骨髓间充质干细胞与组织损伤修复的研究进展★

学术背景：骨髓间充质干细胞是骨髓中存在的除造血干细胞外的另一类干细胞, 广泛存在于胎儿和成人的各种组织和脏器中, 其中骨髓中的含量最多, 具有多向分化潜能, 可迅速扩增, 易于获取, 能长期存活等特点。 目的: 分析骨髓间充质干细胞在组织损伤修复中作用机制, 归纳较理想的修复条件, 为组织修复与恢复功能的研究提供科学依据。 检索策略: 应用计算机检索Pubmed1995-01/2007-10期间的相关文章，检索词为“tissue repairing, mesenchymal stem cells, immune, immigration,differentiation”，并限定文章语言种类为English。同时计算机检索万方数据库2001-01/2007-10期间的相关文章，检索词为“间充质干细胞，组织修复,免疫，迁移，分化”，并限定文章语言种类为中文。对资料进行初审，并查看每篇文献后的引文。纳入标准：文章所述内容应与间充质干细胞分化,组织修复等研究进展中的应用相关。排除标准：重复研究或Meta分析类文章。共收集到76篇相关文献，30篇文献符合纳入标准，排除的46篇为内容陈旧或重复文献。 文献评价: 符合纳入标准的30篇文献中，14篇涉及骨髓间充质干细胞的迁移归巢，6篇涉及骨髓间充质干细胞的分化及对受损组织修复可能机制，9篇汲及骨髓间充质干细胞与宿主间的免疫反应,1篇涉及存在的问题与展望。 资料综合: 骨髓间充质干细胞具有高可塑性、可移植性, 可用作心肌,神经元等细胞修复的治疗方法。当组织损伤发生后,骨髓会迅速动员多种干细胞迁移至病变处,产生自然的代偿性修复;通过各种途径引入体内的外源性骨髓干细胞,也会定向归巢至损伤处,发挥治疗作用, 有助于组织损伤的修复,但骨髓间充质干细胞参与受损组织的修复过程是复杂的,其作用机制主要表现在归巢至受损部位,局部微环境下的正确定向分化和抑制宿主的免疫等方面。 结论: 随着研究的进一步深入, 骨髓间充质干细胞参与受损组织修复作用机制将会逐步被明确,有望更好的用于指导临床治疗工作。     

骨髓间充质干细胞治疗肾缺血再灌注损伤的旁分泌机制*☆

背景：骨髓间充质干细胞治疗肾缺血再灌注损伤的机制除了干细胞分化成肾细胞参与损伤修复外,还存在其他多种机制参与保护受损的肾脏。 目的：探讨骨髓间充质干细胞治疗肾缺血再灌注损伤的旁分泌机制。 方法：体外培养、纯化并体外DAPI标记大鼠骨髓间充质干细胞,并将其移植入肾缺血再灌注损伤模型大鼠体内,观察骨髓间充质干细胞对肾缺血再灌注损伤肾功能的保护作用和在受体鼠体内的迁移情况,并应用免疫组织化学法检测骨髓间充质干细胞治疗后第2天缺血肾脏组织中血管内皮生长因子（VEGF）、肿瘤坏死因子-α(TNF-α)（用中文表示）等细胞因子的表达。 结果与结论：与注射生理盐水的对照组比较,细胞移植组大鼠血清肌酐和尿素氮水平在移植后第1、2天明显降低(P < 0.05),但细胞移植组移植后第1、2天肾组织中均未见DAPI阳性细胞;第3、4天则逐渐可见DAPI阳性细胞。免疫组织化学染色结果显示,与对照组相比,移植后第2天肾组织中可见较多血管内皮生长因子（VEGF）阳性细胞,而肾组织中肿瘤坏死因子-α(TNF-α)阳性细胞明显减少。结果显示旁分泌机制参与了骨髓间充质干细胞治疗肾缺血再灌注损伤。     

骨髓间充质干细胞改善心肌梗死后心功能的机制和诱导因素*

目的：分析骨髓间充质干细胞移植对心肌梗死后心脏功能的影响及其作用机制，探讨骨髓间充质干细胞向心肌样细胞分化过程中的诱导因素。 方法：由本文第一作者检索Pubmed数据库和中国期刊全文数据库有关骨髓间充质干细胞移植治疗心肌梗死的相关文献，共检索到60篇文献，对资料进行初审，纳入标准：①骨髓间充质干细胞移植对心肌梗死后心功能的影响。②骨髓间充质干细胞移植改善心肌梗死后心功能的作用机制。③骨髓间充质干细胞向心肌样细胞分化的诱导因素。排除标准：重复研究和Meta分析类文章。最终纳入23篇文献进行分析讨论。 结果：①骨髓间充质干细胞具有很强的自我更新能力及多项分化潜能，在一定条件下能分化为肌细胞、骨细胞、软骨细胞和脂肪细胞等。②骨髓间充质干细胞可以改变心肌梗死后的心脏功能。其可能的作用机制为能够分化为心肌样细胞，并促进了新血管生成，减少心室重构，即移植后的干细胞在缺血受损伤的心脏微环境中与宿主细胞和细胞外基质之间的相互作用下,分化为有效的心肌组织，同时还可分泌促进血管生成的血管生成素配体和细胞因子,进一步促进新生血管的形成。③体内外环境诱导因素在骨髓间充质干细胞诱导分化为心肌样细胞的过程中起着非常重要的作用，对各种微量因素及微环境中各种细胞因子的调节可以提高干细胞定向分化的效率。 结论：骨髓间充质干细胞在心脏疾病治疗中已表现出独特的优越性，然而其作为心血管疾病治疗的一种全新策略目前还处于初步研究阶段，为了使骨髓间充质干细胞移植更安全有效地应用于临床，仍有很多方面的问题需要进一步探索。     

双重荧光标记验证静脉移植碱性成纤维细胞生长因子基因修饰骨髓间充质干细胞在脑缺血模型大鼠脑内的存活及分化

背景：碱性成纤维细胞生长因子可以促进骨髓间充质干细胞的增殖和向神经细胞方向分化，并被认为是胶质细胞的分裂原。 目的：以双重荧光标记验证静脉移植碱性成纤维细胞生长因子基因修饰的骨髓间充质干细胞在脑缺血模型大鼠脑内的存活及分化情况，及其向神经元样细胞和神经胶质细胞分化的趋势。 设计、时间及地点：随机对照动物实验，于2005-07/2006-03在中南大学实验动物中心实验室完成。 材料：选用50只SD大鼠，按随机数字表法分为4组：假手术组（n=10），脑缺血/再灌注损伤模型组（n=10），骨髓间充质干细胞治疗组（n=15），碱性成纤维细胞生长因子基因修饰的骨髓间充质干细胞治疗组（n=15）。 方法：除假手术组外，其余3组制备局灶性脑缺血再灌注模型。分别将骨髓间充质干细胞或碱性成纤维细胞生长因子基因修饰的骨髓间充质干细胞通过静脉移植至实验性脑缺血大鼠体内，脑缺血再灌注损伤组大鼠注入相同体积的DMEM培养基。 主要观察指标：应用5-溴-2-脱氧尿苷-神经元特异核蛋白及5-溴-2-脱氧尿苷-胶质纤维酸性蛋白双重荧光标记法观察移植细胞在脑内的存活和分化情况，比较各组大鼠脑缺血后的神经功能评分及脑梗死体积变化。 结果：移植7 d后，碱性成纤维细胞生长因子基因修饰的骨髓间充质干细胞组大鼠脑内5-溴-2-脱氧尿苷阳性细胞数、5-溴-2-脱氧尿苷-神经元特异核蛋白双标阳性细胞数均高于骨髓间充质干细胞治疗组（P < 0.05），两组间5-溴-2-脱氧尿苷-胶质纤维酸性蛋白双标阳性细胞数差异无显著性意义（P > 0.05）。再灌注7 d后，静脉移植骨髓间充质干细胞和碱性成纤维细胞生长因子基因修饰的骨髓间充质干细胞均能改善脑缺血后大鼠的神经功能、减少脑梗死体积，碱性成纤维细胞生长因子基因修饰的骨髓间充质干细胞的作用明显优于骨髓间充质干细胞。 结论：碱性成纤维细胞生长因子诱导的骨髓间充质干细胞静脉移植后可在脑内缺血区存活，并分化为比例更合适的神经元和神经胶质细胞，发挥神经修复作用。     

间充质干细胞在眼科领域的研究与应用

目前间充质干细胞治疗主要应用于局部移植、系统移植、结合干细胞的基因治疗以及组织工程领域。基于间充质干细胞易于分离培养、扩增,易于外源基因的导入和表达,在体外长期培养的过程中始终保持多向分化潜能,遗传背景相当稳定,在眼组织工程中具有广泛的应用前景。目前国内外已有大量研究证实间充质干细胞可分化为角膜及视网膜细胞,间充质干细胞在眼前节的应用多借助于某种载体,如羊膜、纤维凝胶膜等,另外温度敏感培养皿实现了无载体的眼表干细胞移植;在眼后节的应用重点集中于寻找治疗视网膜色素变性可行的治疗方法上。间充质干细胞作为种子细胞参与眼部受损组织修复与再生的研究趋于成熟,但其致瘤性及安全性还有待进一步解决。     

肝纤维化大鼠血清体外诱导骨髓间充质干细胞向肝细胞的分化

背景：近年来临床运用骨髓间充质干细胞移植治疗晚期肝纤维化取得了较好疗效,但由于肝源稀少,骨髓干细胞体外分化为成熟肝细胞的技术仍然不成熟。 目的：探讨骨髓间充质干细胞体外诱导其分化为肝细胞的可行性。 方法：构建大鼠肝纤维化模型,分离、纯化并鉴定大鼠骨髓间充质干细胞,制备正常及肝纤维化大鼠血清,取第3代骨髓间充质干细胞分组培养,分别加入以下培养基：DMEM+体积分数10%胎牛血清;肝细胞生长培养基(HGM)+体积分数5%胎牛血清;HGM+5%正常大鼠血清;HGM+5%肝纤维化大鼠血清;HGM+5%肝纤维化大鼠血清+25 μg肝细胞生长因子。观察各组细胞形态变化,MTT法测定细胞生长曲线,采用Realtime-PCR检测甲胎蛋白和细胞角蛋白18的表达,溴甲酚绿法检测上清液中白蛋白水平,ELISA法检测培养上清液中转化生长因子β1表达。 结果与结论：正常大鼠血清能促进骨髓间充质干细胞的增殖;肝纤维化大鼠血清对骨髓间充质干细胞促增殖作用最好,且能有效诱导其向肝细胞分化,联合使用肝细胞生长培养基能提高分化率。     

体外诱导人骨髓间充质干细胞向肝样细胞分化：肝细胞生长因子和表皮细胞生长因子的作用*

背景：研究证实人骨髓间充质干细胞能够诱导分化为肝样细胞,但其具体生物学特性及分化机制尚不清楚,且诱导分化培养体系尚不成熟。 目的：探讨肝细胞生长因子和表皮细胞生长因子体外诱导人骨髓间充质干细胞向肝样细胞分化的可行性。 方法：取食管癌手术患者肋骨,采用密度梯度离心联合贴壁筛选法获取人骨髓间充质干细胞,流式细胞仪检测细胞表面分子的表达。取第3代人骨髓间充质干细胞,分为4组,肝细胞生长因子组加入20 μg/L肝细胞生长因子,表皮细胞生长因子组加入20 μg/L表皮细胞生长因子,联合组同时加入上述两种生长因子,空白对照组不加任何生长因子。倒置显微镜下观察细胞形态的变化,诱导7,14 d RT-PCR检测甲胎蛋白与白蛋白mRNA的表达。 结果与结论：人骨髓间质干细胞不表达造血细胞标志CD34,CD45,强表达β1-整合素CD29和基质受体CD44。肝细胞生长因子组、表皮细胞生长因子组、联合组诱导后,细胞形态由长梭形变为类圆形或多角形,诱导第7,14天甲胎蛋白、白蛋白 mRNA均呈阳性表达;空白对照组未见多角形细胞,甲胎蛋白、白蛋白 mRNA均呈阴性表达。证明肝细胞生长因子、表皮细胞生长因子以及二者联合均具有诱导人骨髓间充质干细胞向肝样细胞分化的能力,至于二者联合是否能增强其分化能力尚待进一步免疫细胞化学染色定量分析予以验证。     

大鼠骨髓间充质干细胞体外诱导为肝细胞样细胞***

背景：目前体外诱导骨髓间充质干细胞分化为肝细胞样细胞的研究主要集中在不同诱导因子的诱导作用,而微环境的诱导作用研究较少。 目的：观察肝细胞生长因子、胎肝细胞对骨髓间充质干细胞体外诱导分化为肝细胞样细胞的影响。 方法：采用密度梯度离心法分离大鼠骨髓间充质干细胞,反复贴壁法纯化扩增骨髓间充质干细胞;采用Ⅰ型胶原酶消化法分离孕3周大鼠胚胎肝脏细胞,差速贴壁法纯化肝脏细胞;阴性对照组骨髓间充质干细胞只加入含体积分数为10%胎牛血清的L-DMEM培养基。诱导组在阴性对照组基础上加入一定浓度肝细胞生长因子或者与胎肝细胞共培养进行诱导分化。 结果与结论：诱导组白蛋白、甲胎蛋白水平均高于非诱导组(P < 0.01),诱导组糖原染色阳性,免疫细胞化学染色CK-18阳性,而非诱导组糖原染色、CK-18均阴性。结果显示肝细胞生长因子和胎肝细胞均可在体外诱导大鼠骨髓间充质干细胞分化为具有肝细胞样细胞表型和功能的类肝细胞。     

人骨髓间充质干细胞移植老年性痴呆大鼠认知能力和海马超微结构的变化

背景：因发病机制不明,目前尚无治愈老年性痴呆的有效方法。现临床上主要是采用药物治疗,而骨髓间充质干细胞的替代治疗尚处于基础研究阶段,其海马移植后对老年性痴呆认知能力的影响未见报道。 目的：探讨人骨髓间充质干细胞移植对老年性痴呆大鼠认知能力和海马超微结构的影响。 方法：老年雄性Wistar大鼠30只,制备自然衰老痴呆模型,造模后随机分为3组,选取双侧海马为移植区,分化细胞移植组注射定向神经细胞诱导分化的人骨髓间充质干细胞悬液4 μL(2×105个细胞),干细胞移植组注射等量常规培养的人骨髓间充质干细胞,模型组注射等量生理盐水。通过Y迷宫试验测定大鼠的学习、记忆能力,透射电镜观察海马区超微结构。 结果与结论：与移植前大鼠学习、记忆分数比较,移植后12周模型组均显著下降(P < 0.01),干细胞移植组均有所提高(P > 0.05),分化细胞移植组均显著提高(P < 0.01)。移植后12周与模型组比较,干细胞移植组、分化细胞移植组大鼠学习、记忆分数均显著提高(P < 0.01)。电镜观察模型组大鼠海马区神经细胞可见明显损伤,干细胞移植组损伤减轻,分化细胞移植组多数神经细胞结构正常。证实骨髓间充质干细胞移植可以提高老年性痴呆大鼠的认知能力,且定向神经诱导分化的骨髓间充质干细胞移植治疗效果优于未分化的骨髓间充质干细胞,提示骨髓间充质干细胞减少海马组织神经细胞变性坏死可能是其改善老年性痴呆大鼠认知功能障碍的作用机制之一。     

骨髓间充质干细胞向神经元样细胞的分化及其在细胞移植中的应用☆

学术背景：骨髓间充质干细胞主要存在于骨髓基质中,具有横向分化潜能,在适当的条件下可以向3个胚层的细胞进行分化。 目的：文章就骨髓间充质干细胞生物学特性、神经元诱导分化及其机制、细胞移植方面的最新进展进行综述。 检索策略：应用计算机检索PubMed1999/2007年相关文章,检索词为“bone marrow mesenchymal stem cells”,并限定文章语言种类为English。同时计算机检索中国期刊全文数据库1999/2007年相关文章,检索词为“骨髓间充质干细胞”,并限定文章语言种类为中文。对资料进行初审,并查看每篇文章后的引文。纳入标准：文章所述内容与骨髓间充质干细胞的发展现状及神经元诱导分化及机制和移植研究相关。排除标准：重复研究或较陈旧的文献。 文献评价：共收集到230篇相关文献,72篇文献符合纳入标准,排除的158篇文献为内容陈旧或重复文献。选取29篇文献进行分析,分别涉及骨髓间充质干细胞的分离培养和鉴定8篇、神经元细胞方向分化及其机制17篇、细胞移植治疗各种神经损伤的实验研究4篇。 资料综合：由于尚未找到骨髓间充质干细胞特异性标记物,其在体内的细胞生物学和分子生物学作用机制仍不清楚,如何限定体内外诱导条件使其向所需细胞或组织定向分化以及移植时机、是否具有致瘤性等问题尚需深入研究。但近来在骨髓间充质干细胞向神经细胞方向分化的诱导方法和机制,以及骨髓间充质干细胞移植治疗神经损伤方面取得很多成果。 结论：骨髓间充质干细胞具有易获得、易培养、低免疫原性等特性,已成为具有细胞治疗和基因治疗临床疾病潜在实用价值的有效载体。     

基质细胞衍生因子-1对间质干细胞迁移的影响

目的:观察基质细胞衍生因子-1(SDF-1)在体内外对大鼠骨髓间质干细胞(rMSCs)的趋化诱导作用,探讨SDF-1对rMSCs迁移影响的可能机制。方法:应用体外细胞迁移实验及大鼠脑梗死模型体内移植,观察SDF-1对rMSCs的迁移影响。流式细胞术与RT-PCR检测rMSCs的CXC趋化因子受体4(CXCchemokinereceptor4,CXCR4)表达。结果:在SDF-1存在时,rMSCs迁移活跃,应用抗体封闭CXCR4后,这种迁移显著减弱。体内移植的rMSCs主要聚集在脑梗死灶周围,但在封闭CXCR4后,这种聚集现象大大减弱。流式细胞术示仅小部分rMSCs表面表达CXCR4,但经TritonX-100处理后,表达CXCR4的rMSCs增加。结论:SDF-1可通过CXCR4对rMSCs起趋化作用,针对这种作用可望调控干细胞向靶组织的趋化聚集量,达到治疗目的。     

Bone marrow mesenchymal stem cells can be mobilized into peripheral blood by G-CSF in vivo and integrate into traumatically injured cerebral tissue

The efficacy of granulocyte colony-stimulating factor (G-CSF) in mobilizing mesenchymal stem cells (MSCs) into peripheral blood (PB) and the ability of PB-MSCs incorporated into injured brain were tested. Colony forming, cell phenotype and differentiation potential of mouse MSCs mobilized by G-CSF (40 μg/kg) were evaluated. Mortality and pathological changes in mice with serious craniocerebral trauma plus G-CSF treatment (40 μg/kg) were investigated. Bone marrow (BM) cells derived from GFP mice were fractionated into MSCs, hematopoietic stem cells (HSCs), and non-MSC/HSCs using magnetic beads and adherent culture. The resultant cell populations were transplanted into injured mice. The in vivo integration and differentiation of the transplanted cells were detected immunocytochemically. The expression of SDF-1 in injured area of brain was tested by Western blot. G-CSF was able to mobilize MSCs into PB (fourfold increase). PB-MSCs possessed similar characteristics as BM-MSCs in terms of colony formation, the expression pattern of CD73, 44, 90, 106, 31 and 45, and multipotential of differentiation. Accumulative total mice mortality was lower in TG group (5/14) than that in T group (7/14). It was MSCs, not HSCs or non-MSC/HSC cells integrated into the damaged cerebral tissue and differentiated into cells expressing neural markers. Increased SDF-1 expression in injured area of brain was confirmed, which could facilitate the homing of MSCs to brain. G-CSF can mobilize MSCs into PB and MSCs in PB can integrate into injured cerebral tissue and transdifferentiated into neural cells and may benefit the repair of trauma.     

Bone marrow stromal cells enhance differentiation of cocultured neurosphere cells and promote regeneration of injured spinal cord

Transplantation of bone marrow stromal cells (MSCs) has been regarded as a potential approach for promoting nerve regeneration. In the present study, we investigated the influence of MSCs on spinal cord neurosphere cells in vitro and on the regeneration of injured spinal cord in vivo by grafting. MSCs from adult rats were cocultured with fetal spinal cord-derived neurosphere cells by either cell mixing or making monolayered-feeder cultures. In the mixed cell cultures, neuroshpere cells were stimulated to develop extensive processes. In the monolayered-feeder cultures, numerous processes from neurosphere cells appeared to be attracted to MSCs. In an in vivo experiment, grafted MSCs promoted the regeneration of injured spinal cord by enhancing tissue repair of the lesion, leaving apparently smaller cavities than in controls. Although the number of grafted MSCs gradually decreased, some treated animals showed remarkable functional recovery. These results suggest that MSCs might have profound effects on the differentiation of neurosphere cells and be able to promote regeneration of the spinal cord by means of grafting.     

Human bone marrow stromal cell cultures conditioned by traumatic brain tissue extracts: growth factor production

Treatment of traumatic brain injury (TBI) with bone marrow stromal cells (MSCs) improves functional outcome in the rat. However, the specific mechanisms by which introduced MSCs provide benefit remain to be elucidated. Currently, the ability of therapeutically transplanted MSCs to replace injured parenchymal CNS tissue appears limited at best. Tissue replacement, however, is not the only possible compensatory avenue in cell transplantation therapy. Various growth factors have been shown to mediate the repair and replacement of damaged tissue, so trophic support provided by transplanted MSCs may play a role in the treatment of damaged tissue. We therefore investigated the temporal profile of various growth factors, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and hepatocyte growth factor (HGF), within cultures of human MSCs (hMSCs) conditioned with cerebral tissue extract from TBI. hMSCs were cultured with TBI extracts of rat brain in vitro and quantitative sandwich enzyme-linked immunosorbent assays (ELISAs) were performed. TBI-conditioned hMSCs cultures demonstrated a time-dependent increase of BDNF, NGF, VEGF, and HGF, indicating a responsive production of these growth factors by the hMSCs. The ELISA data suggest that transplanted hMSCs may provide therapeutic benefit via a responsive secretion of an array of growth factors that can foster neuroprotection and angiogenesis.     

Mesenchymal stem cells and the neuronal microenvironment in the area of spinal cord injury

Cell-based technologies are used as a therapeutic strategy in spinal cord injury(SCI). Mesenchymal stem cells(MSCs), which secrete various neurotrophic factors and cytokines, have immunomodulatory, anti-apoptotic and anti-inflammatory effects, modulate reactivity/phenotype of astrocytes and the microglia, thereby promoting neuroregeneration seem to be the most promising. The therapeutic effect of MSCs is due to a paracrine mechanism of their action, therefore the survival of MSCs and their secretory phenotype is of particular importance. Nevertheless, these data are not always reported in efficacy studies of MSC therapy in SCI. Here, we provide a review with summaries of preclinical trials data evaluating the efficacy of MSCs in animal models of SCI. Based on the data collected, we have tried(1) to establish the behavior of MSCs after transplantation in SCI with an evaluation of cell survival, migration potential, distribution in the area of injured and intact tissue and possible differentiation;(2) to determine the effects MSCs on neuronal microenvironment and correlate them with the efficacy of functional recovery in SCI;(3) to ascertain the conditions under which MSCs demonstrate their best survival and greatest efficacy.     

Beneficial effects of BV2 cell on proliferation and neuron-differentiating of mesenchymal stem cells in the circumstance of injured PC12 cell supernatant

Objective The microglias is the representative of immune cells in the brain. It plays dual roles of both repairing and damaging in injured nervous system, and works as an inevitable component of the circumstance of injured neurons. This study was aiming at the effects of the microglias on the biological activities of mesenchymal stem cells （MSCs） in the circumstance of injured neurons. Methods MSCs were obtained by primary culture. We adopted PC12 cells （PC12） and BV2 cells （BV2） to substitute for neurons and microglias, respectively. PC12 were injured by aged Aβ1-40 and the supernatant of the injured PC12 was used to set up the circumstance of injured neurons. Transwells were used for co-culture of BV2 and MSCs, which allowed the independent detection of cells after co-culture. Immunofluorescence was used to identify MSCs and neuron-differentiating cells with CD44 and neuron specific enolase （NSE） staining, respectively. MTT assay was adopted to measure the proliferation. Results In the circumstance of both BV2 presence and injured PC 12 supernatant incubation, either the proliferation or the differentiation of MSCs reached the highest, which seemed to be contradictory, but we gave our explanations. With the BV2 co-culture, the proliferation of MSCs tend to be higher, but the neuron-differentiating MSCs were similar to those incubated without BV2 co-culture either in normal or injured in PC12 supernatant. With the incubation of injured PC12 supernatant, the neuron-differentiating cells were significantly higher than that of control （P 〈 0.05）. Conclusion In the circumstance of injured neurons, microlgias tend to promote the MSCs proliferation. Although not helpful in neuron-differentiating, microglias did not exert any negative effect either.     

肝细胞生长因子基因修饰的骨髓间充质干细胞移植治疗兔股骨头坏死

背景：课题利用实验室自主研制、已获国家SFDA批准临床试验的重组肝细胞生长因子腺病毒感染骨髓间充质干细胞,结合肝细胞生长因子的促血管新生和抗细胞凋亡以及间充质干细胞的成骨功能,通过细胞治疗的手段进行骨损伤修复。 目的：探讨肝细胞生长因子基因修饰的骨髓间充质干细胞移植对兔股骨头坏死骨缺损的治疗效果。 设计、时间及地点：细胞-材料学体内实验,于2007-09/12在北京放射与辐射医学研究所和解放军66400部队骨病专科医院完成。 材料：清洁级26~28周龄新西兰大白兔18只,由北京开源兔业养殖场提供。骨基质明胶为上海骁博科技发展有限公司产品。 方法：采用贴壁法分离培养兔自体骨髓间充质干细胞,体外鉴定成骨和成脂肪能力。18只兔均建立双侧股骨头坏死骨缺损模型,随机分为3组,6只/组,单纯材料组缺损区仅填塞骨基质明胶,细胞-材料组填塞复合骨髓间充质干细胞的骨基质明胶,基因修饰细胞-材料组填塞复合Ad-HGF感染的骨髓间充质干细胞的骨基质明胶,各组细胞用量为 107个/缺损,骨基质明胶用量为27 mm3/缺损,修复3个月后取材进行组织学检查。骨髓间充质干细胞和Ad-HGF感染的骨髓间充质干细胞经荧光染料二醋酸琥珀酰酯羟基荧光素标记后,氯化钴处理72 h,流式细胞学分析细胞增殖情况。 主要观察指标：骨髓间充质干细胞的诱导分化,骨髓间充质干细胞对兔股骨头坏死的修复效果,Ad-HGF感染的骨髓间充质干细胞抗低氧损伤能力。 结果：培养的细胞在适当诱导条件下可分化为成骨和成脂肪细胞。修复后3个月组织学分析显示,单纯材料组仅见疏松的纤维肉芽组织填充;细胞-材料组可见致密的纤维肉芽组织填充,其间有较多的新生血管,但未见新生骨组织;基因修饰细胞-材料组多为软骨样组织填充,周围可见新骨带形成。各组Lane-Sandhu骨组织学评分比较差异有非常显著性意义(P < 0.01),其中基因修饰细胞-材料组评分最高,即Ad-HGF感染的骨髓间充质干细胞具备最佳的骨修复能力。培养体系中加入氯化钴后,已增殖的Ad-HGF感染的骨髓间充质干细胞比例显著高于未感染骨髓间充质干细胞(P < 0.001)。 结论：在骨缺损模型中Ad-HGF感染的骨髓间充质干细胞具有更强的抗低氧损伤能力,其在体内成骨能力优于未感染的骨髓间充质干细胞,提示肝细胞生长因子基因修饰的骨髓间充质干细胞移植是应用于缺血性骨坏死的有效治疗途径。     

低氧对间充质干细胞生物学特性的影响

间充质干细胞是细胞组织工程、细胞替代治疗、基因治疗及移植领域的研究热点。最近研究表明,氧体积分数的变化将影响间充质干细胞的许多生物学特性,在不同氧体积分数下间充质干细胞具有不同增殖凋亡、分化及迁移趋化能力。低氧是一种常见的病理生理状态,适度低氧可促进间充质干细胞增殖及凋亡,有利于迁移和趋化,其对分化的影响因细胞类型不同而有所差异。低氧影响间充质干细胞的分子机制主要涉及低氧诱导因子1、趋化因子及其受体、基质金属蛋白酶。低氧可活化低氧诱导因子1信号通路,该通路的活化上调基质衍生因子1的表达,并形成适合干细胞存在及生长的微环境,通过基质衍生因子1促进趋化因子受体CXCR4阳性的干细胞黏附、迁移和归巢到低氧部位;同时可通过调节基质金属蛋白酶的表达和基质金属蛋白分泌来促进细胞外基质降解,进而使间充质干细胞迁移能力增强。     

BDNF-expressing marrow stromal cells support extensive axonal growth at sites of spinal cord injury

Bone marrow stromal cells (MSCs) constitute a heterogeneous cell layer in the bone marrow, supporting the growth and differentiation of hematopoietic stem cells. Recently, it has been reported that MSCs harbor pluripotent stem cells capable of neural differentiation and that simple treatment of MSCs with chemical inducing agents leads to their rapid transdifferentiation into neural cells. We examined whether native or neurally induced MSCs would reconstitute an axonal growth-promoting milieu after cervical spinal cord injury (SCI), and whether such cells could act as vehicles of growth factor gene delivery to further augment axonal growth. One month after grafting to cystic sites of SCI, native MSCs supported modest growth of host sensory and motor axons. Cells "neurally" induced in vitro did not sustain a neural phenotype in vivo and supported host axonal growth to a degree equal to native MSCs. Transduction of MSCs to overexpress brain-derived neurotrophic factor (BDNF) resulted in a significant increase in the extent and diversity of host axonal growth, enhancing the growth of host serotonergic, coerulospinal, and dorsal column sensory axons. Measurement of neurotrophin production from implanted cells in the lesion site revealed that the grafts naturally contain nerve growth factor (NGF) and neurotrophin-3 (NT-3), and that transduction with BDNF markedly raises levels of BDNF production. Despite the extensive nature of host axonal penetration into the lesion site, functional recovery was not observed on a tape removal or rope-walking task. Thus, MSCs can support host axonal growth after spinal cord injury and are suitable cell types for ex vivo gene delivery. Combination therapy with other experimental approaches will likely be required to achieve axonal growth beyond the lesion site and functional recovery.