Lymphocyte infiltration following allo-and xenomyoblast transplantation in mdx mice

Human and mouse (C57BL/10SnJ+/+) myoblasts were injected separately in the muscles of C57BL/10SnJ mdx/mdx mice. Mouse myoblasts (C57BL/10SnJ+/+) were also injected in normal mice (C57BL/10SnJ+/+ and BALB/c +/+). Some muscles that received a xenotransplantation (i.e., human myoblasts) were previously injected with a myotoxin, i.e., notexin. This treatment was not used for the allografts (i.e., mouse myoblasts). Human myoblast injections did not increase the number of dystrophin-positive cells above the background level due to backmutation. Moreover, the human myoblasts detected with an anti-HLA antibody decreased rapidly during the 6-week followup. The injection of normal mouse myoblasts in mdx mice did, however, increase the number of dystrophin-positive fibers. Moreover, numerous cells expressing mouse MHC class II, macrophages, granulocytes, neutrophils, natural killer cells, and a subset of T lymphocytes were detected by immunohistochemistry in cryostat sections of myoblast injected muscles. These cells were present within 1 week of the myoblast injection in the muscle regions containing injected human or mouse myoblasts, and progressively decreased during the 6-week follow-up in the human myoblast transplantation. Lymphocyte infiltration reached a significant level following xeno- and alloincompatible transplantations. Antibodies against the human myoblasts and against alloincompatible myoblasts were also detected in the serum of the recipients. These results suggest that humoral and cellular immune reactions are responsible for the poor outcome of myoblast transplantation in mice and could be involved in failure of transplantation in Duchenne muscular dystrophy patients. These results indicate that adequate immunousuppression must be used in these patients.© 1995 John Wiley &Sons, Inc.     

CNTF 联合RA 诱导成肌细胞类神经分化及与REST 的相关性

目的 探讨睫状神经营养因子（CNTF）联合视黄酸（RA）诱导成肌细胞类神经分化的方法以 及该诱导过程与神经元限制性沉默因子（REST）相关性。方法 以CNTF 诱导C2C12 去分化,再以RA 诱导 C2C12 类神经分化。通过细胞形态学、细胞免疫荧光、流式细胞术、Western blot 等技术对去分化及类神经 分化的细胞进行鉴定及相关分析,比较细胞类神经分化前后REST 表达变化。结果 诱导后细胞聚集生长,呈 典型的神经球样。细胞免疫荧光染色显示神经球能够被神经特异性标志物NSE、Sox1、GFAP 标记,神经球 离散为单个细胞接种后生长状态良好,神经干细胞标志物（nestin）呈阳性表达。Western blot 结果显示CNTF 去分化诱导后成肌细胞分化相关蛋白Myogenin、P21 表达降低,类神经分化后NSE、Sox1、GFAP 表达增多, 且成肌细胞特异性标志物Desmin 及REST 表达下降。流式细胞仪分析表面标志物NSE 显示诱导组与对照组 比较细胞有差异,且诱导组特异性阳性细胞率达84% 左右。结论 CNTF 联合RA 可诱导成肌细胞类神经分化, 且该诱导过程可能与REST 表达降低相关。     

骨髓间充质干细胞来源外泌体对成肌细胞缺氧状态的影响北大核心

背景:细胞在缺氧条件下会发生功能的变化,而间充质干细胞分泌的外泌体可以缓解细胞缺氧带来的病理性损伤。目的:探讨不同浓度氯化钴对成肌细胞功能变化的影响,并进一步研究骨髓间充质干细胞来源外泌体对成肌细胞缺氧凋亡和活性氧产生的作用。方法:体外分离SD大鼠骨髓间充质干细胞,通过超速离心法提取外泌体,采用透射电镜、纳米颗粒跟踪分析、Western blot对外泌体进行鉴定。应用0,50,100,200μmol/L氯化钴干预C2C12成肌细胞1,3,5,7 d,CCK-8检测成肌细胞增殖活性变化;干预5 d,qRT-PCR检测成肌分化基因MyHC、MyoD、MyoG的表达水平,改良吉姆萨染色观察成肌细胞融合情况;干预3,5 d,流式细胞仪检测细胞凋亡率;干预3 d,使用DCFH-DA染色观察细胞活性氧水平。成肌细胞分为4组:对照组(0μmol/L氯化钴),氯化钴组(200μmol/L氯化钴),外泌体组(200μmol/L氯化钴+50 mg/L外泌体),NAC组(200μmol/L氯化钴+2 mmol/L抗氧化剂NAC)处理,干预3 d,检测凋亡率和活性氧水平。结果与结论:①外泌体呈杯口状结构,粒径分布在30-150 nm之间,CD9、TSG101表达阳性;②氯化钴对成肌细胞的增殖、分化有显著抑制作用(P<0.05),并且促进了细胞凋亡(P<0.05),高浓度的氯化钴对成肌细胞的功能抑制作用更加明显;③外泌体能够降低200μmol/L氯化钴所诱导的成肌细胞缺氧凋亡和活性氧水平(P<0.05),并与抗活性氧试剂NAC具有相似的作用(P<0.05);④结果表明,氯化钴对成肌细胞的生理功能抑制作用具有一定的浓度和时间依赖性,而骨髓间充质干细胞来源外泌体与NAC具有相似的抗缺氧凋亡和降低活性氧产生的作用,其机制可能为外泌体通过降低活性氧产生的途径而缓解了成肌细胞的缺氧凋亡。     

大鼠脂肪间充质干细胞向成肌细胞诱导分化的研究

目的探索在不使用5-氮杂胞苷的情况下,体外诱导大鼠脂肪间充质干细胞向成肌细胞的分化。方法取SD大鼠附睾处脂肪垫组织,酶消化分离获间充质干细胞,传至第3代,加入成肌细胞诱导液,在体外诱导8周,应用间接免疫荧光法、激光共聚焦显微镜进行成肌细胞的鉴定。结果大鼠脂肪组织来源的间充质干细胞经诱导5d后,显微镜下见细胞形态发生变化。2～8周的诱导细胞Myosin染色阳性。逆转录聚合酶链反应检测诱导3周的细胞有MyoD1和Myosin表达。结论大鼠脂肪组织来源的间充质干细胞在体外经诱导具有向成肌细胞分化的潜能。     

成年大鼠骨骼肌成肌细胞的培养和鉴定

目的探索成年大鼠骨骼肌成肌细胞的高浓度培养方法。方法以成年同种系Wistar大鼠为研究对象,采用两步消化法获取大鼠骨骼肌卫星细胞,进行体外培养,对获得的细胞进行形态学研究,以免疫组织化学方法进行鉴定。结果细胞增殖旺盛,分化良好,可融合成肌管。免疫细胞化学染色显示,骨骼肌卫星细胞呈弱阳性,肌管呈强阳性。结论两步消化法体外培养的骨骼肌卫星细胞具有良好的增殖与分化能力,用此种方法可培养出高纯度的骨骼肌成肌细胞,操作简单、污染少。     

Application of tissue engineering in the treatment of stress urinary incontinence

Stress urinary incontinence is one of the most common diseases in urology. The main treatments for stress urinary incontinence are pharmacotherapy, physicobehavioral therapy and surgery.However, the results of present methods are not satisfactory. Tissue engineering is a newly emerging technology that may provide a novel method for the treatment of stress urinary incontinence.     

Deficiency screen identifies a novel role for beta 2 tubulin in salivary gland and myoblast migration in the Drosophila embryo

The Drosophila embryonic salivary gland is an epithelial organ formed by the coordinated invagination and migration of primordial cells. To identify genes that regulate gland migration we performed a deficiency screen of the third chromosome. Here, we report on the analysis of the beta 2 tubulin isoform (β2t) that maps at 85D15. We show that, in β2t mutant embryos, salivary glands did not complete their posterior migration and that migration of fusion competent myoblasts and longitudinal visceral muscle founder cells between the gland and circular visceral mesoderm was delayed. We also demonstrate that gland migration defects correlate with reduced βPS and αPS2 integrin expression in the surrounding mesoderm and that β2t genetically interacts with genes encoding integrin αPS1 and αPS2 subunits. Our studies reveal for the first time that β2t is expressed in embryogenesis and that β2t plays an important role in salivary gland and myoblast migration, possibly through proper regulation of integrin adhesion proteins. Developmental Dynamics 238:853–863, 2009. © 2009 Wiley‐Liss, Inc.     

微电流脉冲体外诱导神经干细胞增殖分化的研究

目的探讨微电流脉冲对神经干细胞体外增殖分化的诱导作用。方法采用联合培养和微电流脉冲技术改变神经干细胞生长的微环境,通过免疫细胞化学技术及电子显微镜技术观察神经干细胞增殖分化情况。结果骨骼肌成肌细胞及微电流脉冲均可诱导神经干细胞分化(细胞ChAT阳性率分别为5.90%和6.48%),与神经干细胞自然分化组相比差异显著(细胞ChAT阳性率为4.03%)。结论微电流脉冲对神经干细胞的体外增殖分化具有一定的诱导作用。