IL-6在猪肝细胞与骨髓间充质干细胞共培养中的作用

目的 探索IL-6在猪肝细胞与骨髓间充质干细胞(mesenchymal stem cells,MSCs)体外共培养中的作用.方法 自中华实验猪髂前上棘抽取骨髓(3只),采用密度梯度离心法分离单个核细胞,贴壁传代培养至第3代;原位两步胶原酶法分离猪肝细胞后与MSCs按照2:1比例使用Millicell小室培养,检测共培养中肝细胞功能,观察IL-6、TNF-α、TGF-α的分泌水平变化.结果 肝细胞活率＞95%.共培养组肝细胞白蛋白分泌水平和尿素合成能力均显著高于单纯肝细胞组(P＜0.05).共培养组IL-6浓度显著高于对照组(P＜0.01).中和IL-6后共培养体系白蛋白、尿素合成水平明显下降(P＜0.01).结论 MSCs通过分泌IL-6改善共培养体系中肝细胞功能.     

脐带间充质干细胞旁分泌物质对暴发性肝衰竭大鼠肝功能及肝细胞增殖的影响

目的 探索人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,UC-MSCs)旁分泌物质对实验性暴发性肝衰竭大鼠的治疗作用,研究其对大鼠肝功能及肝细胞增殖的影响.方法 体外分离培养人脐带间充质于细胞,流式细胞仪检测UC-MSCs的表面标志,制备含有UC-MSCs旁分泌物质的条件培养基(MSC-CM),腹腔注射D-氨基半乳糖制备暴发性肝衰竭大鼠模型.实验分为3组:MSC-CM组、生理盐水(NS)组和促肝细胞生长素(PHGF)组.在模型制备后24 h从大鼠尾静脉分别注射三组治疗药物.每组8只大鼠治疗后12 h、24 h、36 h、60 h分别经内眦取血测定谷丙转氨酶(ALT)和总胆红素(TBIL)含量.每组另取5只大鼠在治疗后36 h取肝脏组织制备切片进行PCNA免疫组化染色,检测大鼠肝细胞增殖情况.观察并记录各组大鼠的生存状态及生存时间.结果 MSC-CM组及PHGF组大鼠治疗后24 h ALT及TBIL的含量均低于NS组(P＜0.05),MSC-CM组与PHGF组比较差异无统计学意义.大鼠治疗后36 h肝脏切片PCNA染色显示,MSC-CM组和PHGF组PCNA肝细胞阳性数显著高于NS组(P＜0.01),MSC-CM组与PHGF组比较差异无统计学意义.生存分析显示,MSC-CM组和PHGF组大鼠的生存率高于NS组(P=0.049),MSC-CM组与PHGF组比较差异无统计学意义.结论 人脐带间充质干细胞的旁分泌物质可以刺激暴发性肝衰竭大鼠肝细胞增殖,改善暴发性肝衰竭大鼠的肝功能,为暴发性肝衰竭的治疗提供了一种新途径.

Abstract：

Objective To investigate the therapeutic effect of human umbilical cord mesenchymal stem cell-paracrine substance on fulminant hepatic failure (FHF) rat, and to study the effect on liver function and hepatocyte proliferation. Methods Mesenchymal stem cells(MSCs)were separated from human umbilical cord, and surface makers of cells were detected by flow cytometry. Human umbilical cord mesenchymal stem cells-conditioned medium(MSC-CM) was prepared. FHF rat model was induced by intraperitoneal injection of D-galactosamine and they were randomly diveded into three groups: MSC-CM group, NS group, PHGF group. 24 h later, 1 ml MSC-CM, 1 ml 0. 9% NaCl solution and lml PHGF solution was injected into the tail vein of MSC-CM, NS, and PHGF rats, respectively. In each group (n=8 per group), blood samples were collected at 12, 24, 36, and 60 h after treatment from inner canthus for analysis of blood ALT and TBIL levels. We used five rats per group for tissue collection after sacrifice at 36 h after treatment and 10 animals per group for survival analysis. PCNA immunohistochemical staining was used in the sections of liver tissue to detect hepatocyte proliferation. Results 24 h after treatment, the levels of ALT and TBIL in the MSC-CM and PHGF groups were lower than those in the NS group(P＜0. 05), but there was no significant difference between the MSC-CM and PHGF groups. There were more PCNA-positive hepatocytes in the MSC-CM and PHGF groups than in the NS group(P＜0.01), but there was no significant difference between MSC-CM and PHGF group. Survival analysis found that the survival rate of rats in the MSC-CM and PHGF groups was higher than that of rats in the NS group (P=0. 049), but there was no significant difference between the MSC-CM and PHGF group. Conclusions The paracrine substance of human umbilical cord mesenchymal stem cells can stimulate hepatocyte proliferation and improve liver function of FHF rats, potentially creating a new avenue for the treatment of FHF.     

人脐带间充质干细胞分泌物对肝细胞增殖和凋亡的影响

Objective To investigate the effect of human umbilical cord mesenchymal stem cell paracrine substance on proliferation and apoptosis of liver cells in vitro. Methods Mesenchymal stem cells (MSC)were separated from human umbilical cord with type Ⅳ collagenase and trypsogen digestion method and cultured in vitro. The human umbilical cord mesenchymal stem cells-conditioned medium(MSC-CM) which contain paracrine substance of human umbilical cord mesenchymal stem cells (HUCMSC) was prepared. Hepatocytes were isolated from SD rats by low concentration collagenase perfusion procedure. There were three groups in the experiment, control group, 2% MSC-CM group and 8% MSC-CM group. The proliferation of normal hepatocytes were assayed with MTT method. We detected the urea and albumin level in culture supernatant to assay the hepatocyte function under different concentration MSC-CM. Hepatocytes were induced for apoptosis by Actinomycin D and tumor necrosis factor alpha (TNF-α),and the apoptosis effect of different concentration MSC-CM was assayed with LIVE/DEAD Viability/Cytotoxicity Kit. Results The MTT assay showed that the absorbance of 2% MSC-CM group was significantly increased (P＜0. 01), and the urea and albumin levels of 2 % MSC-CM group were also significantly increased when compared with control group(P＜0. 01).LIVE/DEAD Viability/Cytotoxicity Kit revealed that hepatocyte survival rate of 2 % MSC-CM group was increased when compared with control group(P＜0. 05), there were no significant differences in above-mentioned experiments when 8% MSC-CM group compared with control group. Conclusion The low concentration MSC-CM could stimulate normal hepatocyte proliferation, inhibit impaired hepatocyte apoptosis and improve hepatocyte function.     

人脐带间充质干细胞分泌物对肝细胞增殖和凋亡的影响

Objective To investigate the effect of human umbilical cord mesenchymal stem cell paracrine substance on proliferation and apoptosis of liver cells in vitro. Methods Mesenchymal stem cells (MSC)were separated from human umbilical cord with type Ⅳ collagenase and trypsogen digestion method and cultured in vitro. The human umbilical cord mesenchymal stem cells-conditioned medium(MSC-CM) which contain paracrine substance of human umbilical cord mesenchymal stem cells (HUCMSC) was prepared. Hepatocytes were isolated from SD rats by low concentration collagenase perfusion procedure. There were three groups in the experiment, control group, 2% MSC-CM group and 8% MSC-CM group. The proliferation of normal hepatocytes were assayed with MTT method. We detected the urea and albumin level in culture supernatant to assay the hepatocyte function under different concentration MSC-CM. Hepatocytes were induced for apoptosis by Actinomycin D and tumor necrosis factor alpha (TNF-α),and the apoptosis effect of different concentration MSC-CM was assayed with LIVE/DEAD Viability/Cytotoxicity Kit. Results The MTT assay showed that the absorbance of 2% MSC-CM group was significantly increased (P＜0. 01), and the urea and albumin levels of 2 % MSC-CM group were also significantly increased when compared with control group(P＜0. 01).LIVE/DEAD Viability/Cytotoxicity Kit revealed that hepatocyte survival rate of 2 % MSC-CM group was increased when compared with control group(P＜0. 05), there were no significant differences in above-mentioned experiments when 8% MSC-CM group compared with control group. Conclusion The low concentration MSC-CM could stimulate normal hepatocyte proliferation, inhibit impaired hepatocyte apoptosis and improve hepatocyte function.     

血清浓度对人髓核间充质干细胞生物学活性的影响

目的:体外观察、研究不同血清浓度对人正常髓核间充质干细胞(nucleus pulposus mesenchymal stem cells,NPMSCs)生物学活性的影响.方法:选取先天性脊柱侧凸矫形患者手术切除的髓核组织(Pfirrmann分级为Ⅰ级或Ⅱ级),体外分离培养NPMSCs并传代培养.利用流式细胞仪分别对P3...     

联合间充质干细胞培养对大鼠胰岛的保护作用

目的 探讨间充质干细胞与胰岛联合培养在延长胰岛体外存活时间、保护胰岛功能方面的作用.方法 以4周龄Wistar大鼠作为供体,分离纯化骨髓间充质干细胞并培养鉴定;Histopaque-1077一步法分离纯化Wistar大鼠胰岛;将胰岛培养分为单纯胰岛基础培养、单纯胰岛高糖培养、胰岛与间充质干细胞联合基础培养、胰岛与间充质于细胞联合高精培养4组,每组又分3个时间段(3、7、14 d)观察胰岛形态变化及胰岛存活率,酶联免疫吸附法检测胰岛素分泌量及刺激指数.结果 骨髓间充质干细胞传代培养3代后,流式细胞术检测CD45及CD90,二者荧光强度差异有统计学意义(P＜0.05);联合培养组3、7、14 d的胰岛存活率均高于单纯培养组(P＜0.01);高糖刺激下联合培养组7 d的胰岛素分泌量及刺激指数均高于单纯培养组(P＜0.01).结论 间充质干细胞与胰岛联合培养可以明显延长胰岛体外存活时间并保持其活性,对胰岛具有良好的保护作用.

Abstract：

Objective To observe the effect of mesenchymal stem cells (MSCs) on enhancing rat islets viability and function in vitro by a pretransplant co-culture. Methods 4-week-old Wistar rats were used as donors, bone mesenchymal stem cells were isolated and subcultured. Islets of Wistar rats were isolated and purified by one-step single-layer Histopaque-1077. Then islets were divided into four groups randomly, 2 groups co-cultured with mesenchymal stem cells (MSCs) (one using low-glucose medium; the other using high-glucose medium ); 2 groups were cultured alone (low-glucose medium; high-glucose medium), each group was further stratified into 3 subgroups(3, 7, 14 d); the survival and functionality of these islets were observed and evaluated. The amount of glucose stimulated secreted insulin were measured wth a rat/mouse insulin enzyme-linked immunosorbent assay (ELISA) kit and stimulation index was also calculated. Results Compared with those not co-cultured, islets co-cultured with MSCs demonstrated significantly higher survival rates and viability both in 3th, 7th and 14th day ( P ＜ 0. 01 ); furthermore, cocultured islets revealed higher levels of glucose stimulated insulin secretion and secretion indexes in 7th day (P＜0.01). Conclusion Rat islet cells co-cultured with MSCs have longer in vitro survival and better functions.     

骨髓间充质干细胞维持猪肝细胞形态与功能的实验研究

目的 建立猪肝细胞与骨髓间充质干细胞(MSCs)体外共培养体系,为生物人工肝的构建提供理想细胞来源.方法 自中华实验猪(n=3)髂前七棘抽取骨髓,采用密度梯度离心法分离单个核细胞,贴壁传代培养至第3代;原位两步胶原酶法分离猪肝细胞后与MSCs随机混合培养,观察共培养肝细胞形态和功能的变化水平.结果 第3代MSCs纯度>90%,肝细胞活率>95%,纯度>99%.共培养组肝细胞迅速黏附于MSCs表面,在三维空间呈球形聚集生长.异质细胞间出现细胞连接,超微结构与正常肝细胞接近.共培养组肝细胞白蛋白分泌水平和尿素合成能力自第1天培养起均显著高于对照组(P<0.05),并在第2天达到高峰.结论 猪肝细胞与MSCs共培养可维持肝细胞形态与功能,使构建功能性生物人工肝成为可能.     

骨髓间质干细胞培养上清液调控肝细胞的体外研究

目的探讨骨髓间质干细胞(bone marrow mesenchymal stem cells,BMSC)培养上清液对肝细胞增殖、凋亡的影响,初步探讨BMSC治疗肝纤维化的旁分泌机制。方法采用密度梯度离心及贴壁细胞分离相结合提取BMSC。培养48h的BMSC细胞培养上清液按BMSC不同处理(培养)时间和含量两种方法建组。不同处理时间建组:取培养48hBMSC上清液处理肝细胞(完全培养),分为处理24h、48h、72h组,对照组为1640培养基处理24h的肝细胞。不同含量BMSC建组:实验1组为完全BMSC培养上清液(含肝细胞的6孔板中每孔加入BMSC上清液2ml)处理肝细胞;实验2组为部分BMSC培养上清液(每孔加BMSC上清液1ml+1640培养基1ml)处理肝细胞;对照组为完全细胞培养基(每孔加1640培养基2ml)处理肝细胞。用酶联免疫吸附试验(ELISA)试剂盒检测BMSC旁分泌肝细胞生长因子(hepatocyte growth factor,HGF)情况及培养时间对其的影响;用流式细胞仪观察肝细胞细胞分期和检测凋亡细胞数;用蛋白免疫印迹法(Western-blot)检测样本上清液中白蛋白的表达。结果 BMSC分泌HGF,其含量变化具有时间依赖性。加入BMSC培养上清液后,与对照组相比,实验1组中的培养上清液可以明显促进肝细胞增殖、抑制其凋亡,并随着处理时间的延长而增加(处理72h>处理48h>处理24h,均为P<0.05);实验组(1组、2组)的白蛋白分泌较对照组上调,且随着处理时间的延长白蛋白含量增加。结论 BMSC有可能通过分泌HGF促进肝细胞增殖、抑制凋亡,促进白蛋白分泌,从而抑制肝脏纤维化。     

人脐带间充质干细胞应用研究进展

间充质干细胞(mesenchymal stem cells,MSCs)是来源于中胚层的一种具有自我更新多向分化潜能的成体干细胞,存在于全身结缔组织和器官间质中.人脐带间充质干细胞成功克服了骨髓间充质干细胞含量低、受年龄影响大等诸多限制,而且具有来源广泛、免疫原性低等优点.Mitchell[1]首次自脐带华尔通氏胶中成功地分离出成纤维样细胞并证实其具有多能干细胞的分化潜能以后,对于脐带来源的MSCs便引起了人们的广泛关注.近年来实验研究表明脐带间充质干细胞在组织工程、神经系统、肝脏疾病、心血管系统、免疫疾病、肿瘤疾病等方面取得了巨大的进展.本文主要在脐带间充质干细胞应用方面予以综述.     

间充质干细胞移植治疗多器官功能不全综合征的研究

目的 观察骨髓间充质干细胞(mesenchymal stem cells,MSC)移植对多器官功能不全综合征(multiple organ dysfunction syndrome,MODS)的影响,探讨骨髓间充质干细胞用于多器官功能不全综合征治疗的应用前景.方法 建立性休克合并内毒素休克引起的官功能不全综合征模型.兔骨髓间充质干细胞体外扩增、鉴定、分化、慢病毒转基因标记GFP、静脉移植,通过PCR和病理切片观察MSCs对MODS兔的作用.结果 与对照组相比,模型移植组病理切片发现肝、肾、肺等脏器有慢病毒转基因标记有GFP的MSCs.结论 骨髓间充质干细胞具有良好的生物学活性,移植后通过整合能替代凋亡坏死细胞并对多器官功能不全综合征起到治疗作用.     

CD34+细胞在干细胞旁分泌下向内皮细胞分化的研究

目的 探讨人脐血CD34+细胞在脐带间充质干细胞(UC-MSCs)旁分泌作用下向内皮细胞诱导分化的可行性.方法 收集20份脐血,体积(103.80±19.77)ml.免疫磁珠(MACS)分选CD34+细胞;取脐带用消化贴壁法获得UC-MSC.流式鉴定干细胞表型.实验分单纯培养组、诱导组、共培养组.结果 流式鉴定CD34+细胞纯度(95.02±3.81)%.培养14 d流式检测共培养组表达CD31、CD144、VWF分别为(65.43±5.61)%、(54.40±4.13)%、(47.53±3.96)%(与单纯培养组比较P＜0.05),部分表达CD34,阴性表达CD45,这与诱导组及成熟脐静脉内皮细胞表达率一致.结论 UC-MSCs旁分泌作用与外源性细胞因子都具有促分化作用,均能使脐血CD34+细胞向内皮细胞分化.

Abstract：

Objective To study whether the paracrine action of umbilical cord-derived mesenchymal stem cells (UC-MSC) can induce differentiation of human umbilical cord blood-derived CD34+ cells into endothelial cells in vitro. Methods The 20 fresh umbilical cord blood samples were collected with volume of (103. 80 ± 19. 77) ml. CD34+ cells were isolated from the mononuclear cells by magnetic activated cell sorting system (MACS) , and mesenchymal stem cells (MSCs) were isolated from umbilical cord by collagenase and trypsin digestion. Three groups were set up: CD34+ cells pure culture group, cytokineinduced group and two stem cells co-culture group with noncontact. Results The average purity of enriched CD34+ cells as assessed by FACS was (95. 02 ± 3. 81) %. Freshly isolated CD34+ cells were small and round which suspended in culture medium. Attached cord-like structure cells of CD34+ cells appeared after 7 days coculture with noncontacted MSC, and when the CD34+ cells grew into large number, they formed colonies. These cells expressed endothelial specific markers, including CD144 (54. 40 ±4. 13)% ,vWF (47. 53 ± 3. 96) % , CD31 (65. 43 ± 5. 61) % ( P ＜ 0. 05, as compared with CD34+ cells pure culture group) , partially expressed CD34 and, the leukocyte common antigen CD45 was negatively expressed.Conclusion Human umbilical cord blood-derived CD34+ cells could be induced into endothelial cells under the paracrine action of umbilical cord-derive mesenchymal stem cells which has the same effect of cytokines.     

人脐带间充质干细胞获得和纯化及向软骨细胞分化能力的研究

目的 研究脐带间充质干细胞获得纯化的高效方法及向软骨细胞的分化能力.方法 取人的正常分娩或剖腹产胎儿的脐带,用胶原酶和胰酶初步消化后,将流式细胞仪筛选得到的CD45(-)、CD90(+)细胞作为原代细胞进行培养,并用复合胶原酶差速消化法逐级传代、纯化细胞,每次传代后取部分细胞用作流式细胞仪分析细胞的CD45、CD90阳性比率;对P3代细胞向软骨细胞进行诱导.结果 流式细胞仪检测提示,采用流式细胞仪进行初次筛选并用复合胶原酶差速消化法逐级传代获得的P1、P2、P3代细胞中CD90的阳性率为(80.86±7.85)%、(95.86±3.28)%、(97.15±1.43)%,而CD45的阳性率为(2.53±0.28)%、(0.97±0.48)%、(0.05±0.01)%;向软骨细胞诱导结果提示P3代细胞有向终末软骨细胞分化能力,具有干细胞的特性.结论 采用流式细胞仪进行初次筛选并用复合胶原酶差速消化法能快速获得高纯度的具有向终末软骨细胞分化能力的人脐带间充质干细胞.     

hBMP-2基因改良修饰骨髓间充质干细胞和人脐血间充质干细胞研究

目的研究hBMP-2基因改良修饰骨髓和人脐血间充质干细胞的方法,并比较修饰结果。 方法联合应用密度梯度离心法和贴壁培养法分离、培养骨髓和人脐血间充质干细胞,通过高效非脂质体试剂X-treme GENE介导重组hBMP-2质粒转染骨髓和人脐血间充质干细胞,倒置荧光显微镜检测荧光强度并计算转染效率,qPCR检测hBMP-2及软骨连接蛋白在两种细胞中的表达,免疫组化检测细胞中Ⅱ型胶原的变化。 结果成功分离、培养出骨髓和人脐血间充质干细胞,两种细胞具有不同的生长特性。高效非脂质体试剂介导的重组hBMP-2质粒成功转染骨髓和人脐血间充质干细胞,转染骨髓间充质干细胞效率[（18.44±5.94）%]低于人脐血间充质干细胞[（27.74±7.59）%],且差异有统计学意义（t=3.027,P<0.05）。转染后的两种细胞均检测到hBMP-2、软骨连接蛋白及Ⅱ型胶原的表达。 结论hBMP-2基因可以有效改良修饰骨髓和人脐血间充质干细胞,且能促进其向软骨细胞方向分化。     

血小板裂解液对人脐带间充质干细胞体外成软骨分化的影响

目的探讨血小板裂解液(platelet lysate,PL)在体外定向诱导人脐带间充质干细胞(human umbilical cord derived mesenchymal stem cells,hUCMSCs)分化成软骨细胞中的作用。方法取健康产妇自愿捐赠脐带,采用胶原酶消化法分离hUCMSCs,体外培养扩增,流式细胞仪进行细胞表型鉴定。根据加入诱导培养基成分不同将实验分为以下3组:A组为H-DMEM培养基、10%FBS及10%PL,B组为H-DMEM培养基、10%FBS、10 ng/mL TGF-β1、1×10-7 mol/L地塞米松、50μg/mL维生素C及1%胰岛素铁硒传递蛋白(insulin-transferrin-selenium,ITS),C组为H-DMEM培养基、10%FBS、10 ng/mL TGF-β1、1×10-7 mol/L地塞米松、50μg/mL维生素C、1%ITS及10%PL。诱导培养2周,甲苯胺蓝染色检测各组软骨细胞基质的分泌,免疫荧光检测软骨特异性Ⅱ型胶原表达,半定量RT-PCR检测蛋白聚糖(Aggrecan)和Ⅱ型胶原表达。结果分离得到的hUCMSCs不表达造血细胞的表面标记CD45、CD34和HLA-DR,而表达黏附分子和MSCs表面标记CD44、CD105和CD146。甲苯胺蓝染色和Ⅱ型胶原免疫荧光染色示C组呈阳性,B组呈弱阳性,而A组均呈阴性。半定量RT-PCR检测示Aggrecan和Ⅱ型胶原在B、C组中均有表达,A组中未见表达;C组Aggrecan mRNA和Ⅱ型胶原mRNA表达明显高于B组,差异均有统计学意义(P<0.05)。结论单纯10%PL不能诱导hUCMSCs成软骨分化,但它可当作成软骨诱导培养基的辅助添加剂,对hUCMSCs成软骨分化有明显促进作用,为构建组织工程软骨提供了新的可利用条件。     

淫羊藿苷诱导人脐带间充质干细胞分化为成骨细胞的实验研究

目的 研究淫羊藿苷对脐带间充质干细胞的生物学作用,以及诱导其向成骨细胞分化.方法体外分离、培养及扩增人脐带间充质干细胞,免疫组化技术检测人脐带间充质干细胞(huCMSCs)表面特异标志物表达.取第3代细胞,设立2组,空白对照组加入基础培养基,实验组加入淫羊藿苷诱导.用倒置光学显微镜、碱性磷酸酶染色、碱性磷酸酶活性测定细胞.进行对比,研究人脐带间充质干细胞增殖和分化规律.结果 免疫组化结果显示huCMSCs表面标记CD44、CD34阴性,检测结果符合huCMSCs的特征.随着培养天数的增加,淫羊藿苷组诱导后细胞液中成骨细胞特征性碱性磷酸酶强阳性表达及钙沉积.淫羊藿苷组细胞内ALP含量与对照组比较差异有统计学意义(P＜0.05).结论 huCMSCs可以在体外采用组织块贴壁培养法培养成功,淫羊藿苷对人脐带间充质干细胞的增殖和成骨分化具有促进作用,可作为骨性诱导因子.

Abstract：

Objective To study the biological effect of Icariine on human umbilical cord derived mesenchymal stem cells(huCMSCs) and inducing them to differentiate into osteoblasts. Methods MSCs were isolated and expanded in vitro and their surface antigens of huCMSCs were detected by immunohistochemistry. HuCMSCs were assigned into two groups. In the blank control group, huMSCs were incubated in basic medium. In the experimental group huMSCs were incubated in Icariine medium, The proliferation and differentiation of huMSCs were examined by inverted microscope, Alkaline phosphatase (ALP) staining and the determination of ALP activities. Results HuCMSCs were strongly positive for CD44, and negative for CD34; strongly positive expression for ALP and calcium deposition was detected from the second day to the fourth day in Icariine group; blank control group and Icariine group had significant differences (P ＜ 0.05).Conclusion HuCMSCs can be successfully cultured from the adherent tissue pieces, Icariine enhances proliferation and osteogenic differentiation of huCMSCs and be used as an osteoinductive factor.     

人脐带间充质干细胞复合去细胞神经基膜管修复大鼠坐骨神经缺损

目的 探讨诱导后的脐带间充质干细胞作为组织工程种子细胞修复大鼠坐骨神经缺损的可行性.方法 从正常足月新生儿脐带中分离培养间充质干细胞并诱导分化为神经样细胞,与去细胞神经基膜管共培养以构建组织工程神经;用30只健康成年雄性SD大鼠建立坐骨神经缺损(10 mm)的动物模型并随机分成3组:A组为脐带间充质干细胞复合去细胞神经基膜管组,B组为单纯去细胞神经基膜管组,C组为自体神经桥接组.术后10周通过神经电生理检测、组织学观察等评测效果.结果 在局部观察和肌肉测量、神经电生理检测、组织学观察等方面,脐带间充质干细胞复合去细胞神经基膜管组(A组)神经再生及肢体功能情况良好,效果接近于自体神经桥接组(C组),明显优于单纯去细胞神经基膜管组(B组).结论 脐带间充质干细胞复合去细胞神经基膜管构建的组织工程神经可有效促进大鼠坐骨神经缺损(10 mm)的修复.     

Human umbilical cord mesenchymal stem cells co-culture ameliorates podocytic apoptosis: a possible role of HGF

Objective To explore the effects of human umbilical cord mesenchymal stem cells(HUC-MSCs) on podocytic apoptosis and injury induced by high glucose (HG) and the underlying mechanisms. Methods Podocytes were divided into six groups according to treatment: ⑴ normal glucose group (NG); ⑵ high glucose group (HG); ⑶ mannitol control group (NG+Ma); ⑷ HUC-MSC co-culture group (HUC-MSCs); ⑸ recombinant human hepatocyte growth factor treatment group (rhHGF); ⑹ neutralizing antibody group(HGF-NtAb). Cytometry and Hoechst staining were used to detect the apoptosis rates. Western blot was used to measure the ratio of active PARP to total PARP and the level of Bcl-2. Immunofluorescence was used to study podocytic apoptosis and injury. Neutralizing antibody (NtAb) was used to block its function and the recombinant cytokine was added to induce its function. Results High glucose induced podocytic apoptosis in a time-dependent manner, HUC-MSCs co-culture decreased the podocytic apoptosis rate and the expression of PARP (all P﹤0.05), increased the expression of Bcl-2, prevented the reduced expression and maintained the normal arrangement of podocytic podoplanin. The rhHGF prevented podocytic apoptosis and injury similarly to HUC-MSCs, the beneficial effect of HUC-MSC decreased when blockade of HGF. Conclusions HUC-MSCs co-culture ameliorates podocytic apoptosis and injure induced by HG, probably through secreting soluble HGF.     

Distribution of infused umbilical cord mesenchymal stem cells in a rat model of renal interstitial fibrosis

Abstract

Aims: Stem cell transplantation for the treatment of kidney diseases is dependent on choice of transplant pathway. We evaluated the safety of human umbilical cord mesenchymal stem cells through peripheral infusion and their distribution in a rat model of renal interstitial fibrosis (RIF). Method: Cryopreserved umbilical cord mesenchymal stem cells were infused via tail vein injection into rats with unilateral ureteral obstruction and Sham-operated. Blood, kidney, heart, liver, spleen and lung were collected at 14, 21, and 28 days after infusion. Testing included microscopic observation of kidney morphological changes and immunohistochemical testing to identify and count the number of MAB1281 (labeled human cells) positive cells in the heart, liver, spleen, lungs, and kidneys of different treatment groups. Results: There was no significant difference in the Sham-operated group and Sham-operated?+?cell transplantation group at different time points. Human cells were identified mainly in the lungs, spleen, and kidney. The number of human umbilical cord mesenchymal stem cells in the kidney was greater in the unilateral ureteral obstruction?+?cell transplantation group, compared to the Sham-operated?+?cell transplantation group. human umbilical cord mesenchymal stem cells were mainly located in the interstitium of the left kidney. These results suggest that infused mesenchymal stem cells were primed to engraft a damaged kidney, especially damaged renal interstitium. Conclusions: Intravenous infusion of exogenous umbilical cord mesenchymal stem cells is feasible and safe. Infused mesenchymal stem cells can reach damaged kidney tissues with obstructive RIF after a vein graft.     

脐带间充质干细胞分化为内皮细胞促进血管新生

目的 探讨脐带间充质干细胞能否在体内外分化为内皮细胞,参与血管新生.方法 体外实验采用内皮细胞生长因子和碱性成纤维细胞生长因子对脐带间充质于细胞进行诱导,观察其形态变化.体内实验将脐带间充质干细胞移植到小鼠后肢缺血模型中,采用免疫组织化学鉴定细胞在体内的迁移和分化,激光多普勒血流成像仪鉴定缺血局部血流恢复情况.结果 在体外脐带间充质干细胞能够形成血管网样结构.在体内脐带间充质干细胞能够分化为内皮细胞,表达CD31抗体,参与实验组的动物血管重建.与小鼠的血管网络发生整合,实验组的动物后肢血流灌注明显好于对照组.结论 脐带间充质干细胞能够分化为内皮细胞,参与血管新生,为治疗性血管新生提供新的细胞选择.