Therapeutic capacities of human and mouse skeletal muscle-derived stem cells for a long gap peripheral nerve injury

An overview of a long-gap peripheral nerve therapy： A long- gap peripheral nerve transection injury is an irreparable injury to the living body, and mostly leads to permanent loss of re- lated motor and sensory functions. In such long gap injuries, nerve end-to-end suture is physically impossible. Therefore, bridging a long nerve-gap is critical to re-establish adequate mechanical support for separated nerve ends, and prevent the diffusion of neurotrophic and neurotropic factors secreted by transected stumps （Deumens et al., 2010）.

Protein hairy enhancer of split-1 expression during differentiation of muscle-derived stem cells into neuron-like cells

Muscle-derived stem cells were isolated from the skeletal muscle of Sprague-Dawley neonatal rats aged 3 days old. Cells at passage 5 were incubated in Dulbecco＇s modified Eagle＇s medium supplemented with 10% （v/v） fetal bovine serum, 20 IJg/L nerve growth factor, 20 pg/L basic fibroblast growth factor and 1% （v/v） penicillin for 6 days. Cells presented with long processes, similar to nerve cells. Connections were formed between cell processes. Immunocytochemical staining with neuron specific enolase verified that cells differentiated into neuron-like cells. Immunofluorescence cytochemistry and western blot results revealed that the expression of protein hairy enhancer of split-1 was significantly reduced. These results indicate that low expression of protein hairy enhancer of split-1 participates in the differentiation of muscle-derived stem cells into neuron-like cells.

Research progress in muscle-derived stem cells Literature retrieval results based on international database

OBJECTIVE： To identify global research trends of muscle-derived stem cells （MDSCs） using a bibliometric analysis of the Web of Science, Research Portfolio Online Reporting Tools of the National Institutes of Health （NIH）, and the Clinical Trials registry database （ClinicalTrials.gov）. DATA RETRIEVAL： We performed a bibliometric analysis of data retrievals for MDSCs from 2002 to 2011 using the Web of Science, NIH, and ClinicalTrials.gov. SELECTION CRITERIA： Inclusion criteria： （1） Web of Science： （a） peer-reviewed articles on MDSCs that were published and indexed in the Web of Science. （b） Type of articles： original research articles, reviews, meeting abstracts, proceedings papers, book chapters, editorial material and news items. （c） Year of publication： 2002-2011. （d） Citation databases： Science Citation Index-Expanded （SCI-E）, 1899-present; Conference Proceedings Citation Index-Science （CPCI-S）, 1991-present; Book Citation Index-Science （BKCI-S）, 2005-present. （2） NIH： （a） Projects on MDSCs supported by the NIH. （b） Fiscal year： 1988-present. （3） ClinicalTrials.gov： All clinical trials relating to MDSCs were searched in this database. Exclusion criteria： （1） Web of Science： （a） Articles that required manual searching or telephone access. （b） We excluded documents that were not published in the public domain. （c） We excluded a number of corrected papers from the total number of articles. （d） We excluded articles from the following databases： Social Sciences Citation Index （SSCI）, 1898-present; Arts ＆ Humanities Citation Index （A＆HCI）, 1975-present; Conference Proceedings Citation Index - Social Science ＆ Humanities （CPCI-SSH）, 1991-present; Book Citation Index - Social Sciences ＆ Humanities （BKCI-SSH）, 2005-present; Current Chemical Reactions （CCR-EXPANDED）, 1985-present; Index Chemicus （IC）, 1993-present. （2） NIH： （a） We excluded publications related to MDSCs that were supported by the NIH. （b） We limited the keyword search to studies that included MDSCs within the title or abstract. （3） ClinicalTrials.gov： （a） We excluded clinical trials that were not in the ClinicalTrials.gov database. （b） We excluded clinical trials that dealt with stem cells other than MDSCs in the ClinicalTrials.gov database. MAIN OUTCOME MEASURES： （1） Type of literature; （2） annual publication output; （3） distribution according to journals; （4） distribution according to country; （5） distribution according to institution; （6） top cited authors over the last 10 years; （7） projects financially supported by the NIH; and （8） clinical trials registered. RESULTS： （1） In all, 802 studies on MDSCs appeared in the Web of Science from 2002 to 2011, almost half of which derived from American authors and institutes. The number of studies on MDSCs has gradually increased over the past 10 years. Most papers on MDSCs appeared in journals with a particular focus on cell biology research, such as Experimental Cell Research, Journal of Cell Science, and PLoS One. （2） Eight MDSC research projects have received over US$6 billion in funding from the NIH. The current project led by Dr. Johnny Huard of the University of Pittsburgh-＂Muscle-Based Tissue Engineering to Improve Bone Healing＂-is supported by the NIH. Dr. Huard has been the most productive and top-cited author in the field of gene therapy and adult stem cell research in the Web of Science over last 10 years. （3） On ClinicalTrials.gov, ＂Muscle Derived Cell Therapy for Bladder Exstrophy Epispadias Induced Incontinence＂ Phase 1 is registered and sponsored by Johns Hopkins University and has been led by Dr. John P. Gearhart since November 2009. CONCLUSION： From our analysis of the literature and research trends, we found that MDSCs may offer further benefits in regenerative medicine.

Negative effects of Notch1 on the differentiation of muscle-derived stem cells into neuronal-like cells

We cultured rat muscle-derived stem cells in medium containing nerve growth factor and basic fi-broblast growth factor to induce neuronal-like cell differentiation.Immunocytochemical staining and reverse transcription-PCR showed that the differentiated muscle-derived stem cells exhibited processes similar to those of neuronal-like cells and neuron-specific enolase expression,but Notch1 mRNA and protein expression was decreased.Down-regulation of Notch1 expression may facilitate neuronal-like cell differentiation from muscle-derived stem cells.

TGF-β1-induced Synthesis of Collagen Fibers in Skeletal Muscle-Derived Stem Cells

The aim of this study was to investigate the mechanism of deposition of extracellular matrix induced by TGF-β1 in skeletal muscle-derived stem cells （MDSCs）. Rat skeletal MDSCs were obtained by using preplate technique, and divided into four groups： group A （control group）, group B （treated with TGF-β1, 10 ng/rnL）, group C （treated with TGF-β1 and anti-connective tissue growth factor （CTGF）, both in 10 ng/mL）, and group D （treated with anti-CTGF, 10 ng/mL）. The expression of CTGF, collagen type- I （COL- I ） and collagen type-III （COL-III） in MDSCs was examined by using RT-PCR, Western blot and immunofluorescent stain. It was found that one day after TGF-β1 treatment, the expression of CTGF, COL- I and COL-Ⅲ was increased dramatically. CTGF expression reached the peak on the day 2, and then decreased rapidly to a level of control group on the day 5. COL- I and COL-Ⅲ mRNA levels were overexpresed on the day 2 and 3 respectively, while their protein expression levels were up-regulated on the day 2 and reached the peak on the day 7. In group C, anti-CTGF could partly suppress the overexpression of COL-I and COL-Ill induced by TGF-131 one day after adding CTGF antibody. It was concluded that TGF-β1 could induce MDSCs to express CTGF, and promote the production of COL- I and COL-III. In contrast, CTGF antibody could partially inhibit the effect of TGF-β1 on the MDSCs by reducing the expression of COL- I and COL-III. Taken together, we demonstrated that TGF-β1-CTGF signaling played a crucial role in MDSCs synthesizing collagen proteins in vitro, which provided theoretical basis for exploring the methods postponing skeletal muscle fibrosis after nerve injury.

Skeletal Muscle-derived Stem Cells Exhibit Cardiocyte Competences

Adult stem cells from skeletal muscle cells were induced to differentiate into cardiocytes to see if stem cells from another different but histologically-comparable tissues can differentiate to the target cells. Skeletal muscles-derived stem cells （MDSCs） were isolated from adult skeleton muscle tissues by differential adhesion, and immunocytochemically identified by using Sca-1. In order to induce the proliferation but not differentiation of MDSCs, the cells were cultured in Dulbecco’s modified Eagle’s medium/F12 （DMEM/F12） supplemented with 1：50 B27, 20 ng/mL basic fibroblast growth factor （bFGF）, 20 ng/mL epidermal growth factor （EGF） in a suspension for 6 days. Then these stem cells were treated with 5 μmol/L 5-azacytidine for 24 h in an adherence culture. The characteristics of induced cells were examined by immunocytochemistry, quantitative real time RT-PCR and morphological observation of cell phenotype. Our results showed that the appearance of some cells gradually changed from spindle-shape into polygonal or short-column-shape. Some of these post-treated cells could contract spontaneously and rhythmically. The expression of GATA-4 and cTnT was increased 1 and 2 week（s） after the treatment. And about 16.6% of post-treated cells were cTnT-positive. Therefore, we are led to conclude that skeletal muscle-derived stem cells could differentiate into cardiocyte-like cells, which exhibited some characteristics of cardiocytes.

Can muscle-derived stem cells serve as seed cells to repair spinal cord injury?

Muscle-derived stem cells （MDSCs） can come from a number of different sources, which are easy to isolate and culture, and are also useful in the transformation and expression of exogenous genes. Therefore, MDSCs could possibly be used for gene therapy in the treatment of neurological diseases. However, research on MDSCs has focused on identifying phenotypes and induced differentiation, with few in vivo animal experiments conducted. In this study, MDSCs were selected as seed cells and implanted into the rat spinal cord injury area. Results demonstrated that the MDSCs survived, migrated, and were distributed along the spinal nerves. Moreover, the motor function of rat lower limbs improved significantly, suggesting that MDSCs could be used as seed cells to repair spinal cord injury.

Adipose-derived stem cell therapy for severe muscle tears in working German shepherds: Two case reports

Injuries to muscle in the elite athlete are common and may be responsible for prolonged periods of loss of competitive activity. The implications for the athlete, his/her coach and team may be catastrophic if the injury occurs at a critical time in the athlete's diary. Imaging now plays a crucial role in diagnosis, prognostication and management of athletes with muscle injuries. This article discusses the methods available to clinicians and radiologists that are used to assess skeletal muscle injury. The spectrum of muscle injuries sustained in the elite athlete population is both discussed and illustrated.

Similarities and differences between mesenchymal stem/progenitor cells derived from various human tissues

BACKGROUND Mesenchymal stromal/stem cells (MSCs) constitute a promising tool in regenerative medicine and can be isolated from different human tissues. However, their biological properties are still not fully characterized. Whereas MSCs from different tissue exhibit many common characteristics, their biological activity and some markers are different and depend on their tissue of origin. Understanding the factors that underlie MSC biology should constitute important points for consideration for researchers interested in clinical MSC application. AIM To characterize the biological activity of MSCs during longterm culture isolated from: bone marrow (BM-MSCs), adipose tissue (AT-MSCs), skeletal muscles (SMMSCs), and skin (SK-MSCs). METHODS MSCs were isolated from the tissues, cultured for 10 passages, and assessed for: phenotype with immunofluorescence and flow cytometry, multipotency with differentiation capacity for osteo-, chondro-, and adipogenesis, stemness markers with qPCR for mRNA for Sox2 and Oct4, and genetic stability for p53 and c-Myc;27 bioactive factors were screened using the multiplex ELISA array, and spontaneous fusion involving a co-culture of SM-MSCs with BM-MSCs or AT-MSCs stained with PKH26 (red) or PKH67 (green) was performed. RESULTS All MSCs showed the basic MSC phenotype;however, their expression decreased during the follow-up period, as confirmed by fluorescence intensity. The examined MSCs express CD146 marker associated with proangiogenic properties;however their expression decreased in AT-MSCs and SM-MSCs, but was maintained in BM-MSCs. In contrast, in SK-MSCs CD146 expression increased in late passages. All MSCs, except BM-MSCs, expressed PW1, a marker associated with differentiation capacity and apoptosis. BM-MSCs and AT-MSCs expressed stemness markers Sox2 and Oct4 in long-term culture. All MSCs showed a stable p53 and c-Myc expression. BM-MSCs and AT-MSCs maintained their differentiation capacity during the follow-up period. In contrast, SK-MSCs and SM-MSCs had a limited ability to differentiate into adipocytes. BM-MSCs and AT-MSCs revealed similarities in phenotype maintenance, capacity for multilineage differentiation, and secretion of bioactive factors. Because AT-MSCs fused with SM-MSCs as effectively as BM-MSCs, AT-MSCs may constitute an alternative source for BM-MSCs. CONCLUSION Long-term culture affects the biological activity of MSCs obtained from various tissues. The source of MSCs and number of passages are important considerations in regenerative medicine.

肌源干细胞通过TGF-β1/CTGF信号通路减轻压力性尿失禁大鼠的排尿障碍和尿道损伤

目的基于转化生长因子β1/结缔组织生长因子(TGF-β1/CTGF)信号通路探究肌源干细胞(MDSCs)对压力性尿失禁(SUI)大鼠排尿功能和尿道损伤的影响。方法分离、培养MDSCs,免疫细胞化学法检测MDSCs标志物Desmin和Sca-1表达。40只SD雌性大鼠按照随机数字表法分为4组:NC组(不作任何处理)、SUI组(SUI模型)、MDSCs组(SUI模型+MDSCs治疗)、MDSCs+SB431542组(SUI模型+MDSCs治疗+通路抑制剂SB431542处理),每组10只。比较4组大鼠喷嚏阳性率、尿动力学指标[膀胱最大容积(MBC)、漏尿点压(LPP)、腹压漏尿点压(ALPP)、排尿量、残余尿量、排尿效率];HE染色观察4组尿道组织病理学变化;蛋白免疫印迹法检测4组尿道组织中TGF-β1、CTGF蛋白表达。结果成功获得Desmin和Sca-1均呈阳性表达的MDSCs。与NC组比较,SUI组喷嚏阳性率、MBC、LPP、ALPP、排尿量、残余尿量均增加,排尿效率、TGF-β1和CTGF蛋白表达均降低(P<0.05),伴有严重尿道损伤;与SUI组比较,MDSCs组喷嚏阳性率、MBC、LPP、ALPP、排尿量、残余尿量均降低,排尿效率、TGF-β1和CTGF蛋白表达均增加(P<0.05),尿道损伤减轻;与MDSCs组比较,MDSCs+SB431542组喷嚏阳性率、MBC、LPP、ALPP、排尿量、残余尿量均增加,排尿效率、TGF-β1和CTGF蛋白表达均降低(P<0.05),尿道损伤加重。结论MDSCs可改善SUI大鼠的排尿功能,减轻尿道损伤,其可能是通过调控TGF-β1/CTGF信号通路发挥作用。

骨粘连蛋白促骨髓来源间充质干细胞成骨分化在青少年特发性脊柱侧凸中的作用

目的探究骨骼肌分泌的骨粘连蛋白(Osteonectin)促骨髓来源间充质干细胞(BMSC)成骨分化在青少年特发性脊柱侧凸(AIS)中的作用。方法纳入30例AIS患者,根据X射线片测量的Cobb角,将其分为轻度AIS组和重度AIS组。比较2组患者腰椎和骨骼肌骨密度(BMD)。收集2组患者BMSC,通过流式细胞术检测CD73和CD90蛋白表达,qRT-PCR检测Runx2、Osterix和Osteocalcin mRNAs表达水平,ELISA检测碱性磷酸酶(ALP)活性。收集2组患者骨骼肌,检测Osteonectin mRNA和蛋白表达水平。ELISA检测2组患者血清中Osteonectin水平。体外培养BMSC,分为BMSC组(无特殊处理)和BMSC+Osteonectin组(添加Osteonectin),检测Runx2、Osterix和Osteocalcin mRNAs表达水平及ALP活性。结果与轻度AIS组比较,重度AIS组患者腰椎和骨骼肌BMD降低(P<0.05),BMSC中Runx2、Osterix和Osteocalcin mRNAs表达水平降低(P<0.05),ALP活性降低(P<0.05);骨骼肌中Osteonectin mRNA和蛋白表达水平降低(P<0.05);血清中Osteonectin水平降低(P<0.05)。与BMSC组比较,BMSC+Osteonectin组细胞Runx2、Osterix和Osteocalcin mRNAs表达水平升高(P<0.05),ALP活性升高(P<0.05)。结论AIS患者骨骼肌分泌Osteonectin减少,BMSC的成骨分化能力降低。外源性补充Osteonectin可改善BMSC的成骨分化能力,可能有助于AIS病情的恢复。

BMP-2诱导肌源性干细胞转化为软骨样细胞的体外实验研究

目的体外定向诱导大鼠肌源性干细胞(MDSCs)向软骨细胞转化,并对诱导细胞进行鉴定。方法用差速贴壁的方法对SD大鼠MDSCs进行分离和培养。用骨形态发生蛋白2(BMP2)将MDSCs进行诱导分化,9d后取出标本,行甲苯胺蓝染色检测软骨基质的分泌,免疫组织化学检测软骨特异性Ⅱ型胶原表达。RT-PCR检测Ⅱ型胶原和聚集蛋白聚糖(aggrecan)的mRNA表达。结果5d后镜下见细胞形态由梭形向多边多角形转化。9d后诱导组甲苯胺蓝染色阳性,Ⅱ型胶原免疫组化检测阳性,RT-PCR检测Ⅱ型胶原和aggrecan mRNA呈阳性表达。结论BMP-2可诱导MDSC向软骨细胞转化。

当归补血汤对体外造血微环境中小鼠肌卫星细胞增殖及c-kit表达的影响

目的观察当归补血汤对体外造血微环境中小鼠肌卫星细胞增殖及c-kit表达的影响。方法分离培养小鼠卫星细胞并鉴定。制备含有不同剂量当归补血汤的大鼠载药血清及对应剂量的骨髓基质细胞条件培养基。将肌卫星细胞随机分为8组:正常大鼠血清组、当归补血汤载药血清1～3组、正常大鼠血清条件培养基组、条件培养基1～3组,MTT法检测细胞增殖活性,免疫组化法检测细胞c-kit的表达情况,荧光实时定量PCR检测细胞c-kit mRNA的表达。结果培养的肌卫星细胞呈Desmin免疫阳性;与正常大鼠血清组相比,载药血清各组及条件培养基各组细胞增殖显著,载药血清3组、条件培养基各组阳性细胞c-kit蛋白及mRNA表达量有显著性差异,随当归补血汤载药血清浓度增大c-kit表达量增多,条件培养基也呈同样的变化趋势。结论当归补血汤载药血清及含载药血清的条件培养基可促进肌卫星细胞增殖及c-kit的表达。

Wnt/β-catenin信号途径促进肌源性干细胞向神经细胞分化

目的探讨Wnt/β-catenin信号途径在肌源性干细胞(muscle derived stem cells,MDSCs)分化过程中的调控作用。方法对照组单纯体外培养MDSCs;实验组采用丙戊酸(valproic acid,VA)诱导培养MDSCs分化为类神经细胞;抑制剂组在实验组的基础上采用Dickkopf-1(DKK-1)和sFRP抑制Wnt/β-catenin信号途径。免疫细胞化学检测各组MDSCs的分化情况。RT-PCR检测MDSCs中Wnt3a mRNA表达的变化,Western blot检测MDSCs中GSK-3β、β-catenin蛋白含量的变化。结果实验组和抑制剂组MDSCs经VA诱导后出现神经细胞分化现象,抑制剂组分化水平明显低于实验组,对照组未见分化。与对照组相比,实验组和抑制剂组Wnt3a mRNA和β-catenin表达水平增加,实验组明显高于抑制剂组。实验组和抑制剂组GSK-3β表达水平低于对照组。结论 MDSCs向类神经细胞分化过程中Wnt/β-catenin信号通路被激活,Wnt/β-catenin信号通路阻断能够部分抑制MDSCs向类神经细胞分化。

胚胎血管发育早期PDGF-BB对血管平滑肌细胞趋化的影响

目的:建立胚胎血管发育早期血管平滑肌细胞(VSMCs)趋化模型,并观察胚胎血管发育早期血小板源性生长因子-BB(PDGF-BB)对VSMCs趋化的影响。方法:采用转染平滑肌特异性蛋白SM22α启动子控制下表达增强型绿色荧光蛋白(GFP)报告基因载体的胚胎干细胞制备拟胚体,然后接种于under-agarose凝胶作为平滑肌细胞趋化模型。用慢速视频显微摄像技术观察拟胚体分化后表达GFP的VSMCs,分析比较不同浓度PDGF-BB对VSMCs分化和移行的影响。结果:在under-agarose凝胶中,拟胚体在无外源性生长因子存在的条件下可自发向心肌细胞、内皮细胞和VSMCs分化。拟胚体贴壁分化20 d时,对照组及4种浓度PDGF-BB(5μg/L、10μg/L、20μg/L、50μg/L)组VSMCs的平均迁移速率分别为(94.07±23.80)μm/h、(118.08±31.63)μm/h、(173.53±24.58)μm/h、(380.74±39.56)μm/h和(335.62±32.16)μm/h,峰值浓度为20μg/L。经浓度大于10μg/LPDGF-BB处理后,VSMCs向PDGF-BB孔方向移行,对照组、低浓度PDGF-BB组VSMCs呈不规则性迁移。结论:该模型能够模拟胚胎血管发育早期全过程,可用于研究不同生长因子对VSMCs趋化的影响。外源性PDGF-BB能定向诱导胚胎血管发育早期VSMCs迁移,其定向趋化作用在一定浓度范围内呈量效关系。

5-氮胞苷诱导大鼠脂肪间充质干细胞分化成肌细胞

目的:观察并检测5-氮胞苷(5-Aza)诱导大鼠脂肪间充质干细胞(AMSCs)分化成肌细胞的过程中,细胞形态及相关基因、蛋白的表达情况,探讨5-Aza对大鼠AMSCs分化的诱导作用.方法:分离大鼠AMSCs,在含有10μmol/L5-Aza的培养液中培养,观察细胞形态的变化,并通过逆转录-聚合酶链式反应(RT-PCR)检测诱导后MyoG的表达,免疫细胞化学染色法检测诱导后Desmin和α-Sarcometric Actin的表达.结果:用10μmol/L5-Aza诱导后,细胞形态逐渐伸展变长,呈现肌细胞形态;RT-PCR结果显示,诱导后15h左右开始表达MyoG;免疫细胞化学染色结果显示,诱导10d后Desmin染色呈弱阳性,14d阳性程度增强,且此时部分细胞开始表达α-sarcomeric Ac-tin,并随时间延长表达增强,至28d呈强阳性.结论:采用10μmol/L5-Aza可以诱导大鼠AMSCs向肌细胞分化.

人尿源性干细胞的分离培养及诱导分化为平滑肌细胞

背景:传统尿路修复重建手段受限于供体短缺、并发症多以及生理功能恢复不理想等问题,组织工程策略为此领域提供了新方向。鉴于尿路主要由肌性组织构成,其中关键在于发掘适合的种子细胞并高效诱导分化为平滑肌细胞,但关于不同平滑肌细胞诱导方案效能的对比研究仍较为匮乏。目的:旨在分离、培养及鉴定人尿源性干细胞,并比较两种不同成平滑肌诱导方案的效果。方法:采用多次离心法从11份健康成人志愿者尿液中分离提取尿源性干细胞,使用流式细胞仪进行表面标志物的鉴定,通过成骨、成脂诱导分化来验证尿源性干细胞的多向分化潜能。尿源性干细胞分别在含转化生长因子β1以及转化生长因子β1联合血小板衍生生长因子的成平滑肌细胞诱导分化培养基中诱导分化14 d,采用免疫荧光染色和Western blot检测平滑肌特异性蛋白(α-SMA、SM22)的表达差异。结果与结论:①成功从8份健康人尿液中分离出尿源性干细胞,细胞呈“米粒”样,具有很好的分裂增殖能力;②尿源性干细胞高表达间充质干细胞表面标志物CD73、CD90和CD44,极低表达造血干细胞表面标志物CD34和CD45,不表达CD19、CD105和HLA-DR;③经成骨和成脂诱导分化后,可见明显的钙结节和脂滴形成,茜素红染色和油红O染色结果呈阳性;④成平滑肌诱导培养14 d,免疫荧光染色显示转化生长因子β1/血小板衍生生长因子组尿源性干细胞成平滑肌诱导分化率显著高于转化生长因子β1组(P<0.005);⑤成平滑肌诱导培养14 d,Western blot检测显示转化生长因子β1/血小板衍生生长因子组α-SMA和SM22蛋白表达量显著高于转化生长因子β1组(P<0.005)。结果表明:尿源性干细胞可以通过多次离心法无创分离获取;相较于单纯转化生长因子β1,转化生长因子β1/血小板衍生生长因子联合应用能显著提高尿源性干细胞诱导分化为平滑肌细胞的效率。

不同胚层来源成体干细胞修复周围神经损伤

背景:成体干细胞疗法是周围神经损伤修复与再生领域的研究热点之一。中胚层被视为成体干细胞的理想来源,间充质干细胞具有获得率高、来源广、增殖快等优异性能。而外胚层来源成体干细胞,尤其是神经嵴干细胞,具有神经源性,越来越受到研究人员的关注。目的:对来自外胚层和中胚层的多功能成体干细胞在周围神经损伤修复与再生中的作用及机制进行简要综述,探究不同来源成体干细胞的研究进展与应用前景,并结合临床研究,探讨成体干细胞疗法潜在的应用价值以及亟待解决的问题。方法:第一作者于2024年2月应用计算机在PubMed和SinoMed数据库检索2001年12月至2024年2月相关文献,以“ectodermal stem cells,mesenchymal stem cells,peripheral nerve injury,repair,regeneration”为英文检索词,以“外胚层干细胞、间充质干细胞、周围神经损伤、修复、再生”为中文检索词,最终纳入69篇文献进行分析论述。结果与结论:①外胚层来源成体干细胞具有优异的分化和再生潜能,尤其是毛囊神经嵴干细胞、嗅干细胞、牙外胚层干细胞等,具有神经源性,可在体外表达神经特异性标志物,但目前缺少临床试验研究。②中胚层来源成体干细胞种类多、易获得及纯化,其中骨髓间充质干细胞和脐带间充质干细胞在周围神经损伤修复的应用疗效及安全性方面有相关临床试验支持,能改善感觉及运动神经传导,且在随访中未出现并发症和明显不良反应。骨髓间充质干细胞的获取需行侵入性外科手术且要求患者与捐赠者骨髓配型吻合,应用受到一定限制;而脐带间充质干细胞虽无需侵入性获取,但分离较困难且表型不稳定。③内胚层来源成体干细胞常难以在体外生长,应用受限,目前应用于临床的可能性低。④综合来看,骨髓间充质干细胞仍为周围神经损伤干细胞治疗的首选细胞,适用于无外科手术禁忌且符合配型要求的情况,其次为脐带间充质干细胞,辅以分离方法的改进和表型稳定性的提高策略。⑤牙外胚层干细胞以及脂肪间充质干细胞具有较高应用潜能,有待进一步临床试验,其他外胚层、中胚层来源成体干细胞各以其优异特性在动物及细胞实验研究中具有显著优势。

小鼠肌源性干细胞高表达CD34、Sca-1、Bcl-2和desmin

目的:探讨小鼠肌源性干细胞(MDSCs)的生物学特性和表型。方法:取小鼠骨骼肌,用酶分步消化,应用差速贴壁法进行纯化获取MDSCs,观察细胞的形态,对MDSCs分别进行增殖能力、克隆形成率和融合情况等鉴定;通过流式细胞术、免疫荧光法及免疫印迹法(Western blotting)等检测初步确定细胞表型。结果:经差速贴壁法获取的MDSCs为圆形或短梭形细胞,聚集成簇,呈对数生长;细胞倍增时间(9.69±2.08)h,克隆形成率(13.35±2.54)%;细胞在高汇合度(>50%)或低血清(2%FBS)培养时,极易融合成肌管或肌细胞链;流式细胞术和免疫荧光技术鉴定:CD34、Sca-1、Bcl-2和desmin在原代MDSCs中的表达均>90%;Western blotting检测显示随着细胞的纯化,desmin表达越来越强,α-SMA表达越来越弱。结论:MDSCs通过差速贴壁法获取时纯度较高,它具有高水平表达CD34、Sca-1、Bcl-2和desmin的生物学特性,这4种蛋白可作为鉴定MDSCs的标志物。MDSCs增殖旺盛,可在体外大量扩增,是组织工程研究的一种新型种子细胞。

基质细胞衍化因子-1α对大鼠骨髓间充质干细胞分化的诱导

目的探讨基质细胞衍化因子(SDF)-1α诱导大鼠骨髓间充质干细胞分化的作用。方法密度梯度离心法分离培养原代大鼠骨髓间充质干细胞,经流式细胞术鉴定后传代培养,继而用重组SDF-1α及其中和抗体孵育48h,以Western blot及免疫细胞化学法检测骨髓间充质干细胞表面标志表达变化,以Transwell小室法检测其迁移细胞数量的变化。结果大鼠骨髓间充质干细胞经SDF-1α孵育后显著表达平滑肌细胞标志α-SMA、SM-22α和Calponin;与对照组相比,其迁移能力显著提高[(8 375±1 776)vs.(3 950±875),P<0.05]。而在接受SDF-1α孵育同时添加SDF-1α中和抗体的骨髓间充质干细胞中,上述平滑肌细胞标志表达不明显,迁移力较对照组无明显变化[(4 250±1 816)vs.(3 950±875),P>0.05]。结论 SDF-1α可显著诱导骨髓间充质干细胞向平滑肌细胞分化,提示其可能是移植器官动脉硬化增生内膜的重要来源之一。