Ghrelin对缺氧复氧状态下脂肪间充质干细胞保护作用的研究

目的：探讨缺氧复氧损伤环境下Ghrelin对脂肪来源的间充质干细胞（AD-MSCs）的保护作用,以寻求AD-MSCs心肌内移植的有利因素。方法：采用胶原酶消化法分离小鼠AD-MSCs,流式细胞术鉴定其标志。建立缺氧/复氧细胞模型,分3组：①对照组;②缺氧/复氧组（H/R）;③H/R＋Ghrelin（浓度分别为10-9、10-8、10-7mol/L）干预组。MTT法测定各组细胞增殖,TUNEL法检测细胞凋亡。结果：流式细胞术结果显示AD-MSCs CD44及CD90阳性,CD34、CD45阴性。AD-MSCs MTT分析显示在缺氧环境中,Ghrelin相比于单纯H/R组能够显著促进AD-MSCs的存活与增殖,并抑制其凋亡（P〈0.05）。结论：Ghrelin可以明显提高缺氧复氧环境下AD-MSCs的生存与增殖,抑制缺氧诱导的凋亡发生,有望为心肌梗死的干细胞移植治疗创造新的有利因素。     

瑞舒伐他汀促进缺氧复氧间充质干细胞增殖的机制研究

目的:探讨瑞舒伐他汀对缺氧复氧损伤后脂肪来源间充质干细胞增殖的影响及机制。方法:酶消化法分离小鼠的脂肪间充质干细胞(AD-MSCs),流式细胞术检测CD90、CD44、CD34、CD45等细胞标志物。建立缺氧(H)6h/复氧(R)42h细胞模型,AD-MSCs分为3组:①对照组;②缺氧/复氧组(H/R);③H/R+瑞舒伐他汀干预组(浓度分别为10-8、10-7、10-6mol/L)。MTT法测定各组细胞增殖,免疫印迹法检测细胞内Akt、Erk及其磷酸化的表达水平。结果:流式细胞术结果显示脂肪间充质干细胞CD44及CD90阳性,CD34、CD45阴性。MTT实验显示在缺氧环境中,瑞舒伐他汀的干预可显著增加AD-MSCs的增殖(P<0.05)。Westernblot检测pAkt及pErk的表达在瑞舒伐他汀干预组明显高于对照组和H/R组。(P<0.05)。结论:瑞舒伐他汀可通过Akt、Erk信号途径促进H/R损伤后AD-MSCs的增殖。     

瑞舒伐他汀促进缺氧复氧间充质干细胞增殖的机制研究

目的：探讨瑞舒伐他汀对缺氧复氧损伤后脂肪来源间充质干细胞增殖的影响及机制。方法：酶消化法分离小鼠的脂肪间充质干细胞（AD-MSCs）,流式细胞术检测CD90、CD44、CD34、CD45等细胞标志物。建立缺氧（H）6h/复氧（R）42h细胞模型,AD-MSCs分为3组：①对照组;②缺氧/复氧组（H/R）;③H/R＋瑞舒伐他汀干预组（浓度分别为10-8、10-7、10-6mol/L）。MTT法测定各组细胞增殖,免疫印迹法检测细胞内Akt、Erk及其磷酸化的表达水平。结果：流式细胞术结果显示脂肪间充质干细胞CD44及CD90阳性,CD34、CD45阴性。MTT实验显示在缺氧环境中,瑞舒伐他汀的干预可显著增加AD-MSCs的增殖（P〈0.05）。Westernblot检测pAkt及pErk的表达在瑞舒伐他汀干预组明显高于对照组和H/R组。（P〈0.05）。结论：瑞舒伐他汀可通过Akt、Erk信号途径促进H/R损伤后AD-MSCs的增殖。     

缺氧对于间充质干细胞分化的影响

间充质干细胞(mesenchymal stem cell,MSC),是来源于中胚层的具有自我更新能力和多向分化潜能干细胞,在体内外可以分化成骨、软骨、脂肪、肌腱和肌细胞等.由于其强大的分化潜能,MSC在组织工程与再生医学方面具有广泛的应用前景.MSC存在于高度受调控的被称为"壁龛"的微环境中.干细胞壁龛处于一个缺氧的环境中,氧分压可以低至7.2 mmHg.同时MSC是肿瘤微环境的重要的细胞组成成分,肿瘤微环境也是存在于一个缺氧的环境中.了解MSC在缺氧状态下的分化能力,对于组织工程、再生医学和肿瘤的发生发展研究具有重要的意义.缺氧相关的信号转导参与MSC定向分化能力的过程.目前MSC在缺氧状态下的成脂和成骨分化的研究存在着差异,这些研究结果的差异可能是由于MSC的异质性以及实验操作不同所引起.     

腺苷对缺氧/复氧心肌细胞的保护作用

本研究旨在探讨腺苷 (adenosine ,ADO)对缺氧 /复氧 (hypoxia/reoxygenation ,H/R)心肌细胞的保护作用及其分子机制。将原代培养的新生大鼠心肌细胞分成H/R对照组和ADO (1 0 μmol/L)保护组。用倒置相差显微镜观察心肌细胞的生长状态。检测两组培养基质乳酸脱氢酶 (LDH)活性和心肌细胞Ca2 + 和丙二醛 (MDA)浓度。用ELISA法检测肿瘤坏死因子 (TNF α)的表达 ,并用凝胶电泳迁移率改变法 (EMSA)测定核因子 (NF κB)结合活性。所得结果如下 :(1)心肌细胞H/R培养后皱缩、变圆 ,伪足减少 ,ADO组心肌细胞的形态变化小于对照组 ;(2 )ADO减少缺氧和复氧期间心肌细胞LDH的漏出 (bothP <0 0 1) ;(3 )ADO降低缺氧和复氧期间心肌细胞内的Ca2 +浓度 (bothP <0 0 1) ;(4)ADO降低缺氧和复氧期间心肌细胞MDA浓度 (bothP <0 0 1) ;(5 )ADO抑制缺氧和复氧期间TNF α的表达 (bothP <0 0 1) ;(6)ADO抑制缺氧和复氧期间心肌细胞NF κB结合活性 (bothP <0 0 1)。以上结果提示 :(1)外源性ADO可减轻心肌细胞的H/R损伤 ;(2 )外源性ADO抑制H/R期间心肌细胞TNF α的表达 ;(3 )外源性ADO可能通过抑制心肌细胞NF κB结合活性下调TNF α的表达     

超氧化物歧化酶对内皮细胞缺氧复氧损伤的防护作用

体外培养扔兔胸主动脉内皮细胞缺氧３０ｍｉｎ后复氧１０ｍｉｎ,可以发现缺氧后复氧可引起细胞乳酸脱氢酶释放量,细胞悬液丙二醛含量增加,谷胱甘肽过氧化酶活性降低,细胞合成释放一氧化氮减少,细胞内钙离子浓度明显升高;ＥＣ的这些损伤在缺氧期间即有表现,复氧后更为加剧。而在缺氧前预先加入终浓度为２００Ｕ／ｍｌ的超氧化物歧化酶可改善细胞的抗氧化能力,减轻缺氧复氧对ＥＣ的损伤。     

骨髓间充质干细胞对环磷酰胺诱导卵巢损伤的保护作用

目的:研究骨髓间充质干细胞(MBSc)对注射环磷酰胺(CTX)引起的大鼠卵巢损伤的保护作用。方法:将30只SD大鼠按随机数字表随机分为三组,即对照组,模型组及细胞移植组,分别经尾静脉接受生理盐水,CTX和CTX+MBSc移植。监测大鼠体重,动情周期的变化,用药结束后一周处死大鼠,测定血清雌二醇(E2),卵泡刺激素(FSH),黄体生成素(LH)的变化,观察各组大鼠卵巢形态学及卵泡数量变化,并利用原位细胞凋亡TUNEL法检测3组大鼠卵巢中细胞凋亡指数(AI)。结果:与对照组比较,模型组大鼠体重下降,动情周期延长,卵泡数量下降,E2下降,FSH及LH升高,组织细胞AI增加。与模型组比较,细胞移植组大鼠体重增加,动情周期缩短,卵泡数量增加,尤其是中大卵泡数增加,E2上升,LH下降,FSH接近正常。结论:MBSc移植能通过减少卵巢组织AI而在一定程度上减少环磷酰胺引起的卵巢功能损伤。     

间充质干细胞过滤分离器制备人羊膜间充质干细胞的研究

自然存在的间充质干细胞数量少,限制了其研究应用。依靠自主发明的间充质干细胞过滤分离器,分离制备了人羊膜间充质干细胞,并对制备的干细胞进行了三维培养扩增。结果表明,制备的干细胞形态长势良好,并能诱导分化为类胰岛样组织。与常规方法相比,干细胞收获率提高了8倍以上,且细胞活性状态良好。间充质干细胞过滤分离器可以批量制备高质量的各种间充质干细胞,有利于高效率地建设各种间充质干细胞库,以促进间充质干细胞的研究应用。     

脂肪间充质干细胞对乳腺癌进展的影响

肥胖会导致包括癌症在内的多种恶性疾病。流行病学研究表明,过度肥胖会增加患乳腺癌的风险,并使患者的预后恶化。肥胖患者脂肪组织功能障碍的特点是白色脂肪细胞的肥大和增生。脂肪间充质干细胞是从白色脂肪组织中分离出来的一种间充质干细胞,具有很强的增殖和分化能力。脂肪间充质干细胞作为乳腺癌潜在的肿瘤启动子,通过激活多种细胞内信号促进肿瘤进展和侵袭。然而,关于脂肪间充质干细胞与乳腺癌细胞相互作用的报道并不一致,其可能的分子机制还有待进一步探讨。该综述将重点总结近年来脂肪间充质干细胞影响乳腺癌进展的相关研究,以期为乳腺癌的治疗提供新的策略。     

间充质干细胞移植对放射性肠损伤修复作用的实验研究

探讨了人间充质干细胞(mesenchymal stem cells,MSCs)移植对NOD/SCID小鼠放射性肠损伤的修复作用．将雄性NOD/SCID小鼠随机分为3组,每组6只,即A组为空白对照组,B组为模型组,C组为治疗组．B组和C组小鼠全腹接受5 Gy ⁶⁰Co γ射线单次照射,剂量率为100 cGy/min．照射后B组小鼠经尾静脉注射生理盐水,C组小鼠移植MSCs．于移植后第15天取小鼠空肠标本,通过免疫荧光方法检测MSCs在受损肠道的定植和分化情况．结果表明,治疗组小鼠的生存状况明显好于模型组小鼠,病理切片显示小肠黏膜得到修复,免疫荧光结果显示MSCs可定植于辐射损伤的肠道,并表达波形蛋白(vimentin)和α-SMA．MSCs移植入肠损伤的小鼠体内后可在受损肠道定植,并向间质细胞分化,参与辐射损伤的修复.     

Ghrelin联合三氧化二砷对骨髓间充质干细胞增殖与成骨分化的影响

该文主要探究Ghrelin对三氧化二砷(As2O3)导致的骨髓间充质干细胞(BMSCs)增殖和成骨分化的影响。BMSCs设为对照组、As2O3组、Ghrelin组和联合(As2O3+Ghrelin)组。MTT法检测细胞增殖能力;成骨诱导的第7天和第14天,Real-time PCR及Western blot分别检测成骨相关因子OPN、ALP、RUNX2的mRNA及蛋白表达;第21天,茜素红染色分析钙盐沉积情况。结果显示,细胞增殖能力Ghrelin组>对照组>联合组>As2O3组。与对照组比,As2O3组各因子表达均显著下调(P<0.05),Ghrelin组第14天OPN蛋白表达无显著变化,其余因子均上调(P<0.05);联合组与As2O3组比,第14天OPN基因表达和第7天ALP蛋白表达无显著差异,其余均显著上调(P<0.05)。钙盐沉积:Ghrelin组>对照组>联合组>As2O3组。提示0.5μmol/L As2O3抑制BMSCs增殖和成骨分化,600 ng/mL Ghrelin增强细胞增殖和成骨分化;且Ghrelin能减弱As2O3导致的BMSCs增殖和成骨分化抑制作用。     

脂肪干细胞促进肌腱损伤修复研究进展

脂肪干细胞（adipose-derived stem cells, ADSCs）具有来源广泛、易于获取、体外扩增、免疫原性低等许多优于其他组织来源间充质干细胞（mesenchymal stem cells, MSCs）的特点,越来越多的研究开始关注如何将其应用于组织损伤的治疗与修复。就近几年来国内外ADSCs的发现与分离、向肌腱谱系分化能力以及在肌腱损伤修复方面的应用进行了综述,分析了ADSCs应用于肌腱损伤修复时的优势和面临的问题,并对未来的研究方向进行了展望。     

人脂肪来源间充质干细胞植入受损耳蜗后定向归巢到并分化为毛细胞样细胞

目的：通过将人脂肪来源间充质干细胞（human adipose-derived mesenchymal stem cells,hAD-MSCs）移植到受损小鼠,探讨hAD-MSCs替代缺失/受损毛细胞的可行性。方法：将hAD-MSCs经尾静脉移植入药物致聋后的小鼠体内,用免疫染色及RT-PCR等方法检测移植后hAD-MSCs在耳蜗内的归巢和分化。结果：移植的hAD-MSCs能够定向归巢到受损耳蜗内,并至少存活2周,未观察到对移植细胞的免疫排斥反应。有少量细胞定位于耳蜗感觉上皮并表达毛细胞特异性抗体myosin 7a。结论：hAD-MSCs移植入药物性致聋小鼠后,能够定向归巢到耳蜗内,并分化为内耳毛细胞样细胞,是一种针对内耳损伤及退变性疾病治疗的潜在细胞来源。     

Mesenchymal Stem Cells Attenuate Peritoneal Injury through Secretion of TSG-6

Background

Mesothelial cell injury plays an important role in peritoneal fibrosis. Present clinical therapies aimed at alleviating peritoneal fibrosis have been largely inadequate. Mesenchymal stem cells (MSCs) are efficient for repairing injuries and reducing fibrosis. This study was designed to investigate the effects of MSCs on injured mesothelial cells and peritoneal fibrosis.

Methodology/Principal Findings

Rat bone marrow-derived MSCs (5 ×10⁶) were injected into Sprague-Dawley (SD) rats via tail vein 24 h after peritoneal scraping. Distinct reductions in adhesion formation; infiltration of neutrophils, macrophage cells; number of fibroblasts; and level of transforming growth factor (TGF)-β1 were found in MSCs-treated rats. The proliferation and repair of peritoneal mesothelial cells in MSCs-treated rats were stimulated. Mechanically injured mesothelial cells co-cultured with MSCs in transwells showed distinct increases in migration and proliferation. In vivo imaging showed that MSCs injected intravenously mainly accumulated in the lungs which persisted for at least seven days. No apparent MSCs were observed in the injured peritoneum even when MSCs were injected intraperitoneally. The injection of serum-starved MSCs-conditioned medium (CM) intravenously reduced adhesions similar to MSCs. Antibody based protein array of MSCs-CM showed that the releasing of TNFα-stimulating gene (TSG)-6 increased most dramatically. Promotion of mesothelial cell repair and reduction of peritoneal adhesion were produced by the administration of recombinant mouse (rm) TSG-6, and were weakened by TSG-6-RNA interfering.

Conclusions/Significance

Collectively, these results indicate that MSCs may attenuate peritoneal injury by repairing mesothelial cells, reducing inflammation and fibrosis. Rather than the engraftment, the secretion of TSG-6 by MSCs makes a major contribution to the therapeutic benefits of MSCs.     

Bone Mesenchymal Stem Cells Contributed to the Neointimal Formation after Arterial Injury

Objectives

Recent findings suggest that in response to repair-to-injury bone marrow mesenchymal stem cells (BMSCs) participate in the process of angiogenesis. It is unclear what role BMSCs play in the structure of the vessel wall. In present study, we aimed to determine whether BMSCs had the capacity of endothelial cells (ECs).

Methods

BMSCs were separated and cultured. FACS and RT-PCR analysis confirmed the gene expression phenotype. The capacity of migration and adhesion and the ultrastructure of BMSCs were examined. The effect of BMSCs transplantation on the vascular repair was investigated in a murine carotid artery-injured model.

Results

BMSCs could express some markers and form the tube-like structure. The migration and adhesion capacity of BMSCs increased significantly after stimulated. In addition, BMSCs had the intact cell junction. In vivo the local transfer of BMSCs differentiated into neo-endothelial cells in the injury model for carotid artery and contributed to the vascular remodeling.

Conclusion

These results showed that BMSCs could contribute to neointimal formation for vascular lesion and might be associated with the differentiation into ECs, which indicated the important therapeutic implications for vascular diseases.     

Adipose-Derived Mesenchymal Stem Cells Transplantation Alleviates Renal Injury in Streptozotocin-Induced Diabetic Nephropathy

Previous studies have illustrated that bone marrow-derived mesenchymal stem cell (BMMSC) transplantation has therapeutic effects on diabetes and can prevent mice from renal damage and diabetic nephropathy (DN). Moreover, adipose-derived MSCs possess similar characteristics to BMMSCs. We investigated the effect of ADMSC transplantation on streptozotocin (STZ)-induced renal injury. Diabetes was induced in rats by STZ injection. After ADMSC treatment, renal histological changes and cell apoptosis were evaluated as were the expression of apoptosis-related proteins, Wnt/β-catenin pathway members, and klotho levels. We found that ADMSCs improved renal histological changes. Next, NRK-52E cells were exposed to normal glucose (NG; 5.5 mM glucose plus 24.5 mM mannitol)/high glucose (HG) or ADMSCs, and then measured for changes in the aforementioned proteins. Similarly, changes in these proteins were also determined following transient transfection of klotho siRNA. We found that both ADMSC transplantation and co-incubation reduced the rate of cellular apoptosis, decreased Bax and Wnt/β-catenin levels, and elevated Bcl-2 and klotho levels. Interestingly, klotho knockdown reversed the effects of ADMSCs on the expression of apoptosis-related proteins and Wnt/β-catenin pathway members. Taken together, ADMSCs transplantation might attenuate renal injury in DN via activating klotho and inhibiting the Wnt/β-catenin pathway. This study may provide evidence for the treatment of DN using ADMSCs.