【作者】 谭琦；

【导师】 黄政德；

【作者基本信息】 湖南中医药大学 ， 中医内科学， 2011， 博士

【摘要】 目的：探讨加味丹参饮(Jia Wei Dan Shen Yin JWDSY)在体外能否诱导骨髓间充质干细胞(bone mesenchymal stem cells BMSCs)向心肌样细胞分化,以及JWDSY对BMSCs移植心肌梗死心血瘀阻型大鼠心肌的作用。方法：实验一：采用全骨髓贴壁法培养大鼠BMSCs,并进行传代、扩增,对细胞形态学和生长特性进行观察,通过流式细胞术对细胞表面抗原CD90、CD11b、CD45进行表型鉴定。采用水提醇沉法制备JWDSY的提取物并通过MTT法检测不同浓度JWDSY提取物对BMSCs的毒性作用及增殖的影响。实验二：体外诱导BMSCs,将培养的BMSCs分为4组：正常对照组、5-Aza组、JWDSY组和JWDSY+5-Aza组进行诱导分化。通过免疫组化的方法检测心肌细胞特有的结蛋白Desmin、肌球蛋白MHC和肌钙蛋白T的表达,用RT-PCR的方法检测心肌转录因子GATA-4 mRNA的表达。实验三：建立大鼠急性心梗心血瘀阻模型,正常组大鼠10只,60只造模成功大鼠分为4个组：模型组、JWDSY组、BMSCs组和JWDSY+BMSCs组。移植28d后,免疫组化双染法检测大鼠心肌组织中心肌结蛋白Desmin和肌球蛋白重链MHC的表达,Masson三色染色法检测大鼠心肌组织心室肌瘢痕面积百分比和心肌血管新生情况,RT-PCR法检测大鼠心肌组织中心肌细胞转录因子GATA-4 mRNA的表达。结果：实验一：经流式细胞术鉴定通过全骨髓贴壁法培养的BMSCs纯度较高,细胞表面抗原CD90阳性细胞达到95%以上,CD11b和CD45阳性细胞小于5%,符合实验要求。MTT结果显示,0.625mg/mL的JWDSY为进行BMSCs诱导的工作浓度。实验二：体外对BMSCs进行诱导后,正常对照组中未发现MHC、Desmin知CTnT阳性细胞；JWDSY组、5-Aza组和JWDSY+5-Aza组免疫组化染色均可见阳性免疫复合物。经JWDSY+5-Aza联合诱导后的BMSCs其MHC表达量高于JWDSY组和5-Aza组,差异有统计学意义(P<0.05),JWDSY组与5-Aza组两组间差异无统计学意义(P>0.05)。经JWDSY+5-Aza联合诱导后的BMSCs其Desmin表达量高于JWDSY组和5-Aza组,差异有统计学意义(P<0.05),JWDSY组与5-Aza组两组间差异无统计学意义(P>0.05)。经JWDSY+5-Aza联合诱导后的BMSCs其CTnT表达量高于JWDSY组和5-Aza组,差异有统计学意义(P<0.05),JWDSY组与5-Aza组两组间差异无统计学意义(P>0.05)。JWDSY+5-Aza组、JWDSY组和5-Aza组GATA-4 mRNA相对光密度值与正常对照组比较差异具有统计学意义(P<0.05)。实验三：移植后28d,Masson染色切片结果显示,正常组未见梗死灶和纤维形成,其余各组均出现大小不等的梗死灶,JWDSY组+BMSCs组、JWDSY组和BMSCs组的心室肌瘢痕面积百分比均较模型组显著减小(P<0.01)；JWDSY组+BMSCs组较JWDSY组和BMSCs组瘢痕面积百分比显著减小(P<0.05),JWDSY组和BMSCs组两者之间比较,无显著差异(P>0.05)。与正常组比较,各造模组的心肌毛细血管数均增高,差异有统计学意义(P<0.05)。JWDSY组、BMSCs组和JWDSY+BMSCs组的心肌毛细血管数较模型组显著增加(P<0.01)；JWDSY+BMSCs组与JWDSY组和BMSCs组比较,心肌毛细血管数增加有显著性意义(P<0.05),JWDSY组和BMSCs组两者之间比较,差异无显著性意义(P>0.05)。在模型组和JWDSY组梗死边缘未见有MHC-Brdu、Desmin-Brdu双染表达；BMSCs组和JWDSY+BMSCs组均可见猩红色Brdu抗体着色和棕色MHC蛋白的同时表达以及猩红色Brdu抗体着色和棕色Desmin蛋白的同时表达。JWDSY+BMSCs组的阳性细胞计数高于BMSCs组(P<0.05)。RT-PCR检测GATA-4 mRNA的表达结果显示,正常组和模型组未见到阳性条带,JWDSY组阳性条带较弱,BMSCs组和JWDSY+BMSCs组可见阳性条带,对其IOD值半定量分析JWDSY+BMSCs组高于BMSCs组(P<0.05)。结论：(1)JWDSY在体外能促进BMSCs向心肌样细胞分化,并可以加强5-Aza诱导BMSCs向心肌样细胞分化的作用。(2)在心梗心血瘀阻证大鼠模型中JWDSY组、BMSCs组和JWDSY+BMSCs均能促进新生血管,增加移植细胞的血供,能缩小梗死面积。而JWDSY能促进BMSCs移植后的血管新生和减少瘢痕形成。(3)在心梗心血瘀阻证大鼠模型中JWDSY能促进移植的BMSCs向心肌样细胞分化,JWDSY与BMSCs移植联合应用的疗效较单用更佳。更多还原

【Abstract】 Objective:To explore the differentiation possibility of bone mesenchymal stem cells (BMMSCs) into myocardium-like cell induced by Jia Wei Dan Shen Yin(JWDSY) in vitro as well as therapeutic effects made by JWDSY on rats in cardiac blood stasis syndrome present with myocardial infarction after BMMSCs transplantation.Methods:Test 1:Cultivate BMMSCs of rats by bone marrow adherent method, perform passage culture and amplification, inspect cellular morphology and growth characteristics, analysize phenotypes of CD90, CD11b, CD45 with flow cytometer. Prepare extractive of JWDSY with decoction and alcohol sedimentation technique, test toxic effect and proliferation of BMMSCs affected by different JWDSY concentrations in MTT way.Test 2:Induce BMMSCs in vitro, divide them into 4 groups:normal control group,5-Aza group, JWDSY group as well as combined JWDSY and 5-Aza group, induce their differentiation. Detect expressions of peculiar cardiomyocyte bindin Desmin, myosin MHC along with troponin T by immunohistochemistry approaches, check myocardial transcription factor GATA-4 mRNA expression with RT-PCR.Test 3:Set up acute myocardial infarction model in cardiac blood stasis syndrome of the rats, group 60 successfully molded rats into model set, JWDSY group, BMSCs group and combined JWDSY and BMSCs group beyond 10 normal rats. Observe expressions of myocardial bindin Desmin and myosin heavy chain(MHC) in rats’ myocardial tissues with double staning immunohistochemical way 28 days after transplantation. Masson trichrome staning is employed in evaluations of ventricular scar proportion and myocardial angiogenesis in myocardial tissues of the rats, take RT-PCR to measure myocardiocyte expression of GATA-4 mRNA.Results:Test 1:Flow cytometry evaluated that BMSCs fineness by panmyelo-adherent culture was higher, positive cell surface antigen CD90 was above 95% while positive CD11b and CD45 cells were below 5% which fit the experiment requirement. MTT outcomes revealed that 0.625mg/ml JWDSY was optimal concentration for proliferation of BMMSCs.Test 2:MHC, Desmin and CTnT positive cells were absent in normal control group after in vitro BMMSCs induction by JWDSY; whereas positive immunocomplex were present in JWDSY group,5-aza group and combined JWDSY and 5-aza group in immunohistochemical stainig. MHC expression induced by combined JWDSY and 5-Aza group was greater than two single ones, the difference has statistical significance (P<0.05), but there was no statistical significance between the two single groups(P>0.05), so did Desmin and CTnT. Significant statistical differences of relative optical density values of GATA-4 mRNA existed in combined JWDSY and 5-Aza group, JWDSY group and 5-aza group compared to the normal control group (P<0.05).Test 3:Twenty-eight days after transplant, Masson stained slices results suggested that neither infarct locus nor fiber is present in normal control group, while infarctions with different sizes appeared in other groups, ventricle muscular scar areas of combined JWDSY and BMSCs group, JWDSY group and BMSCs group showed notable decreases than model group all(P<0.01); in addition, the combined group performed a more significant scar area proportion decline rather than the two later groups(P<0.05), but the apparent differences were absent between the two single groups(P>0.05).The amount of myocardial blood capillary of every model group elevated compared to the control group with statistical significance (P<0.05). JWDSY group, BMSCs group and the combined group obtained remarkable increases than model group(P<0.01); compared with the two single group, the combined group revealed a significant statistics difference(P<0.05). No distinct significance existed in the two former group. Associated MHC-Brdu and Desmin-Brdu expression was unseen either in the model group or the JWDSY group; but scarlet Bru antibody and brown MHC protein dyes were available in infarct edge of both model group and JWDSY group as well as simultaneous expressions of scarlet Brdu antibody dye and brown Desmin protein. Positive cell count in combined group was greater than BMSCs group (P<.05). Positive stripe was absent in model group or control group after RT-PCR detection for GATA-4 expressions, but present in BMSCs group and the combined group and it was lesser in JWDSY group, what’s more,the combined group was higher than the BMSCs group by semi quantitative analysis of IOD value(P<0.05).Conclusions:(1) JWDSY is able to promote myocardium-like differentiation of BMSCs in vitro and subserve 5-Aza to induce myocardium-like differentiation of BMSCs.(2) In rat models present with myocardial infarction and cardiac blood stasis syndrome, JWDSY group, BMSCs group and combined BMSCs and JWDSY group are all capable to promote vascular neogenesis, increase blood supply of transplanted cells, and shrink infarct area. JWDSY can precipitate angiogenesis after BMSCs transplant, and reduce cicatrization.(3)JWDSY is able to promote myocardium-like differentiation of BMSCs in cardiac blood stasis syndrome rat models present with myocardial infarction, Combined JWDSY and BMSCs transplant showed a better therapeutic effect than single use of each approach.更多还原