摘要
目的：以往研究将长有一层心肌细胞的半透膜放于骨髓间充质干细胞培养皿中，或用培养过心肌细胞的培养基来培养骨髓间充质干细胞，均未发现心肌细胞特异蛋白表达。为此体外模拟心肌微环境诱导骨髓间充质干细胞向心肌细胞分化，并与常规诱导剂5-氮杂胞苷的诱导效果进行比较。方法：实验于2007-03／09在山西医科大学中心实验室完成。①动物：Wistar大鼠20只，新生1d龄Wistar乳鼠10只，均由山西医科大学动物房提供，清洁级，实验过程中对动物的处置符合动物伦理学标准。诱导剂5-氮杂胞苷为美国Sigma公司产品。②实验方法：自新生Wistar乳鼠心脏分离培养心肌细胞，取第1、2代制成1×10^9L^-1细胞悬液，-70℃放置4～5h后融化，吸管吹打，反复3次，离心取上清，过滤后即为心肌细胞冻融液，以此作为培养基体外模拟心肌微环境。自成年Wistar大鼠骨髓分离骨髓间充质干细胞，取生长良好的第2代制成1×10^8L^-1细胞悬液，分为4组：血清对照组加入含15％胎牛血清的DMEM／F12培养基；5-氮杂胞苷组加入10μmol／L 5-氮杂胞苷37℃避光孵育24h后，换DMEM／F12培养基继续培养；5-氮杂胞苷＋心肌细胞冻融液组加入10μmol／L 5-氮杂胞苷诱导24h后，在培养体系中加入相当于4倍骨髓间充质干细胞数量的心肌细胞冻融液；心肌细胞冻融液组加入相当于4倍骨髓间充质干细胞数量的心肌细胞冻融液。③实验评估：诱导1周后观察骨髓间充质干细胞的形态变化，利用免疫细胞化学技术鉴定心肌特异性肌钙蛋白T、连接蛋白43、α-横纹肌肌动蛋白、CD31的表达情况。结果：①体外模拟心肌微环境情况：培养7d时心肌细胞形成细胞簇或细胞单层，呈放射状排列的同心圆状或峰谷样生长，细胞簇搏动呈明显同步性。传代后的心肌细胞仍具有自主收缩的特性。②骨髓间充质干细胞的体外诱导结果：诱导处理1周后，5-氮杂胞苷组多数细胞呈杆状，紧密平行排列生长，细胞体积变大，可见肌丝样结构形成；心肌细胞冻融液组细胞有聚集生长趋势，形成大量的子细胞，可见大量的肌丝样的结构；5-氮杂胞苷＋心肌细胞冻融液组脱落或降解的细胞数明显少于5-氮杂胞苷组，也形成肌丝样结构，但部分细胞内有脂肪空泡形成。③免疫细胞化学鉴定结果：诱导培养1周后，血清对照组仅表达α-横纹肌肌动蛋白；5-氮杂胞苷组表达心肌特异性蛋白T、α-横纹肌肌动蛋白、连接蛋白43，其阳性率分别为20％，28％，25％，抗CD31染色呈阴性；心肌细胞冻融液组上述蛋白阳性表达率分别为23％，32％，28％，明显高于5-氮杂胞苷组（P〈0．05），同时抗CD31染色呈阳性；5-氮杂胞苷＋心肌细胞冻融液组上述蛋白阳性表达率略低于心肌细胞冻融液组，抗CD31染色呈阳性。结论：①以心肌细胞冻融液体外模拟心肌微环境，可高效诱导骨髓间充质干细胞分化为心肌细胞。②部分骨髓间充质干细胞表达CD31，即可向血管内皮细胞分化，提示与5-氮杂胞营单一的肌细胞两导作用相比，心肌细胞冻融液更能提供一个心肌再生所需的自然条件。
AIM： In former studies, semipermeable membrane coated with cardiomyocytes is laid in culture dish with bone marrow mesenchymal stem cells （BMSCs）, or BMSCs are cultured in media cultured with cardiomyocytes. No specific protein in cardiomyocytes is found. The study simulated the cardiac microenvironment in vitro to observe the effects of cardiac microenvimnment on the differentiation of BMSCs into cardiomyocytes, and compare the inductive effect with inductor 5-azacytidine （5-aza）.
METHODS： Experiments were performed at the Center Laboratory of Shanxi Medical University from March to September 2007. ① Twenty Wistar rats and ten Wistar rats aged 1 day were provided by Animal Room of Shanxi Medical University. These animals were at clean grade. Animal intervention in the experiment was accorded with animal ethical standards. 5-aza was employed in the study （Sigma, USA）. ②Cardiomyocytes were isolated from neonatal rat hearts. The first and second passage of cells were made into 1 × 10^9 L^-1 cell suspension, which was lysed 4-5 hours later at -70 ℃, and beaten with pipette, three times. Supernatant was obtained after centrifuging. Cardiomyocyte freezing and thawing liquid was gotten after filtering. It was considered to be cardiomyocyte microenvironment for in vitro culture. BMSCs isolated from adult Wistar rats were cultured and the second passage of cells were made into 1 × 10^8 L^-1 cell suspension. Cell suspension in the serum control group was incubated in the DMEM/F12 media containing 15% fetal bovine serum （FBS）. Cell suspension in the 5-aza group was treated with 10μmol/L 5-aza at 37 ℃ for 24 hours, and then incubated in DMEM/F12 media. Cell suspension in the 5-aza and freezing thawing liquid group were treated with 10 μmol/L 5-aza for 24 hours, and freezing and thawing liquid as 4 times as BMSCs quantity. ③Morphologic changes of BMSCs were observed one week later. The immunocytochemical technique was used to measure Troponin T （cTnT）, Connexin 43, α -Sarcomeric Actin and CD31 expression.
RESULTS： ①Simulation of cardiac microenvironment in vitro： At day 7, cardiomyocyte cluster or monolayer was seen, showing concentric citcle or peak valley. Cell cluster beating was synchronic. Cardiomyocytes after passage were self-contracted. ②in vitro induction of BMSCs： One week later, a majority of cells was stick-like, paralleled closely and the cell body was bigger, forming myofilament-like structure in the 5-aza group. Cardiomyocytes in the freezing and thawing liquid group grew in cluster, and a mass of daughter cells was formed, showing myofilament-like structure. Cardiomyocytes in the 5-aza and freezing thawing liquid group dropped out or degraded and the cell numbers were significantly less than in the 5-aza group, showing myofilament-like structure. Fat vacuole was found in some cardiomyocytes. ③Immunocytochemical determination： One week later, α -Sarcomeric Actin expression was found in the serum control group. Troponin T （cTnT）, α -Sarcomeric Actin and Connexin43 were found in the 5-aza group, and the positive rate was 20%, 28% and 25%. Cells were negative for CD31. The positive rates of Troponin T （cTnT）, α -Sarcomeric Actin and Connexin43 were 23%, 32% and 28% in the freezing and thawing liquid group, which were higher than in the 5-aza group （P 〈 0.05）. Cells were positive for CD31. The positive rate of above-mentioned protein was lower in the 5-aza and freezing thawing liquid group than in the freezing and thawing liquid group. Cells were positive for CD31.
CONCLUSION： ①Simulating the cardiac microenvironment in vitro with freezing and thawing liquid can effectively induce the differentiation of BMSCs into cardiomyocytes. ②Some BMSCs are positive for CD31, and differentiate into vascular endothelial cells. It is indicated that the induction of freezing and thawing liquid surpasses the 5-aza obviously.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第8期1489-1492,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
