發(fā)布時(shí)間：2018-02-03 22:26

  本文關(guān)鍵詞： 血管平滑肌細(xì)胞 胸主動(dòng)脈 膠原酶消化法 組織塊貼壁法 細(xì)胞生長(zhǎng) 細(xì)胞增殖 細(xì)胞分化 大鼠　出處：《山東醫(yī)藥》2017年28期 　論文類型：期刊論文

【摘要】：目的比較膠原酶消化法、組織塊貼壁法培養(yǎng)的大鼠胸主動(dòng)脈平滑肌細(xì)胞(RASMCs)生長(zhǎng)、增殖及分化的影響。方法選擇6~8周齡雄性SD大鼠6只,取胸主動(dòng)脈及主動(dòng)脈弓部,保留中膜備用。分別采用膠原酶消化法、組織塊貼壁法培養(yǎng)RASMCs。采用α-肌動(dòng)蛋白免疫熒光染色法鑒定RASMCs,并比較兩種方法培養(yǎng)第0、24、36、48、60、72、84、96 h RASMCs生長(zhǎng)情況。采用CCK-8法檢測(cè)細(xì)胞增殖情況,流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期。采用實(shí)時(shí)定量PCR法檢測(cè)細(xì)胞表型標(biāo)志基因表達(dá),包括收縮型標(biāo)志基因α-平滑肌肌動(dòng)蛋白(α-SMA)、轉(zhuǎn)膠蛋白(TAGLN)、肌球蛋白重鏈11(MYH-11)、轉(zhuǎn)錄因子1(SP-1)及合成型標(biāo)志基因骨橋蛋白(OPN)。結(jié)果膠原酶消化法、組織塊貼壁法均成功分離出RASMCs,細(xì)胞純度均在95%以上。培養(yǎng)36、48、60、72 h時(shí),組織塊貼壁法分離、培養(yǎng)的RASMCs計(jì)數(shù)多于膠原酶消化法(P均0.05)。培養(yǎng)36、48、60、72 h時(shí),膠原酶消化法分離、培養(yǎng)的RASMCs活力優(yōu)于組織塊貼壁法(P均0.05)。兩種方法分離、培養(yǎng)的RASMCs G_1、G_2、S期細(xì)胞所占比例比較差異均無(wú)統(tǒng)計(jì)學(xué)意義(P均0.05)。膠原酶消化法分離、培養(yǎng)的RASMCs中α-SMA、TAGLN、MYH-11 mRNA相對(duì)表達(dá)量均高于組織塊貼壁法,OPN mRNA相對(duì)表達(dá)量低于組織塊貼壁法(P均0.05)。結(jié)論膠原酶消化法、組織塊貼壁法均成功分離、培養(yǎng)出RASMCs。膠原酶消化法分離、培養(yǎng)的RASMCs生長(zhǎng)、增殖較慢,但保持收縮表型;組織塊貼壁法分離、培養(yǎng)的RASMCs生長(zhǎng)、增殖較快,但有向合成表型轉(zhuǎn)化的趨勢(shì)。
[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 偽 -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 偽 -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 偽 -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.
【作者單位】： 第二軍醫(yī)大學(xué)附屬長(zhǎng)海醫(yī)院;
【基金】：上海市衛(wèi)生系統(tǒng)重要疾病聯(lián)合攻關(guān)項(xiàng)目計(jì)劃(2014ZYJB0401)
【分類號(hào)】：R54
【正文快照】： 主動(dòng)脈夾層(AD)是一類病死率較高的疾病[1,2]。研究顯示,約80%未經(jīng)醫(yī)學(xué)干預(yù)的StanfordA型AD患者在發(fā)病2周內(nèi)會(huì)因主動(dòng)脈夾層破裂死亡[3]。目前關(guān)于AD的發(fā)病機(jī)制尚不明確。血管平滑肌細(xì)胞(VSMCs)是構(gòu)成血管壁中膜的主要結(jié)構(gòu)及物質(zhì)基礎(chǔ),可保持血管壁完整性、維持血管壁張力[4],同

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