TSA对骨髓间充质干细胞定向分化的促进作用及机制探讨

发布时间：2018-06-27 22:46

本文选题：骨髓间充质干细胞 + 诱导分化　；参考：《重庆医科大学》2009年硕士论文

【摘要】： 目的明确TSA是否对MSCs向心肌细胞分化有促进作用;探讨MSCs向心肌细胞分化过程中乙酰化特异性调控的作用机制。材料与方法 1.细胞培养:取Wistar大鼠的股骨和胫骨骨髓,用贴壁法分离、培养、扩增体外MSCs;取Wistar大鼠新生鼠(1～3天)的心脏,用消化分离法、差速贴壁法及化学试剂抑制法分离、纯化、培养心肌细胞。 2.诱导实验:本实验分为两组即原始对照组(MSCs)、诱导组(包括与心肌细胞共培养和5-aza诱导两种方式)。检测指标:(1)采用基于SYBR.GREENI法的实时荧光定量PCR检测心肌细胞早期转录因子GATA-4、NKx2.5、MEF2c;(2)免疫荧光观察心肌特异性蛋白MHC、CX43、cTnT表达变化;(3)Western-blot分析心肌特异性肌钙蛋白T(cardiac muscle Troponin T cTnT)表达量。 3.诱导后干预实验:( 1)5-氮胞苷诱导M SCs细胞;( 2)用transwell插入式培养皿构建MSCs和心肌细胞共培养模式。两组细胞分别用不同浓度的去乙酰化酶抑制剂TSA进行干预。检测指标同上。 4.机制探讨实验:应用染色质免疫共沉淀技术(ChIP)沉淀出与Gcn5蛋白结合的心肌细胞相关基因,设计针对心肌细胞特异基因GATA-4、NKx2.5、MEF2c的DNA引物,实时荧光定量PCR检测几种基因的表达。结果 1. MSCs及心肌细胞培养状况S Cs接种24小时后开始贴壁生长,可见稀少的呈长梭形的贴壁细胞3～4天后开始集落样增殖,梭形贴壁细胞开始增多,集落周围的贴壁细胞逐渐开始融合;传代后细胞呈束状、漩涡状排列,随着换液与传代, MSCs逐渐得到纯化。分离的心肌细胞贴壁生长第3天细胞相互接触交织,逐渐形成细胞簇或细胞单层,镜下不同视野下可见搏动的细胞团(含单个自发搏动的细胞),搏动呈同步性,搏动频率在40～60次/ min。l周后大量细胞成团收缩,同时成纤维细胞开始生长。 2.共培养诱导组: MSCs与心肌细胞共培养1周,实时荧光定量PCR检测心肌细胞早期转录因子,结果显示心肌细胞早期转录因子GATA-4、NKx2.5和MEF2c出现表达;免疫荧光显色心肌细胞特异蛋白,在MSCs胞浆可见心肌特异蛋白cTnT及CX43和MHC表达; Western-blot检测出现c TnT的表达。5-aza诱导组: MSCs经5-aza诱导实时荧光定量PCR显示心肌细胞早期转录因子GATA-4、NKx2.5和MEF2c出现表达; Western-blot检测MSCs也出现cTnT的表达。 3.诱导后干预实验:共培养和经不同浓度TSA干预:实时荧光定量PCR检测心肌细胞早期转录因子,TSA500nmol/L浓度与其余两组不同浓度TSA比较表达最高,差异有显著性(P0.05);而因子GATA-4和MEF2c在100nmol/L与300nmol/L组间比较没有显著差异性。NKx2.5的表达在一定浓度范围随TSA浓度增加呈上调趋势差异有显著性(P0.05);免疫荧光检测MSCs向心肌样细胞分化率,胞浆可见心肌特异性蛋白cTnT和CX43出现表达,随培养时间延长和TSA干预浓度提高,MSCs的分化率逐步提高,但提高幅度较低。Western-blot检测cTnT的表达量,共培养及干预组都比MSCs组有提高,共培养组与干预组间比较,100nmol/L、300nmol/L、500nmol/L都比共培养组有提高;TSA干预组间比较,500nmol/L浓度与其余两组不同浓度TSA比较表达最高,差异有显著性(P0.05);100nmol/L与300nmol/L之间比较没有显著差异性。5-aza诱导和不同浓度TSA干预:心肌早期转录因子表达水平和心肌特异性蛋白cTnT组间比较,差异有显著性(P0.05),在一定浓度范围表达随TSA浓度增加呈正相关。Western-blot检测cTnT的表达量,经三种不同浓度(100nmol/L、300nmo/L、500nmol/L)TSA干预后与5-aza诱导组比较,在一定浓度范围内蛋白表达量上调,各组间比较TSA100 nmol/L与300 nmol/L;TSA100 nmol/L与TSA500 nmol/L;TSA300 nmol/L与TSA500 nmol/L组间比较差异有显著性(P0.05)。 4.利用ChIP技术沉淀出与Gcn5蛋白一起结合的核酸-蛋白复合物,实时荧光定量PCR检测到该核酸中有心肌早期发育特异基因GATA-4、NKx2.5的DNA启动子表达。结论 1.HDAC酶抑制剂TSA对MSCs向心肌样细胞分化有促进作用,在一定浓度范围内,细胞分化与TSA浓度呈正相关。 2.Gcn5乙酰化复合体调控MSCs向心肌细胞分化的作用机制之一,是启动了心肌细胞早期发育特异性基因的表达。
[Abstract]:Objective to clarify whether TSA can promote the differentiation of MSCs into cardiomyocytes, and explore the mechanism of acetylation specific regulation of MSCs during cardiomyocyte differentiation.
Materials and methods
1. cell culture: the bone marrow of the femur and tibia of Wistar rats was taken from the bone marrow of the femur and tibia. The MSCs was cultured and expanded in vitro, and the heart of the neonatal rat (1~3 days) from Wistar rats was taken. The isolation, purification and culture of cardiac myocytes were obtained by the digestion separation method, differential adherence method and chemical reagent inhibition method.
2. induction experiment: this experiment was divided into two groups: primary control group (MSCs), induction group (including co culture with cardiac myocytes and 5-aza induction in two ways). Detection indexes: (1) detection of myocardial cell early transcription factor GATA-4, NKx2.5, MEF2c by real-time fluorescence quantitative PCR based on SYBR.GREENI method; (2) immunofluorescence observation of cardiac specific protein MHC, CX 43, cTnT expression changes; (3) Western-blot analysis of cardiac specific troponin T (cardiac muscle Troponin T cTnT) expression.
3. induced intervention experiment: (1) 5- azacytidine induced M SCs cells; (2) the co culture mode of MSCs and cardiomyocytes was constructed with Transwell insert culture dish. The two groups were treated with different concentrations of deacetylase inhibitor TSA.
4. mechanism study: using chromatin immunoprecipitation (ChIP) to precipitate the related genes associated with Gcn5 protein, and design the DNA primers for GATA-4, NKx2.5, MEF2c of cardiomyocyte specific gene, and detect the expression of several genes by real time fluorescence quantitative PCR.
Result
1. MSCs and cardiomyocyte culture condition S Cs inoculation began to adhere to the wall after 24 hours. It can be seen that the small spindle shaped adherent cells began to proliferate in 3~4 days, and the spindle shaped adherent cells began to increase, and the adherent cells around the colony gradually began to fuse; after the passage, the cells were bundled and swirled, with the change of fluid and passage, MSCs by the passage. The isolated cardiomyocytes were interwoven with each other for third days, gradually forming a cell cluster or cell monolayer. A pulsating cell group (including a single spontaneous pulsating cell) was visible under the microscope, and the pulsation was synchronized. The pulsation frequency of a large number of cells contracted after 40~60 times / min.l weeks, and the fibroblasts were formed at the same time. Start to grow.
2. co culture induction group: MSCs and cardiac myocytes were co cultured for 1 weeks. Real time fluorescence quantitative PCR was used to detect the early transcription factors of cardiac myocytes. The results showed that the early transcription factor GATA-4, NKx2.5 and MEF2c were expressed in cardiac myocytes; the specific protein of cardiac myocytes was detected by immunofluorescence, and the expression of cardiac specific protein cTnT, CX43 and MHC in MSCs cytoplasm; Weste; Weste. Rn-blot detected the expression of.5-aza induced group in the expression of C TnT: MSCs was induced by 5-aza in real-time quantitative PCR to show the early transcription factor GATA-4 of cardiac myocytes, NKx2.5 and MEF2c expression, and Western-blot detection MSCs also appeared.
3. the intervention experiment after induction: co culture and different concentrations of TSA intervention: real-time fluorescence quantitative PCR detection of early transcription factors of cardiac myocytes, TSA500nmol/L concentration and the other two groups of different concentrations of TSA were the highest, the difference was significant (P0.05), but the factor GATA-4 and MEF2c were not significantly different between 100nmol/L and 300nmol/L.NKx2.5. The expression of the expression in a certain concentration range increased with the increase of TSA concentration (P0.05). Immunofluorescence detected the differentiation rate of MSCs to cardiac muscle like cells, and the expression of specific protein cTnT and CX43 in the cytoplasm. With the prolongation of culture time and the increase of TSA intervention concentration, the differentiation rate of MSCs increased gradually, but the increase of.Wester was lower.Wester. N-blot detection of the expression of cTnT, co culture and intervention group is higher than the MSCs group, the co culture group and the intervention group, 100nmol/L, 300nmol/L, 500nmol/L are higher than the co culture group, TSA intervention group, the concentration of 500nmol/L and the remaining two groups of different concentrations of TSA compared to the highest, the difference is significant (P0.05); 100nmol/L and 300nmol/L There was no significant difference between.5-aza induction and different concentrations of TSA intervention: the expression level of early myocardial transcription factors and the comparison of cardiac specific protein cTnT groups were significant (P0.05). The expression of cTnT in a certain concentration range was positively correlated with the increase of TSA concentration by.Western-blot, after three different concentrations (100nmol/L, 3) 00nmo/L, 500nmol/L) compared with the 5-aza induced group, the protein expression was up regulated in a certain concentration range. The TSA100 nmol/L and 300 nmol/L were compared in each group, and TSA100 nmol/L and TSA500 nmol/L were compared between each group, and there was a significant difference between the TSA100 nmol/L and the TSA500 nmol/L.
4. the nucleic acid protein complex combined with Gcn5 protein was precipitated by ChIP technique, and the real time fluorescence quantitative PCR was detected in the nucleic acid of the nucleic acid, the expression of the early development specific gene GATA-4 of the myocardium, and the DNA promoter of NKx2.5.
conclusion
1.HDAC enzyme inhibitor TSA promoted the differentiation of MSCs into cardiomyocyte like cells. In a certain concentration range, cell differentiation was positively correlated with TSA concentration.
One of the mechanisms by which 2.Gcn5 acetylated complex regulates the differentiation of MSCs into cardiomyocytes is to initiate the expression of specific genes in early development of cardiomyocytes.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

【参考文献】