摘要
背景:心肌梗死(myocardial infarction,MI)后机体存在自然修复心肌和血管组织的过程,但不足以弥补MI造成的大范围心肌细胞缺失,亦不能防止心室重构和心功能衰竭的发生。心血管药物治疗目前虽有很多突破,但这些治疗方法都存在着无法逆转梗死期坏死心肌的缺陷,不能从根本上解决心肌细胞(cardiomyocytes,CMs)减少的问题。近年来发展的采用骨髓间充质干细胞(bone marrow mesenchymal stemcells,BMSCs)代替病变心脏中无收缩功能的瘢痕组织,形成新的心肌细胞,为治疗缺血性心脏病提供了新的方向。骨髓中由于BMSCs数量很少,必须体外扩增才能满足细胞移植的需要,而BMSCs增殖能力有限,本研究探讨巨噬细胞集落因子(macrophage colony-stimulating factor,M-CSF)对大鼠BMSCs的体外增殖和分化的影响及M-CSF扩增的BMSCs移植对心肌梗死的实验研究。
第一部分大鼠间充质样干细胞的分离、培养及其生物学和电生理特性研究
目的:从大鼠骨髓中分离培养骨髓间充质干细胞并进一步分析其生物学和电生理特性。
方法:采用密度梯度离心和贴壁培养方法分离、培养大鼠BMSCs,细胞记数绘制BMSCs生长曲线,流式细胞仪检测BMSCs的CD44、CD29、CD45和CD34的表达及其细胞周期,5-氮杂胞苷(5-azacytidine,5-aza)诱导培养细胞,免疫组化检测诱导后心肌特异性分子肌钙蛋白I(cardiac-specific troponin I,cTnI)和肌球蛋白重链(myosin heavy chain,MHC)的表达,膜片钳技术检测诱导前后BMSCs电生理特性。
结果:培养的BMSCs呈梭形贴壁生长,可连续传代培养;高表达CD44和CD29,而不表达CD45和CD34;细胞在接种后第3d即进入对数生长期,传代后培养3天的细胞周期检测结果显示G0+G1、S和G2+M期的比例分别为66.2%、23.8%和10.1%;经5-aza诱导3w后的第三代BMSCs细胞表达心肌特异性蛋白cTnI和MHC:第三代BMSCs诱导前记录到快速激活、长时开放的外向K+通道电流,未能记录到内向电流,经5-aza诱导3w后可记录到钠离子和钙离子通道电流。
结论:BMSCs具有心肌细胞分化的潜能,5-aza诱导后BMSCs可由非兴奋性细胞分化为兴奋性细胞。
第二部分M-CSF对大鼠BMSCs体外增殖和分化的作用
目的:研究M-CSF对BMSCs体外增殖的作用,并分析经M*CSF扩增的BMSCs心肌分化潜能及其电生理特性。
方法:细胞记数绘制不同浓度M-CSF对BMSCs生长增殖作用的生长曲线;流式细胞仪检测经M-CSF扩增的BMSCs CD44、CD29、CD45、CD34的表达及其细胞周期:5-aza诱导经M-CSF扩增的BMSCs,细胞免疫组化检测诱导后心肌特异性分子cTnI和MHC的表达;膜片钳技术检测M-CSF扩增的8MSCs经5-aza诱导3w后的心肌样细胞膜电流。
结果:M-CSF浓度达到5ng/m1时对BMSCs有显著促进增殖作用;经M-CSF扩增BMSCs与自然生长的BMSCs相比,其CD44、CD29、CD45、CD34的表达未出现明显变化,但细胞周期显示其增殖能力增强;M-CSF扩增后的BMSCs经5-aza诱导2w后心肌细胞特异性蛋白cTnI和MHC均为阳性。M-CSF扩增后的BMSCs经5-aza诱导3w后,可记录到钠离子和钙离子通道电流。
结论:M-CSF能促进BMSCs体外有效的增殖,而且体外扩增的BMSCs具有心肌分化的潜能。
目的:模拟心肌环境及分析心肌环境对体外扩增的BMSCs分化的影响,同时建立大鼠心肌梗死(myocardial infarction,MI)模型,在体研究经M-CSF扩增的BMSCs移植对心梗后心功能的改善作用及其可能机制。
方法:经M-CSF扩增后的BMSCs分别与心肌细胞和心肌条件培养液共培养1w后免疫组化检测cTnI的表达;制作大鼠心肌梗死模型,心脏超声检测心功能,BrdU标记移植细胞,心肌梗死区周边多点注射,BMSCs移植20d后心肌HE染色,病理组织学检查梗死区移植细胞cTnI表达和评价新生血管密度。
结果:(1)经M-CSF扩增的与心肌细胞共培养2w后可诱导表达cTnI,而与心肌条件培养液共培养2w的BMSCs未见cTnI的表达;(2)C组(MSCs注射组)(SV=0.633±0.045ml,EF=(0.748±0.054)%)、D组(5-aza诱导BMSCs扩增组)(SV=0.641±0.065ml,EF=(0.756±0.058)%)和E组(BMSCs扩增组)(SV=0.651±0.062ml,EF=(0.762±0.069)%)与对照组(SV=0.459±0.052ml,EF=(0.646±0.025)%)相比心功能均明显改善(P＜0.05),而C组、D组和E组间并无明显差别(P＞0.05);(3)BrdU标记显示M-CSF扩增的BMSCs移植体内后,可表达心肌特异性蛋白cTnI;(4)梗死区新生血管密度M-CSF扩增BMSCs组(31±4条/视野)明显高于对照组(19±4条/视野)。
结论:与心肌细胞的直接接触性培养可使M-CSF扩增的BMSCs分化为心肌细胞,M-CSF扩增的BMSCs移植对MI后心功能有明显改善作用;在梗死区周边注射WCSF扩增的BMSCs,移植细胞诱导或不诱导均不影响对心功能的改善作用。梗死区周边注射的M-CSF扩增的BMSCs可在梗死区中分化为心肌样细胞;M-CSF扩增的BMSCs可提高梗死区新生血管密度。因此,M-CSF可作为BMSCs有效生长体外促增殖因子,M-CSF扩增的BMSCs具有潜在的临床运用价值。
综上所述,BMSCs具有心肌细胞分化的潜质,5-aza诱导后BMSCs可表达功能性的钠和钙离子通道,由非兴奋性细胞分化为兴奋性细胞。体外实验显示M-CSF是BMSCs的有效生长体外促增殖因子,而且扩增的BMSCs可具有心肌分化潜能。体外模拟心肌环境研究显示,直接接触可促使M-CSF扩增的BMSCs分化为心肌细胞。移植M-CSF扩增的BMSCs细胞对MI后心功能有明显改善作用,移植细胞诱导或不诱导均不影响对心功能的改善作用。M-CSF扩增的BMSCs细胞移植对MI后心功能有明显改善的作用机制可能与BMSCs在梗死区中分化为心肌样细胞和提高梗死区新生血管密度有关。
Background:Although several repair mechanisms have been described in the animals and human heart,all fall too limit to prevent clinical heart disease in most acute or chronic pathological cardiac conditions.Moreover,despite many breakthroughs in cardiovascular medicine,the complications of myocardial infarction remain a serious problem.Bone marrow mesenchymal stem cells could provide a promising strategy against myocardial infarctions and postinfarct congestive heart failure,based on that bone marrow mesenchymal stem cells can generate new cardiomyocytes in animals and human beings.
However,due to its poor replicative capacity and short proliferative longevity,it is difficult to isolate enough MSCs from the bone marrow required for large areas of injured tissues.Thus,the expansion of MSCs in vitro is essential for cell transplantation.The purpose of this study was to identify macrophage colony-stimulating factor(M-CSF) contributing to production of MSCs and maintenance of their cardiomyocyte differentiation.
PartⅠIsolation,Characterization and Cardiomyocyte Differentiative Potential of Rat BMSCs in vitro
Objective:To isolate and expand a highly purified population of adult rat BMSCs, and to analyze its biological and electrophysiological characteristics.
Methods:BMSCs were isolated from rat bone marrow with density gradient centrifugation and cultured in low-glucose DMEM supplemented with 10%fetal calf serum.Trypan blue staining and cell counting from day 1-7 were used for the cell concentration.Flow cytometry(FCM) was used to analyze the expression of CD44、CD29、CD45、CD11,and the cell cycle.To induce differentiation,BMSCs were treated with 5-azacytidine(5-aza) for 24h and then cultured with DMEM without 5-aza. Immunohistochemistry analysis were used to test the expression of MHC and cTnI. Patch-clamp recording technique was used to analyze electrophysiological characteristics of BMSCs.
Result:FCM analysis showed that the BMSCs were CD44,CD29 positive,and CD45,CD11 negative.S phase of the passage 2 BMSCs is 23%.The passage 3 BMSCs that were induced by 5-aza could express MHC and cTnI.Before BMSCs were induced by 5-aza,only the outward currents of I_(K+) were recorded by Patch-clamp.The inward currents of I_(Ca2+) and I_(Na+) were be recorded after BMSCs induced by 5-aza for three weeks.
Conclusion:BMSCs have cardiomyocyte differentiation potential and can be differentiated into excitability cells from unexcitability cells by 5-azacytidine.
PartⅡThe effect of M-CSF on Proliferation and Cardiomyocyte Differentiation Potential of BMSCs in Vitro
Objective:To investigate the effect of M-CSF on proliferation of rat BMSCs,and to analyze the cardiomyocyte differentiation potential of the BMSCs expanded by M-CSF.
Methods:Trypan blue staining and cell counting were used to determine the effect of the different concentration of M-CSF on proliferation of rat BMSCs.FCM was used to analyze the expression of CD44、CD29、CD45、CD11,and the cell cycle of the BMSCs expanded by M-CSF.To analyze the cardiomyocyte differentiation potential of the BMSCs expanded by M-CSF,5-aza was used.Immunohistochemistry analysis was used to test MHC and cTnI expression of the induced BMSCs expanded by M-CSF.Patch-clamp recording was used to analyze electrophysiological characteristics of the BMSCs expanded by M-CSF.
Result:M-CSF markedly accelerated proliferation of the BMSCs at the concentration of 5ng/ml of M-CSF.FCM analysis showed the BMSCs expanded by M-CSF were CD44,CD29 positive,and CD45,CD11 negative.S phase cells of the BMSCs cultured by M-CSF is markedly higher than those of the control.The BMSCs cultured by M-CSF induced by 5-aza could express MHC and cTnI.The inward I_(Ca2+) and I_(Na+) currents of BMSCs expanded by M-CSF were recorded after BMSCs were treated with 5-aza three weeks.
Conclusion:BMSC proliferation could be markedly accelerated by M-CSF,and M-CSF stimulated BMSCs could retain the cardiomyocyte differentiation potential in Vitro.
PartⅢBMSCs expanded by M-CSF as Transplantation Cells for Myocardial Ischemia Therapy
Objective:To test the cardiac environment for BMSCs expanded by M-CSF and evaluate the effect of the cardiac environment on cardiomyocyte differentiation potential of the BMSCs.To investigate BMSCs expanded by M-CSF as transplantation cells to improve cardiac functions in rat MI model,and to explore the underlying mechanism.
Methods:The BrdU-BMSCs expanded by M-CSF were co-cultured with rhythmically beating CM or cultured in the presence of conditioned media for one week. Immunohistochemistry analysis was used to test cTnT of the BrdU-BMSCs.Rat myocardial infarction model was established by ligating the left coronary artery.Cardiac function of MI was assessed by echocardiographic-Doppler.BrdU labeled BMSCs were injected directly into cardiac muscle around the infarct zone.After 20 days, immunohistochemistry analysis was used to test the cTnT expression of the transplantation BMSCs and capillary density in the infracted area.
Results:The M-CSF expanded BrdU-BMSCs which were co-cultured with rhythmically beating CM showed cTnI but BrdU-BMSCs cultured with culture media without cardiomyocytes did not.Before the cellular implantation,the stroke volume(SV) and ejection fraction(EF) decreased in the MI heart(SV=0.382±0.033 ml,EF=(0.562±0.052)%) compared with the control group(SV=0.459±0.052ml,EF=(0.646±0.025) %).Twenty days after the M-CSF expanded BrdU-BMSCs implantation,the cardiac function was significantly improved in both induced(SV=0.641±0.065ml,EF=(0.756±0.058)%) and uninduced groups(SV=0.651±0.062ml,EF=(0.762±0.069)%) compared with the control(SV=0.459±0.052ml,EF=(0.646±0.025)%).However, cardiac functions didn't differ between the induced and uninduced groups.The infarct site had more cardiac-like tissue islands.The transplanted BMSCs were found in the infarct zone and the capillary desity was markedly higher than that of the control.
Conclusions:Direct cell-cell contact between BMSCs and CMs was the prerequisite for the cardiomyocyte differentiation of BMSCs expanded by M-CSF.The M-CSF expanded BMSCs as transplantation cells can improve the cardiac function of MI. No matter whether the BMSCs were induced or not,the improvement of the cardiac function might result from myocardial regeneration and renascent capillary vein by engrafted cells.
In summary,BMSCs have cardiomyocyte differentiation potential and can develop into excitability cells.BMSCs showed the inward currents of I_(Ca2+) and I_(Na+) induced by 5-azacytidine.M-CSF is an efficient growth factor in vitro for BMSCs and can retain the cardiomyocyte differentiation potential in Vitro.Direct cell-cell contact between BMSCs and CMs was the prerequisite for the BMSCs cardiomyocyte differentiation.The BMSCs expanded by M-CSF can improve the cardiac function after the cells were implanted around the infarct zone and its mechanism might result from myocardial regeneration and renascent capillary vein by engrafted cells.
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1. Hou M. Yang KM. Zhang H. Zhu WQ. Duan FJ. Wang H. Song YH. Wei YJ. Hu SS. Transplantation of mesenchymal stem cells from human bone marrow improves damaged heart function in rats. International Journal of Cardiology.2007; 115(2):220-8.
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