【作者】 姚燕；

【导师】 陆东风；

【作者基本信息】 广州医学院 ， 心血管内科学， 2011， 硕士

【摘要】 目的检测心肌微环境下,骨髓间充质干细胞(BMSCs)向心肌细胞(CM)转分化过程中组蛋白去乙酰化酶1(HDAC1)的时相表达,初步探讨成体干细胞定向转分化的乙酰化调控机制。方法1.采用全骨髓贴壁法体外分离、培养、纯化SD大鼠骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs),当细胞达80-90%融合时,用胰酶-EDTA(0.25%:0.05%)消化细胞进行传代培养。取第3代细胞进行流式细胞术细胞表面标记检测,并诱导BMSCs向成骨细胞、成脂肪细胞分化。茜素红染色鉴定BMSCs成骨分化的能力,油红O染色鉴定BMSCs成脂肪分化的能力。2. 293A细胞培养,当细胞达50-70%融合时,用0.25%胰酶消化细胞,以1:10-1:20传代扩增培养。3. Ad-EGFP以MOI 100转染90-100%融合的293A细胞进行病毒扩增,转染3天后,细胞出现病变效应,收集细胞。4. Ad-EGFP以MOI 0、10、50、100、200、400转染BMSCs,流式细胞术检测转染效率,并得出最佳转染效率的MOI值。5.新生乳鼠心肌细胞(cardiomyocytes,CM)分离、培养,72 h后免疫荧光染色法鉴定心肌肌钙蛋白T(CTnT)的表达情况。6.将Ad-EGFP转染后的BMSCs与CM以1:1比例直接接触共培养。7.依据共培养的时间,将实验分为六组,即BMSCs组(空白对照组)、共培养3天、6天、9天、12天组、心肌细胞(CM)组,收集细胞(>1×107),流式细胞仪488 nm波长分选EGFP+BMSCs。8.免疫荧光染色法检测EGFP+BMSCs中心脏肌钙蛋白T(CTnT)的表达。9. FQ-PCR检测组蛋白去乙酰化酶1(histone deacetylase 1,HDAC1)的mRNA表达。10. Western Blot检测组蛋白去乙酰化酶1(HDAC1)的蛋白表达。11.数据分析采用SPSS 13.0统计软件,数据用均数±标准差( x±s)表示,两组间比较采用独立样本t检验,多组之间比较采用单因素方差分析,P<0.05为差异有统计学意义。结果1.原代及传代的骨髓间充质干细胞均呈成纤维细胞样的长梭形,第3代骨髓间充质干细胞不表达造血标志CD34(3.04%)、CD45(1.14%) ,强表达CD29 (99.86%)、CD44 (99.28%)、CD90(99.74%)等基质细胞/间质细胞表面标志。成骨诱导21 d后,可见大量橘红色矿化结节形成。成脂诱导2-3周后,可见细胞的胞浆内出现大量红染脂滴。2. 293A细胞贴壁后呈成纤维细胞样,Ad-EGFP转染293A细胞第3天,细胞变圆、脱落,呈葡萄串样聚集,荧光显微镜下观察,293A细胞发出绿色荧光。3. EGFP在Ad-EGFP转染BMSCs 24 h后开始表达,72 h后达到峰值。MOI 10-100时,转染效率不足60%;MOI 400时,转染效率达80-85%,但BMSCs出现细胞病理效应(CPE);MOI 200时,细胞无明显形态改变,转染效率近80%。4.心肌细胞分离培养24 h后出现自主收缩,呈三角形或扁平不规则形,72 h后,免疫荧光染色提示心脏肌钙蛋白T(CTnT)阳性率达90%以上。5. BMSCs与CM共培养2-3天后,可见与CM直接接触的长梭形BMSCs逐渐缩短、增粗,4-5天后荧光显微镜下显示部分EGFP+BMSCs表达CTnT,12天后终止共培养实验,未见EGFP+BMSCs自发搏动。流式细胞术检测分选的EGFP+BMSCs阳性率可达97%以上。6. HDAC1 mRNA在各组标本均有表达,在共培养3天、6天、9天、12天4个时点组和CM组中的表达量均显著低于对照(BMSCs)组,P<0.05,BMSCs组中的HDAC1 mRNA的表达量约是CM组的14倍。7.与对照组(BMSCs组)比较, HDAC1蛋白的表达水平在共培养3天、6天、9天组有明显降低(P<0.05),BMSCs组中HDAC1蛋白的表达量约是CM组的8倍。结论HDAC1负性调节微环境下BMSCs向心肌细胞的转分化。更多还原

【Abstract】 ObjectiveTo investigate the characteristics of histone deacetylase 1 during the transdifferent- -iation of bone mesenchymal stem cells (BMSCs) into myocardial-like cells in the microenvironment provided by cardiomyocytes(CM), and initially explore the acetylation mechanism involved in the transdifferentiation of adult stem cells.Methods1. BMSCs from Sprague-Dawley (SD) rats were isolated and cultured in vitro by using the whole bone marrow adherence method. When 80-90% confluency, BMSCs were digested by trypsin- EDTA(0.25%:0.05%). Cell morphology was observed under the inverted phase contrast microscope. At the third passage, surface markers of BMSCs were determined by flow cytometry. And BMSCs were induced to differentiate into osteoblasts and adipocytes. Osteogenic ability was examined by alizarin red staining. Adipogenic ability was measured by oil red O staining.2. 293A cells were cultured. When 50-70% confluency,cells were digested by 0.25% trypsin and then passage cultured in a ratio of 1:10-1:20. 3.When 80-90 % confluency , enhanced green fluorescent protein adenovirus (Ad-EGFP) was transfected into 293A cells with MOI 100 for virus amplification. After 3 days, cells showed cytopathic effect (CPE) and were collected.4. Ad-EGFP was transfected into BMSCs with MOI 0, 10, 50, 100, 200, 400. The trans- -fection efficiency was examined by flow cytometry, and the optimum MOI was obtained.5. Neonatal rat myocardial cells (cardiomyocytes, CM) were isolated, cultured. The expression of cardiac troponin T(CTnT) was identified by immunofluorescence staining after 72 h.6. BMSCs which were marked by EGFP co-cultured with CM in a ratio of 1:1.7. According to co-culture time, we divided six groups. Namely, BMSCs group (control group) ,co-culture for 3 days, 6 days, 9 days, 12 days-group, respectively, cardiomyocyte (CM) group. BMSCs which expressed enhanced green fluorescent protein (EGFP-positive BMSCs) were sorted from the co-culture system by FACS.8. The expression of cardiac troponinT (CTnT) in EGFP-positive BMSCs was exami- -ned by Immunofluorescence staining.9. The mRNA expression of histone deacetylase 1(HDAC1)was assessed by FQ-PCR.10. The protein expression of HDAC1 was assessed by Western Blot.11. Data was analyzed using SPSS 13.0 statistical software. P<0.05 indicated the diff- -erence Statistically significant.Rusults1.The primary cells and the passage cells were mostly fusiform in shape, and were similar to fibroblasts. At the third passage, BMSCs strongly expressed the surface markers of stromalcells: CD29 (99.86%), CD44 (99.28%), CD90 (99.74%) , but were negative for CD34(3.04%), CD45(1.14%), which were the surface markers of hematopoietic cells. Following 21 days of osteogenic induction, cell alizarin red staining showed that alizarin red was positive in osteoblasts. Following 2 weeks of adipogenic induction, oil red O staining showed that red lipid droplets existed in adipocytes.2.After adherence, 293A cells were fibroblast-like. Ad-EGFP was transfected into 293A cells. Then cells became round and detached on the third day, like grapes gathered. 293A cells all emitted green fluorescence under fluorescence microscope.3.EGFP was expressed after 24 h. With MOI 10-100, the transfection efficiency was less than 60%,and was 80-85% with MOI 400, but the pathological effects (CPE) appeared. With MOI 200, the transfection efficiency was 80 % without any significant morphological changes.4. Cardiomyocytes were isolated and cultured. After 24 h, cells showed triangle or flat irregular forms and began the voluntary contraction. The positive rate of cardiac troponinT (CTnT) was above 90%by immunofluorescence staining.5. After co-cultured for 2-3 days, the spindle BMSCs gradually shortened, thickened. CTnT can be detected on the 4th day after co-culture. The transdifferentiation of BMSCs into spontaneously beating cells was not observed. The EGFP+BMSCs sorted by FACS had positive rate of above 97%.6. HDAC1 mRNA was expressed in each group cells and showed a downward trend with co-culture time. P<0.05. HDAC1 mRNA of BMSCs group was about 14 times as much as that of CM group.7. Contrast to the control group (BMSCs group), the protein of HDAC1 in other groups was significantly reduced. The protein expression of HDAC1 also showed a downward trend with co-culture time. P<0.05. The protein of HDAC1 of BMSCs group was about 8 times as much as that of CM group.Conclusion HDAC1 played a negative role in the transdifferentiation of bone marrow mesenchymal stem cells into the cardiac phenotype.更多还原