人脐带间充质干细胞研究进展及应用

人脐带间充质干细胞是一类具有自我更新和多向分化潜能的成体干细胞。随着细胞技术的提高,人们对于脐带间充干细胞的研究越发深入,在医学的各个领域都取得了长足的进步,如脐带间充质干细胞分离技术及脐带间充质干细胞的诱导分化技术。本文就脐带间充质干细胞的生物学特性、诱导分化潜能、免疫原性进行综述,重点讨论其最新的临床应用。     

人胚胎生殖干细胞移植对大鼠心肌梗死的影响

目的:探讨直接注射移植人胚胎生殖干细胞对大鼠心肌梗死的影响.方法:缝扎SD大鼠左冠状动脉前降支,建立心肌梗死模型.取5～10周人胚胎生殖腺嵴,组织块体外培养,生物学鉴定为人胚胎生殖干细胞(hEG).将其直接注射于大鼠急性心肌梗死边缘.于移植后1 d、1、2、4周处死大鼠,免疫组织化学方法观察心肌特异转录因子GATA-4和抗人细胞核抗体MAB1281在移植细胞的表达.结果:免疫组织化学显示移植组MAB1281检测阳性,GATA-4在移植细胞阳性表达.结论:hEG细胞直接注射移植大鼠心肌梗死处,细胞能存活并呈现向心肌细胞分化的表现,显示出正常心肌细胞的光镜结构.     

人脐带源间充质干细胞静脉移植治疗大鼠肝纤维化

背景：近来国内外研究证明,来自其他组织的干细胞能够归巢到肝脏,并可能参与肝组织的再生,这为发展干细胞治疗肝脏疾病提供了新的希望。 目的：探究人脐带源间充质干细胞的分离、培养方法,观察脐带间充质干细胞移植对大鼠肝纤维化模型的修复作用,为脐带间充质干细胞的临床应用提供可靠的理论依据。 方法：自然贴壁法分离、纯化人脐带间充质干细胞并进行体外培养和扩增,用皮下多点注射CCl₄制备肝纤维化大鼠模型。将22只模型大鼠随机分为模型损伤组(n=11)和细胞移植组(n=11)。细胞移植组在模型制备成功后的第1,2,3周经尾静脉给予1×10⁶脐带间充质干细胞治疗,4周后将大鼠处死,收集各组大鼠血液检测肝功能;摘取肝脏行苏木精-伊红染色,观察病理变化;免疫组化法观察库普弗细胞的数量及分布;免疫组化法观察治疗组脐带间充质干细胞的定位情况。 结果与结论：脐带间充质干细胞经尾静脉移植入肝硬化大鼠后,大鼠的肝功能均明显改善,与对照组比较,差异有显著性意义(P < 0.05);肝组织苏木精-伊红染色提示,肝纤维化程度明显改善;免疫组化法观察库普弗细胞的数量提示,库普弗细胞数量明显减少;免疫组化方法利用抗BrdU抗体在治疗组大鼠肝脏观察到BrdU标记的脐带间充质干细胞。说明脐带间充质干细胞移植可以改善大鼠外周血液的血生化特性和肝的组织学结构。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

脐血干细胞移植治疗心肌梗死的作用与机制

背景：多项基础和临床研究表明,干细胞移植可以改善心肌梗死后心力衰竭患者的心脏功能。 目的：总结脐血干细胞移植治疗心肌梗死的的作用与机制。 方法：由第一作者检索2000/2009 PubMed数据库及CNKI、万方数据库有关脐血干细胞移植治疗心肌梗死基础研究方面的文献。 结果与结论：干细胞移植在治疗急性心肌梗死方面已表现出传统治疗方法无法比拟的优势,而目前一系列基础研究证实人脐血干细胞有望成为更为理想的细胞源,但因其尚处于探索性研究阶段,仍有许多问题有待解决,诸如人脐血干细胞的体外分离、培养,最适宜的移植治疗时间,最有利的移植途径,最佳的移植数量,移植细胞的存活率如何,移植后能否分化为心肌细胞,分化程度如何以及其疗效和安全性等。     

脐血干细胞移植对帕金森病大鼠旋转行为的影响

背景：目前帕金森病的临床治疗还是以药物为主,细胞移植实验也多见于骨髓间充质干细胞,脐血来源干细胞移植能否改善帕金森病的旋转行为报道较少。 目的：观察脐血间充质干细胞移植对帕金森病大鼠旋转行为的影响。 方法：帕金森病模型大鼠随机分成实验组和对照组。实验组大鼠纹状体内植入用Hoechst33258标记的第4代脐血间充质干细胞,对照组注射PBS。此后每周腹腔注射阿扑吗啡以观察大鼠的旋转行为;并在移植后3,6,9周用免疫荧光双标法检测间充质干细胞的存活、迁移情况以及胶质纤维酸性蛋白、神经元特异性烯醇化酶、酪氨酸羟化酶和突触素的表达。 结果与结论：移植脐血间充质干细胞后大鼠的旋转行为与对照组相比有明显改善(P < 0.05);间充质干细胞可在大鼠脑内存活,随时间延长迁移范围扩大,分布于纹状体、胼胝体和皮质;胶质纤维酸性蛋白、神经元特异性烯醇化酶、酪氨酸羟化酶都有表达,突触素无表达。结果可见移植脐血间充质干细胞后能明显改善帕金森病大鼠旋转行为,有望成为治疗帕金森病的种子细胞。      

不同孕周人脐带间充质干细胞移植改善心肌梗死模型心脏功能的比较

BACKGROUND: Stem cells have multi-directional differentiation and self-replication abilities, under certain conditions, which can differentiate into myocardial cells to repair the damaged myocardium. OBJECTIVE: To investigate the effects of transplantation of umbilical cord mesenchymal stem cells derived at different gestational weeks on infarct size and angiogenesis in the infarct region of experimental rabbits with myocardial infarction. METHODS: Ten full-term umbilical cord samples and 10 umbilical cord samples of aborted fetuses at 10-12 gestation weeks were selected to in vitro isolate umbilical cord mesenchymal stem cells that were subjected to BrdU labeling. HLA-G expression was detected in the cells. Thirty white rabbits were selected to make myocardial infarction models, and 2 weeks after modeling, the model rabbits were randomized into aborted cell transplantation group, full-term cell transplantation group and control group (n=10 per group). Then, BrdU-labeled cells were injected correspondingly into the infarct region of rabbits in the two cell transplantation groups. Rabbits in the control group were subjected to an equal volume of serum-free. Four weeks after transplantation, heart function of rabbits was monitored using electrocardiogram, and myocardial tissues were taken to measure infarct size and blood capillary density. RESULTS AND CONCLUSION: HLA-G expression was different in different sources of umbilical cord mesenchymal stem cells: high HLA-G expression was found in the aborted umbilical cord mesenchymal stem cells, and meanwhile, low HLA-G expression was found in the full-term umbilical cord mesenchymal stem cells. Compared with the control group, the left ventricular end-diastolic volume and left ventricular ejection fraction of aborted and full-term cell transplantation groups were significantly improved, especially in the aborted cell transplantation group (P < 0.05). BrdU-positive cells were found in the infarct site in both transplantation groups. Compared with the control group, the infarct size and capillary density were improved most significantly in the aborted cell transplantation group followed by the full-term cell transplantation group (P < 0.05). Electrocardiogram findings showed significant improvement in both cell transplantation groups compared with the control group (P < 0.05), especially in the aborted cell transplantation group. These findings indicate that umbilical cord mesenchymal stem cells derived at low gestational weeks improve the heart function more significantly than the full-term umbilical cord mesenchymal stem cells, which have the potential to become a better source of cardiomyocytes for transplantation.     

大鼠胚胎神经干细胞黑质内移植后的存活及分化

目的：观察胚胎神经于细胞脑内移植后的存活及生长分化状况。方法：从孕11d(E11d)大鼠胚胎神经管获取神经上皮细胞，经神经巢蛋白(nestin)染色鉴定干细胞；同时植入同种大鼠黑质内。于移植后7d、14d取脑，用神经元特异烯醇化酶(NSE)、酪氨酸羟化酶(TH)免疫组化方法检测移植细胞的存活及分化状况。结果：E11d神经管上皮细胞多数呈nestin染色阳性，黑质内移植后增殖形成细胞团并随时间延长而增大。免疫组化染色显示移植细胞团内有NSE及TH免疫阳性细胞。结论：胚胎神经上皮细胞多数为神经干细胞，黑质内移植后可以存活并分化为多巴胺能神经元。     

大鼠心肌匀浆上清液对骨髓间质干细胞诱导分化的影响

目的：探讨心肌匀浆上清液对大鼠骨髓间质干细胞（MSCs）诱导分化的影响。 方法： 体外分离培养大鼠MSCs，检测纯度。于原代培养第3 d加入自体心肌匀浆上清液持续作用1周。3周后，观察细胞形态，采用免疫细胞化学检测肌球蛋白重链和心肌特异性肌钙蛋白-T，RT-PCR方法检测Nkx2.5、α-MHC和ANP基因的表达。 结果： 大鼠MSCs经诱导后，表达肌球蛋白重链、心肌特异性肌钙蛋白-T和Nkx2.5、α-MHC基因，但未表达ANP基因，也未观察到肌管和闰盘样结构。 结论： 心肌匀浆上清液对MSCs具有定向诱导分化作用，但诱导不完全，具体机制仍需深入研究。     

干细胞及组织移植治疗甲状腺功能减退症

背景：干细胞及甲状腺组织移植治疗甲状腺功能减退症已经取得了一定的成果。 目的：回顾分析干细胞与组织移植治疗甲状腺功能减退症的基础和临床研究进展。 方法：由作者检索1980/2010 PubMed 数据库及万方数据库有关干细胞与甲状腺移植治疗甲状腺功能减退症的基础和临床研究。 结果与结论：自体甲状腺组织移植治疗不可逆性甲状腺功能减退症的作用是肯定的,但存在不足之处,比如需要移植多少甲状腺组织才能使甲状腺功能保持在正常状态,移植后的长期效果还需要更多样本、更长期的跟踪随访来评价。胚胎干细胞在体外环境下可诱导分化为甲状腺细胞,但受伦理学和细胞移植排斥反应等原因困扰。脐血间充质干细胞具有来源丰富、易于采集、保存和运输、无异体排斥、避免伦理争议等诸多优点,在不同诱导条件下能够向不同谱系分化,已被广泛用于治疗各种疾病,但能否通过多种途径诱导其分化为甲状腺细胞,并通过移植方法来治疗甲状腺功能减退症仍需深入研究。     

人脐带间充质干细胞肌肉注射健康大鼠心肌组织微血管及胶原纤维的表达

BACKGROUND:To date, it is still unclear whether the intramuscular injection of heterogeneous umbilical cord mesenchymal stem cells (UC-MSC) can cause cardiac ectopic pathological angiogenesis as well as increase collagen synthesis to promote myocardial fibrosis. OBJECTIVE:To explore the effects of intramuscular injection of human UC-MSCs on myocardial micrangium and collagen expression in normal Wistar rats. METHODS:After 2 weeks of feeding, 60 male SPF Wistar rats were randomly assigned to receive intramuscular injection of PBS (normal group), DMEM (culture medium group), human UC-MSCs supernatant (supernatant group), 0.25×10⁵, 1.0×10⁵, 4.0×10⁵ human UC-MSCs (low-, moderate- and high-dose groups), respectively (n=10 per group). All the rats were subjected to second injection (same dose) at 4 weeks after first intramuscular injection. Then, the rats were killed under anesthesia at 4 weeks after second injection, to take heart tissues from the left ventricle for pathological observation, immunohistochemical examination and Masson staining. RESULTS AND CONCLUSION: No alteration of the response, activity, victualage, faeces, weight growth, and fur was found, and there was no death in rats during the experiment. All the rats had no symptoms of molt, inflammation, skin ulcer, scleroma. Strong positive expression of CD34 for the micrangium in the myocardial tissue was observed, and positive expression of the collagen in the myocardial tissue observed by Masson staining. There were no significant differences in the microvessel density and collagen expression in the myocardium among the groups (F=0.110 and 0.585, P > 0.05). To conclude, hUC-MSCs or its supernatant via intramuscular injection has no effect on the micrangium and collagen expression in normal rats.     

人脐带间充质干细胞的分离鉴定及多向诱导分化

背景：脐带间充质干细胞取材方便、无创,不受伦理学限制,比一般干细胞原始,免疫原性小,其应用前景广阔,是一种理想的种子细胞。 目的：分离鉴定脐带间充质干细胞,并诱导其向成骨细胞和成脂细胞分化。 方法：组织块贴壁法分离纯化脐带间充质干细胞,取对数生长期的第3代细胞,观察细胞形态、生长方式;流式细胞仪检测干细胞表型CD90、CD105、CD34和CD45的表达情况,并在体外检测能否将其诱导分化为成脂细胞及成骨细胞。 结果与结论：用组织块法成功分离培养出脐带间充质干细胞,流式细胞学鉴定显示细胞强表达CD90和CD105,不表达CD34和CD45;能在体外将其成功诱导为脂肪细胞和成骨细胞。结果显示组织块贴壁法能够从人脐带中分离出间充质干细胞,该细胞可向成脂细胞及成骨细胞分化。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

脐带血间充质干细胞的研究进展

脐带血中存在着丰富的造血干细胞,在移植方面发挥重要作用,在脐血中是否还存在间充质干细胞却有争议,有的学者认为其中有间充质干细胞,且与骨髓间充质干细胞(mesenchymalstemcells,MSC)的形态、表面标志及分化潜能非常相似,有的学者认为含量较低,难以传代培养扩增,总结了近5年来国内外关于脐血间充质干细胞的研究情况,为脐血的充分利用提供更多的资料。     

人脐血间充质干细胞体外诱导分化为神经元样细胞

目的从人脐血中分离单个核细胞(MNCs)、体外培养扩增间充质干细胞(MSCs)并研究其生物学特性和定向诱导能力。方法无菌条件下采集脐血,肝素抗凝,分离单个核细胞。用DMEM/F12培养基进行纯化和扩增培养,分别向神经元样细胞诱导,获取MSCs,显微镜下观察它的形态、尼氏体染色;取扩增2、5、7代的MSCs,免疫细胞化学法检测nestin表达和神经元样细胞特异性标记。结果诱导扩增后的MSCs尼氏体染色阳性,第2、5、7代MSCs的nestin的阳性表达率分别为(51.2±3.2)%、(34.6±2.7)%、(11.3±3.3)%;神经元特异性烯醇化酶(NSE)在原代细胞无表达,而在2、5、7代MSC的阳性表达率分别为(11.4±2.3)%、(21.78±3.1)%、(40.7±3.4)%。结论人脐血MSCs具有神经干/祖细胞的特性,在一定条件下能向神经元样细胞分化。     

体外定向诱导人脐带间充质干细胞分化为胰岛样细胞

背景：人脐带间充质干细胞具有获取容易、免疫原性低等优点,目前尚缺乏体外诱导其分化为胰岛样细胞的成熟方案。 目的：探讨人脐带间充质干细胞在体外一定条件下分化为胰岛样细胞的可能性。 方法：无菌条件下分离培养人脐带间充质干细胞,细胞传3代后,采用两步法诱导其向胰岛样细胞分化：第1步,加入含100 μg/L β-神经生长因子,4 nmol/L ActivinA,10 mmol/L 尼克酰胺,25 μg/L表皮生长因子,体积分数为10%胎牛血清的DMEM/F12培养基中培养7 d;第2步,将诱导液换为含10 mmol/L尼克酰胺,10 μg/L碱性成纤维细胞生长因子,1%胰岛素-转铁蛋白-硒的DMEM/F12培养基,诱导时间为14 d。对照组单纯加入DMEM/F12培养基进行培养。 结果与结论：诱导2周后脐带间充质干细胞开始聚集成团,诱导3周后,葡萄糖刺激试验阳性、PDX-1和Insulin基因表达。而对照组细胞无胰岛素分泌,胰岛细胞相关基因表达阴性。实验成功地将脐带间充质干细胞诱导成了胰岛样细胞。     

人脐带间充质干细胞的快速分离、纯化及冻存

目的:建立在一般实验条件下快速分离、纯化及冻存人脐带间充质干细胞(UC-MSCs)的简便有效方法并研究其冻存复苏后的增殖及多向分化能力。方法:用胶原酶Ⅱ、胰蛋白酶次序消化及二步离心法从人的完整脐带中快速分离UC-MSCs,通过把握传代时的消化程度及酸化培养基来快速纯化UC-MSCs,以流式细胞仪检测表面抗原进行鉴定;用简易二步法(-4℃停留30 min,-20℃存放2 h,-80℃长期保存)冻存UC-MSCs,半年后复苏,扩增、传代,检测其胚胎干细胞特异性抗原Oct4、SSEA-4和人端粒酶逆转录酶hTERT的表达情况,并诱导其向神经元样细胞、成肌细胞和肝细胞样细胞分化。结果:通过双酶次序消化结合二步离心法,2 h可提取全脐带中的MSCs,原代培养7 d即可传代,通过适度消化传代和选用弱酸性培养基培养,第3代即可得到纯化的UC-MSCs,UC-MSCs表达CD29和CD44,不表达CD34和CD45,极低表达HLA-DR、CD80、CD86和CD106。简易法冻存半年后复苏的UC-MSCs表达Oct4、SSEA-4和hTERT,可向三个胚层来源的细胞分化。结论:双酶次序消化结合二步离心法是从人的完整脐带中快速分离UC-MSCs的一种简便有效方法,偏酸性环境有利于UC-MSCs的扩增及纯化;简易二步法冻存对UC-MSCs增殖和多向分化能力没有明显影响。     

瑞舒伐他汀联合脐血间充质干细胞移植改善急性心肌梗死后心功能

BACKGROUND:In recent years, it has been a hot topic that stem cell transplantation is used to improve cardiac insufficiency after acute myocardial infarction by inducing regeneration of cardiomyocytes in the infarction regions. OBJECTIVE:To observe the effect of rosuvastatin combined with umbilical cord blood mesenchymal stem cells transplantation on rat cardiac function after acute myocardial infarction. METHODS:Forty-five Sprague-Dawley rats were enrolled to prepare myocardial infarction models by ligaturing the left anterior descending coronary artery. Then they were equivalently divided into model group, transplantation group and combination group. At 7 days after modeling, rats in the combination group were given injection of 300 μL umbilical cord blood mesenchymal stem cells (15.0×10⁸) via the tail vein and by gavage once a day for 28 days with 1 mg/kg rosuvastatin; rats in the transplantation group and model group were injected with 300 μL umbilical cord blood mesenchymal stem cell suspension through the tail veins or the same amount of LG-DMEM medium, respectively, followed by intragastrical administration of the same amount normal saline. At 5 weeks after modeling, indexes of cardiac function, level of plasma Lp-PLA2 and heat shock protein 70 in the infarction regions were detected by color Doppler ultrasound, enzyme-linked immunosorbent assay and western blot assay, respectively. In addition, pathological changes of myocardial tissues were observed using hematoxylin-eosin staining. RESULTS AND CONCLUSION:Left ventricular ejection fraction and left ventricular end-systolic pressure were significantly higher in the combination group than in the transplantation group as well as higher in the transplantation group than the model group (P < 0.05); compared with the transplantation group, left ventricular end-diastolic pressure was significantly decreased in the combination group, but significantly increased in the model group (P < 0.05); the number of cardiomyocytes in the infarction regions was significantly higher in the combination group than the other groups. Additionally, expression of heat shock protein 70 in the infarction regions was significantly increased in the combination group (P < 0.05). To conclude, rosuvastatin combined with umbilical cord blood mesenchymal stem cell transplantation can significantly improve rat cardiac function after myocardial infarction.     

Immunomodulatory function of whole human umbilical cord derived mesenchymal stem cells

Bone marrow derived mesenchymal stem cells (MSCs) play a critical role in immune modulation. However, immunomodulatory function of whole human umbilical cord derived mesenchymal stem cells (UC-MSCs) remains unclear. In this study, UC-MSCs were separated from whole umbilical cord using a single enzyme digestion. UC-MSCs (CD73⁺, CD90⁺, CD105⁺, and CD34⁻, CD45⁻, HLA-DR⁻) were differentiated into adipocytes, osteocytes and chondrocytes in vitro under specific stimulatory environments. UC-MSCs suppressed umbilical cord blood lymphocyte proliferation stimulated by mitogen, and ELISA showed that the secretion of INF-γ was downregulated, and the secretion of IL-4 was upregulated, with CD8⁺ T cells markedly decreased and CD4⁺ T cells changed lightly. Moreover, the infusion of UC-MSCs in recipient mice transplanted with donor bone marrow cells ameliorated acute graft-versus host disease (aGVHD) and extended survival. In conclusion, UC-MSCs might negatively modulate immunoreactions, and have application potential in the treatment of aGVHD caused by allogeneic stem cells transplantation.     

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。     

携带人胰岛素基因腺病毒载体转染人脐带间充质干细胞及其成脂分化

背景：胰岛素是成脂诱导剂的重要组成成分。然而胰岛素存在半衰期,会随着体内代谢不断灭活及降解,植入体内的细胞或材料无法像体外那样以更换培养液来达到目的。实验拟通过转染胰岛素基因,使转染后的干细胞稳定分泌胰岛素,促进其成脂分化。 目的：探讨携带人胰岛素基因腺病毒载体转染人脐带间充质干细胞后对其成脂分化能力的影响。 方法：取第3代人脐带间充质干细胞,以感染复数为20转染腺病毒载体。实验分为4组：对照组为第4代人脐带间充质干细胞;实验组1为腺病毒转染的第4代人脐带间充质干细胞;实验组2为第4代人脐带间充质干细胞+成脂诱导液;实验组3为腺病毒转染的第4代人脐带间充质干细胞+成脂诱导液。 结果与结论：转染48 h后实验组1和实验组3的人脐带间充质干细胞在荧光显微镜下显现弱荧光,72 h后荧光较强。经脂肪诱导培养液培养14 d后,实验组1,2,3油红O染色后显微镜下呈红色,对照组未见脂滴形成,实验组1可见一些细小脂滴形成;实验组3与实验组2相比,脂滴大且多,定量分析显示,实验组3油红O染色阳性的面积和吸光度大于实验组2(P < 0.05)。由此说明携带人胰岛素基因腺病毒载体转染人脐带间充质干细胞后可促进其成脂能力。