Human umbilical cord blood-derived mesenchymal stem cells promote regeneration of crush-injured rat sciatic nerves

Several studies have demonstrated that human umbilical cord blood-derived mesenchymal stem cells can promote neural regeneration following brain injury. However, the therapeutic effects of human umbilical cord blood-derived mesenchymal stem cells in guiding peripheral nerve regeneration remain poorly understood. This study was designed to investigate the effects of human umbilical cord blood-derived mesenchymal stem cells on neural regeneration using a rat sciatic nerve crush injury model. Human umbilical cord blood-derived mesenchymal stem cells （1 ~ 106） or a PBS control were injected into the crush-injured segment of the sciatic nerve. Four weeks after cell injection, brain-derived neurotrophic factor and tyrosine kinase receptor B mRNA expression at the lesion site was increased in comparison to control. Furthermore, sciatic function index, Fluoro Gold-labeled neuron counts and axon density were also significantly increased when compared with control. Our results indicate that human umbilical cord blood-derived mesenchvmal stem cells promote the functinnal r~.RcJv^rv nf P.n I^h-inillr^4 ~r^i~tit, n^r~e

Functional recovery and microenvironmental alterations in a rat model of spinal cord injury following human umbilical cord blood-derived mesenchymal stem cells transplantation

BACKGROUND： Transplantation of human umbilical cord blood-derived mesenchymal stem cells （MSCs） has been shown to benefit spinal cord injury （SCI） repair. However, mechanisms of microenvironmental regulation during differentiation of transplanted MSCs remain poorly understood. OBJECTIVE： To observe changes in nerve growth factor （NGF）, brain-derived neurotrophic factor （BDNF）, and interleukin-8 （IL-8） expression following transplantation of human umbilical cord-derived MSCs, and to explore the association between microenvironment and neural functional recovery following MSCs transplantation. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Department of Orthopedics, First Affiliated Hospital of Soochow University from April 2005 to March 2007. MATERIALS： Human cord blood samples were provided by the Department of Gynecology and Obstetrics, First Affiliated Hospital of Soochow University. Written informed consent was obtained. METHODS： A total of 62 Wister rats were randomly assigned to control （n = 18）, model （n = 22, SCI ＋ PBS）, and transplantation （n = 22, SCI ＋ MSCs） groups. The rat SCI model was established using the weight compression method. MSCs were isolated from human umbilical cord blood and cultured in vitro for several passages. 5-bromodeoxyuridine （BrdU）-Iabeled MSCs （24 hours before injection） were intravascularly transplanted. MAIN OUTCOME MEASURES： The rats were evaluated using the Basso, Beattie and Bresnahan （BBB） locomotor score and inclined plane tests. Transplanted cells were analyzed following immunohistochemistry. Enzyme-linked immunosorbant assay was performed to determine NGF, BDNF, and IL-8 levels prior to and after cell transplantation. RESULTS： A large number of BrdU-positive MSCs were observed in the SCI region of the transplantation group, and MSCs were evenly distributed in injured spinal cord tissue 1 week after transplantation. BBB score and inclined plane test results revealed significant functional improvement in the transplantation group compared to the model group （P 〈 0.05）, which was maintained for 2-3 weeks. Compared to the model group, NGF and BDNF levels were significantly increased in the injured region following MSCs transplantation at 3 weeks （P 〈 0.05）, but IL-8 levels remained unchanged （P 〉 0.05）. CONCLUSION： MSCs transplantation increased NGF and BDNF expression in injured spinal cord tissue. MSCs could promote neurological function recovery in SCI rats by upregulating NGF expression and improving regional microenvironments.

Germline cells derived from mesenchymal stem cells, with the focus on Wharton's jelly

Previous attempts have indicated that mesenchymal stem cells (MSCs) are a valuable source and candidate and new approach for tissue engineering and reproductive medicine. MSCs have this potential to be induced and differentiated in an appropriatein vivoandin vitro condition toward various cell lineages and then they can be applied in cell therapies and clinical applications. During recent two decades, various sources have demonstrated they are a great source for MSCs, including bone marrow, the human umbilical cord as well as Wharton's jelly. Due to discarding after birth, easily accessible cells and less ethical concerns, these cells have attracted more and more scientists' attention. Infertility and reproduction diseases have provided special opportunity to examine the efficiency of MSCs in this kind of application. Based on recent investigations, MSCs embedded in Wharton's jelly tissue are more appealing for cell therapies, especially in infertility treatment purposes. So, differentiation of MSCs embedded in Wharton's jelly tissue into germ layer cells for cell-based therapy purposes is now under intensive study.

<i>In vitro</i>differentiation of human umbilical cord-derived mesenchymal stem cells into CD34⁺cells via CD34 antibody

CD34+cells differentiated from mesenchymal stem cells (MSCs) have a strong biological function in cardiovascular regeneration. However, the molecular mechanisms of and the methods to improve the CD34+ cell differentiation from MSCs, especially from human MSCs (hUC-MSCs) are still unclear. In the current study, the effect of CD34 antibody on the CD34+ cell differentiation from human umbilical cord (UC)-derived MSCs (hUC-MSCs) is determined. The results have demonstrated that the expression of cd34 protein is significantly increased in hUC-MSCs treated with CD34 antibody. In addition, the cell proliferation is increased in hUC-MSCs after treatment with CD34 antibody. Moreover, the expression of PI3K, AKT, p-AKT proteins, which are signaling molecules related to stem cell differentiation, is increased by CD34 antibody. The results suggest that CD34 antibody could promote the differentiation of hUC-MSCs into CD34+ cells and PI3K/AKT may be involved in this important process.

Therapeutic effects of human umbilical cord-derived mesenchymal stem cells against acute tubular necrosis quantified through measures of iNOS, BMP-7 and Bcl-2

Introduction: Acute tubular necrosis (ATN) is the most prevalent cause of acute renal failure (ARF). Mesenchymal stem cell transplantation has been studied as a potential treatment for renal dysfunction due to ATN. Inducible nitric oxide synthase (iNOS), bone morphogenetic protein-7 (BMP-7) and B-cell lymphoma 2 (Bcl-2) are surrogate markers of renal tubular epithelial regeneration and subsequent recovery of renal function following ATN. Methods: Serum creatinine (Scr) and blood urea nitrogen (BUN), as well as expression of iNOS, BMP-7 and Bcl-2 in gentamycin-induced ATN rat kidneys was investigated after human umbilical cord-derived mesenchymal stem cell (HUC-MSC) transplantation. Immunohistochemical staining was performed in 3 groups of rats: gentamycin-induced ATN treated with HUC-MSC, gentamycin-induced ATN without HUC-MSC, and untreated rats not receiving any treatments. Results: HUC-MSC transplantation led to a reduction in Scr and BUN in the kidneys of rats with gentamycin-induced ATN. Expression of iNOS in the HUC-MSC treated group occurred later and the expression levels were much lower during gentamycin-induced ATN compared to rats with ATN that were not treated with HUC-MSC. The expression of BMP-7 and Bcl-2 in the MSC-transplanted group was significantly increased compared to both control groups of rats with injured and healthy renal tubules. Conclusions: HUC-MSCs induce renal protection in a rat model of gentamycin-induced ATN, which is associated with reduced iNOS expression and up-regulation of Bcl-2 and BMP-7.

Human umbilical cord mesenchymal stem cells promote peripheral nerve repair via paracrine mechanisms

Human umbilical cord-derived mesenchymal stem cells （hUCMSCs） represent a promising young-state stem cell source for cell-based therapy. hUCMSC transplantation into the transected sciatic nerve promotes axonal regeneration and functional recovery. To further clarify the para-crine effects of hUCMSCs on nerve regeneration, we performed human cytokine antibody array analysis, which revealed that hUCMSCs express 14 important neurotrophic factors. Enzyme-linked immunosorbent assay and immunohistochemistry showed that brain-derived neurotrophic factor, glial-derived neurotrophic factor, hepatocyte growth factor, neurotrophin-3, basic fibroblast growth factor, type I collagen, fibronectin and laminin were highly expressed. Treatment with hUCMSC-conditioned medium enhanced Schwann cell viability and proliferation, increased nerve growth factor and brain-derived neurotrophic factor expression in Schwann cells, and enhanced neurite growth from dorsal root ganglion explants. These ifndings suggest that paracrine action may be a key mechanism underlying the effects of hUCMSCs in peripheral nerve repair.

Transplantation of human umbilical cord blood mesenchymal stem cells to treat a rat model of traumatic brain injury

In the present study, human umbilical cord blood mesenchymal stem cells were injected into a rat model of traumatic brain injury via the tail vein. Results showed that 5-bromodeoxyuridine-labeled cells aggregated around the injury site, surviving up to 4 weeks post-transplantation. In addition, transplantation-related death did not occur, and neurological functions significantly improved. Histological detection revealed attenuated pathological injury in rat brain tissues following human umbilical cord blood mesenchymal stem cell transplantation. In addition, the number of apoptotic cells decreased. Immunohistochemistry and in situ hybridization showed increased expression of brain-derived neurotrophic factor, nerve growth factor, basic fibroblast growth factor, and vascular endothelial growth factor, along with increased microvessel density in surrounding areas of brain injury. Results demonstrated migration of transplanted human umbilical cord blood mesenchymal stem cells into the lesioned boundary zone of rats, as well as increased angiogenesis and expression of related neurotrophic factors in the lesioned boundary zone.

Methods of isolation, expansion, differentiating induction and preservation of human umbilical cord mesenchymal stem cells

Objective This literature review aims to summarize the methods of isolation, expansion, preservation of human umbilical cord mesenchymal stem cells （hUCMSCs）, for comprehensive practical use in preclinical research and clinical trials. differentiation and understanding and Data sources All the literature reviewed was published over the last 10 years and is listed in PubMed and Chinese National Knowledge Infrastructure （CNKI）. Studies were retrieved using the key word ＂human umbilical cord mesenchymal stem cells＂. Results Explants culture and enzymatic digestion are two methods to isolate hUCMSCs from WJ and there are modifications to improve these methods. Culture conditions may affect the expansion and differentiating orientations of hUCMSCs. In addition, hUCMSCs can maintain their multi-potential effects after being properly frozen and thawed. Conclusion Considering their multi-potential, convenient and non-invasive accessibility, low immunogenicity and the reported therapeutic effects in several different preclinical animal models, hUCMSCs have immense scope in regeneration medicine as a substitute for MSCs derived from bone marrow or umbilical cord blood.

人脐带Wharton's Jelly来源间充质干细胞和人牙周膜干细胞复合PHBHHx、PHBV支架成骨分化能力的体外研究

目的:探讨人脐带Wharton's Jelly来源间充质干细胞(h UCWJMSCs)和人牙周膜干细胞(h PDLSCs)复合聚(3-羟基丁酸酯-3-羟基己酸酯)(PHBHHx)、聚羟基丁酸-羟基戊酸酯(PHBV)支架成骨分化的能力。方法:将2种干细胞分别接种在2种支架中,MTT法及粘附率分析2种干细胞和PHBHHx、PHBV的生物相容性;成骨诱导后ALP活性检测、茜素红染色和扫描电镜观察研究两种干细胞在两种支架上的成骨分化情况。结果:2种干细胞与PHBV具有更好的黏附效果及增殖效率(P <0. 05),且h UCWJMSCs组优于h PDLSCs组; h UCWJMSCs和h PDLSCs与PHBV组ALP活性检测、茜素红染色阳性表达明显高于与PHBHHx组; h UCWJMSCs与2种支架组的表达高于h PDLSCs组。扫描电镜显示2种干细胞与PHBV组可见大量矿化基质形成。结论:h UCWJMSCs与PHBV生物相容性和成骨能力优于h PDLSCs组。

人脐带间充质干细胞源胞外囊泡对屋尘螨诱导的哮喘小鼠气道炎症的影响

目的:探讨人脐带间充质干细胞源胞外囊泡(human umbilical cord mesenchymal stem cells-derived extracellular vesicles, hUCMSC-EVs)对屋尘螨(house dust mite, HDM)诱导的哮喘小鼠气道炎症的影响。方法:超高速离心法提取hUCMSC-EVs;透射电镜观察hUCMSC-EVs形态;蛋白质印迹法检测胞外囊泡标志物肿瘤易感基因101(tumor susceptibility gene 101,TSG101)和热休克蛋白70(heat shock protein 70,HSP70)表达。将15只雌性BALB/C小鼠随机均分为3组,每组5只,分别为对照组(不做任何处理)、HDM组(HDM处理)和HDM+EVs组(HDM+hUCMSC-EVs处理);苏木精-伊红(HE)和过碘酸-雪夫(PAS)染色法分别观察各组小鼠肺脏气道炎性细胞浸润和杯状细胞化生;收集各组小鼠支气管肺泡灌洗液(BALF)并对总细胞和嗜酸性粒细胞进行计数;ELISA法检测BALF中IL-4和IL-13含量;蛋白质印迹法检测肺脏中E-钙黏蛋白(E-cadherin)和闭锁连接蛋白1(ZO-1)表达。将人支气管上皮BEAS-2B细胞株分为对照组和EVs组(DIO标记的hUCMSC-EVs处理),共聚焦显微镜观察细胞对DIO标记的hUCMSC-EVs的内吞情况。另将BEAS-2B细胞分为3组:对照组、HDM组(HDM处理)、HDM+EVs组(HDM+hUCMSC-EVs处理),蛋白质印迹法检测细胞中E-cadherin和ZO-1表达。结果:hUCMSC-EVs呈杯托样膜性结构且表达HSP70和TSG101。与HDM组相比,HDM+EVs组小鼠气道炎性评分、PAS评分、BALF中总细胞和嗜酸性粒细胞数、IL-4和IL-13含量均显著下调(P均<0.01),而肺脏组织中E-cadherin和ZO-1表达明显增加(P<0.05)。体外实验显示hUCMSC-EVs可被人支气管上皮BEAS-2B细胞内吞;与HDM组相比,HDM+EVs组BEAS-2B细胞中E-cadherin和ZO-1表达明显增加(P<0.01或<0.05)。结论:hUCMSC-EVs可显著改善HDM诱导的小鼠气道周围炎性细胞浸润和气道杯状细胞化生。

人脐带Wharton’s Jelly来源间充质干细胞与人牙周膜干细胞成骨分化能力的对比研究

目的:比较人脐带Wharton’s Jelly来源间充质干细胞(human umbilical cord Wharton’s Jelly-derived mesechymal stem cells,h UCWJMSCs)与人牙周膜干细胞(periodontal mesenchymal stem cells,h PDLSCs)成骨分化能力。方法:体外培养h UCWJMSCs和h PDLSCs。MTT法检测细胞增殖情况;成骨诱导后测定细胞的ALP活性,茜素红染色检测细胞矿化能力,Real-time PCR分析OPN和Runx2基因的表达。结果:hUCWJMSCs增殖能力高于hPDLSCs;经矿化诱导后hPDLSCsALP表达、矿化结节形成高于hUCWJMSCs(P<0.05);Runx2在hPDLSCs中表达高于hUCWJMSCs(P<0.05);而hUCWJMSCs中OPN表达高于hPDLSCs(P<0.05)。结论:h UCWJMSCs、hPDLSCs均具有成骨分化能力,hPDLSCs成骨分化能力较强。

长期传代人脐带间充质干细胞的特性及功能

目的:探讨传至10代(P10)的人脐带来源间充质干细胞(P10-hUC-MSC)的生物学特性及功能。方法:人脐带来源于厦门弘爱医院(伦理批号:HAXM-MEC-20201012-037-01),分离、收集、培养hUC-MSC并传代培养,收集P1-、P10-hUC-MSC,FCM检测hUC-MSC表型,细胞衰老β-半乳糖苷酶染色法及FCM法检测终末期细胞衰老与凋亡情况,秋水仙碱处理检测细胞染色体稳定性,体外成脂、成骨诱导实验检测其多向分化能力,以不同比例与外周血单个核细胞(PBMC)混合培养后FCM检测T细胞亚群及表型变化。结果:成功分离和培养的P10-hUC-MSC与P1-hUC-MSC的表型相似,表现为CD45、CD34、HLA-DR表达阴性而CD105、CD90阳性率≥95%。终末期的P1-hUC-MSC和P10-hUC-MSC均表现出β-半乳糖苷酶表达阳性和早期凋亡特征,细胞染色体核型一致且保持稳定,未发生转化现象。P1-、P10-hUC-MSC在体外都可被诱导分化成脂肪、成骨细胞。P10-hUC-MSC与PBMC以1∶1混合培养7 d后,可显著上调CD4^(+)/CD8^(+)T细胞比值、CD4^(+)Treg细胞比例和PD-1表达(均P<0.01)。结论:长期传代的P10-hUC-MSC仍然保持其生物学特性和安全性,并具备多向分化能力及免疫调节能力,这为最大限度发挥hUC-MSC的临床放疗损伤修复与预防作用提供了前期实验依据和指导。

hUMSCs外分泌蛋白治疗实验性自身免疫性葡萄膜炎模型大鼠的机制初探

目的:探讨腹腔注射人脐带间充质干细胞外分泌蛋白(hUMSCs-S)治疗实验性自身免疫性葡萄膜炎(EAU)模型大鼠的可能性及其相关作用机制。方法:6~8周龄Lewis大鼠72只,用随机数字表分方法均分为3组,分别为正常对照(CTRL)组、EAU模型组及hUMSCs-S治疗组,每组24只。EAU组与hUMSCs-S组大鼠采用光感受器间维生素A结合蛋白(IRBP)联合弗氏完全佐剂建立EAU模型,hUMSCs-S组大鼠在建模的同时予以腹腔注射hUMSCs-S,EAU组及CTRL组大鼠均腹腔注射等量PBS。裂隙灯显微镜拍摄记录各组大鼠眼前段情况,根据Caspi评分标准评价眼前段炎症程度;于免疫后第7天(初发期)、14天(高峰期)、21天(恢复期)取眼球组织制成石蜡切片进行HE染色、免疫组化及免疫荧光染色,分别用于眼球组织病理学评分、前房浸润细胞情况评估,观察眼球组织中CD3^(+)T细胞及紧密连接蛋白ZO-1的表达。无菌操作下采腹主动脉血,制备血清,行ELISA法检测各组大鼠血清中白细胞介素10(IL-10)和IL-17的浓度。结果:(1)通过裂隙灯显微镜观察和比较,EAU组大鼠出现明显眼前段炎症反应,在建模后第11~17天,hUMSCs-S治疗有降低眼前段Caspi评分的作用(P<0.05);(2)眼球组织切片HE染色结果显示,建模后第14天,hUMSCs-S组大鼠虹膜睫状体和视网膜结构破坏及前房炎细胞浸润数量均低于EAU组(P<0.05);(3)眼球组织切片免疫组化及免疫荧光染色结果显示,hUMSCs-S组大鼠虹膜睫状体、前房和视网膜中CD3^(+)T细胞浸润较EAU组减少,视网膜及视网膜色素上皮(RPE)层ZO-1表达量较EAU组增加;(4)ELISA法检测大鼠外周血血清IL-17及IL-10浓度结果显示,与EAU组相比,hUMSCs-S组大鼠外周血IL-17表达下降、IL-10表达增加(P<0.05)。结论:hUMSCs外分泌蛋白对EAU模型大鼠治疗有效;腹腔注射hUMSCs外分泌蛋白可影响EAU大鼠外周血中炎症因子表达,减少眼内炎症细胞浸润,减轻RPE层紧密连接蛋白的破坏程度。

3D培养人脐带间充质干细胞来源的外泌体对成骨细胞分化的作用

目的比较三维立体(3D)培养与传统贴壁培养(2D)人脐带间充质干细胞(hUC-MSCs)来源的外泌体促进成骨细胞分化能力的差异,探索前者在促进骨形成相关治疗中应用的可能性。方法将hUC-MSCs分为2D组与3D组分别进行培养,在普通光学显微镜下观察细胞的形态特征,同时应用钙黄绿素-AM/PI活死细胞双染法检测3D组细胞的活性;通过转录组测序筛选两组差异表达基因并进行GO富集分析。提取2D组与3D组细胞分泌的外泌体并分为2D外泌体(2D-Exo)组与3D外泌体(3D-Exo)组,通过透射电子显微镜、纳米颗粒跟踪分析及Western blotting对外泌体进行表征。在体外应用2D-Exo及3D-Exo干预乳鼠颅骨成骨细胞并进行成骨诱导分化,通过茜素红染色、碱性磷酸酶染色及RT-qPCR鉴定2D-Exo、3D-Exo对成骨分化能力的影响。结果与2D组比较,3D组细胞大小均等,聚拢成球形生长;钙黄绿素-AM/PI活死细胞双染可见3D培养的hUC-MSCs具有较高活性;转录组测序结果显示,与2D组比较,3D组上调基因富集于骨矿化、软骨发育、细胞外基质的组成、成骨细胞分化、血管生成、细胞增殖的正向调控及基因表达的正向调控等通路,下调基因富集于细胞增殖的负向调控、细胞迁移的负向调控、细胞凋亡等通路。透射电子显微镜观察可见两组外泌体的直径均为100 nm左右,呈现外泌体典型的杯状形貌特征,且均表达外泌体相关标志蛋白CD9、CD63和CD81。乳鼠颅骨成骨细胞染色结果显示,与2D-Exo组比较,3D-Exo组茜素红着色的钙结节数量及碱性磷酸酶染色强度均明显升高;RT-qPCR检测结果显示,与2D-Exo组比较,3D-Exo组成骨分化相关基因Bglap、Runx2、Alp、Col1a1、Spp1 mRNA相对表达量明显升高,差异有统计学意义(P<0.05)。结论3D培养的hUC-MSCs来源的外泌体可上调成骨相关基因Bglap、Runx2、Alp、Col1a1、Spp1的表达,相较于2D培养来源的外泌体具有更强的促成骨细胞分化的能力。

hUC-MSCs对大鼠膝骨软骨缺损的修复作用

目的研究关节腔注射人脐带来源间充质干细胞(hUC-MSCs)对大鼠膝关节软骨缺损的修复作用及部分机制。方法舒泰50麻醉大鼠后,在大鼠股骨滑车沟采用电钻造成2 mm×2 mm的骨软骨缺损。术后一周将动物分为模型组和hUC-MSCs移植组,另设假手术组作为对照。hUC-MSCs移植组关节腔注射hUC-MSCs(2×10^(6)个细胞,50μl),假手术组和模型组关节腔注射等量生理盐水作为对照。大鼠在细胞移植后第10周麻醉处死。通过体视显微镜、组织病理学和免疫荧光分析评估hUC-MSCs对受损软骨的修复作用。体外培养大鼠原代软骨细胞,Transwell法检测hUC-MSCs对软骨细胞的增殖与迁移作用。结果关节腔注射hUC-MSCs可提高ICRS评分,缺损部位修复相对完整,有大量软骨细胞和基质填充,具有明显的修复作用。HE、番红固绿及Masson染色结果显示,hUC-MSCs组损伤部位与周围正常关节软骨组织差异无显著性。与模型组相比,hUC-MSCs组Ⅰ型胶原(ColⅠ)水平下降,Ⅱ型胶原(ColⅡ)水平上升,增殖细胞核抗原(PCNA)阳性细胞数增加,性别决定区域Y相关的高迁移率族框9(SOX9)表达水平及入核增加。体外共培养结果显示,hUC-MSCs可促进软骨细胞增殖和迁移。结论关节腔移植hUC-MSCs可促进关节软骨缺损修复,其机制可能与保护软骨基质和促进软骨细胞增殖迁移有关。

左归丸靶向C-X-C趋化因子受体4对人脐带间充质干细胞体外迁移的影响

目的:探讨左归丸通过靶向调控C-X-C趋化因子受体4(CXCR4)表达对人脐带间充质干细胞(hUC-MSCs)体外迁移的影响。方法:采用组织块法分离培养hUC-MSCs并进行流式鉴定。利用CCK-8检测不同浓度左归丸对hUC-MSCs增殖的影响,细胞划痕和Transwell实验分析左归丸对hUC-MSCs体外迁移的影响,实时聚合酶链反应(PCR)、蛋白印迹法和流式细胞术分析hUC-MSCs中CXCR4表达变化。结果:流式细胞术结果显示,hUC-MSCs表面高表达CD73和CD90,极低表达CD34和CD45。CCK-8结果显示,0.1、0.2 mg/mL左归丸均能明显促进hUC-MSCs增殖(P<0.05)。细胞划痕和Transwell实验结果显示,0.1、0.2 mg/mL左归丸能明显促进hUC-MSCs迁移,最佳浓度为0.2 mg/mL,CXCR4拮抗剂普乐沙福则能抑制其促迁移作用(P<0.05)。实时PCR、蛋白印迹法和流式细胞术结果显示,左归丸能上调hUC-MSCs中CXCR4 mRNA和蛋白表达水平,并促进CXCR4蛋白在细胞膜表达(P<0.05)。结论:左归丸通过上调CXCR4表达促进hUC-MSCs的体外迁移。

胶原/丝素蛋白支架联合人脐带源间充质干细胞干预犬创伤性脑损伤

背景:迄今为止,创伤性脑损伤颠覆性的治疗手段非常有限,医学与工程的结合为中枢神经系统神经修复与再生带来了新的前景。目的:探讨可携带种子细胞、兼具安全性与多孔隙的胶原/丝素蛋白支架联合人脐带间充质干细胞对犬创伤性脑损伤的治疗作用。方法:(1)将第3代人脐带间充质干细胞接种至胶原/丝素蛋白支架上,倒置相差显微镜、扫描电镜、免疫荧光染色、苏木精-伊红染色观察人脐带间充质干细胞生长情况。(2)将24只比格犬利用随机数字法分为4组:创伤组只建立创伤性脑损伤模型不做其他处理,干细胞组造模后移植人脐带间充质干细胞,支架组造模后移植胶原/丝素蛋白支架,联合组造模后移植人脐带间充质干细胞与胶原/丝素蛋白支架,移植物均在损伤灶局部移植。术后1 d以及1,4,8,12,16,20,24周分别进行改良格拉斯哥昏迷评分评估,术后1,3,6个月进行运动诱发电位检测,术后6个月行核磁共振成像弥散张量成像和脑组织修复RNA原位杂交,评估创伤性脑损伤恢复情况。结果与结论:(1)人脐带间充质干细胞贴附胶原/丝素蛋白支架表面生长良好并伸出许多伪足;(2)术后1,4,8,12,16,20,24周,联合组改良格拉斯哥评分显著高于创伤组、干细胞组、支架组(P<0.05);(3)不同恒压刺激下,1,3,6个月时联合组运动诱发电位潜伏期、振幅均极其显著优于创伤组(P<0.01);(4)核磁共振成像弥散张量成像显示:联合组皮质脊髓束完整性好于创伤组、干细胞组和支架组,损伤侧可见皮质新生;(5)脑组织修复RNA原位杂交结果:联合组Syn、MAP-2、vWF、NEFM、MBP表达量多于创伤组、干细胞组和支架组;(6)结果表明:人脐带间充质干细胞联合胶原/丝素蛋白支架对犬创伤性脑损伤后皮质脊髓束新生、运动功能恢复、血管新生与突起生长发挥了积极作用。

脐带间充质干细胞通过上调神经营养因子表达改善Aβ损伤大鼠的学习记忆能力

目的观察人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,h UCMSCs)对β-样淀粉蛋白(amyloidβ,Aβ)损伤大鼠学习记忆能力的影响,并探讨神经营养因子(neurotrophin,NT)在h UCMSCs改善大鼠学习记忆能力中的作用。方法实验分5组,分别为:空白对照组(control,con)、Aβ溶剂对照组(v-con)、人脐带间充质干细胞对照组(h UCMSCs-con)、Aβ损伤组(injury),以及人脐带间充质干细胞治疗组(h UCMSCs)。双侧海马CA1区定位注射Aβ制备阿尔茨海默样学习记忆障碍大鼠模型;通过Morris水迷宫检测大鼠学习记忆能力;通过硫堇尼氏体染色观察海马CA1区细胞区形态;通过ELISA分析神经营养因子含量。结果侧脑室注射脐带间充质干细胞可改善Aβ损伤大鼠的空间学习记忆能力,对抗Aβ损伤大鼠海马CA1区锥体细胞损伤,增加Aβ损伤大鼠海马组织神经营养因子表达。结论脐带间充质干细胞可对抗Aβ的神经毒性作用,改善Aβ损伤大鼠的空间学习记忆能力,其神经保护作用与增加海马组织中神经营养因子表达有关。

hUC-MSCs和白藜芦醇对AD小鼠学习记忆能力及脑内SIRT1信号通路的影响

目的:探讨人脐带间充质干细胞(h UC-MSCs)移植联合白藜芦醇(RES)灌胃对AD小鼠学习记忆能力的影响及对脑内SIRT1信号通路的调控作用。方法:40只AD小鼠随机分为AD组、h UC-MSCs组、RES组、h UC-MSCs联合RES组(联合组),每组10只。干预8周后,用Morris水迷宫评估小鼠学习记忆能力;取小鼠脑组织,采用免疫组化法检测Nestin的表达,TUNEL检测神经细胞凋亡,qRT-PCR检测神经营养因子BDNF、NGF和NT-3 mRNA的表达水平,Western blot法检测SIRT1、PCNA、P53、P21和P16的表达。结果:h UC-MSCs与RES均可使AD小鼠逃避潜伏期缩短,穿越平台次数增多,目的象限停留时间增加;海马区再生细胞增多,凋亡细胞减少,BDNF、NGF和NT-3mRNA表达水平升高,SIRT1和PCNA蛋白的表达增强,P53、P21、P16蛋白的表达减弱(P<0.05)。h UC-MSCs和RES联合对AD小鼠学习记忆能力、神经细胞再生、BDNF以及SIRT1蛋白的表达具有协同作用(P<0.05)。结论:h UC-MSCs移植和RES灌胃可能通过调控脑内SIRT1信号通路共同促进AD小鼠学习记忆能力的改善。

人脐带间充质干细胞的原代培养及多向分化潜能的研究

目的探讨人脐带间充质干细胞（hUCMSC）的体外分离培养鉴定方法及多向分化潜能。方法应用Ⅱ型胶原酶和透明质酸酶联合消化法及组织贴块培养法对hUCMSC进行体外培养。在倒置显微镜下观察不同方法获得的hUCMSC的生长特点；台盼蓝法测定细胞传代成活率；采用生长曲线、MTT法测定hUCMSC的增殖、流式细胞术（FCM）测定细胞周期变化及细胞免疫表型变化；采用相应试剂盒鉴定其向成脂细胞、成骨细胞的分化，采用免疫荧光细胞化学技术检测向成心肌样细胞的分化、采用荧光标记技术检测hUCMSC向血管内皮细胞的分化。结果酶消化法分离培养的细胞1d后，在倒置相差显微镜下可见细胞贴壁呈圆形生长，4d后生长迅速呈长梭形，7d后由中心向周围生长且增殖明显，10d后达80％融合即可传代；贴块法培养的细胞7d后，可见细胞从组织块边缘长出，10d后细胞数量逐渐增多，16d后细胞生长迅速呈单层致密排列即可传代，细胞传代成活率均为96％以上。2种方法获得的第3代细胞生长曲线近似“S”形、MTr法显示3～5d细胞增殖较明显；酶消化法培养的细胞G0／G1期和s＋G2／M期所占比例分别为88．78％和10．21％，组织贴块法培养的细胞G0／G1期和S＋G2／M期所占比例分别为84．82％和13．87％，细胞DNA周期无明显差异；2种方法获得的细胞经FCM检测CD90、CD105、CD73阳性率均为99％以上为高表达；CD45、CD34、CD14、CD11b、CD79a、CD19、HLA—DR低表达；2种方法获得的细胞在体外诱导都能向成骨细胞、成脂细胞、成心肌样细胞及血管内皮细胞分化，其诱导阳性率均为90％以上。结论酶消化法和贴块法均可高效获得hUCMSC，与贴块法培养相比较，酶消化法能更有效的获得扩增迅速、细胞成分单一的细胞。