紫杉醇联合骨髓基质干细胞种植体外修复内皮及对血管平滑肌增生的影响

目的探讨紫杉醇联合骨髓基质干细胞种植体外修复内皮的可行性及对血管平滑肌细胞增生的影响。方法培养兔主动脉内皮、平滑肌和人骨髓基质干细胞,通过细胞共培养将内皮/骨髓基质干细胞接种于下室、平滑肌细胞接种于上室模拟血管内皮修复过程,分别用3H TdR掺入和Westernblot检测紫杉醇(1,10,100nmol/L)干预20min后第10天平滑肌DNA合成和PCNA蛋白表达,用免疫荧光细胞化学法观察与紫杉醇干预内皮共培养的骨髓基质干细胞vWF和Flk1蛋白表达。结果骨髓基质干细胞种植组平滑肌细胞3H TdR掺入和PCNA蛋白光密度相对值均高于融合内皮组(n=6,P<0.05),低于对数内皮组(n=6,P<0.05)。共培养前骨髓基质干细胞不表达vWF和Flk1蛋白,与紫杉醇干预内皮共培养10天时vWF染色阴性,但部分骨髓基质干细胞开始表达Flk1蛋白。结论骨髓基质干细胞种植能部分抑制紫杉醇引起的平滑肌细胞延迟增生,与紫杉醇干预内皮共培养的骨髓基质干细胞有向内皮分化的能力。     

Cd1234人骨髓基质干细胞及其分化细胞表面糖基化相关分子的研究

目的 分析人骨髓基质干细胞及其分化的成骨细胞表面糖链,探讨是否可以用于判断干细胞的分化状态.方法 原代培养人骨髓基质干细胞,诱导分化为成骨细胞,利用流式细胞术观察两种细胞表面路易斯寡糖.X、SSEA-4和多聚N-乙酰半乳糖胺糖链的差异.结果 人骨髓基质干细胞表面路易斯寡糖.X和SSEA-4的平均荧光强度均高于其分化的成骨细胞;人骨髓基质干细胞表面多聚N-乙酰半乳糖胺的平均荧光强度与其分化的成骨细胞之间未见差异.结论 分析细胞表面糖链的差异可以判断人骨髓基质干细胞的分化状态.     

人骨髓基质干细胞的分子表型特征及微环境依赖向内皮分化的能力

目的 :探讨人骨髓基质干细胞的分子表型特征及与成熟内皮共培养时向内皮分化的能力。方法 :采用密度梯度离心法分离培养人骨髓基质干细胞 ,用荧光激活细胞分选法分析其CD34、CD10 5和CD16 6表达率 ,用免疫荧光细胞化学法观察与成熟兔主动脉内皮共培养的人骨髓基质干细胞Flk -1和vWF蛋白表达 ,并分析anti VEGF抗体对骨髓基质干细胞Flk -1表达的影响。结果 :分离培养的骨髓基质干细胞CD34表达率为 (4 .16± 0 . 16 ) % ,与阴性对照 (4. 0 6± 0 . 2 3) %相比无统计学差异 ,CD10 5表达率为(90 .2 0± 2 . 35 ) % ,CD16 6表达率为 (82. 30± 3. 2 2 ) % ,均明显高于阴性对照 (n =6 ,P <0 .0 5 ) ;与成熟内皮细胞共培养 5d时 ,vWF染色仍为阴性 ,但部分骨髓基质干细胞开始表达Flk -1;anti VEGF抗体呈浓度依赖地抑制Flk- 1阳性骨髓基质干细胞计数 (n =6 ,P <0 .0 5 )。结论 :与成熟内皮共培养的骨髓基质细胞具有微环境依赖向内皮细胞分化的能力。     

滋养层细胞与原代白血病细胞共培养体系的建立及其意义

目的:建立一套体外培养前B急性淋巴细胞白血病(ALL)原代细胞的方法,并尝试进行难治性白血病克隆的鉴定。方法:采用来源于骨髓的基质细胞HS-5作为滋养层细胞与原代白血病细胞进行双相培养,并通过药敏和凋亡检测进行双相培养体系生物动力学评估。结果:双相滋养层培养体系能够耐受细胞毒药物的毒性;8例前B急淋白血病原代细胞标本在基质滋养层支持下离体72 h存活率20.4%~68.3%不等,与无滋养层支持或基质细胞上清液支持的标本相比存活率有显著性提高;其中1例标本的体外生长与有否滋养层支持无关,怀疑为难治性白血病克隆。结论:骨髓基质细胞来源的滋养层具有一定的体外支持白血病细胞存活的能力,但存在个体差异,与肿瘤细胞本身生物学特征有关;利用双相培养体系可以在体外早期初步鉴别出难治性白血病克隆并早期制定干预措施。     

大鼠骨髓间充质干细胞体外定向诱导分化为心肌样细胞的实验研究

目的研究大鼠骨髓间充质干细胞的体外培养及向心肌样细胞转化的条件。方法获取成年大鼠胫骨干骨髓,采用贴壁法进行间充质干细胞的培养、传代,观察经5-氮胞苷诱导后骨髓间充质干细胞的生长和分化。结果大鼠骨髓基质细胞贴壁呈集落生长,5-氮胞苷诱导骨髓基质细胞转化为心肌样细胞。结论骨髓基质细胞能够在体外被诱导分化为心肌样细胞,为自体心肌细胞移植提供了一种良好的来源。     

人骨髓基质细胞的培养及鉴定

目的:探讨骨髓基质细胞的分离、体外培养方法,为基因治疗提供载体细胞.方法:采用密度梯度离心法结合贴壁筛选法分离人骨髓进行体外培养扩增,用倒置显微镜观察细胞形态学特征并用流式细胞仪鉴定其CD90表达.结果:分离培养的骨髓基质细胞似成纤维状,原代细胞呈集落生长,并可表达CD90,传代后细胞形态较一致,且随着传代次数的增加CD90表达越高,第3代可达94.31%.结论:骨髓基质细胞可通过体外培养纯化获得,随着传代次数增加,纯度越高,该方法是一种较理想的骨髓基质细胞培养方法.     

骨髓间充质干细胞体外定向诱导分化为心肌样细胞的实验研究

目的 研究大鼠骨髓间充质干细胞的体外培养及向心肌样细胞转化的条件。方法 获取成年大鼠胫骨干骨髓，采用贴壁法进行间充质干细胞的培养、传代，观察经5-氮胞苷诱导后骨髓间充质干细胞的生长和分化。结果 大鼠骨髓基质细胞贴壁旱集落生长，5-氮胞苷诱导骨髓基质细胞转化为心肌样细胞，结论 骨髓基质细胞能够在体外被诱导分化为心肌样细胞，为自体心肌细胞移植提供了一种良好的来源。     

骨髓基质干细胞对H_2O_2诱导的肝细胞损伤保护作用的研究

背景:骨髓基质干细胞是一种多能干细胞,不但可分化为肝细胞,并且可通过旁分泌的方式分泌细胞因子,从而促进肝再生。目的:探讨骨髓基质干细胞对H_2O_2诱导的肝细胞损伤的保护作用。方法:以500μmol/L H_2O_2诱导人肝细胞株THLE-3损伤模型,24h后与骨髓基质干细胞共培养。倒置相差显微镜下观察细胞形态改变,检测细胞毒性、caspase-3/7活性以及YO-PRO-1阳性细胞率。结果:与骨髓基质干细胞共培养后,H_2O_2损伤的THLE-3细胞形态明显改善,细胞毒性显著减少(P0.05),caspase-3/7活性显著下降(P0.05),YO-PRO-1阳性细胞率显著降低(P0.05)。结论:骨髓基质干细胞通过降低细胞毒性、caspase-3/7活性和细胞凋亡而对H_2O_2诱导的THLE-3细胞损伤起保护作用。     

成年犬自体骨髓基质干细胞经冠状动脉移植在心梗后心肌组织内分化

目的　探讨经冠状动脉移植成年犬自体骨髓基质干细胞在心梗后心肌组织内的存活、分化情况。方法　结扎犬冠状动脉前降支制备心肌梗死模型 ;细胞移植组将体外培养、诱导的自体骨髓基质干细胞标记后在心肌梗死后 4周经冠状动脉植入心脏 ;对照组植入等量培养基 ;细胞移植 4周后取犬心脏 ,行组织学检查、免疫组织化学检查及电镜检查。结果　细胞移植组植入骨髓基质干细胞在心肌组织内存活 ;免疫组织化学检查结蛋白、肌钙蛋白I染色阳性 ;电镜示心肌疤痕组织中可见胞浆丰富的异源性细胞 ,圆形或椭圆形 ,细胞体积较大 ,核糖体丰富 ,线粒体较丰富 ,有肌丝样结构 ,单核。对照组心脏标本中可见疤痕组织 ;心肌细胞结蛋白染色阴性 ,肌钙蛋白I染色阳性 ;电镜未发现异源性细胞。结论　成年犬骨髓基质干细胞经冠状动脉移植后在心肌组织内存活并分化为肌源性细胞。     

人骨髓组织相容性抗原DR阴性C盒细胞阴性细胞体外分化为肝细胞样细胞

骨髓中含有大量可分化为各种细胞类型的干细胞，主要包括造血干细胞和间充质干细胞两大类型。这两类细胞均有向肝细胞样细胞分化的潜能。本实验在体外扩增并特异性诱导人骨髓组织相容性抗原DR阴性C盒细胞阴性(HLA-DR^-C-Kit^-)细胞向肝细胞样细胞分化，旨在寻找一种适合人骨髓HLA-DR^-G-Kit^-细胞向肝细胞样细胞大量转化的体外环境，从而为其在肝脏疾病的临床应用打下基础。     

A Stro-1(+) human universal stromal feeder layer to expand/maintain human bone marrow hematopoietic stem/progenitor cells in a serum-free culture system

OBJECTIVE: To compare the ability of allogeneic versus autologous purified human Stro-1(+) mesenchymal stem cell (MSC) populations from different human donors to support the ex vivo expansion and maintenance of human hematopoietic stem/progenitor cells (HSCs). Furthermore, we compared the results obtained with MSC as a feeder layer to traditional allogeneic stromal layers grown in long-term bone marrow culture media (LT-ST). METHODS: Adult human bone marrow CD34(+)-enriched cells were cultured in serum-free medium for 2 to 3 weeks over the respective MSC-irradiated feeder layers or over traditional allogeneic LT- ST stromal layers in the presence of stem cell factor, basic fibroblast growth factor, leukemia inhibitory factor, and Flt-3 and analyzed every 2 to 4 days for expansion, phenotype, and clonogenic ability. RESULTS: There was a progressive expansion of total numbers of cells in all the experimental groups; however, allogeneic MSCs were more efficient at expanding CD34(+)CD38(-) cells and showed a higher clonogenic potential than both allogeneic LT-ST and autologous MSCs. The differentiative potential of cells cultured on both MSC and LT-ST was primarily shifted toward myeloid lineage; however, only MSCs were able to maintain/expand a CD7(+) population with lymphocytic potential. Importantly, transplantation into preimmune fetal sheep demonstrated that the HSCs cultured over MSCs retained their engraftment capability. CONCLUSION: These results indicate that purified Stro-1(+) MSCs may be used as a universal and reproducible stromal feeder layer to efficiently expand and maintain human bone marrow HSCs ex vivo.     

Oxygen tension plays a critical role in the hematopoietic microenvironment in vitro

Background

In the bone marrow mesenchymal stromal cells and osteoblasts form functional niches for hematopoietic stem and progenitor cells. This microenvironment can be partially mimicked using in vitro co-culture systems. In this study, we examined the oxygen tension in three distinct compartments in a co-culture system of purified CD34⁺ cells and mesenchymal stromal cells with regard to different spatial localizations.

Design and Methods

Hypoxic cells in the co-culture were visualized by pimonidazole staining. Hematopoietic cell distribution, and functional and phenotypic characteristics were analyzed by flow cytometry. The secretion of vascular endothelial growth factor and stromal-derived factor-1 by mesenchymal stromal cells in low oxygen co-cultures was determined by an enzyme-linked immunosorbent assay. The effect of co-culture medium on the hematopoietic cell migration potential was tested in a transwell assay.

Results

In co-cultures under atmospheric oxygen tension, regions of low oxygen tension could be detected beneath the feeder layer in which a reservoir of phenotypically more primitive hematopoietic cells is located in vitro. In low oxygen co-culture, the adhesion of hematopoietic cells to the feeder layer was decreased, whereas hematopoietic cell transmigration beneath mesenchymal stromal cells was favored. Increased vascular endothelial growth factor-A secretion by mesenchymal stromal cells under low oxygen conditions, which increased the permeability of the monolayer, was responsible for this effect. Furthermore, vascular endothelial growth factor-A expression in low oxygen mesenchymal stromal cells was induced via hypoxia-inducible factor signaling. However, stromal cell-derived factor-1 secretion by mesenchymal stromal cells was down-regulated under low oxygen conditions in a hypoxia-inducible factor-independent manner.

Conclusions

We demonstrate for the first time that differences in oxygen tension cause selective modification of hematopoietic cell and mesenchymal stromal cell interactions in a co-culture system, thus confirming that oxygen tension plays a critical role in the interaction between hematopoietic cells and the niche environment.     

Bone marrow mesenchymal stromal cells non-selectively protect chronic myeloid leukemia cells from imatinib-induced apoptosis via the CXCR4/CXCL12 axis

Background

Residual chronic myeloid leukemia disease following imatinib treatment has been attributed to the presence of quiescent leukemic stem cells intrinsically resistant to imatinib. Mesenchymal stromal cells in the bone marrow may favor the persistence and progression of leukemia by preserving the proliferation and self-renewal capacities of the malignant progenitor cells.

Design and Methods

BV173 or primary chronic myeloid leukemia cells were co-cultured with human mesenchymal stromal cells and imatinib-induced cell death was then measured. The roles of pro-and anti-apoptotic proteins and chemokine CXCL12 in this context were evaluated. We also studied the ability of BV173 cells to repopulate NOD/SCID mice following in vitro exposure to imatinib and mesenchymal stromal cells.

Results

Whilst imatinib induced dose-dependent apoptosis of BV173 cells and primary chronic myeloid leukemia cells, co-culture with mesenchymal stromal cells protected both types of chronic myeloid leukemia cells. Molecular analysis indicated that mesenchymal stromal cells reduced caspase-3 activation and modulated the expression of the anti-apoptotic protein Bcl-XL. Furthermore, chronic myeloid leukemia cells exposed to imatinib in the presence of mesenchymal stromal cells retained the ability to engraft into NOD/SCID mice. We observed that chronic myeloid leukemia cells and mesenchymal stromal cells express functional levels of CXCR4 and CXCL12, respectively. Finally, the CXCR4 antagonist, AMD3100 restored apoptosis by imatinib and the susceptibility of the SCID leukemia repopulating cells to the tyrosine kinase inhibitor.

Conclusions

Human mesenchymal stromal cells mediate protection of chronic myeloid leukemia cells from imatinib-induced apoptosis. Disruption of the CXCL12/CXCR4 axis restores, at least in part, the leukemic cells’ sensitivity to imatinib. The combination of anti-CXCR4 antagonists with tyrosine kinase inhibitors may represent a powerful approach to the treatment of chronic myeloid leukemia.     

脐血干细胞与骨髓干细胞联合移植治疗大鼠急性肝衰竭实验研究

目的：观察脐血干细胞和自体骨髓干细胞共同移植治疗D-半乳糖苷所致急性肝衰竭大鼠的疗效。方法采集大鼠脐血和骨髓中单个核细胞,以促肝细胞生长因子和干细胞因子培养3w,采用免疫细胞化学法检测白蛋白和甲胎蛋白表达情况;以D-半乳糖苷腹腔注射法建立大鼠急性肝衰竭模型,24 h后每日经鼠尾静脉分别注入脐血干细胞,或骨髓干细胞,或混合的脐血干细胞和骨髓干细胞,对照组注射等量生理盐水,治疗7d,观察4组大鼠存活率、肝功能和肝组织病理学变化。结果在促肝细胞生长因子及干细胞因子的诱导下,脐血干细胞和骨髓干细胞可以在体外扩增并分化为肝细胞;脐血干细胞移植、骨髓干细胞移植和联合细胞移植组大鼠9d 存活率分别为55.6%、50.0%和77.8%,均明显高于生理盐水对照组（16.7%,P〈0.01）;联合移植组存活率高于任何一种单纯干细胞移植（P〈0.01）。结论脐血干细胞和骨髓干细胞移植对大鼠急性肝损伤有一定的保护作用,两者联合移植有协同作用。     

Bi-directional activation between mesenchymal stem cells and CLL B-cells: implication for CLL disease progression

It was hypothesized that contact between chronic lymphocytic leukaemia (CLL) B-cells and marrow stromal cells impact both cell types. To test this hypothesis, we utilized a long-term primary culture system from bone biopsies that reliably generates a mesenchymal stem cell (MSC). Co-culture of MSC with CLL B-cells protected the latter from both spontaneous apoptosis and drug-induced apoptosis. The CD38 expression in previously CD38 positive CLL B-cells was up-regulated with MSC co-culture. Upregulation of CD71, CD25, CD69 and CD70 in CLL B-cells was found in the co-culture. CD71 upregulation was more significantly associated with high-risk CLL, implicating CD71 regulation in the microenvironment predicting disease progression. In MSC, rapid ERK and AKT phosphorylation (within 30 min) were detected when CLL B-cells and MSC were separated by transwell; indicating that activation of MSC was mediated by soluble factors. These findings support a bi-directional activation between bone marrow stromal cells and CLL B-cells.     

两种类型的干细胞经门静脉注入治疗失代偿期肝硬化患者近期疗效观察

目的 比较自体骨髓干细胞和脐带血干细胞经门静脉注入治疗失代偿期肝硬化患者的安全性及对肝功能和PTA的近期改善作用。方法 选择失代偿期肝硬化患者59例,随机分为骨髓组31例和脐血组28例。骨髓组患者经门静脉注入自体骨髓干细胞治疗,脐血组经同样途径注入脐带血干细胞治疗。治疗8周后检测两组患者血清ALT、AST、TBil、PTA、ALB和AFP水平变化。同时观察对比患者临床症状的改善情况及术后的不良反应。结果 细胞治疗3天,两组患者乏力、纳差症状均有改善,差异均无统计学意义（P均〉0.05）。治疗8周,骨髓组和脐血组ALB水平分别上升至（34.8±6.3）g/L和（36.8±8.1）g/L,差异无统计学意义（P〉0.05）;PTA水平上升至（54.3±13.8）%和（57.0±15.2）%,差异无统计学意义（P〉0.05）;骨髓组血清ALT、AST、TBil和AFP分别为（43.2±13.8）U/L、（50.8±14.2）U/L、（34.5±14.7）μmol/L、（10.0±3.1）μg/L,脐血组分别为（46.3±12.3）U/L、（49.1±15.0）U/L、（31.4±12.5）μmol/L、（8.8±3.2）μg/L,两组差异均无统计学意义（P均〉0.05）。结论 经门静脉注入自体骨髓干细胞和脐带血干细胞治疗失代偿期肝硬化患者有一定的安全性及疗效,脐带血干细胞疗效优于自体骨髓干细胞,但两组疗效差异无统计学意义。     

骨髓基质干细胞对肝细胞与肝纤维化细胞增殖影响的体外研究

目的探讨微环境对骨髓基质干细胞（BMSCs）分化的影响以及BMSCs对肝纤维化细胞（CFSC）和肝细胞增殖的作用。方法分离、培养大鼠原代BMSCs和肝细胞,用PKH26荧光标记BMSCs,将标记后的BMSCs与肝细胞/CFSC进行接触和非接触共培养,用抗白蛋白抗体/抗α、平滑肌抗体对BMSCs进行检测;BMSCs条件培养液与CFSC共培养,倒置显微镜下计数CFSC的细胞数量变化。结果BMSCs与肝细胞共培养72h,白蛋白染色均出现阳性,且接触共培养的BMSCs白蛋白染色阳性率高于非接触共培养（P〈0.01）。肝细胞与BMSCs非接触共培养48h后,肝细胞的数量明显多于对照组（P〈0.01）。BMSCs与CVSC共培养体系中BMSCs的形态无明显变化,且αSMA染色未出现阳性。BMSCs条件培养液能抑制CFSC的增殖,作用时间越长,抑制作用越明显（P〈0.01）。结论肝细胞形成的局部微环境能诱导BMSCs向肝样细胞分化,BMSCs能够促进肝细胞生长并对CFSC的增殖有明显的抑制作用。     

重组腺相关病毒介导人血管内皮生长因子165基因在大鼠骨髓间充质干细胞中的表达

目的探讨以腺相关病毒(rAAV)为血管内皮细胞生长因子165基因(VEGIF165)载体,在体外转染大鼠骨髓间充质干细胞(MSCs),并研究其相关特性。方法 (1)全骨髓培养法提取培养MSCs,利用免疫组化法检测MSCs表面标志CD34、CD44;流式细胞分析法检测CD90。(2)rAAV-VEGF165转染MSCs,采用ELISA及PCR检测VEGF的表达,比较转染前后细胞的变化情况,观察VEGF165基因转染对MSCs的影响。结果 (1)成功培养出MSCs,CD44阳性表达,CD34阴性表达,CD90阳性表达。(2)在转染rAAV-VEGF165后转染组上清中VFGF165分泌水平明显高于未转染组(p<0.05),5 d时达到高峰,此后表达开始下降。琼脂糖凝胶电泳可见高亮度条带。表明rAAv-VEGF165成功转染进MSCs细胞中,绘制生长曲线,显示rAAV-VEGF165基因转染后对MSC生长无影响。结论rAAV-VEGF表达载体可有效感染MSCs,并在体外高效表达,为MSCs联合基因治疗提供了实验依据。     

经肝动脉自体骨髓或脐带血干细胞移植治疗失代偿期肝硬化患者的安全性和近期疗效比较

比较经肝动脉途径注入自体骨髓干细胞或脐带血干细胞治疗失代偿期肝硬化患者的安全性,以及对患者肝功能和凝血酶原活动度的近期改善作用。方法选择失代偿期肝硬化患者65例,随机分为骨髓组33例和脐血组32例;骨髓组患者经股动脉插管至肝固有动脉注入自体骨髓干细胞移植治疗,脐血组经同样途径注入脐带血干细胞治疗;治疗后8周检测血清谷丙转氨酶、总胆红素、凝血酶原活动度、白蛋白和甲胎蛋白水平变化,同时观察临床症状的改善情况及术后的不良反应。结果治疗后第3天两组患者乏力、纳差症状均有改善,两组间差异无显著性。治疗后8w,骨髓组和脐血组白蛋白水平分别由（31.0±4.6） g/L 上升至（34.6±7.1）g/L和由（34.6±7.1） g/L上升至（37.8±8.3） g/L,凝血酶原活动度上升由（48.8±13.4）%上升至（55.5±11.2）%和由（47.5±12.5）上升至（58.9±14.0）%,但两组间改善程度的差异无显著性;在治疗8w末血清谷丙转氨酶、总胆红素和甲胎蛋白在骨髓组分别为（45.6±12.3） IU/L、（28.1±13.5）μmol/L和（11.3±4.1）μg/L,在脐血组分别为（47.2±11.8） IU/L、（30.7±14.8）μmol/L和（9.8±3.5）μg/L,两组间与基础水平相比改善程度的差异也无显著性。结论自体骨髓干细胞或脐带血干细胞经肝动脉途径移植治疗失代偿期肝硬化患者的近期疗效及安全性均良好,但两种细胞治疗的改善水平无显著差异。     

骨髓基质细胞移植治疗脑梗死

骨髓基质细胞是一类多能干细胞,具有自我更新和多向分化潜能,为多种疾病的细胞和基因治疗提供了基础.许多实验研究证实,骨髓基质细胞移植对脑梗死具有显著的治疗作用.文章就近年来骨髓基质细胞对脑梗死的治疗方法和效果进行了综述.