【作者】 傅发源；

【导师】 陈良龙；

【作者基本信息】 福建医科大学 ， 内科学， 2009， 博士

【摘要】 冠脉微栓塞(Coronary microembolization,CME)是急性冠脉综合征及冠脉介入治疗中常见的事件[1],CME的发生意味着患者不良的预后和更高的死亡率,目前未有理想的治疗方法[2]。本研究利用注射自体微血栓法复制大鼠CME模型,探讨骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs)移植联合瑞舒伐他汀治疗CME的效果及可能的机制,为临床有效的治疗CME提供新思路及理论依据。一、大鼠骨髓间充质干细胞体外分离、培养与鉴定目的:建立稳定的体外分离、扩增和纯化技术体系以获取足够多的大鼠BMSCs,为后序的移植治疗奠定良好的基础。方法:分离幼年大鼠骨髓,采用贴壁法培养扩增BMSCs;对连续传代的第一(P1)、第三(P3)、第五代(P5)BMSCs进行绝对计数并描绘生长曲线;收集P3 BMSCs进行流氏细胞仪检测及体外成脂、成骨分化鉴定。结果:从P1至P5,BMSCs增殖活力呈递减趋势,传至P3时即可得到活力强、纯度高的BMSCs;大鼠BMSCs基本表达CD44、CD90,不表达CD11b、CD34、CD45,并在体外诱导下能分化为脂肪细胞、成骨细胞。结论:通过全骨髓贴壁分离法可以获取活力强、纯度高的大鼠BMSCs。二、单纯骨髓间充质干细胞移植对大鼠冠脉微栓塞的影响目的:利用带绿荧光蛋白(green florescent protein,GFP)的慢病毒pGC FU-GFP-LV感染BMSCs,在注射自体微血栓法复制大鼠CME模型的基础上,观察单纯BMSCs移植治疗大鼠CME的效果及机制。方法:以携带GFP的pGC FU-GFP-LV慢病毒为媒介,以复感染指数(MOI)2、8、32的病毒感染P1 BMSCs,倒置相差荧光显微镜观察GFP阳性细胞的感染率,确定最佳感染条件,按最佳MOI值大规模感染P1 BMSCs,传至P3以备移植用;采用注射自体微血栓法复制大鼠CME模型,64只清洁级雄性SD大鼠随机均分为假手术组、CME组、BMSCs低数量组和BMSCs高数量组,低数量组移植的细胞数为2×105,高数量组为2×106,每组根据不同观察时间点再分为术后3天、7天、28天分为3个亚组,每个亚组6只;以酶联免疫法(ELISA)及免疫组织化学染色检测肿瘤坏死因子a(TNF-a)、白介素1β(IL-1β)、白介素4(IL-4)、白介素10(IL-10)的表达,多重免疫荧光染色技术观察移植细胞存活情况、评价移植细胞分化,逆转录聚合酶链反应(RT-PCR)及蛋白电泳(Western blot)法检测心肌组织血管内皮细胞生长因子(VEGF)、成纤维细胞生长因子(bFGF)mRNA及蛋白表达,免疫组织化学Ⅷ因子染色评价细胞移植后毛细血管生成,伊红-苏木素(HE)染色评价细胞移植后心肌病理变化,麦松(Masson)染色检测细胞移植后心肌胶原纤维含量变化,超声心动图观察心功能改变。结果:以MOI值为8的病毒感染大鼠P1 BMSCs四天后,细胞均基本表达GFP,且传至P3未有明显变化。CME后早期心肌细胞表达炎症因子TNF-a、IL-1β明显增加;HE染色显示心肌内多中心坏死灶形成,伴明显的炎性细胞浸润,多分布于心内膜下;Masson染色显示大量胶原纤维增生;心脏超声检测显示CME后,左室舒张末期内径(LVEDD)、收缩末期内径(LVESD)渐进扩大,左室射血分数(LVEF)、左室短轴缩短率(LVFS)维持于低水平,上述结果符合CME的病理改变。移植后28天,无论BMSCs低数量组还是高数量组都几乎难以见到存活的移植细胞;与CME组比较,无论BMSCs低数量组还是高数量组心肌组织TNF-a、IL-1β、IL-4、IL-10及VEGF、bFGF的表达,均无明显差异(P>0.05),微梗死区炎症细胞浸润和瘢痕化也未见减轻;与CME组比较,BMSCs低数量组和高数量组毛细血管密度均未见增加(P>0.05),心功能指标也未见明显改善(P>0.05)。结论:CME后早期心肌局部的炎症微环境可能不利于干细胞的存活,单纯BMSCs移植治疗在植入早期可能就有大量的移植细胞死亡,最终无法减轻CME后心脏重塑,改善心功能。三.瑞舒伐他汀干预对大鼠冠脉冠脉微栓塞后炎症反应的影响目的:探讨瑞舒伐他汀干预对CME后早期炎症因子表达及心功能的影响,明确瑞舒伐他汀抑制CME炎症反应的有效剂量,为下一步BMSC移植联合瑞舒伐他汀治疗大鼠CME奠定基础。方法:48只清洁级雄性SD大鼠均分为假手术组、CME组、瑞舒伐他汀小剂量组(0.5mg/kg.d)和瑞舒伐他汀大剂量组(3.0mg/kg.d),每组12只,每组根据不同观察时间点再分为术后3天、28天分为2个亚组,每个亚组6只,瑞舒伐他汀干预的时间从术前7天到术后7天;以ELISA法及免疫组织化学染色检测心肌细胞炎症因子TNF-a、IL-1β、IL-4、IL-10的表达,HE染色评价心肌组织病理变化,Masson染色检测心肌组织胶原纤维含量变化,超声心动图观察心功能改变。结果:与CME组比较,瑞舒伐他汀低剂量组心肌组织TNF-a、IL-1β、IL-4、IL-10的表达无明显差异(P>0.05),而高剂量组TNF-a、IL-1β表达降低(P<0.05),IL-10表达有所升高(P<0.05);HE染色显示瑞舒伐他汀低剂量组心肌组织病理学改变与CME相似,而高剂量组心肌组织微梗死灶数量有所减少,炎症细胞浸润程度也减轻;瑞舒伐他汀高剂量组能减少心肌胶原纤维增生,降低LVESD、LVEDD(P<0.05),增加LVEF、LVFS(P<0.05),而低剂量组则不能。结论:高剂量(3.0mg/kg.d)瑞舒伐他汀能一定程度上抑制CME后心肌局部炎症反应,减轻心脏重塑,改善心功能。四.骨髓间充质干细胞移植联合瑞舒伐他汀干预对大鼠冠脉微栓塞的影响目的:探讨BMSCs移植联合瑞舒伐他汀治疗CME是否能在减轻CME后心肌局部炎症反应基础上,更有利于移植的BMSCs存活,产生叠加的治疗效果;以及上述治疗效果可能的机制。方法:90只清洁级雄性SD大鼠随机均分为假手术组、CME组、BMSCs组、瑞舒伐他汀组和BMSCs+瑞舒伐他汀组,BMSCs移植的细胞数为2×105,瑞舒伐他汀干预的剂量为3.0mg/kg.d,干预的时间从术前7天到术后7天;每组根据不同观察时间点再分为术后3天、7天、28天分为3个亚组,每个亚组6只;以ELISA法及免疫组织化学染色检测心肌细胞TNF-a、IL-1β、IL-4、IL-10的表达,多重免疫荧光染色技术观察移植细胞存活情况、评价移植细胞分化,RT-PCR及Western blot法检测心肌组织VEGF、bFGF mRNA及蛋白表达,免疫组织化学Ⅷ因子染色评价细胞移植后毛细血管生成,HE染色评价细胞移植后心肌病理变化,Masson染色检测细胞移植后心肌胶原纤维含量变化,超声心动图观察心功能改变。结果:与瑞舒伐他汀组比较,BMSCs+瑞舒伐他汀组心肌组织TNF-a、IL-1β表达进一步减少(P<0.05),IL-10表达增多(P<0.05);移植后28天,BMSCs+瑞舒伐他汀联合组存活的细胞数明显增多,约是BMSCs组的45倍,除少许移植细胞与宿主心肌细胞融合外未见明显分化为心肌样的细胞;与BMSCs组比较,BMSCs+瑞舒伐他汀组心肌组织VEGF、bFGF表达明显增多(P<0.05);与瑞舒伐他汀组比较,微梗死区炎症细胞浸润和瘢痕化也进一步减轻,毛细血管密度增加(P<0.05),心功能指标显示,BMSCs+瑞舒伐他汀组LVESD、LVEDD更小(P<0.05),LVEF、LVFS进一步改善(P<0.05)。结论:BMSCs移植联合瑞舒伐他汀能在瑞舒伐他汀基础上进一步抑制CME后心肌局部炎症反应,有利于更多的移植细胞存活并可能通过旁分泌效应最终更为有效的减轻心脏重塑,改善心功能。更多还原

【Abstract】 Coronary microembolization (CME) is a frequent and important event in acute coronary syndromes and during coronary interventions,which leads to bad prognosis and higher mortality for patients. But effective therapies have been limited so far. Here, we aim to copy a model of CME with homologous microthrombi and observe the curative effect of bone marrow mesenchymal stem cells ( BMSCs) transplantation in combination with rosuvastain for treatment of CME in rats.PartⅠIsolation,cultivation and identification of rat BMSCs in vitroObjective To establish a effective method of isolation, cultivation and identification of rat BMSCs in vitro. Methods BMSCs were seperad from the bone marrow of rats based on their adherence to tissue culture surface. The growth properties were compared by cell counting between successively passaged BMSCs( P1, P3, P5). P3 BMSCs were further identified by fluorescence-activated cell sorter (FACS) for CD11b,CD34,CD44,CD45,CD90 antibodies.Simultaneously, osteoblastic and adipocytic differentiation were also induced and observed. Results A gradual loss of replication ability was companied with BMSCs passaged from P1 to P5. P3 BMSCs were showed active proliferative ability and good purification.They were also showed CD44,CD90-positive and CD11b, CD34,CD45-negtive.Further, Osteoblastic and adipocytic differentiation were got positive. Conclusion The attachment culture method can be used to isolate and purify BMSCs of rats, which helps for further study.PartⅡThe effect of transplantation with bone marrow mesenchymal stem cells following coronary micoembolization in ratsObjective To copy the model of CME with homologous microthrombi and observe the curative effect of BMSCs transplantion for treatment of CME in rat. Methods pGC FU-GFP-LV, a lentiviral vector with green fluorescent protein(GFP)used as a tool, P1 BMSCs were infected at MOIs of 2, 8 and 32 respectively. The GFP-positive cells were detected by fluorescence microscope. After infected by the virus according to appropriate MOI, P1 BMSCs were passaged to P3 for transplantation. A total of 64 male Sprague Dawley(SD) rats were randomized into control group, CME group, low-dose BMSCs group(2×105) and high-dose BMSCs group (2×106) averagely. The model of CME was copied by injection of homologous microthrombotic particle suspension into left ventricle when clamping the ascending aorta. The rats were sacrificed at day 3,7,and 28 after operation. Surviving cells were tracked and their differentiation into cardiomyocyte-like cells was evaluated by immunofluorescent staining after transplantation. Expression of TNF-a, IL-1β, IL-4, IL-10 in myocardium were assay by Enzyme linked immunosorbent assay(ELISA), immunostaining and VEGF, bFGF were detected by RT-PCR, western blot. Pathological changes were detected by HE staining and angiogenesis was quantified by immunnostaining forⅧfactor antibody. Collagen deposition was detected by Masson staining and recovery of cardiac function was assessed by echocardiography. Results More than 90% P1 BMSCs were GFP-positive at MOIs 8 and fluorescent intensity was no changes when P1 BMSCs passaged to P3. Compared with control group, higher expression of TNF-a, IL-1βin myocardium, multi-focal myocardial necrosis with inflammatory cells infiltration, fibrosis in subendocardial region and significant cardiac dysfunction were observed in CME group, indicating it was a successful model. No BMSCs survival were observed nearly at 28 days after transplantation in both of BMSCs groups . Whether high-dose BMSCs group or low-dose BMSCs group, the expression of TNF-a, IL-1β, IL-4, IL-10, VEGF, bFGF in myocardium were no difference when compared with CME group(p>0.05). Inflammatory cell infiltration, collagen deposition and capillary density in the farct zone in both of BMSCs groups were as same as in CME group. Further, recovery of cardiac function was also not found in both of transplantation groups when compared with CME group(p>0.05). Conclusion Lots of BMSCs may be lost in a few days after transplantation due to serious inflammation in myocardium following CME. BMSCs transplantation alone can not prevent cardiac remodeling and dysfunction in rats with CME.PartⅢThe effect of rosuvastatin for treatment of myocardial inflammation following coronary microembolization in ratsObjective To evaluate the effect of rosuvastatin on myocardial inflammation and cardiac dysfunction following CME in rats. Methods A total of 48 male SD rats were randomized into control group, CME group, low-dose rosuvastatin group(0.5mg/kg.d) and high-dose rosuvastatin group(3.0mg/kg.d) averagely. Rosuvastatin was dissolved into saline fed in perioperation for two weeks and the rats were sacrificed at day 3 and 28 after operation. Expression of TNF-a,IL-1β,IL-4,IL-10 in myocardium were assay by ELISA, immunostaining and pathological changes were detected by HE staining. Collagen deposition was detected by Masson staining and recovery of cardiac function was assessed by echocardiography. Results The expression of TNF-a,IL-1β,IL-4,IL-10 in myocardium were no difference between low-dose rosuvastatin group and CME group(p>0.05).In contrast, the levels of TNF-a and IL-1βwere lower, and IL-10 was higher in high-dose rosuvastatin group compared with CME group(p<0.05). High-dose but not low-dose rosuvastatin also reduced inflammatory cell infiltration and collagen deposition in farct zone,decreased LVEDD,LVESD and increased LVEF,LVFS. Conclusion In rats with CME, perioperative therapy with high-dose rosuvastatin prevents cardiac remodeling and dysfunction. This benefit may be partly derived from reducing myocardial inflammation. PartⅣCombination of bone marrow mesenchymal stem cells transplantation and rosuvastatin for treatment of coronary microembolization in ratsObjectct To observe the curative effect of BMSCs transplantation in combination with rosuvastatin for treatment of CME in rats, and to compare with the effect of BMSCs transplantation or rosuvastatin alone. Methods A total of 48 male SD rats were randomized into control group, CME group, BMSCs group(2×10~6), rosuvastatin group(3.0mg/kg.d) and BMSCs+rosuvastatin group averagely. The rats were sacrificed at day 3 ,7 and 28 after operation. Surviving cells were tracked and their differentiation into cardiomyocyte-like cells was evaluated by immunofluorescent staining after transplantion. Expression of TNF-a,IL-1β,IL-4,IL-10 in myocardium were assay by ELISA, immunostaining and VEGF,bFGF were detected by RT-PCR ,western blot. Pathological changes were detected by HE staining and angiogenesis was quantified by immunnostaining forⅧfactor antibody. Collagen deposition was detected by Masson staining and recovery of cardiac function was assessed by echocardiography. Results BMSCs+rosuvastatin group increased about 45-fold of the cellular survival at 28 days when compared with BMSCs group(p<0.05). Only few transplanted BMSCs fusion with host cardiomyocytes but not differrention were detected. Expression of TNF-a,IL-1βin myocardium were lower,and IL-10 was higher in BMSCs+rosuvastatin group than in rosuvastatin group or BMSCs group(p<0.05). Expression of VEGF,bFGF were also higher in BMSCs+rosuvastatin group than in BMSCs group(p<0.05). Further, BMSCs transplantation in combination with rosuvastatain significantly inhibited inflammatory cell infiltration , collagen deposition in the infarct zone, decreased LVEDD,LVESD and increased LVEF,LVFS when compared with rosuvastatin group or BMSCs group alone. Conclusion BMSCs transplantation in combination with rosuvastatain significantly inhibites inflammation in myocardium when compares with rosuvastatain or BMSCs alone, enhances survival of BMSCs under ischemic condition., and significantly prevents cardiac remodeling and dysfunction after CME.更多还原