丹参联合骨髓间充质干细胞移植对兔急性心肌梗死后胶原重构及心功能的影响

【作者】 齐晓云；

【导师】 杨关林；

【作者基本信息】 辽宁中医药大学 ， 中西医结合临床， 2009， 博士

【摘要】 目的:探讨体外培养兔骨髓问充质干细胞(mesenchymal stem cells,MSCs)的方法,为进一步的实验研究打下基础。材料与方法:选择健康日本大耳白兔,取双侧胫骨行骨髓穿刺,提取动物骨髓,采用密度梯度离心法分离提取MSCs,通过贴壁培养筛选、纯化获得的MSCs,传代培养。对MSCs进行形态学观察,通过流式细胞仪,对CD34、CD29二种表面抗原进行鉴定,证明所培养的细胞为MSCs。传代至第3代时,加BrdU抗原,免疫组化方法俭测Brdu标记率。结果:分离培养48h,贴壁生长的原代MSCs数量少,呈长梭形,8d-10d细胞集落扩大,形态呈较均一的长梭形或三角形。达80%融合时消化传代,传代细胞生长旺盛时呈旋涡样。培养的细胞经流式细胞仪鉴定,CD34(-)、CD29(+),证明所培养的细胞为较纯的MSCs。MSCs经Brdu孵育后,BrdU体外标记率可达8096。结论:应用本实验方法,可以分离、纯化培养兔MSCs,所培养的MSCs体外生长稳定、增殖速度快、贴壁率高、可连续传代,可用于MSCs移植治疗心肌梗死的进一步研究。目的:观察丹参联合骨髓间充质干细胞(MSCs)移植对急性心肌梗死模型兔胶原容积和Ⅰ、Ⅲ型胶原比值的影响,探讨两者是否具有协同治疗作用。方法:取日本大耳白兔2只,穿胫骨骨髓,应用贴壁和密度梯度离心法分离、培养MSC,传至第3代,行BrdU标记。10只兔作为正常对照组,另60只建立急性心肌梗死模型,造模成功后随机均分为4组:模型组、丹参组、MSC组、丹参+MSC组。MSC组、丹参+MSC组于造模成功后7d静脉注射MSC2×10~6;丹参组、丹参+MSC联合组于造模成功后静脉注射丹参15.4mg/kg,1次/d,共10d。各组于术后35d光镜、电镜观察心肌组织病理改变;Masson染色及免疫组化测定胶原容积分数(CVF)、Ⅰ、Ⅲ型胶原比值:夹心酶联免疫(ELASA)法检测血清MMP-1、TIMP-1及Ⅰ、Ⅲ型胶原水平。结果:MSC移植后35dMSC组、联合组在梗死及周边区存在Brdu颗粒。光镜下模型组的梗死区可见大片纤维化区,梗死及周边区可见大量炎性细胞浸润,MSC组的梗死纤维化区及梗死边缘区可见血管增生、炎细胞浸润。电镜下梗死模型组、MSCs组的梗死区可见大量纤维母细胞增生。与模型组比较,MSC组、MSC+丹参组,血清Ⅰ型胶原显著增加(p＜0.01),Ⅲ型胶原也增加,但各组间无差异(p＞0.05)。与模型组比较,MSC组、丹参组、MSC+丹参组血清MMP-1均有所下降,TIMP-1增加,但各组间无差异(p＞0.05)。与正常组比较,模型组CVF显著增加(p＜0.05),Ⅰ/Ⅲ显著下降(p＜0.05);与模型组比较,MSC组、丹参组CVF下降(p＜0.05),但MSC+丹参联合组下降显著(p＜0.05);丹参组Ⅰ/Ⅲ增加(p＜0.05),MSC+丹参联合组Ⅰ/Ⅲ增加优于单纯MSC组(p＜0.05)。结论:1.采用结扎冠脉前降支的方法可成功建立兔心肌梗死模型。2.静脉移植骨髓间充质干细胞可以迁移至心肌梗死及周围区。3.骨髓间充质干细胞移植可通过改善Ⅰ、Ⅲ型胶原比值使兔急性心肌梗死后胶原重构减轻。4.中药丹参可以减轻心肌梗死后胶原重构,与干细胞移植联合应用,效果更显著。目的:观察丹参联合骨髓间充质干细胞移植对兔急性心肌梗死后心功能的影响,探讨两者是否具有协同治疗作用,并从炎症、凋亡角度探讨其作用机制。方法:取日本大耳白兔2只,穿胫骨骨髓,应用贴壁和密度梯度离心法分离、培养MSCs,传至第3代,行5-溴脱氧核苷尿嘧啶(5-Bromo-2’-deoxy-uridine,Brdu)标记。正常对照组10只,另60只建立急性心肌梗死模型,造模成功后随机均分为4组,模型组、丹参组、MSC组、丹参+MSC联合组。MSC组、丹参+MSCs组于造模成功后第7d静脉注射MSC2×10~6;丹参组、丹参+MSC组于造模成功后静脉注射丹参15.4mg/kg配制成溶液,1次/d,共10d。各组分别于术后28d行多普勒超声心动图测量左室舒张期末内径(LVEDD)、左室短轴缩短率(FS)。术后35d行病理学检查,原位末端标记法(TUNEL)检测梗死及周围区心肌细胞凋亡指数;RT-PCR和Western blot法检测NF-kb、bcl-2、fas、fash的表达。结果:与模型组比较,丹参组、干细胞组和联合组左室舒张末内径均明显缩小(P＜0.05),左室短轴缩短率明显增加(P＜0.05),联合组变化更显著,但与干细胞组比较无统计学意义。与模型组比较,丹参组、干细胞组和联合组凋亡细胞平均吸光度值明显下降(p＜0.05),联合组比单纯干细胞组下降更显著(P＜0.05)。PCR与Western检测均发现:模型组NF-kb、fas、fasL表达增加,bcl-2表达下降;干细胞组、联合组NF-KB、fas、fasL表达下降,且联合组下降更显著;丹参组bcl-2水平增加,而干细胞及联合组未见增加。结论:1.经静脉移植MSC可改善兔急性心肌梗死后心功能。2.MSC移植可减轻梗死及周围区心肌细胞凋亡相关蛋白fas、fash表达,减轻炎症因子NF-KB表达。3.MSC移植心功能的改善可能与其抑制凋亡和炎症反应作用相关。4.中药丹参注射液可改善兔心肌梗死后心功能,与干细胞联合应用效果更显著,丹参对MSC移植治疗AMI具有协同和促进作用。更多还原

【Abstract】 Popurse:To establish a method of in vitro isolation,purification and amplification of bone marrow derived mesenchymal stem cells of adult rabbits for a further study.Methods:Bone marrow tissue was harvested from healthy Japanese rabbits.Mesenchymal stem cells were isolated by the method of density gradient centrifuge.Then MSCs were proliferated by adhering culture.CD34、CD29 antigen of MSCs were identified by flow cytometry.At the third passage, MSCs were labeled with BrdU(5-Bromo-2’-deoxy-uridine) and the rate of lable was measured by immunohistochemical method.Results:Primary cultured MSCs start adhering to plates 48 hours after seeding,and spindle-shaped MSCs were observed under microscopy.8-10 days after seeding MSCs were homogenous population and exhibited a spindle-shaped or triangle-shaped.Confluence of MSCs reach to 80%and growing at a rapid speed,MSCs were exhibited a whirlpool-shaped.CD34、CD29 were directed by flow cytometry.Cultured MSCs after incubating of BrdU,the rate of in vitro labling was 80%.Conclusion:In this study,MSCs were isolated and proliferated stably and rapidly.Cultured MSCs possessed high activity of growing and amplification, and were appropriate for further research of transplantation in myocardial infarction. Popurse:To observe the effects of miltiorrhiza combined with marrow mesenchymal cells(MSCs) transplantation on the collagen matrix remodeling after acute myocardial infarction in rabbits,and investigate the synergetically therapeutic effects of miltiorrhiza and MSCs transplantation.Methods:MSCs were harvested from bilateral femur of two rabbits using the method of density gradient centrifuge isolation and adhering culture.At the third passage,MSCs were labeled with Brdu(5-Bromo-2’-deoxy-uridine).Ten rabbits served as normal control group,and other 60 rabbits were used for establishing models of acute myocardial infarction.After model establishment, rabbitts were randomly divided into 4 groups,model group,MSCs group, miltiorrhiza group,and MSCs and miltiorrhiza combition group.2×10~6MSCs labeled Brdu were injected into the vein of the MSCs group and MSCs and miltiorrhiza after seven days of successful models.Miltiorrhiza 15.4mg/kg were injected into the vein of the miltiorrhiza group and combition group after successful models,once a DAY for ten days.Light and electron microscope were taken to observe twenty-eight days after cellular transplant respectivly.The collegen volum fraction(CVF) and the ratio of typeⅠandⅢcollegen were measured by means of masson and immunohistochemistry.The levels of serum matrix metalloproteinase enzyme-1(MMP-1),matrix metalloproteinase enzyme inhibitor -1(TIMP-1),and levels of typeⅠandⅢcollegen were detected by means of enzyme linked immunoadsorbnent. Results:MSCs by vein-injection labled Brdu were found in the myocardial infarction and marginal zones in MSC group and combition group after twenty-eight days of AMI.In myocardial infarction groups,by light microscope,a large number of myocadial fibration were found in the infarcted zone.In stem cell groups, a great number of inflammatory cells were found in the infarcted and marginal zone.In stem cell groups,by electron microscope,a great number of fibroblast were found in the infarction zone.Compared with the model group,the levels of serum typeⅠcollegen was significantly increased(P<0.01) in the MSCs group and MSCs and miltiorrhiza combition group,the levels of serum typeⅢcollegen was increased but there was no difference in each group(P>0.05).Compared with the model group,the levels of serum MMP-1 decreased and TIMP-1 increased in MSCs group,miltiorrhiza group,and MSC and miltiorrhiza combition group,but there was no difference in each group(P>0.05).Compared with the normal group, CVF significantly increased(P<0.05) and the ratio of typeⅠandⅢcollegen decreased significantly(P<0.05)in model group,Compared with the model group, CVF decreased significantly(P<0.05) in MSC group,miltiorrhiza group,but combition group was better(P<0.05),the ratio of typeⅠandⅢwas significantly increased(P<0.05),and combition group was more significant than MSCS group.Conclusion:1.The model of myocardial infarction in rabbits was build successfully by ligaturing left anterior descending coronary artery 2.MSCs by vein transplantation were found in the myocardial infarction and marginal zones.3.MSCs transplantation can improve the ratio of typeⅠandⅢcollegen and inhibit the collagen matrix remodeling after acute myocardial infarction in rabbits.4.Miltiorrhiza can inhibit the collagen matrix remodeling of AMI rabbits,and has a better effect combined with MSC transplantation,and with synergetically therapeutic effects on collagen remodeling. Popurse:To observe the effects and mechanism of miltiorrhiza combined with marrow mesenchymal cells(MSCs) transplantation on cardiac function after acute myocardial infarction in rabbits,and investigate the synergetically therapeutic effects of miltiorrhiza and MSCs transplantation.Methods:Mscs were harvested from bilateral shin of two rabbits using the method of density gradient centrifuge isolation and adhering culture.At the third passage,MSCs were labeled with Brdu(5-Bromo-2’-deoxy-uridine).Ten rabbits served as normal control group,and other sixty rabbits were used for establishing models of acute myocardial infarction.After model establishment, rabbitts were randomly divided into four groups,model group,MSCs group, miltiorrhiza group,and MSC and miltiorrhiza combition group.2×10~6MSCs labeled Brdu were injected into the vein of the miltiorrhiza group and combition group after successful models.Miltiorrhiza 15.4mg/kg were injected into the vein of the miltiorrhiza group and combition group after successful models,once a day for ten days.Doppler echocardiography was used to detect left ventricular internal dimension in diastolic(LVDD),fractional shortening(FS) after twenty-eight days of successful models;Myocardium apoptosis was observed at infracted and marginal regions by TUNEL staining.NF-kb、bcl-2、fas、fasL were measured by fluorescent quantitation reverse transcription-polymerase chain reaction(RT-PCR) and western blot methods after thirty-five days of successful models.Results:Compared with the model group,left ventricular end-diastolic diameter were significantly decreased(P<0.01) and shortening fraction were significantly increased(P＜0.01)in the MSCs group,miltiorrhiza group,and MSC and miltiorrhiza combition group.The changes were better in the miltiorrhiza combition group than the MSC group(P<0.05).Compared with the model group, myocardium apoptosis were significantly decreased(P<0.01) in the MSC group, miltiorrhiza group,and MSCs and miltiorrhiza combition group,and combition group was better than MSC group.It was founded that NF-kb,fas,fasL protein expression increased and bcl-2 protein expression decreased in the model group; NF-kb,fas,fasL protein expression were significantly decreased(P<0.01) in the MSC group and combition group,and combition group were more significant than MSCs group.Bcl-2 expression was increased in the miltiorrhiza group,but other groups were not.Conclusion:1.MSCs transplantation by vein injection can improve cardiac function of AMI rabbits.2.MSCs transplantation can reduce the expression of fas,fasL and NF-kb,which were related to apoptosis and inflammation.3.The improvement of cardial function by MSCs transplantation could be related to the inhibition of apoptosis and inflammation.4.Miltiorrhiza can improve cardiac function of AMI rabbits,and has a better effect combined with MSC transplantation,and with synergetically therapeutic effects on myocardial infarction.更多还原