本文選題：骨髓間充質(zhì)干細(xì)胞 + 誘導(dǎo)分化��； 參考：《重慶醫(yī)科大學(xué)》2009年碩士論文

【摘要】： 目的明確TSA是否對MSCs向心肌細(xì)胞分化有促進(jìn)作用;探討MSCs向心肌細(xì)胞分化過程中乙酰化特異性調(diào)控的作用機(jī)制。 材料與方法 1.細(xì)胞培養(yǎng):取Wistar大鼠的股骨和脛骨骨髓,用貼壁法分離、培養(yǎng)、擴(kuò)增體外MSCs;取Wistar大鼠新生鼠(1～3天)的心臟,用消化分離法、差速貼壁法及化學(xué)試劑抑制法分離、純化、培養(yǎng)心肌細(xì)胞。 2.誘導(dǎo)實驗:本實驗分為兩組即原始對照組(MSCs)、誘導(dǎo)組(包括與心肌細(xì)胞共培養(yǎng)和5-aza誘導(dǎo)兩種方式)。檢測指標(biāo):(1)采用基于SYBR.GREENI法的實時熒光定量PCR檢測心肌細(xì)胞早期轉(zhuǎn)錄因子GATA-4、NKx2.5、MEF2c;(2)免疫熒光觀察心肌特異性蛋白MHC、CX43、cTnT表達(dá)變化;(3)Western-blot分析心肌特異性肌鈣蛋白T(cardiac muscle Troponin T cTnT)表達(dá)量。 3.誘導(dǎo)后干預(yù)實驗:( 1)5-氮胞苷誘導(dǎo)M SCs細(xì)胞;( 2)用transwell插入式培養(yǎng)皿構(gòu)建MSCs和心肌細(xì)胞共培養(yǎng)模式。兩組細(xì)胞分別用不同濃度的去乙�；敢种苿㏕SA進(jìn)行干預(yù)。檢測指標(biāo)同上。 4.機(jī)制探討實驗:應(yīng)用染色質(zhì)免疫共沉淀技術(shù)(ChIP)沉淀出與Gcn5蛋白結(jié)合的心肌細(xì)胞相關(guān)基因,設(shè)計針對心肌細(xì)胞特異基因GATA-4、NKx2.5、MEF2c的DNA引物,實時熒光定量PCR檢測幾種基因的表達(dá)。 結(jié)果 1. MSCs及心肌細(xì)胞培養(yǎng)狀況S Cs接種24小時后開始貼壁生長,可見稀少的呈長梭形的貼壁細(xì)胞3～4天后開始集落樣增殖,梭形貼壁細(xì)胞開始增多,集落周圍的貼壁細(xì)胞逐漸開始融合;傳代后細(xì)胞呈束狀、漩渦狀排列,隨著換液與傳代, MSCs逐漸得到純化。分離的心肌細(xì)胞貼壁生長第3天細(xì)胞相互接觸交織,逐漸形成細(xì)胞簇或細(xì)胞單層,鏡下不同視野下可見搏動的細(xì)胞團(tuán)(含單個自發(fā)搏動的細(xì)胞),搏動呈同步性,搏動頻率在40～60次/ min。l周后大量細(xì)胞成團(tuán)收縮,同時成纖維細(xì)胞開始生長。 2.共培養(yǎng)誘導(dǎo)組: MSCs與心肌細(xì)胞共培養(yǎng)1周,實時熒光定量PCR檢測心肌細(xì)胞早期轉(zhuǎn)錄因子,結(jié)果顯示心肌細(xì)胞早期轉(zhuǎn)錄因子GATA-4、NKx2.5和MEF2c出現(xiàn)表達(dá);免疫熒光顯色心肌細(xì)胞特異蛋白,在MSCs胞漿可見心肌特異蛋白cTnT及CX43和MHC表達(dá); Western-blot檢測出現(xiàn)c TnT的表達(dá)。5-aza誘導(dǎo)組: MSCs經(jīng)5-aza誘導(dǎo)實時熒光定量PCR顯示心肌細(xì)胞早期轉(zhuǎn)錄因子GATA-4、NKx2.5和MEF2c出現(xiàn)表達(dá); Western-blot檢測MSCs也出現(xiàn)cTnT的表達(dá)。 3.誘導(dǎo)后干預(yù)實驗:共培養(yǎng)和經(jīng)不同濃度TSA干預(yù):實時熒光定量PCR檢測心肌細(xì)胞早期轉(zhuǎn)錄因子,TSA500nmol/L濃度與其余兩組不同濃度TSA比較表達(dá)最高,差異有顯著性(P0.05);而因子GATA-4和MEF2c在100nmol/L與300nmol/L組間比較沒有顯著差異性。NKx2.5的表達(dá)在一定濃度范圍隨TSA濃度增加呈上調(diào)趨勢差異有顯著性(P0.05);免疫熒光檢測MSCs向心肌樣細(xì)胞分化率,胞漿可見心肌特異性蛋白cTnT和CX43出現(xiàn)表達(dá),隨培養(yǎng)時間延長和TSA干預(yù)濃度提高,MSCs的分化率逐步提高,但提高幅度較低。Western-blot檢測cTnT的表達(dá)量,共培養(yǎng)及干預(yù)組都比MSCs組有提高,共培養(yǎng)組與干預(yù)組間比較,100nmol/L、300nmol/L、500nmol/L都比共培養(yǎng)組有提高;TSA干預(yù)組間比較,500nmol/L濃度與其余兩組不同濃度TSA比較表達(dá)最高,差異有顯著性(P0.05);100nmol/L與300nmol/L之間比較沒有顯著差異性。5-aza誘導(dǎo)和不同濃度TSA干預(yù):心肌早期轉(zhuǎn)錄因子表達(dá)水平和心肌特異性蛋白cTnT組間比較,差異有顯著性(P0.05),在一定濃度范圍表達(dá)隨TSA濃度增加呈正相關(guān)。Western-blot檢測cTnT的表達(dá)量,經(jīng)三種不同濃度(100nmol/L、300nmo/L、500nmol/L)TSA干預(yù)后與5-aza誘導(dǎo)組比較,在一定濃度范圍內(nèi)蛋白表達(dá)量上調(diào),各組間比較TSA100 nmol/L與300 nmol/L;TSA100 nmol/L與TSA500 nmol/L;TSA300 nmol/L與TSA500 nmol/L組間比較差異有顯著性(P0.05)。 4.利用ChIP技術(shù)沉淀出與Gcn5蛋白一起結(jié)合的核酸-蛋白復(fù)合物,實時熒光定量PCR檢測到該核酸中有心肌早期發(fā)育特異基因GATA-4、NKx2.5的DNA啟動子表達(dá)。 結(jié)論 1.HDAC酶抑制劑TSA對MSCs向心肌樣細(xì)胞分化有促進(jìn)作用,在一定濃度范圍內(nèi),細(xì)胞分化與TSA濃度呈正相關(guān)。 2.Gcn5乙酰化復(fù)合體調(diào)控MSCs向心肌細(xì)胞分化的作用機(jī)制之一,是啟動了心肌細(xì)胞早期發(fā)育特異性基因的表達(dá)。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R329

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本文編號：2075550