Effects of Co-grafts Mesenchymal Suspension in the Repair of Spinal Stem Cells and Nerve Growth Factor Cord Injury

To investigate effect of the transplantation of mesenchymal stem cells （MSCs） in combination with nerve growth factor （NGF） on the repair of spinal cord injury （SCI） in adult rats, spinal cord of adult rats （n= 32） was injured by using the modified Allen＇ s method. One week after the injury, the injured cords were injected with Dubeeeo-modified Eagles medium （DMEM , Group Ⅰ ）, MSCs （Group Ⅱ ）, NGF （Group Ⅲ）, and MSCs plus NGF （Group Ⅳ）. One month and two months after the injury, rats were sacrificed and their injured cord tissues were sectioned for the identification of the transplanted cells. The axonal regeneration and the differentiation of MSCs were examined by immunoeytoehemieal staining. At the same time, rats were subjected to behavioral tests by using the open-field BBB scoring system. Immunoeytoehemieal staining showed that axonal regeneration and the transplanted cells partially expressed neuron-specific nuclear protein （NeuN） and glial fibrillary acidic protein （GFAP）. At the same time, significant improvement in BBB locomotor rating scale （P〈0. 05） were observed in the treatment group. More importantly, further functional improvement were noted in the combined treatment group. MSCs could differentiate into neurons and astroeytes. MSCs and NGF can promote axonal regeneration and improve functional recovery. There might exist a synergistic effect between MSCs and NGF.     

Monitoring the source of mesenchymal stem cells in patients after transplantation of mismatched-sex hematopoietic stem cells plus third-party cells

Background In bone marrow transplant patients, the microenvironment in bone marrow is damaged after chemotherapy or radiotherapy. Subsequent to allogenic hematopoietic stem cell transplantation in patients with clinically successful engraftments, the source of mesenchymal stem cells （MSCs） remains controversial. To further verify the stimulatory effect of the simultaneous transplantation of cells from second donors on engraftment success for hematopoietic stem cell transplantation in support of donor MSCs engraftments, the aim of this study is to monitor the dynamics of the engraftment of bone marrow-derived MSCs in patients after transplantation with mismatched-sex hematopoietic stem and third-party cells. Methods In this study, the hematopoietic stem cells from 32 clinical donors of different sexes that resulted in successful engraftments were selected for transplantation and were classified into three groups for research purposes： group A consisted of 14 cases of transplantation with bone marrow and recruited peripheral hematopoietic stem cell transplantation, group B contained 8 cases of simultaneous re-transfusion of MSCs from the second donor, and group C contained 10 cases of simultaneous re-transfusion of umbilical blood from the second donor. The bone marrow from 32 patients with successful engraftments of hematopoietic transplantation were selected and sub-cultured with MSCs. Flow cytometry （FCM） was used to measure the expression of surface antigens on MSCs. Denaturing high-performance liquid chromatography （DHPLC） in combination with polymerase chain reaction amplification of short tandem repeats （STR- PCR） was used to measure the engraftment status of fifth-generation MSCs in patients. Fluorescence in situ hybridization （FISH） revealed the sex origin of the fifth-generation MSCs in 32 patients. Dynamic examinations were performed on patients receiving donor transplantations. Results The progenies of fifth-generation MSCs were successfully cultured in 32 cases. The results of FCM demonstrated that the expression levels of CD14＋ and CD45＋ cells were lower than 0.04% in the fifth-generation MSCs. The analysis using DHPLC and FISH showed similar results. One patient from group B also received a temporary transplantation of MSCs from the donor. The MSCs in the remaining 31 patients all originated from the patients themselves. Conclusions After transplantation, the MSCs present in patients originated from the host. In patients transplanted with MSCs from a second donor, the phenomenon of temporary chimerization of MSCs was observed.     

Functional recovery after rhesus monkey spinal cord injury by transplantation of bone marrow mesenchymal-stem cell-derived neurons

Background The treatment of spinal cord injury is still a challenge. This study aimed at evaluating the therapeutical effectiveness of neurons derived form mesenchymal stem cells (MSCs) for spinal cord injury.Methods In this study, rhesus MSCs were isolated and induced by cryptotanshinone in vitro and then a process of RT-PCR was used to detect the expression of glutamic acid decarboxylase (GAD) gene. The induced MSCs were tagged with Hoechst 33342 and injected into the injury site of rhesus spinal cord made by the modified Allen method. Following that, behavior analysis was made after 1 week, 1 month, 2 months and 3 months. After 3 months, true blue chloride retrograde tracing study was also used to evaluate the re-establishment of axons pathway and the hematoxylin-eosin (HE) staining and immunohistochemistry were performed after the animals had been killed.Results In this study, the expression of mRNA of GAD gene could be found in the induced MSCs but not in primitive MSCs and immunohistochemistry could also confirm that rhesus MSCs could be induced and differentiated into neurons. Behavior analysis showed that the experimental animals restored the function of spinal cord up to grade 2-3 of Tarlov classification. Retrograde tracing study showed that true blue chollide could be found in the rostral thoracic spinal cords, red nucleus and sensory-motor cortex.Conclusions These results suggest that the transplantation is safe and effective.     

Induction of functional recovery by co-transplantation of neural stem cells and Schwann cells in a rat spinal cord contusion injury model

Objective To study the transplantation efficacy of neural stem cells （NSCs） and Schwann cells （SC） in a rat model of spinal cord contusion injury. Methods Multipotent neural stem cells （NSCs） and Schwann cells were harvested from the spinal cords of embryonic rats at 16 days post coitus and sciatic nerves of newborn rats, respectively. The differential characteristics of NSCs in vitro induced by either serum-based culture or co-culture with SC were analyzed by immunofluorescence. NSCs and SCs were co-transplanted into adult rats having undergone spinal cord contusion at T9 level. The animals were weekly monitored using the Basso-Beattie-Bresnahan locomotor rating system to evaluate functional recovery from contusion-induced spinal cord injury. Migration and differentiation of transplanted NSCs were studied in tissue sections using immunohistochemical staining. Results Embryonic spinal cord-derived NSCs differentiated into a large number of oligodendrocytes in serum-based culture upon the withdrawal of mitogens. In cocultures with SCs, NSCs differentiated into neuron more readily. Rats with spinal cord contusion injury which had undergone transplantation of NSCs and SCs into the intraspinal cavity demonstrated a moderate improvement in motor functions. Conclusions SC may contribute to neuronal differentiation of NSCs in vitro and in vivo. Transplantation of NSCs and SCs into the affected area may be a feasible approach to promoting motor recovery in patients after spinal cord injury.     

Spinal cord and other aspects

Modified partial labyrinthectomy petrous apicectomy approach：an microanatomic stody; Intraoperative monitoring of facial aberrant responses during microvascular decompression for hemifacial spasm; Evaluation of 3D-CISS sequence MRI before and after the endoscopic third ventriculostomy; Microneurosurgery of multisegmental intramedullary spinal cord ependymomas;Evaluation of neurological function recovery following human umbilical cord mesenchymal stem cells transplantation to injured spinal cord in rats;     

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, differentiation and preservation of human umbilical cord mesenchymal stem cells (hUCMSCs), for comprehensive understanding and practical use in preclinical research and clinical trials.

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.

     

Improvement of neurological function in rats with spinal cord injury after the transplantation of neural stem cells directly differentiated from bone marrow mesenchymal stem cells

<正>Objective To study the effect and mechanism of neurological function recovery in rats with spinal cord injury ( SCI) rats after transplantation of neural stem cells which are directly differentiated from bone marrow mesenchymal stem cells ( BMSC ) ,and to investigate the suitable engraftment time. Methods BMSC at 3rd passage were differentiated into neural stem cells ( NSC) , and immunofluorescence staining was used to identify     

Brain-derived neurotrophical factor after olfactory ensheathing cells transplantation in spinal cord injury of rats

<正>Objective To observe the expression of brain - derived neurotrophical factor ( BDNF) in injury spinal cord after transplantation olfactory ensheathing cells ( OECs) , and to investigate the mechanism of OECs repairing spinal cord injury. Methods OECs from GFP transgenic rats were separated and cultured for transplantation. Spinal cord injury rats were separated two groups by random     

人脐带间充质干细胞的生物学特性及向软骨细胞、骨细胞分化实验研究

目的 研究人脐带华尔通胶来源间充质干细胞的特性及向软骨细胞、骨细胞的分化能力,为椎间盘再生研究寻找新的细胞来源.方法 取人的正常分娩或剖腹产胎儿的脐带,酶消化法从Wharton胶中分离干细胞,应用复合胶原酶NB4、dispaseⅡ和透明质酸的组合混合酶消化,检测人脐带来源的MSCs的细胞表面标记;分别应用成软骨诱导液、成骨诱导液诱导人脐带来源的MSCs向软骨细胞、骨细胞分化.用免疫细胞化学方法对分化和未分化的细胞进行鉴定.结果 人脐带分离培养的贴壁细胞,体外生长形态类似于成纤维细胞,可以维持在未分化状态稳定增殖,这类细胞MSCs的表面标记CD44、CD105、CD90和D73呈现高表达,而不表达CD45、CD34、CD14、CD19及HLA-DR.向软骨细胞、骨细胞诱导结果提示P3代细胞有向终末软骨细胞、骨细胞分化能力,具有干细胞的特性.结论 人脐带华尔通胶含有丰富的MSCs,易于培养扩增.人脐带来源的MSCs能分化为软骨细胞、骨细胞,这类细胞可能成为椎间盘移植的一个干细胞来源.

Abstract：

Objective To investigate the isolation and expansion of mesenchymal stem cells (MSCS) from human umibilical cord Wharton's jelly and their biological identities , and explore the possibility of inducing human umbilical cord-derived MSCS to differentiate into chondrogenic and osteogenic cells. Methods The hUCMSCs were isolated form human umbilical cord by tissue adherence and digested with collagenaseNB4, dispase Ⅱ and hyaluronidase. The morphology, proliferation and immunophenotype of the 3rd passage cells were analyzed, and then the chondrogenic and osteogenic differentiation was tested and evaluated by specific staining methods. cells were induced to chondrogenic and osteogenic differentiation in vitro. Results The isolation of hUCMSCs by digestion with collagenaseNB4, dispase Ⅱ and hyaluronidase was efficient. After seeded for 24 hours, the adherent cells showed spindle shape and fibroblast cell-like shape and the size of hUCMSCs was homogeneous. Flow cytometry analysis revealed that the hUCMSCs were positive for CD44,CD105, CD90, CD73, but were negative for CD45, CD34, CD14, CD19 and HLA-DR.These cells could be induced to differentiate into chondrogenic and osteogenic cells under proper inducing conditions. The hUCMSCs retained the appearance and phenotype even after being expanded more than 40 passages in vitro. Conclusions The human MSCs could be isolated from human umbilical cord Wharton's jelly ,and it was easy to propagate these MSCs. An in vitro method for isolation and purification of hUCMSCs from human umbilical cord has been established. The cultured cells were composed of only undifferentiated cells and their biological properties were stable. The hUCMSCs are expected to be a new type of stem cells of tissue engineering.     

人脐带间充质干细胞在脊髓损伤修复中的研究进展

脊髓损伤发病率和致残率高,至今尚无完全修复损伤脊髓的治疗方法。随着干细胞移植技术的发展,有望从根本上修复损伤的脊髓。而在所有干细胞中,人脐带间充质干细胞可能是最佳的移植选择。动物研究已证实人脐带间充质干细胞具有巨大的脊髓损伤修复潜力,但临床转化并不顺利。本文将着重讨论人脐带间充质干细胞对脊髓损伤的神经修复机制,以及临床最佳移植途径、剂量、时机和临床安全性、有效性。     

人脐带间充质干细胞在帕金森病的应用进展

帕金森病(Parkinson's disease,PD)是一种复杂的神经系统疾病,其典型病理特征为与黑质多巴胺能(DA)神经元丢失有关的运动系统功能受损。目前尚无有效的治疗方法逆转DA神经元的进行性损失阻止PD的恶化。近年来,干细胞移植意逐渐被临床用于治疗神经退行性疾病,包括间充质干细胞(mesenchvmal stem ce11s,MSCs)移植治疗,其中人脐带间充质干细胞(hUC-MSCs)较其他来源的MSCs优势更明显,已广泛用于多种疾病的治疗。MSC能分化为DA神经元并分泌多种保护因子发挥功能。本文对hUC-MSCs的特性及其应用于PD治疗的研究现状进行综述,分析目前hUC-MSCs治疗研究存在的问题,旨在为干细胞再生医学治疗PD提供新的思路。     

人CD34+脐血干细胞移植治疗大鼠脊髓损伤

目的:探讨人脐血来源的CD34 干细胞治疗脊髓损伤的可行性,为脊髓功能障碍性疾病的临床治疗打下基础.方法:用密度梯度离心及免疫磁珠法分离出人CD34 脐血干细胞,在含有B27、碱性成纤维细胞生长因子(bFGF)、人重组干细胞因子(rhSCF)的营养液中培养,用核荧光染色剂Hoechst33258标记细胞;制作大鼠脊髓横断模型,造成大鼠下肢瘫痪,在脊髓损伤3 d移植荧光标记的CD34 脐血干细胞,观察移植后的动物行为变化、脊髓组织形态学变化等.结果:脊髓损伤大鼠移植CD34 脐血干细胞5～6 d可见瘫痪大鼠的后肢肌力开始恢复,10～15 d后可出现爬行,3～4周后后肢活动活跃;对照组瘫痪的肢体未见恢复.脊髓移植区肉眼可见有增生的组织充填,镜下有大量神经胶质原纤维酸性蛋白、神经元特异性烯醇化酶阳性的细胞,移植区Hoechst33258荧光标记阳性细胞增生.结论:移植的CD34 脐血干细胞可在移植部位增殖,并使脊髓横断大鼠运动功能恢复,有望成为治疗脊髓损伤的有效手段.     

脐血干细胞移植的研究及临床应用

脐血作为干细胞的主要来源,近年来国内外对其进行了大量的基础性研究和临床探索。已经证实脐血中含有大量造血干细胞、丰富的间充质干细胞。临床干细胞移植治疗采用脐血具有诸多优势,如来源丰富,组织配型时间短,抗原表达弱,加之间充质干细胞的免疫调节及加速造血恢复等影响,脐血的移植成功率高,移植反应弱、移植物抗宿主病少见。目前脐血移植治疗的疾病已经达到80余种,治疗的患者全世界已经超过了6000例。脐血移植已经在恶性肿瘤、免疫缺陷、心脏病、神经系统损伤、组织器官修复、糖尿病、血管疾病等的治疗上显示巨大的潜力。     

干细胞移植联合电针治疗对脊髓损伤修复的影响

目的研究人脐血干细胞移植联合电针刺激治疗脊髓损伤过程中对于类胰岛素样生长因子-1（IGF-1）表达的影响。方法将SD大鼠利用随机数字表法随机分成3组,所有大鼠均为脊髓横断损伤。脊髓损伤（SCI）组,干细胞移植（UCBSC）组,干细胞移植＋电针刺激（UCBSC＋EA）组,每组均为12只。UCBSC、UCBSC＋EA组在损伤处注射脐血干细胞,SCI组以等量PBS缓冲液代替,UCBSC＋EA组每天固定时间取损伤处上、下部位的＂大椎＂＂命门＂进行电针刺激。各组均在损伤后的7天、14天和28天于损伤处取材,观察神经元细胞的形态、数量及IGF-1表达。结果常规HE组织学检查发现横断处灰质内神经元细胞变性、减少,并伴星形胶质细胞增生,细胞体肥大。免疫组化显示,损伤后IGF-1表达均上调,UCBSC、UCBSC＋EA两组表达进一步上调,IGF-1的表达在前14天显著上调,14天后表达放缓。Western Blot结果显示,与单纯的人脐带血移植（UCBSC）相比较,UCBSC＋EA更能促进IGF-1的表达,且有统计学意义（P〈0.05）。RT-PCR电泳结果显示UCBSC＋EA组,IGF-1mRNA的表达明显高于其他两组,有显著性差异（P〈0.05）。结论 UCBSC＋EA在大鼠脊髓损伤处可更有效分泌神经营养因子IGF-1,弥补脊髓损伤后维持神经元细胞生存所匮乏的营养因子,促进神经元细胞存活。     

三七皂甙诱导脐血干细胞移植对脊髓损伤患者脑脊液巢蛋白的影响

目的研究中药提取物三七皂甙注射液对脐血源干细胞移植治疗术后早期增殖分化作用的影响。方法以脐血干细胞鞘注治疗30例脊髓损伤患者为受试对象，移植后给予三七皂甙注射液静脉注射，连续30d，观察4周前后脑脊液中巢蛋白变化。结果三七组及对照组脑脊液巢蛋白含量均有增高趋势；三七组脑脊液巢蛋白增高比率明显优于对照组，差异有统计学意义。结论三七皂甙能够诱导干细胞的增殖及分化，但是三七组对神经功能近期疗效并不优于对照组，远期疗效尚需做进一步的观察。     

人脐带华通氏胶间充质干细胞的分离、培养、鉴定及冻存、复苏

目的：探讨从人脐带华通氏胶（Wharton’s jelly,WJ）中分离、培养、鉴定间充质干细胞（mesenchymal stemcells,MSCs）及其冻存、复苏的方法。方法：采用植块法分离、培养间充质干细胞,流式细胞仪检测P3代细胞免疫表型,鉴定其向成骨、成脂方向诱导分化的能力;将P1细胞冻存6个月后复苏,鉴定复苏后细胞的特性。结果：植块法容易从人脐带华通氏胶中获得间充质干细胞;组织块贴壁后6 d可见组织块周围细胞爬出,原代培养14~18 d细胞融合70%~80%;P3代细胞强烈表达CD73、CD90、CD105,不表达CD14、CD34、CD45、CD79a和HLA-DR;成骨诱导分化后10 d,可见明显钙结节;成脂诱导14 d,有明显的脂滴出现,油红O染色阳性。冻存再复苏细胞活力达80%,细胞免疫表型及成骨、成脂诱导显示与冻存前细胞呈相同的特性。结论：组织块培养法可从人脐带华通氏胶中分离、培养出纯度较高间充质干细胞,冻存、复苏不改变其特性。     

谷氨酰胺联合脐血间充质干细胞移植在大鼠肠缺血再灌注损伤中的作用

目的探讨谷氨酰胺联合脐血间充质干细胞(MSCs)移植在大鼠肠缺血再灌注损伤中作用。方法体外复苏并培养脐血间充质干细胞移植前备用,观察CM-Di I荧光标记后脐血间充质干细胞的去向。80只SD大鼠随机分为正常对照组,缺血再灌注损伤组,谷氨酰胺组,MSCs移植组及联合组每组各15只。对照组采用生理盐水灌肠,损伤组采用TNB(S乙醇稀释)灌肠,在TNBS建模后1 h,谷氨酰胺组于尾静脉输入谷氨酰胺0.45 g/kg、MSCs移植组于尾静脉输入1×10~(10)/L脐血间充质干细胞悬液,联合组尾静脉输入谷氨酰胺0.45 g/kg+脐血间充质干细胞悬液1×10~(10)/L。通过ELISA法检测各组大鼠血清中肠脂肪酸结合蛋白(IFABP)、白介素-6(IL-6)、超氧化物歧化酶(SOD)的含量;各组于再灌注1 h、3 h后检测肠组织含水率;通过RT-PCR、Western blot观察大鼠肠黏膜上皮细胞caspase-3、NF-k B、Bcl-2在谷氨酰胺联合MSCs移植后的mRNA和蛋白的表达情况。结果通过荧光示踪法观察到移植的MSCs细胞分布于肠粘膜淋巴组织内和腺上皮细胞间,表明MSCs可能参与了肠缺血再灌注损伤的修复过程。各组大鼠血清中SOD、IFABP、IL-6的含量变化比较,损伤组血清中IFABP、IL-6的含量较对照组显著增加,而谷氨酰胺组,MSCs移植组及联合组与之比较,则显著减少,联合组减少更为明显,损伤组血清中SOD的含量较对照组显著减少,而谷氨酰胺组,MSCs移植组及联合组与之比较,则显著增高,联合组增高更为明显(P0.05)。再灌注1 h和3 h,损伤组肠组织含水率均明显高于对照组;与损伤组相比,谷氨酰胺组、MSCs移植组及联合组肠组织含水率值均显著降低,联合组降低更为明显,而谷氨酰胺组、MSCs移植组差异无统计学意义(P0.05)。与对照组比较,损伤组肠黏膜上皮细胞caspase-3、NF-k B的mRNA和蛋白表达明显上调,Bcl-2的mRNA和蛋白表达明显下调(P0.05),而谷氨酰胺组、MSCs移植组及联合组与之比较,caspase-3、NF-k B的mRNA和蛋白表达明显下调,Bcl-2的mRNA和蛋白表达明显上调(P0.05),谷氨酰胺组及MSCs移植组之间无统计学差异(P0.05),但两组与联合组比较,差异明显(P0.05)。结论谷氨酰胺组及MSCs移植后,明显减轻了大鼠肠缺血再灌注损伤程度,其可能通过抑制caspase-3、NF-k B表达和促进Bcl-2表达减轻肠黏膜缺血再灌注损伤。