携带HGF基因骨髓间充质干细胞移植对大鼠心力衰竭的作用及机制研究
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摘要
本课题研究分为三个部分。第一部分:绿色荧光蛋白示踪技术在大鼠骨髓间充质干细胞心脏移植中的应用研究,对本研究的方法学进行了探讨,研究了本实验所采用的干细胞示踪方法的安全性和可靠性;第二部分探讨了携带HGF基因的腺病毒转染骨髓间充质干细胞对大鼠心力衰竭的疗效和安全性以及作用机制;在第二部分研究结果的基础上,为了探讨更有效的心力衰竭治疗方法,进行了第三部分的研究,联合应用细胞因子G-CSF和携带HGF基因的骨髓间充质细胞治疗大鼠心力衰竭,阐明这两种细胞因子联用具有协同作用。
第一部分:绿色荧光蛋白示踪技术在大鼠骨髓间充质干细胞心脏移植中的应用研究
目的:
骨髓间充质干细胞(mesenchymal stem cells,MSCs)在心肌组织再生和修复方面的应用取得了较多的进展。然而目前对移植到缺血心肌后的干细胞进行体内示踪的方法十分有限。本研究通过探讨绿色荧光蛋白(GFP)标记识别和示踪大鼠骨髓间充质干细胞的(MSCs)的可靠性,为MSCs移植到缺血心肌后体内示踪技术的应用提供实验依据。
方法:
从大鼠骨髓中分离骨髓间充质干细胞后进行培养和扩增,用携带GFP的腺病毒感染MSC,植入心肌梗死后的大鼠心脏梗死边缘区,观察细胞移植后3天、7天、14天、21天、28天标记细胞存活、迁移和分布情况。
结果:
骨髓间充质干细胞在体内、体外均可成功能表达GFP。携带GFP的腺病毒感染MSC的感染效率达到98%,并且基本没有毒性作用。激光共聚焦显微镜可观察到GFP可成功示踪移植的MSCs 28天。
结论:
GFP可以有效地示踪骨髓MSCs移植入心肌梗死大鼠心脏后在体内的分布和转归情况。
第二部分:携带HGF基因骨髓间充质细胞移植治疗大鼠心力衰竭的研究
目的:
缺血性心肌病是引起心力衰竭的主要病因,并且这类患者生存时间比非缺血性心力衰竭短,治疗效果差,急需寻找新的治疗策路。本研究的目的就是研究携带HGF基因的骨髓间充质干细胞心脏移植后,对大鼠心力衰竭的治疗作用,并初步探讨其作用机制。
方法:
通过结扎左冠状动脉的方法建立大鼠心肌梗死模型。4周后根据心脏超声检查结果,将FS≤30%的大鼠随机分为4组,观察时程为4周:①对照组(n=6):心梗边缘注射生理盐水;②MSC组(n=6):心梗边缘注射GFP-Ad感染的MSCs;③HGF组(n=6):心梗边缘注射HGF-Ad感染的MSCs;④HGF+CsA组(n=6):心梗边缘注射HGF-Ad感染的MSCs,并于细胞移植次日腹腔内注射环孢A25mg/kg.d 14天。所有动物腹腔注射BrdU标记新生细胞。心肌梗死8周后,行心脏超声和血流动力学检查检测心功能,将动物处死取材。Western-Blot方法检测HGF在心肌内的表达。用HE染色,免疫荧光染色,TUNEL等方法评价各种干预对心脏形态,血管生成和内皮细胞增殖情况的影响,检测心肌内凋亡相关的蛋白表达,探讨HGF的作用机制。
结果:
携带HGF的骨髓间充质细胞可以在体内成功表达HGF蛋白。
心肌梗死8周后,MSC组与对照组相比,LVEDP(9.76±0.54 vs.10.09±0.68kPa)、LVSP(15.98±0.81vs.15.08±0.88 kPa)、dp/dtmax(362.15±14.00 vs.347.34±19.16 kPa/s)和FS(23.83±2.37 vs.21.33±1.76%)几项指标差别无统计学意义(p＞0.05);HGF组与MSC组相比,LVEDP(7.65±0.52 vs.9.76±0.54 kPa)、(19.76±0.73 vs.15.98±0.81 kPa)、dp/dtmax(503.48±16.97 vs.362.15±14.00 kPa/s)和FS(29.63±2.88 vs.23.83±2.37%)几项指标差别均有统计学意义(p＜0.05)。HGF组的LVEDP显著降低、LVSP和dp/dtmax升高和FS显著增加。
MSC组和对照组相比,心室壁厚度(1.56±0.34 vs.1.53±0.41mm)和血管密度(81.67±6.32 vs.82.00±8.68mm)差别无统计学意义(p＞0.05)。HGF组与MSC组相比,心室壁厚度(1.98±0.50 vs.1.56±0.34 mm)显著增加,存活的心肌也较多(p＜0.05),心梗交界区的血管密度(137.67±15.42 vs.81.67±6.32mm)显著也增加,差别皆具有有计学意义(p＜0.05)。
采用了白细胞标记物CD45、内皮细胞标记物CD34和vWF、心肌细胞标记物心肌肌球蛋白重链蛋白的抗体和BrdU抗体作免疫荧光双染,鉴定新生细胞类型。结果显示几乎所有的BrdU阳性的细胞都是CD34或vWF阳性而MHC、CD45是阴性的,说明绝大多数增生的细胞是内皮细胞,没有发现白细胞和心肌细胞的增生。MSC组与对照组相比,增生的CD34阳性细胞数量(13±2.72vs.10.01±2.10 per mm~2)和vWF阳性的细胞数量(14.17±1.74 vs.11.33±1.97 permm~2)差别无统计学意义(p＞0.05)。HGF组与MSC组相比,增生的CD34阳性细胞数量(27±4.66 vs.13±2.72 per mm~2)和vWF阳性的细胞数量(30.33±2.65vs.14.17±1.74 per mm~2)差别具有统计学意义(p＜0.05),HGF组CD34阳性和vWF阳性的细胞数目明显增加。
通过末端标记脱氧核苷酸转移酶介导的缺口末端标记(TUNEL)法标记凋亡心肌细胞,结果显示MSC组与对照组相比,凋亡指数(2.26±0.45 vs.3.01±0.72)降低,差异具有统计学意义(p＜0.05);HGF组与MSC组相比,凋亡指数(0.90±0.46 vs.2.26±0.45)显著降低,差异具有统计学意义(p＜0.05);CsA组与HGF组相比,凋亡指数(2.06±0.66 vs.0.90±0.46)明显升高,差异具有统计学意义(p＜0.05),但仍较对照组(2.06±0.66 vs.3.01±0.72)低,差异具有统计学意义(p＜0.05)。
细胞凋亡相关蛋白的表达检测结果显示,MSC组与对照组比较,CaN、磷酸化的Akt、Bcl-2、NFAT4的表达没有明显差别(p＞0.05)。HGF组与MSC组比较,CaN、磷酸化的Akt和Bcl-2的表达水平明显增加,差别具有统计学意义(p＜0.05),但是NFAT4的水平没有明显改变(p＞0.05)。CsA组给予CaN的抑制剂CsA处理后,结果发现CsA部分抑制大鼠心肌组织中的CaN蛋白的表达,CsA组与HGF组比较,CaN表达显著降低(p＜0.05),伴随着Akt和Bcl-2的蛋白表达也相应减少(p＜0.05),但NFAT4蛋白的表达无改变(p＞0.05)。
结论:本研究证实了在心梗4周时移植腺病毒介导的过表达HGF的MSCs,可以通过促进血管生成和抑制心肌细胞凋亡,明显延缓心功能的恶化,HGF部分抑制心肌细胞凋亡的保护作用是通过上调CaN的表达来发挥作用的,Bcl-2和Akt是参与了其抗凋亡作用的下游通路的信号分子。
第三部分:G-CSF联合携带HGF基因骨髓间充质干细胞治疗大鼠心力衰竭的研究
目的:
在第二部分研究结果的基础上,为寻找到更有效的心力衰竭的治疗方法,研究联合应用细胞因子HGF和G-CSF对大鼠心力衰竭的治疗效果,并初步探讨其作用机制。
方法:
通过结扎左冠状动脉的方法建立大鼠心肌梗死模型。4周后根据心脏超声检查结果,将FS≤30%的大鼠随机分为4组,观察时程4周:①对照组(n=11)给予心梗边缘注射0.1ml生理盐水;②G-CSF组(n=11)心梗边缘注射0.1ml生理盐水,细胞移植当日起开始腹腔注射G-CSF,100μg/kg.d共5天;③HGF组(n=11)心梗边缘注射0.1ml Ad-HGF感染的MSCs;④HGF+G-CSF组(n=11)心梗边缘注射0.1ml Ad-HGF感染的MSCs,并于同时腹腔内注射G-CSF,100μg/kg.d 5天。心肌梗死8周,心脏超声和血流动力学检查检测心功能后,将动物处死取材。用组织学检查,免疫荧光染色,Western-Blot等方法评价各种干预对心脏形态,血管生成以及VCAM-1、MMP-9蛋白表达的影响。
结果:
心肌梗8周后,评价各组大鼠心功能,G-CSF组和对照组相比,左室舒张末期压力(LVEDP)(10.44±0.46 vs.10.65±0.62 kPa)、左室收缩末期压力(LVSP)(16.31±0.66 vs.15.25±0.90 kPa)、dP/dtmax(368.50±7.72 vs.357.83±10.76 kPa/s)、左室缩短率(FS)(23.83±2.52 vs.21.33±1.76%)指标的差别均没有统计学意义(p＞0.05)。HGF组与G-CSF组相比,的LVEDP(8.55±0.20 vs.10.44±0.46 kPa)、LVSP(19.09±0.67 vs.16.31±0.66 kPa)、dP/dtmax(493.48±10.99 vs.368.50±7.72 kPa/s)和FS(28.63±2.88 vs.23.83±2.52%)指标的差别均具有统计学意义(p＜0.05),HGF组的LVEDP显著降低,而LVSP、dP/dtmax和FS显著升高。HGF+G-CSF组与HGF组相比,LVEDP(6.70±0.36 vs.8.55±0.20 kPa)、LVSP(21.83±1.16 vs.19.09±0.67kPa)、dP/dtmax(527.22±10.24 vs.493.48±10.99 kPa/s)和FS(29.99±2.79 vs.28.63±2.88%)指标的差别均亦具有统计学意义(p＜0.05),HGF+G-CSF组的LVEDP显著降低,LVSP、dP/dtmax和FS显著升高。4组中,HGF+G-CSF组收缩功能和舒张功能都改善最明显。
心梗8周后,心梗面积都在40%左右,组间比较没有统计学差异。G-CSF组和对照组相比,左室内径(6.78±0.36 vs.6.86±0.36 mm)和心梗交界区室壁厚度(1.48±0.14 vs.1.46±0.13mm)差别都没有统计学意义(p＞0.05)。HGF组与G-CSF相比,HGF组左室内径(5.72±0.30 vs.6.78±0.36 mm)和心梗交界区室壁厚度(1.85±0.09 vs.1.48±0.14 mm)差别具有统计学意义,HGF组左室内径较小(p＜0.05),心梗交界区室壁厚度较厚(p＜0.05)。HGF+G-CSF与HGF组相比,左室内径(4.7±0.34 vs.5.72±0.30 mm)和心梗交界区室壁厚度(2.22±0.13 vs.1.85±0.09 mm)差别具有统计学意义(p＜0.05),HGF+G-CSF组左室内径较小,梗死交界区室壁厚度较大(p＜0.05)。HGF+G-CSF组在4组中,左室内径最小,梗死交界区室壁厚度最大。
用掺入假核苷酸BrdU的方法标记心肌内增殖的细胞,细胞移植4周后,用白细胞标记物CD45、内皮细胞标记物CD34和vWF、心肌细胞标记物心肌肌球蛋白重链蛋白的抗体和BrdU抗体作免疫荧光双染,结果显示几乎所有的BrdU阳性的细胞都是CD34或vWF阳性而MHC、CD45是阴性的,说明绝大多数增生的细胞是内皮细胞,仍然没有发现白细胞和心肌细胞的增生。G-CSF组与对照组相比,增生的CD34阳性细胞数(6.83±1.13vs.8.83±1.30 per mm~2)和vWF阳性细胞数量(14.00±1.63 vs.11.00±1.82 per mm~2)差别无统计学意义(p＞0.05),vWF染色计数＜20μm血管密度(93.33±5.7vs.80±6.36 per mm~2)差别也无统计学意义(p＞0.05)。HGF组与G-CSF组相比,增生的CD34阳性细胞数(14.00±1.83vs.6.83±1.13 per mm~2)、vWF阳性细胞数(26.01±2.44 vs.14.00±1.63permm~2)差别都具有统计学意义(p＜0.05),血管密度(111.67±9.54 vs.93.33±5.7per mm~2)差别也具有统计学意义(p＜0.05),HGF组CD34阳性细胞数和vWF阳性细胞数显著增加,血管密度也显著增加。HGF+G-CSF组与HGF组相比,CD34阳性细胞数(20.67±1.82 vs.14.00±1.83 per mm~2)、vWF阳性细胞数(33.00±2.87 vs.26.01±2.44 per mm~2)差别都具有统计学意义(p＜0.05),血管密度(133.67±7.04 vs.111.67±9.54 per mm~2)差别皆具有统计学意义(p＜0.05)。较HGF组,HGF+G-CSF组CD34阳性细胞数和vWF阳性细胞数、血管密度皆显著增加。4组中,HGF+G-CSF组CD34阳性的细胞数、vWF阳性细胞数和血管密度明显较其它3组为多。
为了探索HGF-MSC和G-CSF对心梗交界区心肌组织内环境的影响,检测相关的蛋白的表达。发现HGF组和G-CSF+HGF组的VCAM-1的表达较对照组和G-CSF组明显增加(p＜0.05),但是HGF组和G-CSF+HGF组两组之间相比没有明显差异(p＞0.05),证明VCAM-1的表达增加与HGF的治疗有关,与G-CSF无关。HGF组和G-CSF组MMP-9的表达都较对照组增加(p＜0.05),而HGF+G-CSF组MMP-9蛋白的表达最多,表明HGF和G-CSF都可以促进MMP-9蛋白的表达。
结论:
在心梗4周时给予G-CSF和携带HGF基因的MSCs治疗,可以明显改善大鼠的心功能,促进心梗边界缺血区域的血管生成,其作用机制之一可能是HGF和G-CSF可以通过增加缺血心肌VCAM-1和MMP-9蛋白的表达,改善缺血心肌的内环境。
In this study,we investigated the effectiveness of cytokines on post-ischemic heart failure and the mechanisms underlying the effect.This study was divided into 3 parts.
Part 1:In vivo tracking of green fluorescent protein labeled mesenchymal stem cells in hearts of rats with myocardial infarction
Objective:There are rapid advances in the use of mesenchymal stem cells for tissue regeneration and repair in the heart in recent years.However,there is limited ability to identify and "track" transplanted or migrating stem cells in vivo.In this study,we transplanted bone marrow derived mesenchymal stem cells infected with the recombinant replication-defective adenovirus vector carrying green fluorescent protein into rat heats,to evaluate the effectiveness of green fluorescent protein identifying and tracking implanted cells in vivo in order to provide a effective way to track the delivered mesenchymal stem cells in vivo.
Methods:Mesenchymal stem cells isolated from rat femur marrow were cultured and expanded,then infected by recombinant retrovirus containing green fluorescent protein.Transplant the green fluorescent protein labeled mesenchymal stem cells into rat heart infarct zone.The survival,distribution and migration of labeled mesenchymal stem cells were analyzed at 3 days,7 days,14 days,21 days,28 days after transplantation.
Results:Green fluorescent protein was successfully expressed by mesenchymal stem cells in vitro and in vivo.The adenoviral vectors' infection of mesenchymal stem cells had high efficiency(98%) and low toxicity,green fluorescent protein positive cells were detected by confocal microscope during the initial 28 days after transplantation.Green fluorescent protein labeled mesenchymal stem cells were gradually retained into host tissue and migrated.
Conclusions:Green fluorescent protein can be used to track mesenchymal stem cell location and fate after delivery in the rat heats with myocardial infarction.
Part 2:Locally Overexpressing Hepatocyte Growth Factor Prevents Post-ischemic Heart Failure by Inhibition of Apoptosis via Calcineurin-Mediated Pathway and Angiogenesis
Objective:Myocardial infarction is a significant cause of heart failure.Currently, therapies are limited,and novel revascularization methods might have a role.We investigated the effects of hepatocyte growth factor expressing by bone marrow-derived mesenchymal stem cells(MSCs) on post-ischemic heart failure.
Methods:4 weeks after MI,SD rats(F≤30%) were randomly divided into 1.control group:injection of saline into the infarct zone,n=6;2.MSC group:transplantation of bone marrow derived MSCs transfected with Ad-GFP into the infarct zone,n=11;3, HGF group:transplantation of MSCs transfected with Ad-HGF into the infarct zone, n=6;4.HGF+CsA group:transplantation of MSCs transfected with Ad-HGF into the infarct zone and intraperitoneal injection of cyclosporine A,25mg/kg daily for 14 days,n=6.After another 4 weeks,hearts were analyzed for ventficular geometry, myocardial function,angiogenesis and endothelial cell density,apoptosis and the expression of HGF,calcineurin,Akt and Bcl-2 protein.
Results:we detected the expression of HGF protein at 2days,7days of transplantation in HGF group.We found a significant improvement in left ventricular function in the HGF-MSC group compared with GFP-MSC group.The difference in LVFS,LVEDP, LVSPand dp/dtmax in the MSC-HGF group compared to the saline control group and MSC-GFP group(p＜0.05).Overexpression of HGF was associated with a greater number of CD34and von vWF positive cells proliferated in the infarct zone (27.00±4.66 vs.13.00±2.72,30.33±2.65 vs.14.17±1.74 per mm2,respectively). There were no difference in the number of cardiac myosin heavy chain positive cells between HGF and MSC group.TUNEL assays results showed that about 0.90±0.46‰nuclei were TUNEL-positive in HGF group,whereas 2.26±0.45‰nuclei were TUNEL-positive staining,in MSC group(p＜0.05).However,the antiapoptotic effect of HGF decreased when HGF and cyclosporine A were given to rats simultaneously and 2.06±0.66‰in CsA group(p＜0.05).The effects of HGF on apoptosis were associated with the expression level of calcineurin Akt and Bcl-2 protein.
Conclusion:Our findings suggest that overexpression of HGF improved ischemic cardiac function through angiogenesis and reduction of apoptosis partly mediated by up-regulation of calcineurin.
Part 3:Hepatoeyte growth factor and granulocyte colony-stimulating factor form a combined neovasculogenic therapy for ischemic cardiomyopathy
Objective:Myocardial infarction(MI) is a significant cause of heart failure. Currently,therapies are limited,and novel revascularization methods might have a role.The present study was to investigated whether hepatocyte growth factor(HGF) expressed by genetically modified mesenchymal stem cells(MSCs) combined with granulocyte colony-stimulating factor(G-CSF) 8 weeks from myocardial infarction can offer synergistic therapeutic benefit and the mechanisms underlying the synergistic effect.
Methods:4 weeks after MI,SD(FS＜30%) rats were randomly divided into control group(n=11);HGF group(transplantation MSCs transfected with Ad-HGF into the infarct zone,n=11);G-CSF group(intraperitoneal injection G-CSF,n=11); HGF+G-CSF group(transplantation MSCs transfected with Ad-HGF into the infarct zone and intraperitoneal injection G-CSF,n=11).After another 4 weeks,hearts were analyzed for endothelial cell density and angiogenesis,ventricular geometry, myocardial function and the expression of VCAM-1 and MMP-9 protein.
Results:The rats in HGF+G-CSF group exhibited better LV systolic and diastolic function(p＜0.05),experienced less adverse ventricular remodeling,as manifested by less left ventricular dilatation(4.7±0.34mm vs.6.86±0.36mm in control group, 6.78±0.36mm in G-CSF group,5.72±0.3mm in HGF group,p＜0.05) and increased border-zone wall thickness(2.22±0.13mm vs.1.46±0.13mm in control group, 1.48±0.14mm in G-CSF group,1.85±0.09mm in HGF group p＜0.05) after 8 weeks of MI.Angiogenesis was significantly enhanced in HGF+G-CSF group by inducing proliferation of CD34 positive(20.67±1.82 per mm~2 vs.8.83±1.30 per mm~2 in control group,6.83±1.13 per mm~2 in G-CSF group,14.00±1.83,per mm~2 in HGF group, p＜0.05) and vWF positive(33.00±2.87 per mm~2 vs.11.00±1.82 per mm~2 in control group,14.00±1.63 per mm~2 in G-CSF group,26.01±2.44 per mm~2 in HGF group,p＜0.05) endothelial cells.HGF induced the expression of VCAM-1 and HGF together with G-CSF treatment synergistically stimulated MMP-9 expression in ischemic heart.
Conclusion:The combination of G-CSF and HGF had a significant synergistic effect and enhanced myocardial endothelial density,angiogenesis,geometric preservation, and heart function in the model of ischemic cardiomyopathy.
第一部分:绿色荧光蛋白示踪技术在大鼠骨髓间充质干细胞心脏移植中的应用研究
目的:
骨髓间充质干细胞(mesenchymal stem cells,MSCs)在心肌组织再生和修复方面的应用取得了较多的进展。然而目前对移植到缺血心肌后的干细胞进行体内示踪的方法十分有限。本研究通过探讨绿色荧光蛋白(GFP)标记识别和示踪大鼠骨髓间充质干细胞的(MSCs)的可靠性,为MSCs移植到缺血心肌后体内示踪技术的应用提供实验依据。
方法:
从大鼠骨髓中分离骨髓间充质干细胞后进行培养和扩增,用携带GFP的腺病毒感染MSC,植入心肌梗死后的大鼠心脏梗死边缘区,观察细胞移植后3天、7天、14天、21天、28天标记细胞存活、迁移和分布情况。
结果:
骨髓间充质干细胞在体内、体外均可成功能表达GFP。携带GFP的腺病毒感染MSC的感染效率达到98%,并且基本没有毒性作用。激光共聚焦显微镜可观察到GFP可成功示踪移植的MSCs 28天。
结论:
GFP可以有效地示踪骨髓MSCs移植入心肌梗死大鼠心脏后在体内的分布和转归情况。
第二部分:携带HGF基因骨髓间充质细胞移植治疗大鼠心力衰竭的研究
目的:
缺血性心肌病是引起心力衰竭的主要病因,并且这类患者生存时间比非缺血性心力衰竭短,治疗效果差,急需寻找新的治疗策路。本研究的目的就是研究携带HGF基因的骨髓间充质干细胞心脏移植后,对大鼠心力衰竭的治疗作用,并初步探讨其作用机制。
方法:
通过结扎左冠状动脉的方法建立大鼠心肌梗死模型。4周后根据心脏超声检查结果,将FS≤30%的大鼠随机分为4组,观察时程为4周:①对照组(n=6):心梗边缘注射生理盐水;②MSC组(n=6):心梗边缘注射GFP-Ad感染的MSCs;③HGF组(n=6):心梗边缘注射HGF-Ad感染的MSCs;④HGF+CsA组(n=6):心梗边缘注射HGF-Ad感染的MSCs,并于细胞移植次日腹腔内注射环孢A25mg/kg.d 14天。所有动物腹腔注射BrdU标记新生细胞。心肌梗死8周后,行心脏超声和血流动力学检查检测心功能,将动物处死取材。Western-Blot方法检测HGF在心肌内的表达。用HE染色,免疫荧光染色,TUNEL等方法评价各种干预对心脏形态,血管生成和内皮细胞增殖情况的影响,检测心肌内凋亡相关的蛋白表达,探讨HGF的作用机制。
结果:
携带HGF的骨髓间充质细胞可以在体内成功表达HGF蛋白。
心肌梗死8周后,MSC组与对照组相比,LVEDP(9.76±0.54 vs.10.09±0.68kPa)、LVSP(15.98±0.81vs.15.08±0.88 kPa)、dp/dtmax(362.15±14.00 vs.347.34±19.16 kPa/s)和FS(23.83±2.37 vs.21.33±1.76%)几项指标差别无统计学意义(p＞0.05);HGF组与MSC组相比,LVEDP(7.65±0.52 vs.9.76±0.54 kPa)、(19.76±0.73 vs.15.98±0.81 kPa)、dp/dtmax(503.48±16.97 vs.362.15±14.00 kPa/s)和FS(29.63±2.88 vs.23.83±2.37%)几项指标差别均有统计学意义(p＜0.05)。HGF组的LVEDP显著降低、LVSP和dp/dtmax升高和FS显著增加。
MSC组和对照组相比,心室壁厚度(1.56±0.34 vs.1.53±0.41mm)和血管密度(81.67±6.32 vs.82.00±8.68mm)差别无统计学意义(p＞0.05)。HGF组与MSC组相比,心室壁厚度(1.98±0.50 vs.1.56±0.34 mm)显著增加,存活的心肌也较多(p＜0.05),心梗交界区的血管密度(137.67±15.42 vs.81.67±6.32mm)显著也增加,差别皆具有有计学意义(p＜0.05)。
采用了白细胞标记物CD45、内皮细胞标记物CD34和vWF、心肌细胞标记物心肌肌球蛋白重链蛋白的抗体和BrdU抗体作免疫荧光双染,鉴定新生细胞类型。结果显示几乎所有的BrdU阳性的细胞都是CD34或vWF阳性而MHC、CD45是阴性的,说明绝大多数增生的细胞是内皮细胞,没有发现白细胞和心肌细胞的增生。MSC组与对照组相比,增生的CD34阳性细胞数量(13±2.72vs.10.01±2.10 per mm~2)和vWF阳性的细胞数量(14.17±1.74 vs.11.33±1.97 permm~2)差别无统计学意义(p＞0.05)。HGF组与MSC组相比,增生的CD34阳性细胞数量(27±4.66 vs.13±2.72 per mm~2)和vWF阳性的细胞数量(30.33±2.65vs.14.17±1.74 per mm~2)差别具有统计学意义(p＜0.05),HGF组CD34阳性和vWF阳性的细胞数目明显增加。
通过末端标记脱氧核苷酸转移酶介导的缺口末端标记(TUNEL)法标记凋亡心肌细胞,结果显示MSC组与对照组相比,凋亡指数(2.26±0.45 vs.3.01±0.72)降低,差异具有统计学意义(p＜0.05);HGF组与MSC组相比,凋亡指数(0.90±0.46 vs.2.26±0.45)显著降低,差异具有统计学意义(p＜0.05);CsA组与HGF组相比,凋亡指数(2.06±0.66 vs.0.90±0.46)明显升高,差异具有统计学意义(p＜0.05),但仍较对照组(2.06±0.66 vs.3.01±0.72)低,差异具有统计学意义(p＜0.05)。
细胞凋亡相关蛋白的表达检测结果显示,MSC组与对照组比较,CaN、磷酸化的Akt、Bcl-2、NFAT4的表达没有明显差别(p＞0.05)。HGF组与MSC组比较,CaN、磷酸化的Akt和Bcl-2的表达水平明显增加,差别具有统计学意义(p＜0.05),但是NFAT4的水平没有明显改变(p＞0.05)。CsA组给予CaN的抑制剂CsA处理后,结果发现CsA部分抑制大鼠心肌组织中的CaN蛋白的表达,CsA组与HGF组比较,CaN表达显著降低(p＜0.05),伴随着Akt和Bcl-2的蛋白表达也相应减少(p＜0.05),但NFAT4蛋白的表达无改变(p＞0.05)。
结论:本研究证实了在心梗4周时移植腺病毒介导的过表达HGF的MSCs,可以通过促进血管生成和抑制心肌细胞凋亡,明显延缓心功能的恶化,HGF部分抑制心肌细胞凋亡的保护作用是通过上调CaN的表达来发挥作用的,Bcl-2和Akt是参与了其抗凋亡作用的下游通路的信号分子。
第三部分:G-CSF联合携带HGF基因骨髓间充质干细胞治疗大鼠心力衰竭的研究
目的:
在第二部分研究结果的基础上,为寻找到更有效的心力衰竭的治疗方法,研究联合应用细胞因子HGF和G-CSF对大鼠心力衰竭的治疗效果,并初步探讨其作用机制。
方法:
通过结扎左冠状动脉的方法建立大鼠心肌梗死模型。4周后根据心脏超声检查结果,将FS≤30%的大鼠随机分为4组,观察时程4周:①对照组(n=11)给予心梗边缘注射0.1ml生理盐水;②G-CSF组(n=11)心梗边缘注射0.1ml生理盐水,细胞移植当日起开始腹腔注射G-CSF,100μg/kg.d共5天;③HGF组(n=11)心梗边缘注射0.1ml Ad-HGF感染的MSCs;④HGF+G-CSF组(n=11)心梗边缘注射0.1ml Ad-HGF感染的MSCs,并于同时腹腔内注射G-CSF,100μg/kg.d 5天。心肌梗死8周,心脏超声和血流动力学检查检测心功能后,将动物处死取材。用组织学检查,免疫荧光染色,Western-Blot等方法评价各种干预对心脏形态,血管生成以及VCAM-1、MMP-9蛋白表达的影响。
结果:
心肌梗8周后,评价各组大鼠心功能,G-CSF组和对照组相比,左室舒张末期压力(LVEDP)(10.44±0.46 vs.10.65±0.62 kPa)、左室收缩末期压力(LVSP)(16.31±0.66 vs.15.25±0.90 kPa)、dP/dtmax(368.50±7.72 vs.357.83±10.76 kPa/s)、左室缩短率(FS)(23.83±2.52 vs.21.33±1.76%)指标的差别均没有统计学意义(p＞0.05)。HGF组与G-CSF组相比,的LVEDP(8.55±0.20 vs.10.44±0.46 kPa)、LVSP(19.09±0.67 vs.16.31±0.66 kPa)、dP/dtmax(493.48±10.99 vs.368.50±7.72 kPa/s)和FS(28.63±2.88 vs.23.83±2.52%)指标的差别均具有统计学意义(p＜0.05),HGF组的LVEDP显著降低,而LVSP、dP/dtmax和FS显著升高。HGF+G-CSF组与HGF组相比,LVEDP(6.70±0.36 vs.8.55±0.20 kPa)、LVSP(21.83±1.16 vs.19.09±0.67kPa)、dP/dtmax(527.22±10.24 vs.493.48±10.99 kPa/s)和FS(29.99±2.79 vs.28.63±2.88%)指标的差别均亦具有统计学意义(p＜0.05),HGF+G-CSF组的LVEDP显著降低,LVSP、dP/dtmax和FS显著升高。4组中,HGF+G-CSF组收缩功能和舒张功能都改善最明显。
心梗8周后,心梗面积都在40%左右,组间比较没有统计学差异。G-CSF组和对照组相比,左室内径(6.78±0.36 vs.6.86±0.36 mm)和心梗交界区室壁厚度(1.48±0.14 vs.1.46±0.13mm)差别都没有统计学意义(p＞0.05)。HGF组与G-CSF相比,HGF组左室内径(5.72±0.30 vs.6.78±0.36 mm)和心梗交界区室壁厚度(1.85±0.09 vs.1.48±0.14 mm)差别具有统计学意义,HGF组左室内径较小(p＜0.05),心梗交界区室壁厚度较厚(p＜0.05)。HGF+G-CSF与HGF组相比,左室内径(4.7±0.34 vs.5.72±0.30 mm)和心梗交界区室壁厚度(2.22±0.13 vs.1.85±0.09 mm)差别具有统计学意义(p＜0.05),HGF+G-CSF组左室内径较小,梗死交界区室壁厚度较大(p＜0.05)。HGF+G-CSF组在4组中,左室内径最小,梗死交界区室壁厚度最大。
用掺入假核苷酸BrdU的方法标记心肌内增殖的细胞,细胞移植4周后,用白细胞标记物CD45、内皮细胞标记物CD34和vWF、心肌细胞标记物心肌肌球蛋白重链蛋白的抗体和BrdU抗体作免疫荧光双染,结果显示几乎所有的BrdU阳性的细胞都是CD34或vWF阳性而MHC、CD45是阴性的,说明绝大多数增生的细胞是内皮细胞,仍然没有发现白细胞和心肌细胞的增生。G-CSF组与对照组相比,增生的CD34阳性细胞数(6.83±1.13vs.8.83±1.30 per mm~2)和vWF阳性细胞数量(14.00±1.63 vs.11.00±1.82 per mm~2)差别无统计学意义(p＞0.05),vWF染色计数＜20μm血管密度(93.33±5.7vs.80±6.36 per mm~2)差别也无统计学意义(p＞0.05)。HGF组与G-CSF组相比,增生的CD34阳性细胞数(14.00±1.83vs.6.83±1.13 per mm~2)、vWF阳性细胞数(26.01±2.44 vs.14.00±1.63permm~2)差别都具有统计学意义(p＜0.05),血管密度(111.67±9.54 vs.93.33±5.7per mm~2)差别也具有统计学意义(p＜0.05),HGF组CD34阳性细胞数和vWF阳性细胞数显著增加,血管密度也显著增加。HGF+G-CSF组与HGF组相比,CD34阳性细胞数(20.67±1.82 vs.14.00±1.83 per mm~2)、vWF阳性细胞数(33.00±2.87 vs.26.01±2.44 per mm~2)差别都具有统计学意义(p＜0.05),血管密度(133.67±7.04 vs.111.67±9.54 per mm~2)差别皆具有统计学意义(p＜0.05)。较HGF组,HGF+G-CSF组CD34阳性细胞数和vWF阳性细胞数、血管密度皆显著增加。4组中,HGF+G-CSF组CD34阳性的细胞数、vWF阳性细胞数和血管密度明显较其它3组为多。
为了探索HGF-MSC和G-CSF对心梗交界区心肌组织内环境的影响,检测相关的蛋白的表达。发现HGF组和G-CSF+HGF组的VCAM-1的表达较对照组和G-CSF组明显增加(p＜0.05),但是HGF组和G-CSF+HGF组两组之间相比没有明显差异(p＞0.05),证明VCAM-1的表达增加与HGF的治疗有关,与G-CSF无关。HGF组和G-CSF组MMP-9的表达都较对照组增加(p＜0.05),而HGF+G-CSF组MMP-9蛋白的表达最多,表明HGF和G-CSF都可以促进MMP-9蛋白的表达。
结论:
在心梗4周时给予G-CSF和携带HGF基因的MSCs治疗,可以明显改善大鼠的心功能,促进心梗边界缺血区域的血管生成,其作用机制之一可能是HGF和G-CSF可以通过增加缺血心肌VCAM-1和MMP-9蛋白的表达,改善缺血心肌的内环境。
In this study,we investigated the effectiveness of cytokines on post-ischemic heart failure and the mechanisms underlying the effect.This study was divided into 3 parts.
Part 1:In vivo tracking of green fluorescent protein labeled mesenchymal stem cells in hearts of rats with myocardial infarction
Objective:There are rapid advances in the use of mesenchymal stem cells for tissue regeneration and repair in the heart in recent years.However,there is limited ability to identify and "track" transplanted or migrating stem cells in vivo.In this study,we transplanted bone marrow derived mesenchymal stem cells infected with the recombinant replication-defective adenovirus vector carrying green fluorescent protein into rat heats,to evaluate the effectiveness of green fluorescent protein identifying and tracking implanted cells in vivo in order to provide a effective way to track the delivered mesenchymal stem cells in vivo.
Methods:Mesenchymal stem cells isolated from rat femur marrow were cultured and expanded,then infected by recombinant retrovirus containing green fluorescent protein.Transplant the green fluorescent protein labeled mesenchymal stem cells into rat heart infarct zone.The survival,distribution and migration of labeled mesenchymal stem cells were analyzed at 3 days,7 days,14 days,21 days,28 days after transplantation.
Results:Green fluorescent protein was successfully expressed by mesenchymal stem cells in vitro and in vivo.The adenoviral vectors' infection of mesenchymal stem cells had high efficiency(98%) and low toxicity,green fluorescent protein positive cells were detected by confocal microscope during the initial 28 days after transplantation.Green fluorescent protein labeled mesenchymal stem cells were gradually retained into host tissue and migrated.
Conclusions:Green fluorescent protein can be used to track mesenchymal stem cell location and fate after delivery in the rat heats with myocardial infarction.
Part 2:Locally Overexpressing Hepatocyte Growth Factor Prevents Post-ischemic Heart Failure by Inhibition of Apoptosis via Calcineurin-Mediated Pathway and Angiogenesis
Objective:Myocardial infarction is a significant cause of heart failure.Currently, therapies are limited,and novel revascularization methods might have a role.We investigated the effects of hepatocyte growth factor expressing by bone marrow-derived mesenchymal stem cells(MSCs) on post-ischemic heart failure.
Methods:4 weeks after MI,SD rats(F≤30%) were randomly divided into 1.control group:injection of saline into the infarct zone,n=6;2.MSC group:transplantation of bone marrow derived MSCs transfected with Ad-GFP into the infarct zone,n=11;3, HGF group:transplantation of MSCs transfected with Ad-HGF into the infarct zone, n=6;4.HGF+CsA group:transplantation of MSCs transfected with Ad-HGF into the infarct zone and intraperitoneal injection of cyclosporine A,25mg/kg daily for 14 days,n=6.After another 4 weeks,hearts were analyzed for ventficular geometry, myocardial function,angiogenesis and endothelial cell density,apoptosis and the expression of HGF,calcineurin,Akt and Bcl-2 protein.
Results:we detected the expression of HGF protein at 2days,7days of transplantation in HGF group.We found a significant improvement in left ventricular function in the HGF-MSC group compared with GFP-MSC group.The difference in LVFS,LVEDP, LVSPand dp/dtmax in the MSC-HGF group compared to the saline control group and MSC-GFP group(p＜0.05).Overexpression of HGF was associated with a greater number of CD34and von vWF positive cells proliferated in the infarct zone (27.00±4.66 vs.13.00±2.72,30.33±2.65 vs.14.17±1.74 per mm2,respectively). There were no difference in the number of cardiac myosin heavy chain positive cells between HGF and MSC group.TUNEL assays results showed that about 0.90±0.46‰nuclei were TUNEL-positive in HGF group,whereas 2.26±0.45‰nuclei were TUNEL-positive staining,in MSC group(p＜0.05).However,the antiapoptotic effect of HGF decreased when HGF and cyclosporine A were given to rats simultaneously and 2.06±0.66‰in CsA group(p＜0.05).The effects of HGF on apoptosis were associated with the expression level of calcineurin Akt and Bcl-2 protein.
Conclusion:Our findings suggest that overexpression of HGF improved ischemic cardiac function through angiogenesis and reduction of apoptosis partly mediated by up-regulation of calcineurin.
Part 3:Hepatoeyte growth factor and granulocyte colony-stimulating factor form a combined neovasculogenic therapy for ischemic cardiomyopathy
Objective:Myocardial infarction(MI) is a significant cause of heart failure. Currently,therapies are limited,and novel revascularization methods might have a role.The present study was to investigated whether hepatocyte growth factor(HGF) expressed by genetically modified mesenchymal stem cells(MSCs) combined with granulocyte colony-stimulating factor(G-CSF) 8 weeks from myocardial infarction can offer synergistic therapeutic benefit and the mechanisms underlying the synergistic effect.
Methods:4 weeks after MI,SD(FS＜30%) rats were randomly divided into control group(n=11);HGF group(transplantation MSCs transfected with Ad-HGF into the infarct zone,n=11);G-CSF group(intraperitoneal injection G-CSF,n=11); HGF+G-CSF group(transplantation MSCs transfected with Ad-HGF into the infarct zone and intraperitoneal injection G-CSF,n=11).After another 4 weeks,hearts were analyzed for endothelial cell density and angiogenesis,ventricular geometry, myocardial function and the expression of VCAM-1 and MMP-9 protein.
Results:The rats in HGF+G-CSF group exhibited better LV systolic and diastolic function(p＜0.05),experienced less adverse ventricular remodeling,as manifested by less left ventricular dilatation(4.7±0.34mm vs.6.86±0.36mm in control group, 6.78±0.36mm in G-CSF group,5.72±0.3mm in HGF group,p＜0.05) and increased border-zone wall thickness(2.22±0.13mm vs.1.46±0.13mm in control group, 1.48±0.14mm in G-CSF group,1.85±0.09mm in HGF group p＜0.05) after 8 weeks of MI.Angiogenesis was significantly enhanced in HGF+G-CSF group by inducing proliferation of CD34 positive(20.67±1.82 per mm~2 vs.8.83±1.30 per mm~2 in control group,6.83±1.13 per mm~2 in G-CSF group,14.00±1.83,per mm~2 in HGF group, p＜0.05) and vWF positive(33.00±2.87 per mm~2 vs.11.00±1.82 per mm~2 in control group,14.00±1.63 per mm~2 in G-CSF group,26.01±2.44 per mm~2 in HGF group,p＜0.05) endothelial cells.HGF induced the expression of VCAM-1 and HGF together with G-CSF treatment synergistically stimulated MMP-9 expression in ischemic heart.
Conclusion:The combination of G-CSF and HGF had a significant synergistic effect and enhanced myocardial endothelial density,angiogenesis,geometric preservation, and heart function in the model of ischemic cardiomyopathy.
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