体外培养的骨骼肌卫星细胞生长与分化特性的研究

目的：探讨体外培养的骨骼肌卫星细胞的生长与分化特性。方法：取大鼠肱三头肌,采用二步消化法分离骨骼肌卫星细胞,体外进行原代和传代培养。通过生长曲线、细胞融合率研究骨骼肌卫星细胞的增殖与分化能力,利用免疫细胞化学染色对所获得的细胞进行鉴定。结果：二步消化法适用于大鼠骨骼肌卫星细胞的分离,在生长培养基作用下,细胞增殖旺盛;在分化培养基条件下,细胞分化良好,可融合成肌管。骨骼肌特异性蛋白α-sarcometric actin和myosin免疫化学染色显示,骨骼肌卫星细胞弱阳性,肌管强阳性。结论：体外培养的骨骼肌卫星细胞具有良好的生长与分化能力,适用于骨骼肌组织工程的构建与研究。     

体外培养的骨骼肌卫星细胞生长与分化特性的研究

目的探讨体外培养的骨骼肌卫星细胞的生长与分化特性.方法取大鼠肱三头肌,采用二步消化法分离骨骼肌卫星细胞,体外进行原代和传代培养.通过生长曲线、细胞融合率研究骨骼肌卫星细胞的增殖与分化能力,利用免疫细胞化学染色对所获得的细胞进行鉴定.结果二步消化法适用于大鼠骨骼肌卫星细胞的分离,在生长培养基作用下,细胞增殖旺盛;在分化培养基条件下,细胞分化良好,可融合成肌管.骨骼肌特异性蛋白α-sarcometric actin和myosin免疫化学染色显示,骨骼肌卫星细胞弱阳性,肌管强阳性.结论体外培养的骨骼肌卫星细胞具有良好的生长与分化能力,适用于骨骼肌组织工程的构建与研究.     

肌细胞生成素在分化培养人体肌卫星细胞表达的研究

目的 研究肌细胞生成素(myogenin)在人体肌卫星细胞分化培养中的表达规律，探索肌细胞生成素在肌卫星细胞分化中的作用。方法 取6例正常成人的骨骼肌，消化、分离肌卫星细胞，肌卫星细胞生长培养后进行分化培养。在分化培养过程中，观察肌卫星细胞形态，计算其融合率，应用半定量RT—PCR技术检测细胞总RNA中肌细胞生成素mRNA的表达量。结果 在肌卫星细胞分化培养过程中，肌细胞生成素mRNA的表达增加，其规律与肌卫星细胞分化、融合相一致。结论 肌细胞生成素参与人体肌卫星细胞分化过程。     

原代人胚成肌细胞体外培养增殖及分化特性

目的 分离培养人胚胎成肌细胞,观察原代细胞体外增殖与分化特性。方法 选择健康妇女损赠的胎儿骨骼肌标本,参照Blau等的胰蛋白酶与胶原酶混合、多步消化法分离成肌细胞。经美速贴壁法纯化后,在含20％胎牛血清的生长培养基中培养,以生长曲线评价及其增殖情况;含5％胎牛血清的融合培养基中培养,以磷酸肌酸激酶－MM型合成量及成肌细胞融合率评价及其分化能力。通过光镜、透射电镜、免疫细胞化学方法（小鼠抗人肌球蛋白单克隆抗体）鉴定所得细胞。结果 电镜下可见所得细胞含大量游离核糖体,肌球蛋白免疫细胞化学染色强阳性,能合成磷酸肌酸激酶,能融合形成肌小管,证明其为骨骼肌成肌细胞。在生长培养基中原代细胞倍增时间为4．8天,在融合培养基作用下,肌小管融合率及磷酸肌酸激酶合成量明显增加。结论 多步酶消化法与差速贴壁法能获得足量、纯净成肌细胞,所得细胞增殖能力强,能表达骨骼肌特异的收缩蛋白;融合培养基能促进其体外分化。     

肌肉组织工程的基础研究--高纯度卫星细胞培养方法

目的改进分离培养肌肉卫星细胞的方法.方法选择兔前肢三头肌通过胶原酶消化、提纯肌束,再用胰蛋白酶消化肌束上的卫星细胞.在含有200ml/1胎牛血清的培养液中培养,使用骨骼肌肌动蛋白抗体进行鉴定.结果使用本技术可以得到高纯度的肌肉卫星细胞(＞0.99),骨骼肌肌动蛋白和肌球蛋白可以用于卫星细胞的鉴定.结论使用分离纯化肌束的方法,有效的避免了成纤维细胞的污染,从而得到高纯度卫星细胞,方法简单,可以用于今后的研究工作.     

人体嗜铬细胞培养的实验研究

目的　观察连续 5天体外培养人体肾上腺嗜铬细胞的生长情况和儿茶酚胺分泌功能的变化 ,以便选择细胞移植的适宜时机。方法　取 6例健康成人脑死亡的双侧肾上腺 ,按李氏改良方法 ,培养 5天。每 2 4小时记录培养细胞生长情况 ,更换培养液 ,并采用高效液相色谱技术检测培养液中儿茶酚胺含量。培养第 3天的细胞 ,采用免疫细胞化学 ( SABC)技术检测酪氨酸羟化酶表达阳性细胞 ,并计数阳性细胞百分率。结果　 ( 1)培养第 3天的人体嗜铬细胞形态结构完整 ,生长良好 ;( 2 )免疫组化检测显示酪氨酸羟化酶表达阳性细胞为 72 % ,细胞内嗜铬颗粒清晰 ;( 3 )培养液去甲肾上腺素和肾上腺素含量 ,在培养第 3天均较第 1天明显升高 ( P<0 .0 1)。多巴胺含量在第 2、3天均有升高 ( P<0 .0 5 )。结论　本组人体嗜铬细胞体外培养第 3天是进行细胞移植的适宜时机     

骨形成蛋白对骨骼肌卫星细胞增殖与胶原蛋白合成的影响

目的　探讨骨形成蛋白 (BMP)对骨骼肌卫星细胞增殖与胶原蛋白合成的影响。　方法　体外获取与培养 Wistar大鼠骨骼肌卫星细胞 ,分别用含 BMP浓度为 0、50、1 0 0、50 0和 1 0 0 0 ng/ml的诱导培养基培养 72小时。通过 MTT法测定细胞的增殖 ,光镜观察细胞融合率 ,3 H-脯氨酸掺入法测定细胞胶原蛋白合成量。　结果　 BMP可促进骨骼肌卫星细胞的增殖 ,降低其细胞融合率 ,同时增加胶原蛋白的合成量。这种作用在 BMP浓度为 50 0 ng/ml即可表现出来 ,并随着浓度的增加越明显。　结论　 BMP可促进骨骼肌卫星细胞的增殖 ,抑制成肌表型促进向成骨细胞分化     

GFP转基因鼠肌卫星细胞系的构建及基因介导成骨分化的实验研究

目的 评价肌卫星细胞(Muscle satellite cells,MSCs)体内外成骨分化的能力,探讨肌卫星细胞作为骨组织工程新的种子细胞的可能性.方法 取新生绿色荧光蛋白转基因小鼠颌面部肌肉,采用酶消化法分离出MSCs,体外原代及传代培养,经腺病毒介导的人骨形成蛋白2(Ad-BMP2)基因转染后,行成骨细胞标志物的活性检测及细胞化学染色,并进行细胞体外矿化及体内成骨的检测.结果 转染后细胞碱性磷酸酶(ALP)组织化学染色呈阳性;骨钙素(OC)免疫细胞化学染色呈阳性且OC活性增强(p＜0.05);21天后见钙结节形成;转染后的细胞与材料复合物植入裸鼠后肢肌肉内,4周后见骨组织形成.结论 体外培养的肌卫星细胞体内外可以成骨分化,可以成为骨组织工程的新的种子细胞.     

肌源性干细胞分离培养及诱导分化为平滑肌细胞的研究

目的探讨兔肌源性干细胞分离、鉴定及诱导分化为平滑肌细胞的方法。方法取1．5个月龄新西兰兔大腿肌肉，采用Preplate技术分离培养肌源性细胞，并进行流式细胞仪（FCM）、免疫细胞化学、Western blot等确定细胞表型。采用添加血管内皮生长因子（VEGF）和与兔阴茎海绵体平滑肌细胞（CCSMC）共培养的方法诱导分离而得的细胞分化为平滑肌细胞并运用免疫细胞化学和Western blot检测特异性α-平滑肌肌动蛋白（α—SMA）的表达。结果经过连续6d的差速贴壁分离得小圆形或短梭形的小体积细胞，FCM结果显示这些细胞〉80％为desmin＋，〉70％为bcl-2^＋，〉95％为CD45^-。免疫细胞化学定性结果显示desmin＋。高汇合度（〉50％）或低血清培养时容易融合形成肌管或肌细胞核链。诱导处理后分离的细胞表达α—SMA。结论采用Preplate技术能很好地分离出肌源性干细胞，并能在体外诱导分化为平滑肌细胞，为组织工程提供种子细胞。     

血清对神经干细胞分化的影响北大核心CSCD

目的探讨血清对神经干细胞分化过程的影响。方法取孕14 d SD大鼠胚胎脑组织,分离培养神经干细胞并传代。取第3代细胞倒置相差显微镜下观察细胞形态,并行巢蛋白(Nestin)免疫细胞化学染色鉴定。将鉴定后的细胞分为A、B、C 3组,各组细胞培养基除FBS浓度不同(分别为5%、1%、0)外,其余成分均一致。培养第8天,行活/死细胞染色观察细胞生长情况;培养第4、8天,行免疫细胞化学染色以及实时荧光定量PCR检测,观察胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)、Ⅲ型β神经元微管蛋白(β-Ⅲ Tubulin)表达。结果经细胞形态及免疫细胞化学染色鉴定培养细胞为神经干细胞。培养第8天,活/死细胞染色示A、B、C组细胞均无死亡现象。免疫细胞化学染色示,培养第4、8天,A、B、C组GFAP蛋白表达随FBS浓度降低而逐渐减弱,但β-Ⅲ Tubulin表达逐渐增强;各组第8天时染色均较第4天时增强。实时荧光定量PCR检测示,培养第4天和第8天3组间GFAP mRNA及β-ⅢTubulin mRNA相对表达量比较,差异均有统计学意义(P<0.05)。结论血清能促进神经干细胞向胶质细胞分化,并减缓其向神经元分化速度,且血清浓度越低,该影响越小。     

Potential differentiation of human mesenchymal stem cell transplanted in rat corpus cavernosum toward endothelial or smooth muscle cells

One of the causes of erectile dysfunction (ED) is the damaged penile cavernous smooth muscle cells (SMCs) and sinus endothelial cells (ECs). To investigate the feasibility of applying immortalized human mesenchymal stem cells (MSCs) to penile cavernous ECs or SMCs repair in the treatment of ED, the in vivo potential differentiation of the immortalized human MSCs toward penile cavernous endothelial or smooth muscle was investigated. One clone of immortalized human bone marrow mesenchymal stem cell line B10 cells via retroviral vector encoding v-myc were transplanted into the cavernosum of the Sprague-Dawley rats and harvested 2 weeks later. The expression of CD31, von Willebrand factor (vWF), smooth muscle cell actin (SMA), calponin and desmin was determined immunohistochemically in rat penile cavernosum. Multipotency of B10 to adipogenic, osteogenic or chondrogenic differentiation was found. Expression of EC specific markers (CD31 or vWF protein) and expression of SMC specific markers (calponin, SMA or desmin protein) were demonstrated in grafted B10 cells. When human MSCs were transplanted into the penile cavernosum, they have the potential to differentiate toward ECs or SMCs. Human MSCs may be a good candidate in the treatment of penile cavernosum injury.     

从膀胱癌患者分离并培养人膀胱平滑肌细胞

目的：建立从膀胱癌患者的分离并培养膀胱平滑肌细胞的实验技术.方法：取一小块无明显肿瘤生长的膀胱组织,分离并培养膀胱平滑肌细胞;动态观察细胞形态变化、生长增殖情况以及平滑肌肌动蛋白（SMA）、结蛋白（Desmin）和广谱细胞角蛋白（AE1/AE3）的表达.结果：接种24 h后即有长梭形细胞贴壁生长,10天后长至80%融合,呈典型的＂峰谷＂样形态;传代后1天为潜伏期,2～6天为指数生长期,然后进入融合平台期,需再次传代.第2代细胞的SMA和Desmin表达阳性率分别高达（99.0±0.8）%和（97.0±2.1）%,不表达AE1/AE3.随着传代次数的增加,细胞去分化,细胞形态变成短梭状或椭圆形;SMA和Desmin的表达开始下降,传至第5代时,SMA和Desmin阳性率分别降至（78.0±3.3）%和（74.0±2.6）%;至第7代时,SMA和Desmin阳性率降至（51.0±3.0）%和（49.0±2.6）%.第7代细胞经血清饥饿培养48 h后,细胞又能再分化,形态转变成长梭状,SMA和Desmin阳性率可分别升至（90.0±3.5）%和（88.0±2.5）%,具有显著性差异.结论：本研究所培养的人膀胱平滑肌细胞具有较高的纯度,血清饥饿能促进去分化的细胞再分化,能为构建组织工程膀胱提供种子细胞.     

Smooth muscle differentiation and cell turnover in mouse detrusor development.

PURPOSE: We systematically analyzed detrusor muscle differentiation in normal mice with a focus on cell turnover (proliferation and apoptosis) as well as on expression of the muscle specific proteins alpha-smooth muscle actin and desmin. MATERIALS AND METHODS: The stages examined were embryonic days 14 and 18, and postnatal day 1 and week 6, representing a period spanning organ inception to postnatal maturity. Alpha-smooth muscle actin, desmin and proliferating cell nuclear antigen were assessed by immunohistochemical testing of whole bladders and Western blot analysis of dissected detrusor layers. Apoptosis was detected in tissue sections by end-labeling. RESULTS: Alpha-smooth muscle actin was expressed by the detrusor layer throughout maturation with levels significantly increasing from embryonic days 14 to 18 and cytoplasmic staining gaining in uniformity postnatally. Desmin expression in the detrusor was insignificant at embryonic day 14 but increased progressively thereafter. Proliferating cell nuclear antigen expression in the detrusor was highest at organ inception and fell stepwise at each developmental stage with low levels postnatally. Apoptosis in the detrusor was only detected at embryonic day 14. CONCLUSIONS: These results demonstrate that morphological growth of the mouse detrusor muscle is accompanied by complex serial changes in the expression of muscle specific proteins and in cell turnover. Strikingly, detrusor muscle cell differentiation and proliferation are inversely related. These detailed studies may serve as a comparison for future experiments involving aberrant mouse bladder development.     

成肌分化因子和5-氮杂胞苷联合诱导骨髓间充质干细胞向骨骼肌细胞分化的实验研究

目的研究大鼠骨髓间充质干细胞（mesenchymal stem cells，MSCs）在体外定向分化为骨骼肌细胞的干预条件。方法取健康4周龄Wistar大鼠MSCs制成细胞悬液接种于培养瓶中，置于37C、5％CO2恒温培养箱中培养，倒置相差显微镜下观察，细胞布满瓶底即为1代，取第3代MSCs，以5-氮杂胞苷10μmol／L、成肌分化因子0．1ng／ml、转化生长因子β1 0.1ng／ml、胰岛素样生长因子12ng／ml进行联合诱导，使之分化。诱导后第9天，收集细胞，行MTT比色法、流式细胞仪和免疫组织化学检测鉴定细胞。结果原代培养的MSCs呈贴壁生长，3～5d呈集落样生长，5～7d后有体积不等的多突成纤维细胞、较大的扁平多角形细胞、中等的多角形细胞和较小的三角形细胞。培养12d后，细胞融合，铺满瓶底，MSCs形态变化不明显。联合诱导后，部分细胞死亡，生长缓慢；7d后见细胞明显增殖，体积逐渐增大，呈椭圆形、梭形或不规则形状；14d后，大量增殖的长梭形细胞开始增多；18～22d后，肌管数量增多，体积增大，核数亦明显增多，初生肌管与长梭形成肌细胞呈平行排列，间隔分布。MSCs免疫组织化学法测定CD44反应阳性，胞质呈棕褐颗粒，核周明显，CD34呈阴性反应；诱导后MSCs结蛋白、骨骼肌特异肌球蛋白均为阳性表达。流式细胞仪测定MSCs和诱导后MSCs大部分处于G0／G1期，分别占79．4％、62．9％，而G2／S／M期仅占20．6％、36．1％。根据MTT绘制生长曲线，可见MSCs生长速度明显高于诱导后的MSCs。两种细胞并未达到平台期，都有继续增殖的趋势。结论在体外，MyoD、5-氮杂胞苷可以诱导MSCs向骨骼肌细胞定向分化，并伴有结蛋白和骨骼肌肌球蛋白阳性表达；定向诱导后MSCs增殖期比例大，向骨骼肌细胞分化纯度更高。     

Human prostatic smooth muscle cells in culture: Estradiol enhances expression of smooth muscle cell-specific markers

Smooth muscle cells (SMCs) constitute a major cellular component of prostatic stroma. SMC tension plays an important role in urethral obstruction secondary to benign prostatic hyperplasia (BPH). We have developed an in vitro procedure for the propagation of human prostatic SMCs. Tissue specimens from patients undergoing radical prostatectomy or cystectomy were enzymatically disaggregated and cultured in MCDB-131 medium supplemented with horse serum, insulin, conditioned medium from the tumor cell line CRL-5813, and steroid hormones. The medium was assembled on the basis of the effects these supplements have on the growth of SMC cultures and on the expression of the two markers desmin and smooth muscle myosin. Addition of 0.1 μM of estradiol to the growth medium dramatically increased expression of these SMC-specific markers. Dihydrotestosterone (DHT) and hydrocortisone had a similar, albeit less pronounced effect. At three to five passages, about two thirds of the cells were immunohistologically positive for smooth muscle myosin or desmin. Almost all cells were positive for the myofibroblast marker smooth muscle α-actin throughout 10 passages and more. In SMC cultures, cells staining for smooth muscle myosin and desmin were found to seek direct contact to myofibroblasts. They grew in aggregates on a layer of myofibroblasts which adhered to the surface of the culture vessel. As revealed by transmission electron microscopy the cultured cells exhibited morphological features of myofibroblasts. Characteristics of smooth muscle cells, such as prominent bundles of microfilaments associated with dense bodies, basal laminae investing the cells, and numerous caveolae at the cell surfaces were regularly observed in cultures of low passages. After several passages, these features were markedly decreased and organelles of the biosynthetic system became more prominent. In summary, we present an in vitro model of prostatic SMCs and demonstrate that steroid hormones have characteristic effects on these cells. SMC cultures are expected to facilitate investigation of the functions and properties of human prostatic SMCs. Prostate 30:117–129, 1997. © 1997 Wiley-Liss, Inc.     

骨髓间充质干细胞体外转化为骨骼肌细胞的初步观察

目的观察骨髓间充质干细胞(mesenchymal stem cells，MSCs)经5．氮杂胞苷(5-Aza)诱导分化为成骨骼肌肌细胞的过程。方法分离纯化4～6周龄SD大鼠的骨髓MSCs，以5-Aza作用24h。于作用1周后的不同时相点，观察细胞形态变化，免疫组织化学染色观察细胞内结蛋白(desmin)、骨骼肌肌动蛋白(actin)、肌球蛋白重链(MHC)、肌生成素(myogenin)表达。结果经5-Aza诱导后14d，部分细胞表达desmin；诱导后24d，细胞表达myogenin、MHC、actin。细胞形态和排列方式发生明显变化，可见有肌管样细胞出现。结论MSCs经5-Aza诱导后可以向成骨骼肌细胞定向分化。     

New approaches in the modulation of bladder smooth muscle cells on viable detrusor constructs

Identification of biochemical and mechanical stimuli in order to modulate the function of bladder smooth muscle cells (SMC) in viable detrusor constructs. Human bladder detrusor cells were seeded on bladder acellular matrix and cultured under different conditions. Cell viability and proliferation were assessed by fluorescent microscopic analyses. Histological, immunohistochemical and flow cytometric analyses were performed to compare growth characteristics and differentiation of SMC. The combination of medium conditioned with proliferative urothelium and mechanical stretch resulted in a more densely populated membrane. In this culture system, the expression of α-smooth muscle actin (α-SMA) and desmin were clearly induced after serum elimination. SMC-phenotype can be modulated in viable detrusor constructs by applying selected combinations of urothelial-conditioned media and mechanical stimulation under stepwise reduction and elimination of serum.     

人睾丸圆形精子细胞在体外向长形精子细胞分化

目的:探讨人睾丸生精细胞在体外培养条件下发生的分化。方法:取梗阻性无精子症患者睾丸活检标本,机械法离散睾丸细胞。①睾丸细胞混合培养,每24 h观察,计数分析长形精子细胞比率的变化;②显微操作挑取圆形精子细胞,观察单个细胞在微滴培养过程中变形分化的情况。结果:在添加卵泡刺激素、睾酮的HTF培养液中进行睾丸细胞混合培养,24 h后长形精子细胞比率较培养前显著增加(P<0.05);单个圆形精子细胞培养,可见到圆形精子细胞长出鞭毛转化为长形精子细胞,48 h的转化率为3.54%。Vero细胞条件培养液中人睾丸生精细胞的分化与HTF培养液中相同,两者无显著差异。结论:人睾丸组织的圆形精子细胞在体外可分化为长形精子细胞,Vero细胞条件培养液对此分化无促进作用。