骨髓干细胞对急性梗死心肌修复的实验研究

目的:研究自体骨髓干细胞移植和干细胞动员剂（GM CSF）对急性心肌梗死（AMI）坏死区的修复作用。方法:以同基因背景的雄性日本大耳白兔为研究对象,随机分为3组（对照组,细胞移植组,动员剂组）,均采用结扎冠状动脉的方法制造AMI动物模型。移植组模型建立1周后再次开胸,于梗死区内注射经BrdU标记的自体骨髓干细胞。注射后4周观察移植细胞分化情况和促血管生成作用,并用超声检测心脏功能改变。动员剂组于AMI模型建立后即刻注射特尔立（巨噬-粒细胞集落刺激因子GM CSF）,与细胞移植组在同一试验终点进行上述项目的观察。结果:细胞移植4周后,可以在坏死区内找到增殖的BrdU标记的移植细胞,另外VWF因子染色可见移植组和动员剂组坏死区内有大量的血管新生（24±5/HPE、20±3/HPE）,而对照组中却发现较少（11±1/HP）,两组均与对照组有显著性差异（P<0.05）,超声检查显示移植组心脏功能显著改善,其中射血分数由0.36±0.08增加到0.62±0.07,移植前后有显著性差异（P<0.05）,移植后与对照组比较也有显著性差异（P<0.05）,动员剂组治疗前后心脏功能无明显改善。结论:细胞移植和动员剂注射治疗,均能促进大耳雄兔急性心肌梗死缺血区及其周边区域的毛细血管新生明显,而细胞移植对急性心肌梗死后逆转心室重构、改善心脏功能方面有明显作用     

粒细胞集落刺激因子动员自体骨髓干细胞移植对心梗大鼠慢性心力衰竭的治疗作用

目的探讨粒细胞集落刺激因子动员自体骨髓干细胞移植对心梗大鼠慢性心力衰竭的治疗作用。方法建立Wistar大鼠心肌梗死后心衰模型,随机分为移植组、动员组和对照组各10只。移植组于梗死区内注射经标记后的骨髓间充质干细胞,动员组皮下注射粒细胞集落刺激因子,对照组未行任何处理。4周后采用超声和生理记录仪描记左室收缩压（LVSP）、左室舒张压（LVEDP）、左室压力最大上升或下降速率（±LVdp／dtmax）等指标。取心脏组织做病理检查,观察移植细胞分化、促血管生成、细胞凋亡情况。结果细胞移植4周后,移植组及动员组心脏功能较对照组显著改善（P均〈0．05）,梗死区血管密度明显高于对照组,心肌细胞凋亡指数低于对照组（P均〈0．05）。结论骨髓干细胞动员可能为慢性心力衰竭的治疗提供一种新的无创性手段。     

骨髓动员与细胞移植对直接经皮腔内冠状动脉成形术后急性心肌梗死治疗作用

目的比较骨髓间充质干细胞移植和骨髓动员对急性心肌梗死再通模型的心脏修复作用.方法人重组粒细胞集落刺激因子静注1d,皮下注射连续5d行骨髓动员,骨髓间充质干细胞(4～8)×106经梗死相关冠状动脉注入行细胞移植.3周后观察超声心动图指标;免疫组织化学染色行不同区域血管计数,并观察增殖期内皮细胞和心肌细胞或再生心肌细胞.结果骨髓动员组和细胞移植组心功能明显改善;二组均可见心肌细胞再生;细胞移植组和骨髓动员组均可见大量新生血管,但二者分布区域不同.结论骨髓动员及骨髓间充质干细胞移植均可明显改善心肌梗死后心功能,二者可促进心肌细胞再生且对不同区域的血管再生作用不同.     

兔骨髓间充质干细胞自体移植及血管新生修复梗死心肌的实验研究

目的　了解骨髓间充质干细胞 (MSCs)在心肌梗死的缺血心肌微环境中转化为心肌细胞并改善心功能的可行性。方法　结扎兔左前降支 (LAD)制作急性心肌梗死(AMI)模型 ,骨穿抽取骨髓 ,分离培养扩增骨髓间充质干细胞。术后 2周进行心肌梗死瘢痕内移植 5溴脱氧尿嘧啶(BrdU)标记的骨髓间充质干细胞 ,对照组注射培养基。AMI术前、AMI术后 3d及移植后 4周做超声心动图检测心功能 ,移植后 4周从右颈总动脉插管至左心室测左室压力变化 ,随后处死动物模型 ,取左室标本做冰冻切片 ,采用免疫荧光方法检测BrdU鉴定植入细胞并检测Ⅷ因子相关抗原以测量毛细血管密度。结果　细胞移植组左室切片中可见BrdU染色阳性细胞即植入细胞 ,对照组未见BrdU染色阳性细胞。Ⅷ因子阳性内皮细胞计数表明细胞移植组毛细血管密度大于对照组 (P <0 0 5 )。超声心动图检查证实MSCs移植后 4周左室收缩末期直径 (LVESD)、舒张末期直径 (LVEDD)均小于对照组 (分别为P <0 0 1及P <0 0 5 ) ,小轴缩短率 (ΔD % )、射血分数 (EF)均大于对照组 (均P <0 0 1)。结论　兔缺血心肌内注射骨髓间充质干细胞可依赖组织微环境转化为心肌细胞修复梗死心肌 ,可能伴有移植区域新生血管形成 ,能显著改善心功能 ,可用于心肌梗死的治疗。     

骨髓干细胞动员对高龄大鼠缺血心脏功能的保护作用

目的:了解从老年大鼠中动员出的骨髓干细胞归巢于梗死心肌和分化的能力,以及对缺血心脏功能的影响,并探讨其可能机制。方法:W istar大鼠分为3组:高龄心肌梗死+动员组、高龄心肌梗死组和青年心肌梗死+动员组。结扎大鼠左冠状动脉制作急性心肌梗死（AM I）模型,动员组大鼠用骨髓干细胞动员剂干细胞因子动员自体骨髓干细胞释放并归巢于心肌梗死灶,于建模后24 h、48 h和4周杀死各组大鼠,取出心脏,通过免疫组化、HE染色方法观察老年大鼠心肌梗死灶、边缘区和正常心肌组织CD34阳性细胞浸润及心肌再生的情况,应用流式细胞仪比较动员前后外周血中CD34阳性细胞数量的变化,并应用BL-420E生物机能实验系统检测老年大鼠心功能指标。结果:使用干细胞因子后,高龄动员组及青年动员组大鼠外周血中CD34细胞数量显著升高,两组大鼠心肌梗死灶均可见大量CD34细胞浸润;高龄动员组大鼠在制模4周时心功能指标显著比高龄心肌梗死组改善,而高龄动员组和青年动员组的心功能指标无显著差异,均显著改善。制模后24 h,高龄动员组大鼠和青年动员组大鼠心肌梗死程度轻,可见CD34阳性的幼稚心肌细胞样细胞,4周后瘢痕组织少,缺血心肌的基本结构得到保护。结论:在骨髓干细胞动员剂作用下,从老年大鼠个体中动员出的骨髓干细胞归巢梗死心肌能力仍得到增强,有较多干细胞向梗死灶迁移,并向心肌细胞等分化,保护缺血心肌基本结构,改善心功能。     

骨髓单核细胞移植促进大鼠急性心肌梗死后局部血管新生

目的探讨大鼠急性心肌梗死(AMI)后骨髓单核细胞(BMMNCs)移植对心功能和梗死局部血管新生的影响及其可能的机制。方法利用密度梯度离心法获取BMMNCs,并用4′,6-二脒-2-苯基吲哚(4′,6-diamidino-2-phenylindole,DAPI)标记,结扎大鼠左冠脉前降支血管,建立AMI动物模型,分别于心肌梗死后1h及1w于梗死中央及周边局部注射BMMNCs(2×107个/ml,0.2ml),对照组AMI后1h局部注射等量生理盐水,AMI后8w心脏超声检测3组大鼠心脏功能,然后取材,进行荧光检查及组织学检查。结果心梗1w移植组左室收缩功能明显高于其他两组,荧光检查结果显示所有切片中均未发现DAPI标记的细胞;HE染色血管计数结果显示心梗1w移植组梗死局部毛细血管密度明显高于其他两组。结论大鼠AMI1w后BMMNCs移植可以增加梗死局部血管新生,改善心梗后心功能,而心功能改善及血管新生与BMMNCs的横向转化无关。     

骨髓间充质干细胞移植治疗心力衰竭

目的 探讨骨髓间充质干细胞移植对阿霉素诱导的心力衰竭大鼠心功能的影响.方法 无菌条件下取8周龄F344大鼠的股骨和胫骨,获得骨髓间充质干细胞(MSCs),在体外纯化、扩增后 ,用5-溴-2'脱氧尿苷(BrdU)进行标记,然后注射到心力衰竭模型细胞移植组和对照组大鼠的心肌组织内,细胞移植后4周,采用生理记录仪测量3组大鼠的心功能 ,处死动物,心脏切片行免疫组化了解移植细胞在受体心脏的存活情况.结果 细胞移植后4周,细胞移植组大鼠死亡率为6.2%,明显低于假细胞移植组12.5%(P<0.01);在受体大鼠的心脏切片上有BrdU标记的移植细胞存活.心功能测定显示:与对照组相比,细胞移植组最大左室收缩末压(LVSP)、心率(HR)、左室内压最大(最小)变化速率(LV±dp/dtmax)均明显下降(P<0.01),而左室舒张末压(LVDP)明显升高(P<0.01);与对照组相比,细胞移植组大鼠的LVSP、HR、LV±dp/dtmax均有明显升高(P<0.01),而LVDP明显降低(P<0.01);与假移植组相比,细胞移植组大鼠的HR、 LV±dp/dtmax仍有明显下降(P<0.01),而LVDP明显升高(P<0.01).结论 MSCs移植可有效改善阿霉素诱导的扩张型心力衰竭大鼠的心功能,减少心力衰竭大鼠的死亡率.     

移植冠心病患者骨髓间充质干细胞治疗心肌梗死大鼠的机制探讨

目的:在大鼠心肌梗死模型上移植冠心病患者来源的骨髓间充质干细胞,探讨人骨髓间充质干细胞改善心肌梗死后心功能的作用机制。方法:抽取冠心病患者骨髓,用人骨髓间质细胞专用培养液进行体外培养、扩增、标记。结扎大鼠冠状动脉左前降支制作心肌梗死模型,2周后使用超声筛选出合格的动物模型。然后动物随机分为细胞移植组(n=14)和培养液注射组(对照组,n=13)。在移植前后对心脏的收缩、舒张功能和心室重构指标进行超声评价和对比,取材后进行病理学检查和分子生物学检测。结果:移植后进行超声评价,治疗组较对照组射血分数值和缩短分数值显著改善。免疫组化显示移植细胞在宿主体内可以向肌源性细胞分化。逆转录—聚合酶链反应(RT-PCR)显示细胞治疗组梗死区心肌Ⅰ型、Ⅲ型胶原基因表达水平明显高于对照组,而在非梗死区低于对照组。细胞治疗组梗死区间质细胞源因子、血管内皮生长因子、Bcl-2基因表达水平均明显高于对照组。结论:冠心病患者骨髓间质细胞移植后可以提高心肌梗死后心脏收缩功能,减轻左心室重塑。作用机制可能为新血管生成增加、细胞外基质改变、旁分泌、抗凋亡和心肌再生等共同作用的结果。     

心肌联合注射纤维蛋白封闭剂在细胞移植中应用的研究

目的:探讨心肌注射骨髓间充质干细胞(mesenchymal stemcells,MSCs)联合应用纤维蛋白封闭剂(FibrinSealant,FS),对细胞滞留量的影响。方法:(1)提取、分离、培养、扩增小型猪骨髓MSCs备用;(2)小型猪随机分为2组,成功建立急性心肌梗塞模型,然后分为单纯MSCs移植组和MSCs联合纤维蛋白封闭剂移植组,对比移植6周后细胞的滞留量及新生血管的密度,超声学评价心脏功能。结果:6周后MSCs FS组荧光标记的MSCs数量、新生血管密度均较MSCs组显著增多(P<0.01),MSCs FS组左室短轴缩短率(FS%)、心肌射血分数(EF%)较MSCs组显著提高(P<0.05,<0.01)。结论:经心外膜心肌注射MSCs联合纤维蛋白封闭剂较单纯心肌注射MSCs能够明显提高移植细胞的滞留存活数量,增加新生血管密度,改善心肌收缩功能。     

自体骨髓干细胞动员治疗大鼠实验性心肌梗死

目的研究应用粒细胞集落刺激因子(GCSF)动员自体骨髓干细胞对大鼠实验性心肌梗死的治疗作用。方法应用冠状动脉结扎的大鼠心肌梗死模型,给予GCSF动员自体骨髓干细胞,于大鼠心肌梗死后1周测外周血CD34 细胞含量,并用免疫组化染色观察心肌梗死区的CD34 细胞浸润情况。于梗死后4周用HE和免疫组化染色及血流动力学监测评价实验大鼠的心肌梗死面积,血管密度和心功能。结果心肌梗死后1周,GCSF组大鼠外周血CD34 细胞含量明显高于对照组,心肌梗死交界处见CD34 细胞浸润。4周后,与对照组相比,GCSF组心肌梗死范围减小,新生血管增多,心功能改善。结论应用GCSF动员自体骨髓干细胞对大鼠实验性心肌梗死有明确的治疗作用。     

Phenotypic and functional analysis of bone marrow progenitor cell compartment in bone marrow failure

SUMMARY. Many laboratory findings have demonstrated that the haemopoietic stem cell compartment is defective in aplastic anaemia (AA). AA bone marrow (BM) and peripheral blood (PB) are profoundly deficient in colonyforming cells, and AA progenitors fail to proliferate in longterm assays even in the presence of an intact stroma. Our study was designed to characterize some quantitative and qualitative aspects of the progenitor cell defect in AA. Using flow cytometric analysis of BM from new AA patients and from those recovering after immunosuppressive therapy, we determined that the numbers of CD34⁺ and CD33⁺ cells were markedly decreased in AA. Although PB neutrophil counts did not correlate with BM CD34⁺ cell numbers in acute disease, there was an association between the overall severity of the disease and the degree of CD34⁺ cell reduction. A decrease in BM CD33⁺ cells was a common finding in MDS patients, but reduction in CD34⁺ cells was found only in some hypoplastic MDS cases. Sorting experiments demonstrated lower plating efficiency for purged CD34⁺ cells from AA BM in comparison to controls. Thus, diminished colony formation of total BM appeared to result from both quantitative and qualitative defects. Based on the association between increased cycling and c-kit receptor expression on CD34⁺ cells, we found that the mitotically active CD34⁺ cells bearing the c-kit antigen were reduced in AA. With clinical improvement, CD34⁺ and CD33⁺ cells increased in correlation with PB parameters, but they did not return to normal values. Sorted CD34⁺ cells from recovered patents showed improved plating efficiency. In patients with aplastic anaemia, use of CD34 antigen as a phenotypic marker of progenitor cells may be helpful for the analysis of the early haemopoietic cell compartment and BM recovery.     

骨髓间充质干细胞的免疫学特性

目的 研究骨髓间充质干细胞(MSC)的免疫学特性,为临床应用提供实验依据.方法 培养鉴定MSC,检测其生长动力学、细胞周期.检测单纯和200 U/ml干扰素(IFN)γ干预后MSC的程序性死亡配体(PDL)-1、CD54、CD40、CD80、CD86、主要组织相容性复合物(MHC)-Ⅰ、MHC-Ⅱ的表达情况,并以此为调节细胞进行混合淋巴细胞反应.封闭PDL-1、CD54,观察它们在MSC免疫调节中的作用.检测培养上清液中IFN γ、白细胞介素(IL)-2,IL-4、IL-10的水平.将MSC移植于体内观察肝脏归巢和诱导微嵌合体情况.结果 分离、培养的MSC纯度较高;生长曲线显示第1、2天为潜伏期,第3、4、5天为对数生长期,第6、7天为平台期.细胞周期显示G0/G1期为76.0%±2.0%,S期为13.0%±2.0%,(G2+M)期为10.0%±1.7%.IFN γ上调CD54、PDL-1、MHC-Ⅰ、MHC-Ⅱ的表达,CD40、CD80、CD86为阴性表达.MSC呈剂量依赖性地抑制T淋巴细胞增殖,IFN γ增强其抑制作用.封闭PDL-1、CD54后能减弱MSC对T淋巴细胞增殖的抑制作用.培养上清液中IFN γ、IL-10水平较高,IL-4较低,IL-2未检测到.MSC在肝实质内有多处定居,并能诱导肝移植术后微嵌合体形成.结论 IFN γ能增强MSC对淋巴细胞增殖的抑制作用,PDL-1和CD54可能发挥关键作用;MSC移植后能在体内存活并诱导微嵌合体形成.     

T cell depletion in bone marrow transplantation

Prior to the introduction of T cell depletion graft-versus-host disease (GVHD) was the major cause of transplant-related mortality. Why has the remarkable technical achievement of efficient T cell depletion, which has virtually eliminated severe GVHD over the last few years, failed to reduce mortality and increase disease-free survival? This review examines the value of T cell depletion based on recent clinical experience. It surveys the directions that are being pursued in an effort to solve the problems that have arisen and indicates how this is leading to a greater understanding of the mechanisms involved in GVHD and the graft-versus-leukaemia effect.     

Correlation between characteristics of unrelated bone marrow donor and cell density of total nucleated cell in bone marrow harvest

The relationship between the features of bone marrow donor and the quality of marrow harvest has been unclear because most of bone marrow registries have multiple collection centers with somewhat different harvest procedures. We are able to address this issue for Tzu Chi General Hospital is the only collection center affiliated with Tzu Chi Taiwan Bone Marrow Registry. Between November 1997 and March 2002, data of 286 healthy unrelated donors was analyzed to correlate with the cell density of total nucleated cell in bone marrow harvests. The harvest procedure was standardized by single-hole harvest needle under general anesthesia. The operation staffs were restricted within the members of Oncology–Hematology division. The results showed that the cell density of bone marrow harvest was positively correlated with donor body weight and peripheral white blood cell count P = 0.0475, P < 0.0001, but negatively correlated with the total volume of bone marrow harvest P < 0.0001. We recommend that if multiple human leukocyte antigen-matched donors are available, donor with higher body weight and/or higher white blood cell count be selected for allogeneic bone marrow transplantation.     

Leukemia-stimulated bone marrow endothelium promotes leukemia cell survival

Extensive endothelial cell proliferation and marked neovascularization are the most pronounced microenvironmental changes consistently observed in the bone marrow (BM) of patients with acute lymphoblastic leukemia (ALL). It is not known whether ALL cells induce this phenotype and whether they receive critical signals from the tumor-associated BM endothelium. Here, we show that leukemia cells actively stimulate BM endothelium, promote de novo angiogenesis, and induce neovascularization in the leukemic BM. Soluble factors, present in the leukemic BM microenvironment, promote the proliferation, migration, and morphogenesis of BM endothelial cells, which are critical processes in tumor angiogenesis. We also show in vitro that leukemia cells display directional motion towards assembled BM endothelium and following adherence exhibit cell polarization, pseudopodia, and ultrastructural features that suggest the existence of leukemia-endothelium cross-talk. Finally, we show that BM endothelium promotes leukemia cell survival through a mechanism mediated through the anti-apoptotic molecule bcl-2. These studies indicate that ALL cells actively recruit BM endothelium and mediate the leukemia-associated neovascularization observed in ALL. Therefore, disruption of interactions between leukemia cells and BM endothelium may constitute a valid therapeutic strategy.     

Stem cell self-renewal considerations in bone marrow transplantation

Autologous bone marrow transplantation is being used for an increasing number of patients with malignant diseases including Hodgkin's disease, non-Hodgkin's lymphomas, and leukemia. As the success of this procedure improves, there will be continuing concern for the consequences of such treatment. One such concern is the long-term hematopoietic function of recipients following marrow transplantation. There is evidence that bone marrow stem cells are limited in self-renewal capacity. Under circumstances of exposure to certain cytotoxic agents or to great proliferative stress, and following transplantation of marrow into lethally irradiated recipients, bone marrow stem cells undergo a permanent loss of self-renewal capacity. This loss is not initially reflected in peripheral blood counts or in marrow cellularity, but can be determined by a decrease in marrow stem cell content and by assays measuring self renewal. Animal work suggests that survival may be decreased following this loss in self-renewal. In order to limit the adverse effect of this phenomenon, efforts should be made to optimize both the quantity and quality of donor marrow engrafted. This should be possible by transplanting the largest number of marrow cells feasible, and by avoiding prior exposure to cytotoxic agents that are known to damage early stem cells in those patients who are possible candidates for autologous marrow transplantation. The use of lymphokines and peripheral stem cell harvests in transplantation should be carefully monitored as self renewal of engrafted marrow may also be decreased following these new techniques.