缩型标志基因α-平滑肌肌动蛋白(α-SMA)、转胶蛋白(TAGLN)、肌球蛋白重链11(MYH-11)、转录因子1(SP-1)及合成型标志基因骨桥蛋白(OPN)。结果胶原酶消化法、组织块贴壁法均成功分离出RASMCs,细胞纯度均在95%以上。培养36、48、60、72 h时,组织块贴壁法分离、培养的RASMCs计数多于胶原酶消化法(P均0.05)。培养36、48、60、72 h时,胶原酶消化法分离、培养的RASMCs活力优于组织块贴壁法(P均0.05)。两种方法分离、培养的RASMCs G_1、G_2、S期细胞所占比例比较差异均无统计学意义(P均0.05)。胶原酶消化法分离、培养的RASMCs中α-SMA、TAGLN、MYH-11 mRNA相对表达量均高于组织块贴壁法,OPN mRNA相对表达量低于组织块贴壁法(P均0.05)。结论胶原酶消化法、组织块贴壁法均成功分离、培养出RASMCs。胶原酶消化法分离、培养的RASMCs生长、增殖较慢,但保持收缩表型;组织块贴壁法分离、培养的RASMCs生长、增殖较快,但有向合成表型转化的趋势。
[Abstract]:Objective to compare the effects of collagenase digestion and tissue mass adherence on the growth, proliferation and differentiation of rat thoracic aortic smooth muscle cells. The thoracic aorta and aortic arch were taken and the middle membrane was reserved. RASMCswere cultured by collagenase digestion and tissue mass adherence. The RASMCs was identified by 伪 -actin immunofluorescence staining. The growth of RASMCs was compared between the two methods. The proliferation of the cells was detected by CCK-8 assay. Flow cytometry (FCM) was used to detect cell cycle. The expression of phenotypic marker genes, including 伪 -SMAs, was detected by real-time quantitative PCR. Transglutinin (TAGLN), myosin heavy chain 11 (MYH-11), transcription factor 1 (SP-1) and the synthetic marker gene osteopontin (OPN). Results Collagenase digestion was performed. RASMCs were isolated successfully by tissue mass adherence method, and the purity of RASMCs was above 95%. After 36 cells were cultured for 48 hours, the tissue mass adherent method was used to isolate RASMCs. The RASMCs count of culture was more than that of collagenase digestion (P < 0.05). The activity of RASMCs cultured was better than that of tissue mass adherence method (P < 0.05). The two methods were separated, and the cultured RASMCs G _ (1) / G _ (2). There was no significant difference in the percentage of S phase cells (P < 0.05). Collagenase digestion was used to isolate 伪 -SMA-TAGLN from cultured RASMCs. The relative expression of MYH-11 mRNA was higher than that of tissue mass adherence method (P < 0.05). RASMCs. collagenase digestion method was used to isolate RASMCs. the RASMCs grew slowly, but kept the contraction phenotype. The cultured RASMCs grew faster and proliferated rapidly, but had the tendency of transformation to synthetic phenotype.
【作者单位】：第二军医大学附属长海医院;
【基金】：上海市卫生系统重要疾病联合攻关项目计划(2014ZYJB0401)
【分类号】：R54
【正文快照】： 主动脉夹层(AD)是一类病死率较高的疾病[1,2]。研究显示,约80%未经医学干预的StanfordA型AD患者在发病2周内会因主动脉夹层破裂死亡[3]。目前关于AD的发病机制尚不明确。血管平滑肌细胞(VSMCs)是构成血管壁中膜的主要结构及物质基础,可保持血管壁完整性、维持血管壁张力[4],同

【参考文献】