骨髓干细胞治疗阿霉素诱导的扩张型心肌病的实验研究
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摘要
扩张型心肌病(dilated cardiomyopathy, DCM)发病率近年来呈增高趋势,并且已成为引起充血性心力衰竭的最主要原因之一。目前临床上内科药物治疗、外科手术治疗均效果欠佳,心脏移植手术又存在供体器官有限的缺陷。干细胞治疗对于DCM尤其是终末期心衰患者是一种极富潜力的新的治疗方式。骨髓间充质干细胞(MSC)及骨髓单个核细胞(BMMNC)经研究已证实可明显改善DCM心功能,但尚未见有关二者疗效对比的研究。大多数细胞移植,均是经心外膜、心内膜或冠脉内注射,手术创伤及对操作技术、设备的要求限制了其广泛应用。骨髓自体动员治疗避免了上述缺陷。已有研究证明,骨髓来源的干细胞可迁移至损伤心肌并减轻心肌细胞超微结构的损伤。但关于骨髓自体动员对于阿霉素(ADR)诱导的DCM心脏功能及细胞凋亡的影响则尚未见报道。本实验拟对上述几方面进行研究。
本研究利用ADR(经腹腔注射2.5mg/kg/次,3次/周,共两周)建立ADR诱导的DCM大鼠模型。经超声心动、血流动力学检测及病理组织学检查证实后,将ADR诱导的DCM大鼠随机分组,即细胞移植部分的4个亚组:MSC移植组(移植细胞数5×10~6/50μl,分4点注射于左室前壁)、BMMNC移植组(移植细胞数5×10~6/50μl,分4点注射于左室前壁)、无血清DMEM培养液注射对照组和假手术组及骨髓动员部分的2个亚组:自体骨髓动员组(rhG-CSF50μg/kg/d,连续8d皮下注射)和盐水注射对照组,每个亚组n=10。采用超声心动及生理多导仪检测各组射血分数(EF)、短轴缩短率(FS)、左室收缩末期内径(LVDs)、左室舒张末期内径(LVDd)、左室收缩压峰值(LVSP)、左室舒张末压(LVEDP)以及左室压力最大上升或下降速率(+dp/dtmax, -dp/dtmax)等心功能指标;采用普通病理学及超微结构电镜进行组织病理学检查。应用TUNEL法行细胞凋亡指数测定,Western blot法行凋亡相关蛋白Bc1-2、Bax、Fas检测,免疫组化染色行Ⅷ因子相关抗原检测,免疫荧光双染行移植细胞鉴定与定位检查。
Part Ⅰ and ⅡDilated cardiomyopathy (DCM) is characterized by cardiac enlargement with an impairment of systolic function in one or both ventricles for unknown reasons. DCM is major in nonischemic cardiomyopathy . Nonischemic cardiomyopathy accounts for approximately one-third of the heart failure cases. Cell therapy is becoming increasing important as a potential new therapy for patients with DCM accompanied by advanced heart failure. Bone marrow mesenchymal stem cell(MSC) and bone marrow mononuclear cell (BMMNC) have been testified to have benefical effects on cardiac pump function and regenerated cardiomyocytes .But which is the better one is still unknown. In this study , we compare the effects of MSC transplantation and BMMNC transplantation on the cardiac function and cardiomyocytes regenerations and apoptosis in the rat model of Adriamycin-induced cardiomyopathy.Method:Adriamycin (2.5mg/kg, three times a week for 2 weeks, IP) was introduced into Lewis rats. They were randomly divided into 4 groups at 2 weeks after injection: MSC transplantation group(MSC group, MSC [5><106] implantation, n=10),BMMNC transplantation group(BMMNC group,BMMNC [5×10~6] implantation, n=10), control group (DMEM injection, n=10) and sham group(thoracotomy,n=10).At 4 weeks after implantation, the cardiac function were evaluated by echocardiography and cardiac cathetenzation. Cardiomyocytes apoptosis, apoptosis related protein Bcl-2 and Bax were detected by in situ terminal deoxynucleotidyl transferase assay(TUNEL method) and Western blot respectively ,VIII-factor stained and histologic study including electron-microscopic study were also performed.
Result:The MSC group and BMMNC group showed a significant reduction in systolic left ventricular diameter(LVDs), diastolic left ventricular diameter (LVDd) and left ventricular end-diastolic pressure(LVEDP),whereas fractional shortening(FS),ejection fraction(EF),left ventricular systolic pressure(LVSP), the maximum rate of LV systolic pressure rise(+dp/dtmax) and the maximum rate of LV systolic pressure descend(-dp/dtmax) increased greatly compared with the control group(all PO.05). In comparison with the BMMNC group, the MSC group show a significant redunction in LVDs,LVDd,LVEDP and a significant increase in FS, EF, LVSP, +dp/dtmax, -dp/dtmax.(all P<0.05). Compared to the control group ,the apoptosis of cardiomyocytes in the MSC group and BMMNC group was significantly inhibited(P<0.05).Bcl-2 protein expression was evidently upregulated and Bax protein expression was evidently downregulated in the MSC group and BMMNC group than that in the control group.Conclusion:MSC transplantation and BMMNC transplantation can both contribute to the improvement of cardiac function in ADR-induced cardiomyopathy. MSC transplantation had more benefical effects on cardiac function than BMMNC transplantation. Both MSC transplantation and BMMNC transplantation inhibited the cardiomycytes apoptosis and affected the expression of Bcl-2 and Bax protein ,which contributes to improve cardiac pump function in vivo in ADR-induced DCM rat model.
Part IIINonischemic dilated cardiomyopathy (DCM) accounts for almost one half of new cases of heart failure encountered in clinical practice. Cardiomyocytes apoptosis was reported participating in the occurrence and progression of DCM. Recent reports showed that mobilization with granulocyte colony-stimulating factor (G-CSF) enhanced bone marrow cells migrate to the damaged heart tissue and regenerated cardiomyocytes in the DCM model, however, its influence on both cardiac pump function in vivo and cardiomyocytes apoptosis has not been studied.Method: Lewis rats were randomly grouped into ADR+N.S, ADR+G-CSF group (n=10). Adriamycin(2.5 mg/kg, three times a week for 2 weeks) was administered intraperitoneally in all rats . After a two weeks wash-off period, G-CSF (50 M-g/kg/day for 8 days) was administered subcutaneously in ADR+G-CSF group and was replaced by N.S in ADR+N.S group. Four weeks later, the cardiac functions were evaluated by echocardiography and cardiac catheterization. Cardiomyocytes apoptosis, apoptosis related protein Fas were detected by in situ terminal deoxynucleotidyl transferase assay (TUNEL method) and Western blot, respectively.Result: In comparison with the ADR+N.S group, the ADR+G-CSF group showed significant increase in percent of fractional shortening (56.16±3.61% versus 36.68±1.25%, p<0.05), ejection fraction(EF)(81.9±3.48% vs 67.9±3.47%,P<0.05),left ventricular systolic pressure(l 18.38±5.53 mm Hg versus 85.25±7.50 mm Hg, PO.05), the maximum rate of LV systolic pressure rise (+dp/dt) (7751.8±674.39 mm Hg/s versus 6439±592.52 mm Hg/s, PO.05) and LV systotic pressure descend(-dp/dp) (5387.2±531.42 mm Hg/s versus 4415±280.29 mm Hg/s, PO.05). Left ventricular end-diastolic pressure (LVEDP) in ADR+G-CSF was reduced significantly compared with ADR+N.S group
本研究利用ADR(经腹腔注射2.5mg/kg/次,3次/周,共两周)建立ADR诱导的DCM大鼠模型。经超声心动、血流动力学检测及病理组织学检查证实后,将ADR诱导的DCM大鼠随机分组,即细胞移植部分的4个亚组:MSC移植组(移植细胞数5×10~6/50μl,分4点注射于左室前壁)、BMMNC移植组(移植细胞数5×10~6/50μl,分4点注射于左室前壁)、无血清DMEM培养液注射对照组和假手术组及骨髓动员部分的2个亚组:自体骨髓动员组(rhG-CSF50μg/kg/d,连续8d皮下注射)和盐水注射对照组,每个亚组n=10。采用超声心动及生理多导仪检测各组射血分数(EF)、短轴缩短率(FS)、左室收缩末期内径(LVDs)、左室舒张末期内径(LVDd)、左室收缩压峰值(LVSP)、左室舒张末压(LVEDP)以及左室压力最大上升或下降速率(+dp/dtmax, -dp/dtmax)等心功能指标;采用普通病理学及超微结构电镜进行组织病理学检查。应用TUNEL法行细胞凋亡指数测定,Western blot法行凋亡相关蛋白Bc1-2、Bax、Fas检测,免疫组化染色行Ⅷ因子相关抗原检测,免疫荧光双染行移植细胞鉴定与定位检查。
Part Ⅰ and ⅡDilated cardiomyopathy (DCM) is characterized by cardiac enlargement with an impairment of systolic function in one or both ventricles for unknown reasons. DCM is major in nonischemic cardiomyopathy . Nonischemic cardiomyopathy accounts for approximately one-third of the heart failure cases. Cell therapy is becoming increasing important as a potential new therapy for patients with DCM accompanied by advanced heart failure. Bone marrow mesenchymal stem cell(MSC) and bone marrow mononuclear cell (BMMNC) have been testified to have benefical effects on cardiac pump function and regenerated cardiomyocytes .But which is the better one is still unknown. In this study , we compare the effects of MSC transplantation and BMMNC transplantation on the cardiac function and cardiomyocytes regenerations and apoptosis in the rat model of Adriamycin-induced cardiomyopathy.Method:Adriamycin (2.5mg/kg, three times a week for 2 weeks, IP) was introduced into Lewis rats. They were randomly divided into 4 groups at 2 weeks after injection: MSC transplantation group(MSC group, MSC [5><106] implantation, n=10),BMMNC transplantation group(BMMNC group,BMMNC [5×10~6] implantation, n=10), control group (DMEM injection, n=10) and sham group(thoracotomy,n=10).At 4 weeks after implantation, the cardiac function were evaluated by echocardiography and cardiac cathetenzation. Cardiomyocytes apoptosis, apoptosis related protein Bcl-2 and Bax were detected by in situ terminal deoxynucleotidyl transferase assay(TUNEL method) and Western blot respectively ,VIII-factor stained and histologic study including electron-microscopic study were also performed.
Result:The MSC group and BMMNC group showed a significant reduction in systolic left ventricular diameter(LVDs), diastolic left ventricular diameter (LVDd) and left ventricular end-diastolic pressure(LVEDP),whereas fractional shortening(FS),ejection fraction(EF),left ventricular systolic pressure(LVSP), the maximum rate of LV systolic pressure rise(+dp/dtmax) and the maximum rate of LV systolic pressure descend(-dp/dtmax) increased greatly compared with the control group(all PO.05). In comparison with the BMMNC group, the MSC group show a significant redunction in LVDs,LVDd,LVEDP and a significant increase in FS, EF, LVSP, +dp/dtmax, -dp/dtmax.(all P<0.05). Compared to the control group ,the apoptosis of cardiomyocytes in the MSC group and BMMNC group was significantly inhibited(P<0.05).Bcl-2 protein expression was evidently upregulated and Bax protein expression was evidently downregulated in the MSC group and BMMNC group than that in the control group.Conclusion:MSC transplantation and BMMNC transplantation can both contribute to the improvement of cardiac function in ADR-induced cardiomyopathy. MSC transplantation had more benefical effects on cardiac function than BMMNC transplantation. Both MSC transplantation and BMMNC transplantation inhibited the cardiomycytes apoptosis and affected the expression of Bcl-2 and Bax protein ,which contributes to improve cardiac pump function in vivo in ADR-induced DCM rat model.
Part IIINonischemic dilated cardiomyopathy (DCM) accounts for almost one half of new cases of heart failure encountered in clinical practice. Cardiomyocytes apoptosis was reported participating in the occurrence and progression of DCM. Recent reports showed that mobilization with granulocyte colony-stimulating factor (G-CSF) enhanced bone marrow cells migrate to the damaged heart tissue and regenerated cardiomyocytes in the DCM model, however, its influence on both cardiac pump function in vivo and cardiomyocytes apoptosis has not been studied.Method: Lewis rats were randomly grouped into ADR+N.S, ADR+G-CSF group (n=10). Adriamycin(2.5 mg/kg, three times a week for 2 weeks) was administered intraperitoneally in all rats . After a two weeks wash-off period, G-CSF (50 M-g/kg/day for 8 days) was administered subcutaneously in ADR+G-CSF group and was replaced by N.S in ADR+N.S group. Four weeks later, the cardiac functions were evaluated by echocardiography and cardiac catheterization. Cardiomyocytes apoptosis, apoptosis related protein Fas were detected by in situ terminal deoxynucleotidyl transferase assay (TUNEL method) and Western blot, respectively.Result: In comparison with the ADR+N.S group, the ADR+G-CSF group showed significant increase in percent of fractional shortening (56.16±3.61% versus 36.68±1.25%, p<0.05), ejection fraction(EF)(81.9±3.48% vs 67.9±3.47%,P<0.05),left ventricular systolic pressure(l 18.38±5.53 mm Hg versus 85.25±7.50 mm Hg, PO.05), the maximum rate of LV systolic pressure rise (+dp/dt) (7751.8±674.39 mm Hg/s versus 6439±592.52 mm Hg/s, PO.05) and LV systotic pressure descend(-dp/dp) (5387.2±531.42 mm Hg/s versus 4415±280.29 mm Hg/s, PO.05). Left ventricular end-diastolic pressure (LVEDP) in ADR+G-CSF was reduced significantly compared with ADR+N.S group
引文
1. Kalon KLH, Joan LP, William BK, et al. The epidemiology of heart failure : The Framingham Study. J Am Coll Cardiol, 1993,22(4):6A-13A
2. Agbulut O, Menot ML, Li ZL, et al. Temporal patterns of bone marrow cell differentiation following transplantation in doxorubicin-induced cardiomyopathy. Casdiovascular Research, 2003,58:451-459
3. Baldasseroni S, Opasich C, Gorini M, et al. Left bundle branch block is associated with increased 1-year sudden and total mortality rate in 5517 patients with congestive heart failure,A report from the Italian network on congestive heart failure, Am Heart J, 2002,143(3):398-405
4. Grimm W, Rudolph S, Christ M, et al. Prognostic significance of endomyocardial biopsy analysis in patients with idiopathic dilated cardiomyopathy. Am Heart J, 2003,146(2):372-376
5. Pennington DG, Oaks TE, Lohmann DP. Pemanent ventricular assist device support versus cardiac transplantation. Ann Thorac Surg ,1999,67:729-733
6. Brann WM, Bennett LE, Keck BM, et al. Morbidity, functional status and immunsuppressive therapy after heart transplantation:an analysis of the Joint International Society for heart and lung transplantation/United Network for Organ Sharing Thoracic Registry. Heart Lung Transplant, 1998,17:374-382
7. Friman G, Wesslen L, Fohlman J, et al. The epidemiology of infectious myocarditis, lymphocytic myocarditis and dilated cardiomyopathy. Eur Heart J, 1995,16(Suppl) 0:36-47
8. Gage FH. Cell therapy. Nature, 1998,392:18-24
9. Kohl GY,Klug MG, Soonph MH, et al. Differentiation and long-term survival of CIC12 myoblasts grafts in heart. J Clin Invest, 1993,93:1548-1554
10. Li RK,Jia ZQ, Weisel RD, et al. Smooth muscle cell transplantation into myocardial scar tissue improves heart function. J Mol Cell Cardiol, 1999,31:513-522
11. Taylor DA, Atkins BZ, Hungsprengs P, et al. Regenerating functional myocardium improved performance after skeletal myoblast transplantation. Nat Med, 1998,4(8):929-933
12. Connold AL, Frischknecht R, Dimitrakos M, et al. The survival of embryonic cardiomyocytes transplanted into damaged host rat myocardium. J Muscle Res Cell Motil, 1997,18:63-70
13. Tomita S, Li RK, Weisel RD, et al. Autologous transplantation of bone marrow cells improves damaged heart function. Circulation, 1999,100(19suppl):II247-256
14. Wang JS, Shum-Tim D, Gulipeau J, et al. Marrow stromal cells for cellular cardiomyoplastyrfeasibility and potential clinical advantages. J Thorac Cardiovasc Surg, 2000,120:999-1006
15. Orlic D, Kajstura J, Chimenti S, et al. Bone marrow cells regenerate infarcted myocardium. Nature, 2001,410:701-705
16. Makino S, Fukuda K, Miyoshi S, et al. Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest ,1999,103:697-705
17. Strauer BE, Brehm M, Zens T, et al. Intracoronary human autologous stem cell transplantation for myocardial regeneration following myocardial infarction. Detsch Med Wochenschr ,2001,126(34-35):932-938
18. Menaschc P, Hagege A, Scorsin M, et al. Myoblast transplantation for heart failure. Lancet, 2001,357(9252):279-280
19. Siminak T, Kalawski R, Fiszer D, et al. Skeletal myoblast transplantation during bypass grafting in the treatment of heart failure-phase I clinical trial feasibility study and early results. Heart Surg Forum , 2002,6(1 ):3-7
2. Agbulut O, Menot ML, Li ZL, et al. Temporal patterns of bone marrow cell differentiation following transplantation in doxorubicin-induced cardiomyopathy. Casdiovascular Research, 2003,58:451-459
3. Baldasseroni S, Opasich C, Gorini M, et al. Left bundle branch block is associated with increased 1-year sudden and total mortality rate in 5517 patients with congestive heart failure,A report from the Italian network on congestive heart failure, Am Heart J, 2002,143(3):398-405
4. Grimm W, Rudolph S, Christ M, et al. Prognostic significance of endomyocardial biopsy analysis in patients with idiopathic dilated cardiomyopathy. Am Heart J, 2003,146(2):372-376
5. Pennington DG, Oaks TE, Lohmann DP. Pemanent ventricular assist device support versus cardiac transplantation. Ann Thorac Surg ,1999,67:729-733
6. Brann WM, Bennett LE, Keck BM, et al. Morbidity, functional status and immunsuppressive therapy after heart transplantation:an analysis of the Joint International Society for heart and lung transplantation/United Network for Organ Sharing Thoracic Registry. Heart Lung Transplant, 1998,17:374-382
7. Friman G, Wesslen L, Fohlman J, et al. The epidemiology of infectious myocarditis, lymphocytic myocarditis and dilated cardiomyopathy. Eur Heart J, 1995,16(Suppl) 0:36-47
8. Gage FH. Cell therapy. Nature, 1998,392:18-24
9. Kohl GY,Klug MG, Soonph MH, et al. Differentiation and long-term survival of CIC12 myoblasts grafts in heart. J Clin Invest, 1993,93:1548-1554
10. Li RK,Jia ZQ, Weisel RD, et al. Smooth muscle cell transplantation into myocardial scar tissue improves heart function. J Mol Cell Cardiol, 1999,31:513-522
11. Taylor DA, Atkins BZ, Hungsprengs P, et al. Regenerating functional myocardium improved performance after skeletal myoblast transplantation. Nat Med, 1998,4(8):929-933
12. Connold AL, Frischknecht R, Dimitrakos M, et al. The survival of embryonic cardiomyocytes transplanted into damaged host rat myocardium. J Muscle Res Cell Motil, 1997,18:63-70
13. Tomita S, Li RK, Weisel RD, et al. Autologous transplantation of bone marrow cells improves damaged heart function. Circulation, 1999,100(19suppl):II247-256
14. Wang JS, Shum-Tim D, Gulipeau J, et al. Marrow stromal cells for cellular cardiomyoplastyrfeasibility and potential clinical advantages. J Thorac Cardiovasc Surg, 2000,120:999-1006
15. Orlic D, Kajstura J, Chimenti S, et al. Bone marrow cells regenerate infarcted myocardium. Nature, 2001,410:701-705
16. Makino S, Fukuda K, Miyoshi S, et al. Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest ,1999,103:697-705
17. Strauer BE, Brehm M, Zens T, et al. Intracoronary human autologous stem cell transplantation for myocardial regeneration following myocardial infarction. Detsch Med Wochenschr ,2001,126(34-35):932-938
18. Menaschc P, Hagege A, Scorsin M, et al. Myoblast transplantation for heart failure. Lancet, 2001,357(9252):279-280
19. Siminak T, Kalawski R, Fiszer D, et al. Skeletal myoblast transplantation during bypass grafting in the treatment of heart failure-phase I clinical trial feasibility study and early results. Heart Surg Forum , 2002,6(1 ):3-7