骨髓间充质干细胞移植治疗大鼠扩张型心肌病

1泸州医学院附属医院急诊科,四川省泸州市 646000;2哈尔滨医科大学附属第二医院心内科,黑龙江省哈尔滨市  150000 背景：扩张型心肌病所致的心肌纤维化是心力衰竭的病理基础,目前药物治疗、介入治疗和外科手术均不能替代坏死心肌和彻底改善心脏功能。 目的：观察异体骨髓间充质干细胞移植对大鼠扩张型心肌病心脏功能的作用和心肌纤维化的影响。 方法：40只Wistar大鼠随机数字表法分为细胞移植组(n=15)、对照组(n=15)和空白组(n=10),前2组建立大鼠扩张型心肌病模型。造模成功4周后细胞移植组注射骨髓间充质干细胞悬液150 μL(含3×106个细胞),对照组和空白组注射等量培养液。 结果与结论：与空白组相比,细胞移植组和对照组移植前左室收缩末期内径增加,射血分数和缩短分数明显下降(P < 0.01);移植后4周,细胞移植组超声心动图检查和移植前相比,左室收缩末期内径下降、射血分数和缩短分数明显升高(P < 0.01)。细胞移植组心脏胶原的表达低于对照组(P < 0.05)。与对照组相比,其他2组基质金属蛋白酶2及基质金属蛋白酶9表达明显下降(P < 0.05)。提示骨髓间充质干细胞移植后可改善心肌纤维化及扩张型心肌病鼠的心脏功能。      

静脉移植骨髓间充质干细胞对心肌病心力衰竭心功能的影响

BACKGROUND:Bone marrow mesenchymal stem cell transplantation can effectively improve decreased cardiac function caused by heart failure, but there is a lack of research about the effect in bone marrow mesenchymal stem cell transplantation on cardiac function in heart failure induced by cardiomyopathies. OBJECTIVE:To explore the effect of bone marrow mesenchymal stem cell transplantation on cardiac function in patients with cardiomyopathies accompanied by heart failure. METHODS:Totally 40 Sprague-Dawley rats were enrolled, and bone marrow mesenchymal stem cells were isolated from 10 rats, and the remaining rats were equivalently randomized into normal, model and stem cell transplantation groups. Then rats in the model and stem cell transplantation groups were given intraperitoneal injection of hydrochloric acid doxorubicin to prepare cardiomyopathy-induced heart failure models. At 7 days after modeling, the stem cell transplantation group was treated with bone marrow mesenchymal stem cells through intravenous transplantation, and the model group was treated with equal amount of DMEM medium. Four weeks later, cardiac function of each rat was detected, and the cell survival and differentiation were observed by immunofluorescence method. RESULTS AND CONCLUSION:At 4 weeks after transplantation, compared with normal and stem cell transplantation groups, the left ventricular systolic pressure and maximum rise/fall rate of left ventricular pressure were significantly decreased, but the left ventricular end diastolic pressure significantly increased in the model group (P < 0.05). And there was no significant difference between the normal group and the stem cell transplantation group (P > 0.05). High and dense fluorescence intensity was observed in the host myocardium immediately after transplantation. Subsequently, the fluorescence intensity and density decreased at 4 weeks, but the cell migration could be found, and some cells expressed cardiac troponin T. These results show that intravenous transplantation of bone marrow mesenchymal stem cells can improve cardiac function in rats with heart failure due to cardiomyopathies. Besides, the transplanted cells can survive in the host, and differentiate into cardiomyocyte-like cells.     

人脐血间充质干细胞尾静脉移植治疗扩张型心肌病

背景：体外研究表明人脐血间充质干细胞可分化为自律性跳动的心肌细胞,而经静脉移植人脐血间充质干细胞治疗扩张型心肌病心力衰竭的报道较少。 目的：探讨经尾静脉移植人脐血间充质干细胞对扩张型心肌病大鼠心肌结构、心功能的影响。 方法：Wistar大鼠通过腹腔注射阿霉素诱导扩张型心肌病模型,扩张型心肌病实验组于造模后8周经尾静脉移植人脐血间充质干细胞,扩张型心肌病对照组注射等量DMEM培养基。健康对照组不造模,于相同时间点注射等体积的生理盐水。 结果与结论：与健康对照组相比,扩张型心肌病组心功能明显受损,且扩张型心肌病实验组受损较扩张型心肌病对照组轻;移植的细胞有肌钙蛋白T的表达。结果提示人脐血间充质干细胞移植能促进扩张型心肌病大鼠心功能恢复,使心肌组织病变减轻。     

骨髓间充质干细胞自体移植治疗阿霉素心肌病

目的:探讨骨髓间充质干细胞(MSCs)自体移植入兔阿霉素(ADR)心肌病心肌后的微环境依赖性分化及对心功能的影响。方法:日本大耳白兔随机分为:心肌病自体细胞移植组(n=10),心肌病对照组(n=8)及假手术组(n=6)。将自体MSCs和同体积的培养基分别移植入前两组动物心肌内,假手术组仅作开胸术而无移植。术后4周行心功能及组织学检测。结果:与假手术组相比,心肌病组的心功能明显降低;与心肌病对照组相比,自体移植组的心功能有显著改善,并在移植部位发现移植细胞有心肌肌钙蛋白T的表达。结论:MSCs移植入ADR心肌病的心肌组织中后,可在其微环境中存活并分化为心肌样细胞,并显著改善左室功能。     

自体骨髓间充质干细胞移植修复无功能心肌的对比

背景：目前有关干细胞技术的临床试验多集中在心肌梗死方面,对原发性心肌病研究尚少,且尚未见到两者对比的相关报道。 目的：比较自体骨髓间充质干细胞经冠状动脉内移植修复缺血及非缺血所致无功能心肌的有效性及安全性。 方法：38例拟行择期经皮冠状动脉介入治疗的急性心肌梗死患者随机分为对照A组与试验A组,36例扩张型心肌病患者随机分为对照B组与试验B组。对照组与试验组在介入治疗后分别通过大腔导管于相应冠状动脉内注入等量生理盐水与骨髓间充质干细胞。 结果与结论：移植后1个月试验组左室射血分数较移植前和对照组显著升高(P < 0.05);移植后3个月试验组左室射血分数较移植前和对照组明显升高、心肌灌注缺损面积百分比较移植前和对照组明显降低(P < 0.05),而试验组间仅左室射血分数差异有显著性意义(P < 0.05);试验A组左室舒张末期内径较移植前、对照A组和试验B组均明显减小(P < 0.05),而试验B组仅较移植前明显降低(P < 0.05)。随访期间恶性心血管事件发生率在试验组和对照组间无显著性差异(P > 0.05)。提示经冠状动脉途径行骨髓间充质干细胞移植对无功能心肌的修复是安全有效的,且对急性心肌梗死的疗效优于扩张型心肌病。     

骨髓间充质干细胞经大鼠冠状动脉内移植可行性研究

目的 研究骨髓间充质干细胞经冠状动脉内移植的安全性及可行性。方法 左侧开胸结扎SD大鼠左冠状动脉，2周后取心脏建立Langerndorff模型。将CM-DiI标记的骨髓间充质干细胞悬液注入主动脉根部，同时收集右心房回流的液体，离心后通过流式细胞仪计数细胞数量。观察是否有细胞经冠状静脉回流及数量比例。并在不同时间测LVSP、LVDP、±dp/dt、心率，评价其安全性。在体实验中取心梗后2周的大鼠，经左心室--主动脉途径将细胞悬液注射到临时阻断的主动脉根部，分别在移植后1及24h和1及4周取材观察细胞在心脏内的位置及细胞的迁移情况。结果 离体实验发现经冠状动脉内注射髓间充质干细胞90%以上的细胞在心肌组织内存留，移植后对LVSP、LVDP、±dp/dt、心率没有明显变化。在体内实验中发现细胞移植后早期大部分细胞分布在心外膜下心肌组织内，在心内膜下组织内较少细胞分布，而且大部分细胞在正常心肌组织内，只有少量在心梗区域。而在细胞移植后1~ 4周存活的细胞多在心肌梗死及交界区组织内，在正常组织内很少有移植细胞存在。结论 经冠状动脉内途径进行细胞移植是安全可行的。     

骨髓间充质干细胞静脉移植治疗大鼠脊髓损伤

背景：干细胞移植可以重建中枢神经系统的结构和功能,近年来引起了广泛的关注。 目的：尾静脉移植骨髓间充质干细胞观察其对大鼠脊髓损伤修复的影响并探讨其机制。 方法：密度梯度离心法结合贴壁筛选法分离骨髓间充质干细胞。将成年雌性Wistar大鼠以动脉瘤夹钳夹法制备大鼠脊髓损伤模型后随机分为细胞移植组和对照组,分别干预。 结果与结论：细胞移植组大鼠BBB后肢功能评分恢复优于对照组(P < 0.05)。自损伤后第30天开始,细胞移植组大鼠运动诱发电位潜伏期及体感诱发电位P1波潜伏期的恢复均优于对照组(P < 0.05),并持续至实验结束。细胞移植组大鼠脊髓内在损伤中心及头、尾端距离脊髓损伤中心1 cm处均可见阳性细胞。说明尾静脉注射移植骨髓间充质干细胞可以促进脊髓损伤大鼠神经功能恢复,其机制可能与移植细胞迁移到损伤部位,分化为神经元样和神经胶质细胞样细胞,并分泌或促进宿主分泌神经营养因子有关。     

大鼠骨髓间充质干细胞移植治疗心肌梗死的实验研究

目的　研究骨髓间充质干细胞(BMSCs)移植对心肌梗死大鼠左心功能的影响以及在心肌病大鼠体内存活、分化的情况。 方法　结扎左冠状动脉前降支制备大鼠心肌梗死模型,采用同种异体大鼠BMSCs 体外分离、纯化、扩增,胶体金标记。按照对BMSCs的处理方式和心肌内注射的成分不同随机分为3组:A组接受BMSCs移植治疗;B组移植经5-氮胞苷体外诱导的BMSCs;C组列为对照组,注射等量培养基;每组12只。4周后通过血流动力学各项指标评价BMSCs移植对大鼠心功能的改善情况;免疫组织化学法检测移植细胞的存活和分化。 结果 与对照组C组相比,移植组A, B两组左心功能显著改善（P<0.01）。A, B两组之间无显著性差异。移植组左心室收缩压（LVSP）、左心室等容期压力最大变化速率（±dp/dtmax）明显高于对照组（P<0.01）,左心室舒张末期压（LVEDP）明显低于对照组（P<0.01）。移植的BMSCs能在心外膜下、梗死区及与正常心肌的交界处存活并向心肌细胞分化, 心肌特异性肌钙蛋白T（cTnT）和α－横纹肌肌动蛋白（α-sarcomeric actin）表达阳性。 结论 同种异体BMSCs移植入大鼠心肌梗死区后能明显改善心功能并向心肌细胞转化。     

骨髓间充质干细胞经大鼠冠状动脉内移植的可行性

目的 研究骨髓间充质干细胞经冠状动脉内移植的安全性及可行性.方法 左侧开胸结扎SD大鼠左冠状动脉,2周后取心脏建立Langerndorff模型.将CM-DiI标记的骨髓间充质干细胞悬液注入主动脉根部,同时收集右心房回流的液体,离心后通过流式细胞仪计数细胞数量.观察是否有细胞经冠状静脉回流及数量比例.并在不同时间测LVSP、LVDP、±dp/dt、心率,评价其安全性.在体实验中取心梗后2周的大鼠,经左心室-主动脉途径将细胞悬液注射到临时阻断的主动脉根部,分别在移植后1及24 h和1及4周取材观察细胞在心脏内的位置及细胞的迁移情况.结果 离体实验发现经冠状动脉内注射骨髓间充质干细胞90%以上的细胞在心肌组织内存留,移植后LVSP、LVDP、±dp/dt、心率没有明显变化.在体内实验中发现细胞移植后早期大部分细胞分布在心外膜下心肌组织内,在心内膜下组织内较少细胞分布,而且大部分细胞在正常心肌组织内,只有少量在心梗区域.而在细胞移植后1～4周存活的细胞多在心肌梗死及交界区组织内,在正常组织内很少有移植细胞存在.结论 经冠状动脉途径进行细胞移植是安全可行的.     

骨髓间充质干细胞分化为血管内皮细胞的实验研究

目的 :探讨骨髓间充质干细胞 (MSCs)自体移植后在扩张型心肌病(DCM)微环境中分化为血管内皮细胞的可行性。方法 :以日本大耳白兔为研究对象 ,分离培养扩增MSCs,盐酸阿霉素耳缘静脉注射复制兔DCM模型 ,将 5溴脱氧尿嘧啶 (BrdU)标记的MSCs移植到DCM心肌内 ,4周后观察移植细胞的增殖分化情况。结果 :细胞移植 4周后 ,实验组心肌内有BrdU标记的阳性细胞 ,有一部分参与组成新生血管 ,对照组中没有发现 ,且实验组毛细血管密度大于对照组 ,差异具有显著性 (P<0 .0 1)。结论 :MSCs自体移植到扩张型心肌病后可以分化为血管内皮细胞。     

人骨髓间充质干细胞的生物学特性

目的 建立人胚骨髓间充质干细胞(MSCs)分离及培养的方法，探讨体外培养MSCs的生物学特性．方法 通过密度梯度离心、贴壁法分离培养人胚MSCs，在倒置显微镜下连续观察细胞的形态变化．应用流式细胞术测定细胞周期和CD44、CD34、CD45表达，并研究其增殖及生长特征．结果 原代及传代培养显示，14代以前的MSCs具有活跃的增殖能力，细胞周期分析显示有82％的MSCs处于Gp／Gl期．MSCs阳性表达CD44，阴性表达CD34、CD45．结论 体外培养14代以前的的MSCs生长稳定，增殖较快，可作为组织工程的种子细胞．     

人间充质干细胞表面标志物及特征

评价人间充质干细胞表面标志,对常见的表面标志物进行总结,认识HMSCs表面标志物生物学特性,为分离和鉴定HMSCs提供参考依据.检索2010至2016年PubMed数据库和中国知网数据库收录的与HMSCs表面标志物、生物学特性相关的文献.从细胞表面标记物的本质、实验结果等进行分析总结,总结了一些常见的HMSCs表面标志物.不同组织来源的HMSCs免疫活性可能与HMSCs处于不同组织中的活化状态、组织来源和培养条件的不同有关,从而导致不同源性HMSCs免疫活性也不完全相同,其表面标志物存在差异.利用杂交瘤技术制备的抗体,可将不同来源、不同阶段、不同培养条件的HMSCs分为不同的亚群.不同来源或不同分化阶段的HMSCs表面标记物存在差异.     

Mesenchymal Stem Cells versus Mesenchymal Stem Cells Combined with Cord Blood for Engraftment Failure after Autologous Hematopoietic Stem Cell Transplantation: A Pilot Prospective,Open-Label,Randomized Trial

Engraftment failure (EF) after autologous hematopoietic stem cell transplantation is a serious complication. We prospectively evaluated the effects and safeties of mesenchymal stem cells (MSCs) alone and MSCs combined with cord blood (CB) for EF. Twenty-two patients were randomized to receive MSCs (MSC group; n = 11) or MSCs plus CB (CB group; n = 11). Patients with no response (NR) to MSCs received the therapeutic schedule in the CB group, and those patients with partial response (PR) in the MSC group and patients without complete remission (CR) in the CB group received another cycle of MSC treatment. Patients who did not achieve CR after 2 cycles of treatments received other treatments, including allogeneic HSCT. After the first treatment cycle, response was seen in 7 of 11 patients in the MSC group and in 9 of 11 in the CB group (P = .635), with a significant difference in neutrophil reconstruction between the 2 groups (P = .030). After 2 treatment cycles, 16 patients achieved CR, 3 achieved PR, and 3 had NR. No patient experienced graft-versus-host disease (GVHD). With a median follow-up of 345 d (range, 129 to 784 d) post-transplantation, 18 patients remained alive and 4 had died (3 from primary disease relapse and 1 from cytomegalovirus pneumonia). The 2-year overall survival, disease-free survival, and cumulative incidence of tumor relapse post-transplantation were 75.2% ± 12.0%, 79.5% ± 9.4%, and 20.5% ± 9.4%, respectively. Our data indicate that the 2 strategies are effective for EF and do not result in GVHD or increase the risk of tumor relapse, but the MSC plus CB regimen has a superior effect on neutrophil reconstruction.     

Dysfunctional Bone Marrow Mesenchymal Stem Cells in Patients with Poor Graft Function after Allogeneic Hematopoietic Stem Cell Transplantation

Poor graft function (PGF) is a life-threatening complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT) and is characterized by defective hematopoiesis. Mesenchymal stem cells (MSCs) have been shown to support hematopoiesis, but little is known about the role of MSCs in the pathogenesis of PGF. In the current prospective case-control study, we evaluated whether the number and function of bone marrow (BM) MSCs in PGF patients differed from those in good graft function (GGF) patients. We found that BM MSCs from PGF patients expanded more slowly and appeared flattened and larger, exhibiting more apoptosis and senescence than MSCs from GGF patients. Furthermore, increased intracellular reactive oxygen species, p-p53, and p21 (but not p38) levels were detected in MSCs from PGF patients. Moreover, the ability of MSCs to sustain hematopoiesis was significantly reduced in PGF patients, as evaluated by cell number, apoptosis, and the colony-forming unit–plating efficiency of CD34⁺ cells. In summary, the biologic characteristics of PGF MSCs are different from those of GGF MSCs, and the in vitro hematopoiesis-supporting ability of PGF MSCs is significantly lower. Although requiring further validation, our study indicates that reduced and dysfunctional BM MSCs may contribute to deficient hematopoiesis in PGF patients. Therefore, improvement of BM MSCs may represent a promising therapeutic approach for PGF patients after allo-HSCT.     

Transplantation of Mesenchymal Stem Cells for Acute Spinal Cord Injury in Rats: Comparative Study between Intralesional Injection and Scaffold Based Transplantation

Experimental stem cell therapy for spinal cord injury (SCI) has been extensively investigated. The selection of effective cell transplantation route is also an important issue. Although various types of scaffold have been widely tried as a carrier of stem cells to the injured spinal cord, there was little comparative study to investigate the efficacy of transplantation comparing with conventional transplantation route. A total of 48 Sprague-Dawley rats were subjected to standardized SCI, followed by transplantation of allogeneic mesenchymal stem cells (MSCs), either via intralesional injection (IL group), or via the poly (lactic-co-glycolic acid) (PLGA) scaffold (IP group) or chitosan scaffold (IC group). Engraftment and differentiation of the transplanted cells, expression of neurotrophic factors in the injured spinal cord, and functional recovery were compared with those of the control group. The mean numbers of engrafted MSCs in the IL, IP, and IC groups were 20.6 ± 0.7, 25.6 ± 1.7 and 26.7 ± 1.8 cells/high power filed (HPF), respectively. Results showed higher success rate of MSCs engraftment in the scaffold groups compared to the IL group. Expression of neuroprotective growth factors in the SCI lesions showed no significant differences between the IL, IP, and IC groups. The mean Basso, Beattie and Bresnahan locomotor scales at 6 weeks post-transplantation in the IL, IP, IC, and control groups were 7.9 ± 1.1, 7.9 ± 2.1, 8.7 ± 2.1, and 2.9 ± 1.0, respectively. The functional improvement was most excellent in the IC group. The scaffold based MSC transplantation for acute SCI presented the better cell engraftment and neuroprotective effect compared to the intralesional injection transplantation.     

黄芪多糖注射液对鼠骨髓间充质干细胞增殖的影响

目的探讨黄芪多糖注射液对鼠骨髓间充质干细胞(MSC)增殖的影响。方法体外培养MSC,用活细胞计数法(CCK-8)检测黄芪多糖注射液促进大鼠MSC的增殖作用;在给大鼠体内肌注黄芪多糖注射液的第3、4天腹腔注射5-溴尿嘧啶核苷(Brdu),免疫组化检测MSC细胞的Brdu标记阳性率。结果三种高浓度的药液对MSC的增殖与生理盐水对照组比较有显著性差异;停药后3～5d,黄芪多糖组Brdu标记阳性细胞率与生理盐水组比较有显著差异。结论黄芪多糖注射液具有促进MSC增殖的作用。     

胎盘来源间充质干细胞研究进展

成人骨髓是临床应用时最常见的间充质干细胞(MSC)的来源。由于MSC在成人骨髓的数量以及分化能力随年龄的增长而减低,不同的胎儿组织被用来研究是否存在MSC。胎盘组织中MSC主要分布在羊膜和绒毛膜等来源于胎儿的组织,其抗原表达、分化潜能与骨髓间充质干细胞特征相似。     

慢性心功能不全时经主动脉移植干细胞的可行性研究

心肌梗死或/和慢性心功能不全时,心脏干细胞移植是一项颇有应用前景的新的治疗技术。但是,对于非缺血性心脏病所致的慢性心功能不全,目前所采用的移植路径有一定的局限性。本试验拟研究慢性心功能不全时,封堵主动脉窦上部经主动脉根部移植干细胞是否可行。阿霉素所致的慢性心功能不全兔模型,采用双球囊封堵主动脉窦上部经主动脉根部自体移植骨髓单核细胞,4周后评价心功能。在封堵主动脉窦上部经主动脉根部移植干细胞时,无1例因移植手术死亡。双球囊充盈时造影显示,主动脉窦显影良好,大部分情况下,左、右冠状动脉可显影。骨髓单核细胞移植4周后,左室射血分数改善明显(P=0 .0 34)。所以,慢性心功能不全时,封堵主动脉窦上部经主动脉根部移植干细胞安全可行。