低密度脂蛋白诱导骨髓间充质干细胞向平滑肌样细胞分化的作用研究

发布时间：2018-06-01 21:30

本文选题：骨髓间充质干细胞 + LDL　；参考：《华中科技大学》2008年硕士论文

【摘要】： 背景动脉粥样硬化(artherosclerosis, As)是许多心脑血管疾病发生的前期病理基础。在高脂等因素所致的As发生过程中,平滑肌细胞增生并合成细胞外基质是其重要的环节。传统观念认为,病变中的平滑肌细胞来源于病变部位的动脉中膜。然而最近研究结果表明,病灶部位的平滑肌细胞并非完全来自于中膜,骨髓来源的细胞在一定的情况下可以定向分化为平滑肌细胞参与动脉粥样硬化的发生与发展。目的探讨低密度脂蛋白(low density lipoprotein,LDL)诱导骨髓间充质干细胞(bone marrow stromal cells,BMSC)向平滑肌样细胞分化及myocardin在其分化过程中的作用。方法用差异贴壁法分离纯化原代骨髓间充质干细胞,在不同浓度血清诱导BMSC分化条件下加以LDL刺激,检测BMSC分化过程中myocardin及平滑肌细胞表面标志平滑肌肌动蛋白(α-smooth muscle actin,α-SMA),平滑肌肌球蛋白重链(smooth muscle myosin heavy chain, SM-MHC)以及SM-22α的表达。细胞分为:①常规培养组(含低糖-DMEM培养基,10%胎牛血清,3%谷氨酰胺),②常规培养+LDL刺激组(25mg/L),③2 0%FBS培养组,④20%FBS培养+LDL刺激组。②、④组分别在培养24小时、48小时和72小时后加入LDL,各组细胞均培养96小时。免疫细胞化学检测myocardin、α-SMA及SM-MHC的表达,结合形态学鉴定BMSC向平滑肌样细胞分化的情况;逆转录-聚合酶链反应法(reverse transcription-polymerase chain reaction, RT-PCR)检测myocardin、α-SMA及SM-22αmRNA的表达。结果光镜显示BMSC随着诱导时间延长逐渐由小圆形、多角形转变为长梭形。免疫细胞化学结果显示常规培养+LDL刺激组、20%FBS培养组、20%FBS培养+LDL刺激组分别与常规培养组相比myocardin、α-SMA及SM-MHC蛋白的表达增强,差异有统计学意义(P0. 05, n=9);同种浓度血清培养,LDL作用48小时上述三种蛋白的表达最强;相同LDL作用时间点,20%FBS培养+LDL剌激组与常规培养+LDL剌激组相比上述三种蛋白的表达明显增强,差异有统计学意义(P0. 05, n=9);α-SMA、SM-MHC蛋白的表达与myocardin表达呈正相关(r=0. 9018, P0.05; r=0. 7969,P0.05)。RT-PCR结果显示常规培养+LDL刺激组、20%FBS培养组、20%FBS培养+LDL刺激组分别与常规培养组相比myocardin、α-SMA及SM-22αmRNA表达增强,差异有统计学意义(P0. 05, n=9);同种浓度血清培养,LDL作用48小时myocardin、α-SMA及SM-22αmRNA的表达最强;相同LDL作用时间点,20% FBS培养+LDL刺激组与常规培养+LDL刺激组相比myocardin、α-SMA及SM-22αmRNA的表达均明显增强,差异有统计学意义(P0. 05,n=9)。α-SMA、SM-22αmRNA表达与myocardin表达呈正相关(r=0. 9295, P0.05; r=0. 8940,P0.05)。结论LDL,20%FBS均可诱导BMSC向平滑肌样细胞分化,LDL联合20% FBS诱导BMSC向平滑肌样细胞分化的作用最强。myocardin可能在此过程中起重要作用。
[Abstract]:Background Atherosclerotic atherosclerosis (as) is the early pathological basis of many cardiovascular and cerebrovascular diseases. Smooth muscle cells proliferate and synthesize extracellular matrix (ECM) in the process of as induced by hyperlipidemia and other factors. The traditional view is that the smooth muscle cells in the lesion originate from the medial membrane of the artery at the site of the lesion. However, recent studies have shown that the smooth muscle cells in the lesion are not completely derived from the mesenchyma, and that the cells derived from the bone marrow can be differentiated into smooth muscle cells to participate in the occurrence and development of atherosclerosis. Objective to investigate the differentiation of bone marrow mesenchymal stem cells (BMSCs) into smooth muscle like cells (SMCs) induced by low density lipoprotein (LDL) and the role of myocardin in the differentiation process. Methods Primary bone marrow mesenchymal stem cells were isolated and purified by differential adherent method, and stimulated with LDL under different concentration of serum to induce BMSC differentiation. The expression of myocardin and smooth muscle actinin (伪 -SMAA), smooth muscle myosin heavy chain smooth muscle myosin heavy chain, (SM-MHC) and SM-22 伪 were detected during BMSC differentiation. The cells were divided into two groups: 1: 1 conventional culture group (10% fetal bovine serum containing 10% fetal bovine serum and 3% Glutamine) in 10% low sugar DMEM culture group (LDL stimulation group) 25 mg / L / L ~ (32) S culture group ~ (42) S culture LDL stimulation group. Four groups were cultured for 24 hours, 48 hours and 72 hours, respectively. The cells in each group were cultured for 96 hours. The expression of myocardinin, 伪 -SMA and SM-MHC was detected by immunocytochemistry, and the differentiation of BMSC into smooth muscle cells was identified by morphology, and the expressions of myocardin, 伪 -SMA and SM-22 伪 mRNA were detected by reverse transcription-polymerase chain reactionation (RT-PCR) by reverse transcriptase polymerase chain reaction (RT-PCR). Results the light microscope showed that BMSC gradually changed from small circle and polygonal to long fusiform with the prolongation of induction time. The results of immunocytochemistry showed that the expression of myocardinin, 伪 -SMA and SM-MHC protein in LDL group, 20s culture group and 20s cultured LDL group were significantly higher than those in conventional culture group. The expression of the above three proteins was the strongest in the same concentration of serum culture group at 48 hours, and the expression of the above three proteins in the 20s cultured LDL stimulation group was significantly higher than that in the conventional cultured LDL stimulation group at the same time point of time of LDL, and the expression of the above three proteins was significantly higher in the 20s cultured LDL stimulation group than in the conventional cultured LDL stimulation group. The difference was statistically significant (P0. The expression of 伪 -SMA-SM-MHC protein was positively correlated with the expression of myocardin. 9018, P0. 05; r0. 0. The results of RT-PCR showed that the expressions of myocardinin, 伪 -SMA and SM-22 伪 mRNA were significantly higher in LDL stimulation group, 20 S culture group and 20 S cultured LDL group than in normal culture group, respectively. The expression of myocardinin, 伪 -SMA and SM-22 伪 mRNA in the same concentration serum culture group was the highest at 48 hours, and the expression of myocardinin, 伪 -SMA and SM-22 伪 mRNA in the same LDL treated group was significantly higher than that in the control LDL group, and the difference was statistically significant (P0). The expression of 伪 -SMA-SM-22 伪 mRNA was positively correlated with the expression of myocardin. 9295, P0.05; r0. 0. 8940 (P0.05). Conclusion both LDL and 20s can induce BMSC to differentiate into smooth muscle cells. Myocardin may play an important role in this process.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

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