受体骨髓间质干细胞在大鼠原位肝移植中对免疫排斥的影响

目的:观察受体骨髓间质干细胞(BM-MSCs)在大鼠原位肝移植后体内的分布与作用.方法:以Wistar大鼠为供体,SD大鼠为受体采用双袖套法制作原位肝移植模型,密度梯度离心法分离与贴壁法富集受体BM-MSCs.CFSE标记术后经门静脉注入,荧光显微镜分别观测术后1wk,2 wk,1mo肝脾肺肾组织中BM- MSCs的分布及各时间点检测肝功能及肝组织免疫排斥情况.结果:密度梯度离心所得到的BM-MSCs为比较一致的球形单个核细胞,台盼蓝染色细胞活力达98%左右,其他细胞少见.BM-MSCs体外分离培养扩增至第3代后较为纯化.应用CFSE可快速高效的标记贴壁BM-MSCs.移植的BM-MSCs主要在受体肝脏中聚集,1mo后略有减少,而脾肺肾组织内BM-MSCs在1mo后仅有极少分布.B组无BM-MSCs输入与C组有BM-MSCs输入肝功能比较,C组肝功能有明显好转,B,C组间差异有显著意义(F=63.179,P<0.01),B,C组各时段肝功能差异有显著性意义(F=221.026,P<0.01).C组肝组织免疫排斥反应明显减轻.结论:原位肝移植术后输注受体BM-MSCs可有效缓解免疫排斥反应.     

骨髓间充质干细胞移植在肝再生中的应用

随着当今医学的发展,干细胞的应用逐渐涉及到各个领域,并发挥着无限的潜能。干细胞以其自我更新及多向分化的能力可以对多种疾病进行替代治疗。肝病终末期肝细胞数量减少,肝功能衰竭,通过干细胞的替代治疗,肝功能能够得以改善,肝纤维化得到缓解甚至逆转。干细胞治疗成为有望替代肝移植的新技术。此文就干细胞在肝病治疗中的潜能作一综述,包括干细胞的来源、治疗机制、治疗途径,并对其临床应用进行探讨。     

Adenovirus mediated CTLA4Ig gene inhibits infiltration of immune cells and cell apoptosis in rats after liver transplantation

AIM: To investigate the role of adenovirus-mediated CTLA4Ig gene therapy in inhibiting the infiltration of macrophages and CD8+T cells and cell apoptosis after liver transplantation. METHODS: The rat orthotopic liver transplantation model was applied. The rats were divided into three groups: group I: rejection control (SD-to-Wistar); group II: acute rejection treated with intramuscular injection of CsA 3.0 mg/(kg·d) for 12 d (SD-to-Wistar+CsA); groupIII: injection of 1×109 PFU adenovirus-mediated CTLA4Ig gene liquor in dorsal vein of penis 7 d before liver transplantation (SD-to-Wistar+CTLA4Ig). Immunohistochemistry and transferase-mediated dUTP nick-end labeling (TUNEL) were used to analyze the expression of CTLA4Ig gene in liver, infiltration of macrophages and CD8+T cells, cell apoptosis in grafts at different time-points after liver transplantation. Histopathological examination was done. RESULTS: CTLA4Ig gene expression was positive in liver on d 7 after administering adenovirus-mediated CTLA4Ig gene via vein, and remained positive until day 60 after liver transplantation. Infiltration of macrophages and CD8+T cells in CTLA4Ig-treated group was less than in rejection control group and CsA-treated group. The apoptotic index of rejection group on d 3, 5, and 7 were significantly higher than that of CTLA4Ig-treated group. A good correlation was found between severity of rejection reaction and infiltration of immune activator cells or cell apoptotic index in grafts. CONCLUSION: CTLA4Ig gene is constantly expressed in liver and plays an important role in inducing immune tolerance.     

骨髓间充质干细胞移植治疗大鼠急性肝功能衰竭

目的探讨骨髓间充质干细胞（BMSCs）移植对急性肝功能衰竭的治疗作用。方法大鼠90%肝脏切除制备急性肝功能衰竭模型,实验组大鼠肝内移植2×106个BMSCs,对照组仅注射生理盐水。观察大鼠的生存情况,RT-PCR检测BMSCs在肝脏的植入,血清检测肝功能确认BMSCs移植对肝脏修复的作用。结果移植BM-SCs的实验组大鼠存活80%,对照组大鼠仅存活20%。RT-PCR显示,BMSCs移植后定植于大鼠肝脏内。肝功能检测显示,实验组大鼠的血清丙氨酸氨基转移酶水平、天冬氨酸氨基转移酶水平显著降低,血清白蛋白水平显著升高。结论 BMSCs移植可以显著提高急性功能衰竭大鼠的生存率,促进其肝功能的恢复,对急性肝功能衰竭的治疗具有积极作用。     

SSEA-4 identifies mesenchymal stem cells from bone marrow

Adult bone marrow (BM) contains hematopoietic stem cells (HSCs) as well as a nonhematopoietic, stromal cell population. Within this stromal population are mesenchymal stem cells (MSCs), which not only support hematopoiesis but also differentiate into multiple lineages, including fat, bone, and cartilage. Because of this multipotentiality, the MSC is an attractive candidate for clinical applications to repair or regenerate damaged tissues of mesenchymal origin. However, research progress has been hampered by the limited existing knowledge of the biology of these cells, particularly by the lack of a suitable marker for their prospective isolation. Here, we report that SSEA-4, an early embryonic glycolipid antigen commonly used as a marker for undifferentiated pluripotent human embryonic stem cells and cleavage to blastocyst stage embryos, also identifies the adult mesenchymal stem cell population.     

受者同系骨髓间充质干细胞对大鼠心脏移植急性排斥反应的影响

目的观察受者同系骨髓间充质干细胞（MSC）对大鼠心脏移植急性排斥反应的影响,并初步探讨其免疫调节机制。方法成年雄性Wistar大鼠20只为受体,随机分为空白对照组和MSC处理组,每组10只,行Lewis大鼠→Wistar大鼠颈部心脏移植。空白对照组手术后24h,经尾静脉注射3ml 0.9%氯化钠注射液;MSC处理组手术后24h,经尾静脉注射MSC2×10~6（悬浮在3ml 0.9%氯化钠注射液中）。术后1周,每组随机取4只大鼠,进行组织学检查及下腔静脉血检测血清白介素（IL）-2及IL-10水平。其余6只用以继续观察移植物存活时间。结果 （1）空白对照组大鼠移植心脏存活（7.2±1.3）d,MSC处理组大鼠移植心脏存活（14.8±2.9）d,差异显著（P〈0.01）。（2）血清IL-2空白对照组为（233.99±30.19）ng/L,MSC处理组为（249.11±20.34）ng/L,两组无统计学差异（P〉0.05）;血清IL-10空白对照组为（81.44±11.01）ng/L,MSC处理组为（158.39±18.70）ng/L,差异显著（P〈0.01）。（3）病理学分级,空白对照组3B级1例、4级3例,MSC处理组2级1例、3A级2例、3B级1例,MSC处理组病理分级总体较空白对照组低。结论静脉注射受者同系骨髓MSC,可以延长大鼠颈部移植心脏的存活时间,减轻急性排斥反应的程度。     

自体骨髓间充质干细胞联合促肝细胞生长素治疗失代偿期酒精性肝硬化疗效分析

目的观察自体骨髓间充质干细胞（BMSCs）联合促肝细胞生长素（pHGF）治疗失代偿期酒精性肝硬化（ALC）患者的临床疗效。方法2012年6月~2016年6月我科住院收治的67例失代偿期ALC患者,34例接受常规综合治疗,33例在常规综合治疗基础上接受BMSCs肝动脉移植治疗,并接受HGF注射2月。结果在治疗2月末,观察组血清TBIL、ALB、PT、HA水平分别为（55.9±12.6） μmol/L、（38.8±5.1) g/L、（13.4±3.6） s和（222.2±61.5）ng/ml,与对照组【（66.9±11.0）μmol/L、（33.8±5.8）g/L、（15.8±3.9）s、（284.5±30.5） ng/ml,P<0.01或P<0.05】比,差异显著。结论应用BMSC移植联合HGF治疗ALC患者能较好地改善肝功能指标。     

骨髓间充质干细胞的培养及低氧条件对其影响

目的 骨髓间充质干细胞(BMMSC)的体外培养和低氧对于BMMSC增殖作用的影响.方法 成年雄性SD大鼠断颈处死后于75％酒精中浸泡5 min.用全骨髓贴壁法培养细胞,选取生长状态良好的第3代细胞进行鉴定.以单克隆抗体CD45、CD90行流式细胞术鉴定大鼠BMMSC.用四甲基偶氮唑蓝(MTT)法测定第3代BMMSC以及低氧处理的第3代BMMSC的增殖情况.结果 用全骨髓贴壁法分离并培养SD大鼠BMMSC;流式细胞仪检测显示:第3代BMMSC表面特异性标志CD90表达率为96.0％,而非BMMSC表面标志CD45仅为2.5％.MTT结果显示低氧条件下的BMMSC比常氧条件下增殖速率高,并且光镜下观察到细胞状态均一,折光性更好.结论 用全骨髓贴壁法可以在体外分离、培养出纯度较高的SD大鼠来源BMMSC,低氧环境可以刺激BMMSC的增殖.     

骨髓间充质干细胞移植治疗心肌梗死的研究进展

溶栓、经皮冠状动脉介入治疗及常规药物治疗可挽救许多急性心肌梗死患者的生命,但不能从根本上恢复心肌细胞数量。骨髓间充质干细胞(BM-MSCS)是骨髓中一种具有多向分化潜能的细胞,可被诱导分化为心肌样细胞,改善梗死区的血液供应及心肌重构,为心肌梗死的治疗带来了新的希望。本文就BM-MSCS移植治疗心肌梗死的方式,移植的时机及临床应用等方面作一综述。     

骨髓间充质干细胞治疗肝硬化的动物实验研究

目的了解肝硬化大鼠在输注骨髓间充质干细胞(BM-MSCs)后肝功能生化指标、AFP的变化情况,为临床应用提供实验依据。方法将健康雌性Wistar大鼠随机分为正常组、对照组和实验组;对照组和实验组腹腔注15%的四氯化碳石蜡油溶液建立肝硬化模型。实验组经尾静脉输入用BrdU(Bromodeoxyuridine)标记的健康雄性Wistar大鼠BM-MSCs,于1/2h、1wk、2wk、3wk、4wk处死对照组和实验组大鼠,用PCR检测雄性性别决定基因SRY、免疫组化检测BrdU以了解BM-MSCs的整合情况;用全自动生化分析仪检测血清肝功能指标、放免法检测AFP。结果PCR和BrdU免疫组化初步分析对照组在不同时期均无雄性BM-MSCs,实验组在1/2h处死的5例中均阴性,在第1wk处死的5只中有2例阳性,在第2wk处死组5只中有3只阳性,在第3、4wk处死组5只中均阳性。ALT、AST随着时间推移有所改善,在第4wk时与对照组有显著性差异(28.0±3.74,84.6±36.38vs56.0±26.66,161.2±55.43,P<0.005),ALB仅在第3kw时对照组和实验组有显著性差异(30.3±2.18vs34.4±2.49,P<0.005),但与正常组无显著性差异;GLO在不同时间点均无显著性差异;AFP虽无显著性差异,但有上升的趋势。结论BM-MSCs可以植入肝脏汇管区、肝细胞索,植入细胞最早见于移植后约7d;植入的BM-MSCs能够部分替代肝细胞的功能。     

Protective effect of bone marrow mesenchymal stem cells in intestinal barrier permeability after heterotopic intestinal transplantation

AIM: To explore the protective effect of bone marrow mesenchymal stem cells (BM MSCs) in the small intestinal mucosal barrier following heterotopic intestinal transplantation (HIT) in a rat model.METHODS: BM MSCs were isolated from male Lewis rats by density gradient centrifugation, cultured, and analyzed by flow cytometry. The HIT models were divided into a non-rejection group, saline-treated rejection group (via penile vein), and BM MSC–treated group (via penile vein). Intestinal mucosal barrier injury was estimated by diamine oxidase (DAO) and D-lactic acid (D-LA) expression levels. Tumor necrosis factor-α (TNF-α), interferon-γ (INF-γ), interleukin-10 (IL-10), and transforming growth factor-β (TGF-β) were detected by enzyme-linked immunosorbent assay. Ultrastructural change of tight junctions (TJs) was observed under transmission electron microscope. Expression levels of the TJ proteins occludin and zona occludens (ZO)-1, affected by the inflammatory factors, were measured using real-time polymerase chain reaction and Western blotting.RESULTS: The pathological score at each time point after surgery indicated significantly less serious injury in the BM MSCs-treated group than in the rejection group (P < 0.05). In the former, graft levels of DAO and D-LA were reduced, and TNF-α and INF-γ production was inhibited (at day 7: 10.6473 ± 0.0710 vs 17.2128 ± 0.4991, P < 0.05; 545.1506 ± 31.9416 vs 810.2637 ± 25.1175, P < 0.05). IL-10 and TGF-β production was increased greatly (at day 7: 125.7773 ± 4.7719 vs 80.3756 ± 2.5866, P < 0.05; 234.5273 ± 9.3980 vs 545.1506 ± 31.9416, P < 0.05). There was increased expression of occludin and ZO-1 protein (at day 7: 0.2674 ± 0.0128 vs 0.1352 ± 0.0142, P < 0.05; at day 5: 0.7189 ± 0.0289 vs 0.4556 ± 0.0242, P < 0.05) and mRNA (at day 7: 0.3860 ± 0.0254 vs 0.1673 ± 0.0369, P < 0.05; at day 5: 0.5727 ± 0.0419 vs 0.3598 ± 0.0242, P < 0.05).CONCLUSION: BM MSCs can improve intestinal barrier permeability, repair TJs, and increase occludin and ZO-1 protein expression. With altered cytokine levels, they can protect the intestinal mucosa after transplantation.     

骨髓间充质干细胞在胰腺生理更新和病理再生中的作用

目的:探讨骨髓间充质干细胞在胰腺生理更新和病理再生中的作用. 方法:将60只SD大鼠随机分为5组,每组12 只．A组为正常阴性对照组,不作任何处理,B 组为自体骨髓回输对照组,C组为雨蛙肽致轻度胰腺炎(MAP)模型组,D组为L-精氨酸致重度胰腺炎(SAP)模型组,E组为粒细胞集落因子(G-CSF)预处理组(SAP GSF):在造模前3 d,sc G-CSF 40μg(kg·d),共3 d,其余处理同 D组．造模前3 d用核染料Hoechst33258标记自体骨髓间充质干细胞(MSC),并回输到自体骨髓腔．于骨髓干细胞回输后的2 wk和8 wk,分批处死大鼠,采集胰腺,立即进行冰冻切片, 荧光显微镜下直接观察胰腺组织中是否有呈现黄绿色荧光的细胞,选择有上述黄绿色荧光的冰冻切片进行免疫荧光染色,观察有无 Cytokeratin 19、胰岛素和胰高血糖素染色呈阳性的细胞．结果:A组的胰腺冰冻切片未见有黄绿色荧光．造模后2 wk,B、C、E和D组存活大鼠可见标记的MSC出现在正常胰腺组织和损伤胰腺组织中,在正常胰腺组织中偶见,而在损伤胰腺组织中多见,尤以GSF预处理组最多见, C、D、E组一直持续到8 wk仍可见到黄绿色荧光．A和D组死亡大鼠未见Cytokeratin 19、胰岛素和胰高血糖素染色呈阳性的细胞．造模后2 wk,B、C、E和D组存活大鼠掺入损伤胰腺中的MSC均有分化为Cytokeratin 19染色阳性的细胞,未见胰岛素和胰高血糖素染色呈阳性的细胞．造模后8 wk,胰岛素和胰高血糖素染色呈阳性的细胞在C、D组偶见,E组散见．结论:自体骨髓间充质干细胞参与胰腺的生理更新和病理再生．     

骨髓干细胞移植治疗急性肝衰竭的实验研究

目的探讨骨髓干细胞移植治疗实验性肝衰竭大鼠的效率及可行性。方法以D-氨基半乳糖及内毒素制备雌性大鼠肝衰竭模型,然后动员和富集雄性大鼠骨髓干细胞,并经门静脉移植至雌性大鼠肝脏;移植后观察受体雌鼠临床表现、生化改变、肝脏病变及肝组织Y染色体阳性率。结果受体肝衰竭雌鼠在细胞移植后,精神、食欲逐渐好转,生化异常及肝脏病变得以恢复,肝脏可检出Y染色体阳性的供体源性肝细胞;未接受骨髓干细胞移植的对照组雌鼠则全部死亡。结论骨髓干细胞移植对实验性肝衰竭大鼠具有明确治疗作用。     

兔骨髓间充质干细胞移植后移植细胞凋亡的实验研究

目的利用TUNEL法检测5-氮胞苷体外诱导骨髓间充质干细胞（MSCs）移植后的凋亡情况。方法5-氮胞苷体外诱导骨髓间充质干细胞MSCs向肌源性心肌细胞分化,通过免疫组化,鉴定诱导后的MSCs是否向类心肌细胞转化。建立兔心肌梗死模型,将细胞移植于心梗区域。移值2周后,利用TUNEL法检测植入细胞的凋亡率。结果移植2周后,可见DAPI标记带蓝色荧光的供体细胞核,分布比较广泛,形态呈椭圆形类似心肌细胞核,并与心肌纤维排列方向一致,证明移植细胞已存活。移值细胞表达troponinT,证明移植的MSCs分化为类心肌细胞。移植细胞均出现不同程度细胞凋亡。结论移植的MSCs细胞可在缺血的心肌组织存活,并分化为类心肌细胞,但移植细胞均出现不同程度凋亡。     

骨髓间充质干细胞移植治疗病毒性心肌炎研究的进展

骨髓间充质干细胞(BM-MSCs)移植是一种新兴的治疗病毒性心肌炎(VMC)方法。本文从BM-MSCs的免疫调节、抗氧化、非免疫原性及异基因BM-MSCs的应用等方面阐述其在VMC治疗中的生物学功能,从而为治疗VMC提供新方向。     

Effects of bone marrow derived mesenchymal stem cells transplantation in acutely infarcting myocardium

BACKGROUND: Cellular cardiomyoplasty (CCM) is considered to be a novel therapeutic approach for post-myocardial infarction (MI) heart failure. In this study, the functional effects of cultured mesenchymal stem cells (MSCs) transplantation and the associated histopathologic changes were evaluated in a rat model of MI. METHODS: Rats were subjected to 5 h of coronary ligation followed by reperfusion and, 10 days after MI, animals were randomized into either the MSCs transplantation (MI-MSC, n=8) group or the control (n=8) group. Allogeneic MSCs (3x10(6) cells) or media were epicardially injected into the center and the border area of the infarct scar. RESULTS: Four weeks after the MSCs transplantation, the echocardiogram showed preserved anterior regional wall motion and increases in fractional shortening in the MI-MSC heart relative to the control heart. Left ventricular (LV) end-diastolic pressure was smaller in the MI-MSC than in the control group. Implanted MSCs formed islands of cell clusters on the border of the infarct scar, and the cells were positively immunostained by sarcomeric alpha-actinin and cardiac troponin T. In addition, the number of microvessels on the border area of the infarct scar was greater in the MI-MSC than in the control group. CONCLUSION: Allogeneic MSCs transplanted into the MI scar formed clusters of cell grafts on the border of the infarct, expressed cardiac muscle proteins, increased microvessel formation, and improved regional and global LV function. Our data indicate that CCM using MSCs may have a significant role in the treatment of post-MI heart failure.     

人骨髓间充质干细胞移植在大鼠肝再生衰竭中的作用

骨髓间充质干细胞（MSCs）具有可塑性,包括可以向肝细胞分化的能力。目前诱导MSCs向肝细胞分化的体内外相关实验研究已经由动物向人MSCs过渡。基于人MSCs的人体内实验则较为少见,具体机制尚不明了。因此本实验将人MSCs移植入大鼠肝衰竭模型,对其体内变化机制进行观察。     

骨髓间充质干细胞及Klotho基因移植对慢性缺氧大鼠心脏重构的影响

目的观察骨髓间充质干细胞(BMSCs)及抗衰老基因(Klotho基因)移植对慢性缺氧大鼠心脏重构的影响。方法建立慢性缺氧28 d大鼠心脏重构模型,分为模型组、BMSCs组和Klotho组,并另设正常对照组不给予缺氧处理。分离、培养、鉴定大鼠BMSCs;重组慢病毒介导Klotho基因。模型建立结束后分别通过尾静脉移植BMSCs及Klotho基因至慢性缺氧大鼠体内,4周后用ELISA法检测血清和心肌中Ⅰ型前胶原羟基端肽(P I CP)和Ⅲ型前胶原羟基端肽(PⅢCP)的浓度,HE染色法观察心肌细胞的形态,MASSON染色检测心肌胶原含量,Tunel检测心肌细胞凋亡率。采用SPSS 17.0软件进行数据处理。计量资料以均数±标准差(±s)表示,组间比较采用t检验。结果移植BMSCs和Klotho基因4周后,BMSCs组和Klotho组血清和心肌中的PⅠCP及PⅢCP浓度、胶原容积分数(CVF)和心肌细胞凋亡率均较模型组显著降低(P0.01)。与Klotho组比较,BMSCs组心肌细胞凋亡率显著降低(P0.01)。结论 BMSCs和Klotho基因移植均能有效逆转心脏重构,BMSCs在抗心肌细胞凋亡方面优于Klotho基因。