无血清培养基SYL-SF对间充质干细胞的培养和扩增作用

目的探讨新型无血清培养基SYL-SF对脐带间充质干细胞的体外培养和扩增作用。方法机械分离方法获得脐带间充质干细胞,从P1代开始将含小牛血清的原代培养基SYL-NBCS更换为实验组SYL-SF和对照组MSCM-SF两种无血清培养基对细胞进行培养、传代、扩增和维持。利用Alexa Fluor 488 annexin V检测细胞凋亡率,CCK-8法比较传代细胞的增殖效率,并对细胞进行形态、表型、染色体核型和分化能力等方面的检测和评价。结果分离得到的单个脐带间充质干细胞在原代培养基SYL-NBCS中培养8~15d可获得原代(P0)细胞,更换为无血清培养基后,SYL-SF组细胞凋亡率明显低于MSCM-SF组;在细胞传代过程中SYL-SF组细胞增殖能力较强且维持MSCs特异性表面标志和向脂肪、骨、软骨等多向分化的潜能,并且在传代过程中维持正常染色体核型。结论 SYL-SF无血清培养基可以对UC-MSCs进行良好地体外扩增和维持。     

CM-Dil体外标记人脐带间充质干细胞传代示踪的可行性

背景:掌握人脐带间充质干细胞的移植示踪方法是研究其生物学特性的关键。目的:观察用CM-Dil标记人脐带间充质干细胞及在体外传代示踪的可行性。方法:采用酶消化法体外分离培养人脐带间充质干细胞,通过流式细胞仪检测细胞免疫表型和细胞周期、体外成脂成骨诱导鉴定该细胞。将第5代细胞用CM-Dil标记,并将细胞传代,荧光显微镜观察体外标记情况。结果与结论:第3代人脐带间充质干细胞强表达CD44,CD29,低表达CD106,不表达CD34、CD40;有80%以上的细胞处在G0/G1期,成脂成骨诱导后,油红O染色和碱性磷酸酶染色分别阳性。CM-Dil标记人脐带间充质干细胞细胞标记率达90%以上,体外传代后荧光强度逐渐减退,传8代后,荧光基本消失。说明人脐带间充质干细胞增殖、分化能力强,CM-Dil标记细胞示踪方法简单易行。     

人脐带间充质干细胞体外培养的生物学特性与安全性研究

目的 观察体外长期培养人脐带间充质干细胞的生物活性变化特点及评估其安全性.方法 在体外环境中对人脐带来源的间充质干细胞进行了长期的标准化培养,并延长了培养时间,对细胞的表面标记、端粒酶活性、细胞周期、凋亡率、核型及裸鼠成瘤性进行分析.结果 常规体外培养下,间充质干细胞的表面标记持续稳定.端粒酶分析表明随着传代次数的增加,细胞的端粒酶活性表达逐渐降低,细胞周期正常未出现异常凋亡或癌变.核型分析表明细胞增殖过程中细胞核型无异常变化.裸鼠成瘤性实验表明细胞无成瘤性风险.结论 间充质于细胞在体外标准化培养至第15代,细胞分化能力相对减弱.25代以后生长缓慢细胞生物学特征.培养到第30代,表型及基因型无明显变化,未发生恶性转变.     

人脐带间充质干细胞联合体外受精胚胎移植治疗卵巢早衰成功妊娠一例

近年研究发现,人脐带间充质干细胞可修复损伤的卵巢功能。本文报道了1例人脐带间充质干细胞联合体外受精胚胎移植治疗卵巢早衰并成功妊娠的病例,提出人脐带间充质干细胞治疗可成为卵巢早衰患者一种新的、有效的治疗方法。     

人脐带间充质干细胞在3种不同培养体系中的生长状况及腺病毒感染效率

背景:目前所报道的脐带间充质干细胞体外培养条件及培养效率不尽相同,尚缺乏统一标准。而且由于不同来源的间充质干细胞生物学特征尚有一定差异,因此建立脐带间充质干细胞简便、高效的培养体系十分必要。目的:观察人脐带来源的间充质干细胞在体外不同培养体系中的生长状态,以及不同腺病毒感染的效率。方法:采用胶原酶消化法从正常足月新生儿脐带中分离出间充质干细胞,贴壁法纯化培养,细胞贴壁后利用低糖DMEM,MesenPRO RSTM Medium和STEMPRO MSC SFM这3种培养体系进行体外扩增。取对数生长期的第3~5脐带间充质干细胞,应用腺病毒Ad5-EGFP,Ad5/11-EGFP,Ad5/35-EGFP分别以感染复数=1,10,100进行感染,分别于感染后24,56,72h倒置荧光显微镜观察病毒感染及绿色荧光表达情况。结果与结论:使用低糖DMEM培养的细胞初期融合时间长,STEMPRO MSC SFM培养的细胞虽然连接紧密,但消化传代后不易贴壁,而MesenPRO RSTM Medium培养的细胞在相同时间内能达到较高的细胞密度,更适于脐带间充质干细胞的体外扩增。Ad5/35-EGFP感染脐带间充质干细胞的效率明显高于其他两种腺病毒,但可导致细胞凋亡;腺病毒Ad5/11-EGFP对脐带间充质干细胞的感染效率较佳,随着感染复数的升高,所表达的荧光强度也逐渐增大。     

脐带间充质干细胞促进骨愈合的体外实验及临床应用1例

背景：华通胶来源脐带间充质干细胞比成人间充质干细胞更原始,研究表明其端粒酶活性更高、培养倍增时间更短、分化的细胞谱系更广。 目的：观察脐带间充质干细胞在体外分化为成骨细胞的能力及治疗骨折不愈合的效果。 方法：从脐带Wharton胶获取细胞培养、扩增,取传代细胞行免疫表型测定和成骨细胞诱导分化,并以脐带间充质细胞移植治疗1例骨折感染外露后长期不愈病例。 结果与结论：培养细胞形态类成纤维细胞,可长期稳定培养,传代细胞表达间充质干细胞免疫表型,成骨诱导分化的细胞茜素红染色胞浆中有大量的钙沉积,Von Kossa染色有钙结节形成。患者采用脐带间充质干细胞混悬液外用4次,肉芽组织迅速增生填满窦道并上皮化,12 d创面愈合。说明,脐带间充质干细胞具有高度自我更新能力和分化潜能,能够向成骨细胞分化,将其移植治疗骨不连可以显著改善局部微环境。     

人脐带间充质干细胞冻存复苏后的生物学特征

背景:冻存脐带间充质干细胞,并有效地保持其生物学特性,是储存脐带间充质干细胞以供临床使用的重要工艺之一。目的:观察冻存后脐带间充质干细胞的生物学特性,验证其是否仍具有间充质干细胞的基本特征。方法:从脐带分离得到间充质干细胞后,将其冻存于液氮之中。比较经冻存复苏后的脐带间充质干细胞和新鲜制备的脐带间充质干细胞活率、抑制人外周血单个核细胞分泌γ-干扰素等方面的异同。检验经冻存复苏后的脐带间充质干细胞是否具有多向分化潜能,其表型是否满足间充质干细胞的基本特征。结果与结论:在细胞活率和抑制人外周血单个核细胞分泌γ-干扰素方面,冻存和新鲜的脐带间充质干细胞差异无显著性意义。经冻存复苏后的脐带间充质干细胞保持了间充质干细胞的基本形态,其表型满足间充质干细胞的基本要求,具有多向分化的潜能。     

原代人脐带间充质干细胞培养方法的研究

背景:如何获得大量稳定活力好的细胞是人脐带间充质干细胞培养的难点。目的:通过组织块法、酶消化法和酶解组织块法3种细胞培养方法比较,筛选出最佳的人脐带间充质干细胞的培养方式。方法:分离新鲜脐带10根,分别采用组织块法、酶消化法、酶解组织块法3种方式培养人脐带间充质干细胞,比较3种方式原代人脐带间充质干细胞爬出所需时间、细胞培养成功率、绘制细胞增殖曲线,利用流式细胞仪检测细胞表面标志,并进行多项分化能力检测。结果与结论:酶解组织块培养法原代人脐带间充质干细胞爬出所需时间与酶消化法无显著差异,但明显短于组织块法(P0.01),原代人脐带间充质干细胞培养成功率显著高于其他两组,人脐带间充质干细胞增殖情况3组无显著性差异,酶解组织块培养法培养的第3代人脐带间充质干细胞其细胞表面标志及多项分化能力符合间充质干细胞的特性。酶解组织块培养法能缩短原代人脐带间充质干细胞爬出时间,显著提高原代人脐带间充质干细胞的成功率。     

脐带间充质干细胞促进蜕膜血管内皮生长因子表达改善自然流产小鼠妊娠结局

背景:早期自然流产与母胎界面血管形成障碍或异常有关,人脐带华通氏胶间充质干细胞具有向损伤部位迁移及促血管生成的作用,目前人脐带华通氏胶间充质干细胞作用于早期自然流产蜕膜基质细胞的研究尚未有报道.目的:探讨人脐带华通氏胶间充质干细胞对早期自然流产蜕膜基质细胞增殖、凋亡及血管内皮生长因子表达的影响,以及对早期自然流产小鼠妊...     

大鼠脐带源间充质干细胞的分离及生物学性状

背景:关于大鼠骨髓来源的间充质干细胞用于移植免疫耐受及进行组织修复的研究很多,但尚无脐带来源间充质干细胞的相关研究。目的:建立从大鼠脐带分离间充质干细胞的方法,并观察其生物学性状。方法:大鼠脐带经酶消化和组织块培养两种方法进行分离培养,于DMEM-LG培养基中培养,倒置显微镜观察细胞形态,细胞计数绘制生长曲线,流式细胞仪测定细胞周期及细胞表型,免疫组织化学染色检测其体外诱导成脂肪和成骨分化的能力。结果与结论:两种方法均能成功地从大鼠脐带中获得大量的间充质干细胞:原代培养显示,胶原酶消化法比组织块培养法的效率更高,大约10d就可以进行传代,而组织块培养法要14d才能传代;传代扩增两者之间没有差别。免疫表型分析显示,大鼠脐带源细胞表达黏附分子和基质细胞标记CD90、CD106,不表达造血细胞标记CD34、CD45。体外诱导实验证实,大鼠脐带间充质干细胞具有成脂肪和成骨分化的能力。     

脐带源间充质干细胞与大鼠胰腺细胞共培养向胰岛样细胞分化及移植治疗1型糖尿病

背景：脐带Wharton’s Jelly中间充质干细胞可以向胰岛样细胞诱导分化。 目的：验证脐带源间充质干细胞与大鼠胰腺细胞共培养向胰岛样细胞诱导分化的可能性,并观察移植后对糖尿病大鼠血糖的影响。 方法：分离、诱导、传代脐带Wharton’s Jelly中间充质干细胞,再与大鼠胰腺细胞共培养,诱导成胰岛细胞团样组织。将大鼠分为3组,正常对照组不进行移植及造模;模型组仅制备糖尿病大鼠模型;实验组造模后将胰岛样细胞移植入糖尿病大鼠肾脏包膜。 结果与结论：脐带Wharton’s Jelly细胞培养中有细胞从组织块中爬出,第7天形态发生变化,贴壁细胞部分变成梭形。分离培养的细胞表达具有间充质干细胞表面特有标志CD44、CD29、CD105,不表达CD34、CD45、CD14。诱导第7,10天PDX-1及人胰岛素强染色;胰岛素及C-肽浓度较单纯培养组明显升高;PDX-1及人胰岛素mRNA诱导第7、10天较高表达。移植第1周大鼠尾尖血糖链脲佐菌素实验组明显低于模型组(P < 0.01),但明显高于正常照组(P < 0.01)。8周链脲佐菌素实验组肾脏被膜下发现胞核染棕色染色的Brdu阳性、胞浆棕色染色的胰岛素阳性细胞。结果表明,脐带Wharton’s Jelly中存在脐带源间充质干细胞,与大鼠胰腺细胞共培养可促进间充质干细胞向胰岛样细胞诱导分化,移植入糖尿病大鼠肾脏被膜下,可显著降低糖尿病大鼠血糖。     

两步法分离人脐血单个核细胞的最佳条件

背景：如何获得较为纯化、高活性的干细胞,目前未见深入研究报告,也未见一个标准化操作流程方案。 目的：探讨两步法分离人脐血单个核细胞最佳分离条件。 方法：观察羟乙基淀粉在20,30,40,50,60,70 min不同时间沉淀脐血中红细胞的效果;使用人淋巴细胞分离液,分别在800,700,600,500,400 g/min,离心30,25,20 min的条件下分离人脐血单个核细胞。 结果与结论：6%羟乙基淀粉沉淀脐血60 min效果最好;使用密度为(1.077 0±0.000 1) g/mL人淋巴细胞分离液,在4 ℃条件下以700 g/min离心力,离心30 min,洗涤3次,这样获得的人脐血单个核细胞效果最好,所得细胞沉淀中混杂细胞如红细胞及其他细胞碎片较少,人脐血单个核细胞的细胞得率及活力比较高。提示应用羟乙基淀粉沉淀和人淋巴细胞分离液分离两步法,在最佳时间条件下可提高脐血干细胞的回收率。     

The Effect of Human Cord Blood on SJL/J Mice after Chemoablation and Irradiation and its Possible Clinical Significance

There is evidence from the existing published literature that human umbilical cord blood, when used for purposes of bone marrow transplantation, does not necessarily have to be HLA matched in order to be efficacious. These reports include experimental observations on the ability of human umbilical cord blood to rescue lethally irradiated mice and clinical observations from China wherein HLA mismatched umbilical cord blood has been engrafted successfully in children with malignant disease.

The study reported herein describes an experimental immunocompetent murine model to determine if human umbilical cord blood can be used to improve survival after chemoablation and irradiation. The animals received chemoablation followed by irradiation, and irradiation alone. The presence of human DNA in these mice following injection of human umbilical cord blood cells was determined, and the immunological status of the animals was evaluated. Animals receiving human umbilical cord blood cells after chemoablation and irradiation had a better mean survival at day 50 than animals receiving syngeneic marrow. Human DNA could be found in various organs, particularly the lung, spleen and liver of the mice for the first 30 days. Thereafter, human DNA became more difficult to detect but trace amounts of human DNA could be found up to one year later. The results of mixed lymphocyte reactions and phenotype analyses for murine T cell markers performed after injection of HUCB cells both indicated endogenous repopulation, and relatively intact immune systems in these mice.

Since human umbilical cord blood allowed mice to survive the lethal effects of chemoablation plus irradiation, or irradiation alone, with reconstitution of the animals' own, relatively intact, immune systems, it would appear that HLA mismatched human umbilical cord blood could potentially be used as an adjuvant treatment for patients with advanced malignancies or other diseases for which hematopoietic reconstitution is indicated.     

The Effect of Human Cord Blood on SJL/J Mice after Chemoablation and Irradiation and its Possible Clinical Significance

There is evidence from the existing published literature that human umbilical cord blood, when used for purposes of bone marrow transplantation, does not necessarily have to be HLA matched in order to be efficacious. These reports include experimental observations on the ability of human umbilical cord blood to rescue lethally irradiated mice and clinical observations from China wherein HLA mismatched umbilical cord blood has been engrafted successfully in children with malignant disease.

The study reported herein describes an experimental immunocompetent murine model to determine if human umbilical cord blood can be used to improve survival after chemoablation and irradiation. The animals received chemoablation followed by irradiation, and irradiation alone. The presence of human DNA in these mice following injection of human umbilical cord blood cells was determined, and the immunological status of the animals was evaluated. Animals receiving human umbilical cord blood cells after chemoablation and irradiation had a better mean survival at day 50 than animals receiving syngeneic marrow. Human DNA could be found in various organs, particularly the lung, spleen and liver of the mice for the first 30 days. Thereafter, human DNA became more difficult to detect but trace amounts of human DNA could be found up to one year later. The results of mixed lymphocyte reactions and phenotype analyses for murine T cell markers performed after injection of HUCB cells both indicated endogenous repopulation, and relatively intact immune systems in these mice.

Since human umbilical cord blood allowed mice to survive the lethal effects of chemoablation plus irradiation, or irradiation alone, with reconstitution of the animals' own, relatively intact, immune systems, it would appear that HLA mismatched human umbilical cord blood could potentially be used as an adjuvant treatment for patients with advanced malignancies or other diseases for which hematopoietic reconstitution is indicated.     

人软骨细胞培养上清诱导脐血间充质干细胞向软骨细胞分化

背景：人脐血间充质干细胞作为软骨缺损修复的种子细胞日益受到关注,现有常用诱导方法无论是应用诱导培养基还是诱导因子,因其价格昂贵、用量大等因素限制了实际应用。 目的：验证人软骨细胞培养上清诱导人脐血间充质干细胞向软骨细胞分化的可行性。 方法：利用密度梯度离心法和贴壁培养法分离新生儿脐血,获取并培养人脐血间充质干细胞,流式细胞仪鉴定细胞表面抗原。切取股骨头置换或全髋关节置换患者透明软骨组织,体外分离培养软骨细胞,利用其培养上清培养人脐血间充质干细胞,培养2周后观察细胞表型外观变化,免疫组化染色检测Ⅱ型胶原表达结果。 结果与结论：密度梯度离心法与贴壁培养法可以分离获取人脐血间充质干细胞,流式细胞仪鉴定表面标记CD90,CD105高表达,不表达CD34,CD45。软骨细胞培养上清诱导2周后,人脐血间充质干细胞向多角形、圆形转变,Ⅱ型胶原免疫组化检测表达阳性。提示软骨细胞培养上清可诱导人脐血间充质干细胞表达Ⅱ型胶原,向软骨细胞分化。     

人脐血间充质干细胞尾静脉移植治疗扩张型心肌病

背景：体外研究表明人脐血间充质干细胞可分化为自律性跳动的心肌细胞,而经静脉移植人脐血间充质干细胞治疗扩张型心肌病心力衰竭的报道较少。 目的：探讨经尾静脉移植人脐血间充质干细胞对扩张型心肌病大鼠心肌结构、心功能的影响。 方法：Wistar大鼠通过腹腔注射阿霉素诱导扩张型心肌病模型,扩张型心肌病实验组于造模后8周经尾静脉移植人脐血间充质干细胞,扩张型心肌病对照组注射等量DMEM培养基。健康对照组不造模,于相同时间点注射等体积的生理盐水。 结果与结论：与健康对照组相比,扩张型心肌病组心功能明显受损,且扩张型心肌病实验组受损较扩张型心肌病对照组轻;移植的细胞有肌钙蛋白T的表达。结果提示人脐血间充质干细胞移植能促进扩张型心肌病大鼠心功能恢复,使心肌组织病变减轻。     

去细胞肌肉生物支架与人脐带间充质干细胞的相容性

背景：去细胞肌肉生物支架联合人脐带间充质干细胞移植将是治疗脊髓损伤的一项重要措施。但两者是否具有良好的相容性,人脐带间充质干细胞能否在去细胞肌肉生物支架中长期存活并均匀分布,尚未得到证实。 目的：观察大鼠去细胞肌肉生物支架与人脐带间充质干细胞的相容性。 方法：改良化学法制备大鼠去细胞肌肉生物支架,将第3代人脐带间充质干细胞Hoechest33342荧光标记后分为3组进行实验,细胞+支架组、细胞+支架大鼠体内组和单纯细胞组。分别应用苏木精-伊红、Masson染色方法观察去细胞肌肉生物支架的组织形态,以荧光倒置相差显微镜和扫描电镜观察人脐带间充质干细胞的吸附和生长情况。 结果与结论：人脐带间充质干细胞与去细胞肌肉生物支架充分附着,生长增殖活跃,细胞在支架内分布均匀。细胞+支架体内组与细胞+支架组相比在移植后1-7 d人脐带间充质干细胞数量差异无显著性意义(P > 0.05),在移植14 d细胞+支架体内组人脐带间充质干细胞数量大于细胞+支架组(P < 0.05)。提示去细胞肌肉生物支架与人脐带间充质干细胞有较好的相容性,体内环境更有利于细胞增殖和两者融合。     

胰岛素促进脐带间充质干细胞成骨分化的作用

BACKGROUND: How to effectively and rapidly induce the osteogenic differentiation of human umbilical cord mesenchymal stem cells is the focus of the current stem cell research. Increasing evidence has demonstrated some growth factors, such as bone morphogenetic protein-2, have important effects on the transdifferentiation of umbilical cord mesenchymal stem cells into osteoblasts in vitro. However, widespread use of growth factors is limited because of high cost. Insulin is widely used in the cell culture and induction, but there is no report about the effect of insulin on the osteogenic differentiation of human umbilical cord mesenchymal stem cells. OBJECTIVE: To observe the effect of insulin on osteogenic differentiation of human umbilical cord mesenchymal stem cells and to explore the feasibility of human umbilical cord mesenchymal stem cell transplantation in the treatment of diabetic delayed fracture healing. METHODS: The passage 3 human umbilical cord mesenchymal stem cells were inoculated in two flasks, denoted as experimental group and control group. The insulin (10-7 mmol/L) was added to the experimental group but not to the control group. The proliferative capacity of human umbilical cord mesenchymal stem cells was evaluated by cell count kit-8 and alkaline phosphatase activity. The osteogenic differentiation capacity of human umbilical cord mesenchymal stem cells was evaluated by measuring the protein and mRNA expressions of type I collagen as well as osteocalcin mRNA level. RESULTS AND CONCLUSION: After 1-2 weeks of induction, compared with the control group, insulin could significantly increase the number of human umbilical cord mesenchymal stem cells in the experimental group, the activity of alkaline phosphatase and expressions of type I collagen osteocalcin mRNA (P < 0.05). These data indicate that insulin can promote the proliferation and osteogenic differentiation of human umbilical cord mesenchymal stem cells.       

脐血细胞神经分化与移植的研究进展

自20年前Nakahata等发现人脐血中含有丰富的造血干细胞以来,脐血造血干,祖细胞的研究取得了很大的进展,并已成为一个新的造血干细胞来源。近来研究表明,脐血中的部分细胞在体外培养或体内移植后可分化为神经细胞,并可促进受损动物的神经功能恢复,本文将对脐血源神经细胞的研究进展进行探讨。     

Monosialotetrahexosyl ganglioside at an optimal concentration: Inducing neuron-like differentiation of human umbilical cord mesenchymal stem cells北大核心

BACKGROUND: Studies have confirmed that monosialotetrahexosyl ganglioside can induce human umbilical cord mesenchymal stem cells to differentiate into neuron-like cells, but little is reported on its optimal concentration. OBJECTIVE: To explore the optimal concentration of monosialotetrahexosyl ganglioside that induces human umbilical cord mesenchymal stem cells to differentiate into neuron-like cells in vitro. METHODS: Human umbilical cord mesenchymal stem cells were isolated by using collagenase digestion method, and after expansion, passage 3 cells were randomly allocated into five groups. When 70%-80% of cells were confluent, 50, 100, 150 and 200 mg/L monosialotetrahexosyl ganglioside induction solutions were added in corresponding experimental groups, while cells in the blank control group were cultured in the same volume of L-DMEM medium. Cell morphology was observed under inverted phase contrast microscope. Expression levels of microtubule-associated protein 2, neurofilament protein and glial fibrillar acidic protein were measured by using immunohistochemistry ot 6 hours ofter induction. RESULTS AND CONCLUSION: Human umbilical cord mesenchymal stem cells were isolated successfully and sub-cultured stably. These cells could express surface markers of mesenchymal stem cells. Monosialotetrahexosyl ganglioside at the optimal concentration of 150 mg/L was confirmed to induce the neuron-like differentiation of human umbilical cord mesenchymal stem cells, and differentiated cells could express microtubule-associated protein 2 and neurofilament protein as neuron-specific markers. © 2018, Journal of Clinical Rehabilitative Tissue Engineering Research. All rights reserved.