EGFP和CM-Dil示踪骨髓间充质干细胞构建组织工程骨的体内研究

目的:应用增强型绿色荧光蛋白(Enhanced green fluorescent protein,EGFP)和CM-Dil标记技术,观察组织工程骨在体内形成过程中种子细胞的变化和转归.方法:分别用EGFP慢病毒表达和CM-Dil染料的方法标记比格犬骨髓间充质干细胞(Bone mesenchymal stem cells,BMSCs),MTT法检测标记细胞的体外增殖能力.BMSCs接种珊瑚支架体外成骨诱导7天后,将未标记组、EGFP组和CM-Dil组分别植入裸鼠背部皮下,空白支架作为阴性对照.术后4、8、12周取材,HE染色观察成骨情况,EGFP组采用GFP免疫组化、CM-Dil组冰冻切片荧光显微镜下示踪BMSCs在体内的变化.结果:两种标记技术能高效标记BMSCs,标记前后细胞的体外增殖无显著性差异(P>0.05).细胞-支架复合物植入体内12周后有新生骨形成,标记细胞数量随时间延长而逐渐减少,12周后仍显示有部分标记细胞存活.结论:EGFP和CM-Dil可用于示踪组织工程种子细胞,通过示踪说明BMSCs在体内组织工程骨成骨过程中发挥了重要作用.     

骨髓基质干细胞与软骨脱细胞基质多孔支架异位构建组织工程化软骨的实验研究

目的 探讨骨髓基质干细胞(bone marrow mesenchymal stem cells,BMSCs)与软骨脱细胞基质多孔支架(cartilage ECM-derived porous scaffold,CEDPS)在裸鼠体内异位构建软骨的可行性,并建立利用PKH26荧光和分子荧光活体成像系统无创评估组织工程化细胞-支架复合体在体内生长情况的新方法.方法 PKH26荧光标记成软骨诱导的BMSCs,接种入CEDPS支架,体外进行电镜、Desd/Live荧光染色观察,然后植入裸鼠背部,4周后利用分子荧光活体成像系统无创伤性评估组织工程化组织在裸鼠体内生长情况,取材进行组织学以及Ⅱ型胶原免疫荧光检测,与荧光图像比较.结果 体外培养的BMSCs-CEDPS复合体电镜检查结果表明随着培养时间的增加,细胞在支架中增殖显著,细胞基质分泌增加,Dead/Live染色表明BMSCs在支架内部活性良好.4周后活体荧光示踪显示BMSCs在支架内生长良好,无扩散趋势,BMSCs-CEDPS复合体在裸鼠体内生成软骨样组织,番红"O"、甲苯胺蓝染色、Ⅱ型胶原免疫组化染色阳性,免疫荧光检查表明构建的软骨样组织内的细胞来源为接种的PKH26标记的BM-SCs.结论 利用BMSCs和CEDPS支架能够在裸鼠皮下异位构建类软骨样组织.PKH26标记与分子荧光活体成像系统结合,能够无创伤性评估组织工程化组织的种子细胞在动物体内生长情况与转归.     

体内构建组织工程骨的示踪观察：CM-DiI长效示踪骨髓间充质干细胞的可行性研究

目的探索组织工程骨体内成骨时,CM-DiI长效示踪骨髓间充质干细胞的可行性,为种子细胞的体内转归研究提供标记方法。方法分离比格犬BMSCs,成骨诱导至第2代,用荧光染料CM-DiI进行细胞标记。荧光倒置显微镜观察标记后的细胞形态,MTT比色法测定标记前后BMSCs的增殖状况,检测标记前后Ⅰ型胶原（Col-Ⅰ）、骨形态发生蛋白（BMP-2）、骨钙素（BGLAP）和骨黏连蛋白（SPARC）的表达。最后,CM-DiI荧光标记后的BMSCs与β-TCP复合共培养后植入比格犬背部皮下,8周后取材,荧光显微镜下观察BMSCs体内转归,HE染色观察异位成骨情况。结果CM-DiI标记后早期细胞呈现红色荧光,48 h后荧光增强,72 h内荧光强度无明显减弱。BMSCs在CM-DiI标记前后细胞形态基本一致,两组间细胞的增殖率无明显差别;标记后RT-PCR检测Col-Ⅰ、BMP-2、BGLAP、SPARC表达,显示标记后的BMSCs亦可向成骨方向分化。扫描电镜检测到标记后细胞在β-TCP上增殖良好,细胞基质分泌丰富。细胞-支架复合物植入比格犬皮下,8周后荧光显微镜观察到标记细胞仍存在,且HE染色可见支架孔隙中有类骨基质沉积。结论CM-DiI对BMSCs的生长增殖、成骨分化无明显影响,对体内组织工程骨中BMSCs的示踪时间可长达8周,可用作体内细胞的长效示踪剂。     

PKH26与BrdU体外双重标记兔BMSCs在心肌补片中的实验研究

目的观察PKH26与BrdU体外双重标记兔BMSCs的生物学特性,并体外构建组织工程心肌补片。方法取6月龄新西兰大白兔分离培养BMSCs,并用细胞膜荧光染料PKH26和细胞核标记物BrdU对BMSCs进行标记,通过倒置相差显微镜、荧光显微镜、流式细胞仪、MTT法观察细胞生长状态和荧光标记前后细胞生物学特性的变化;ALP、茜素红、油红O染色及骨钙素、Ⅰ型胶原蛋白等免疫细胞化学技术检测,观察和评价标记前后BMSCs体外分化为成骨细胞和成脂细胞的能力。将标记细胞与小肠黏膜下层(small intestinal submucosa,SIS)复合后共培养5～7d构建组织工程心肌补片,在倒置相差显微镜、荧光显微镜和扫描电镜下观察细胞在SIS上的生长情况,并做活体及石蜡包埋HE染色观察。结果标记后细胞生长状态良好,基本生长特性无明显改变;荧光显微镜下可见标记后细胞膜上红色荧光呈颗粒状分布;细胞表面干细胞标志性抗原表达与标记前无明显差异。标记后的BMSCs成骨诱导后,ALP、茜素红染色阳性,细胞表达骨钙素及Ⅰ型胶原蛋白;成脂诱导后,细胞胞浆内出现明显的脂滴。标记细胞与SIS共培养5～7d后,标记细胞生长状态良好,在材料上呈现出多层细胞结构。活体及石蜡包埋后HE染色可见细胞生长状态良好。结论兔BMSCs能被PKH26和BrdU稳定标记;标记的细胞在体外具有自我更新和多向分化的能力;标记的BMSCs与SIS体外复合培养可以构建组织工程心肌补片。     

应用荧光蛋白示踪组织工程骨体外构建的实验研究

目的应用荧光蛋白标记技术对组织工程骨体外构建过程进行细胞示踪。方法采用逆转录病毒pLEGFP-N1对种子细胞进行荧光蛋白标记,以羟基磷灰石为支架材料,体外构建细胞-材料复合体,测定细胞的粘附率。通过倒置荧光显微镜,定期观察种子细胞在支架材料内的分布。结果逆转录病毒载体pLEGFP-N1成功标记人骨髓基质干细胞(BMSCs),并对组织工程骨的体外构建和4周的体外培养进行了良好示踪;标记细胞的粘附率为(90.3±2.1)%,无标记处理细胞的粘附率为(92.0±1.5)%,两组数据差异无显著性意义(P=0.236)。结论采用逆转录病毒介导方式对种子细胞进行荧光蛋白标记后,细胞在材料内仍维持较高的粘附能力,这有利于对体外构建和长期培养组织工程骨时进行种子细胞示踪。     

骨髓内皮祖细胞输注后在肝纤维化大鼠肝脏内的示踪研究

目的 建立PKH26荧光标记肝纤维化大鼠骨髓内皮祖细胞(EPC)的方法,观察PKH26标记的EPC输注后在肝纤维化大鼠肝内的迁移和分化情况.方法 分离和培养肝纤维化大鼠骨髓源性EPC,并在体外进行PKH26荧光标记,通过荧光共聚焦显微镜观察和采用流式细胞仪检测PKH26的标记率,并观察荧光标记对细胞生长状态的影响;将PKH26荧光标记的EPC经尾静脉输注入肝纤维化大鼠体内,观察其在大鼠肝内的迁移示踪情况,并用免疫荧光法检测内皮细胞标志物CD31和血管性假血友病因子(vWF)的表达.结果 PKH26标记的细胞呈红色荧光,标记率为96.65％;PKH26标记的EPC生长状态良好,与未标记的EPC相比,生长曲线基本一致;EPC迁移入肝后主要存在于沿纤维分布的血管内皮和肝小叶内的窦内皮,并表达内皮细胞特异性抗原CD31和vWF.结论 PKH26可在体外成功标记肝纤维化大鼠骨髓源性EPC,该细胞输注入肝纤维化大鼠体内后,可迁移至肝内血管周围,并向成熟的内皮细胞分化.     

骨髓基质细胞经CM-Dil标记后的活性及移植到损伤脊髓的示踪观察

目的 观察骨髓基质细胞(BMSCs)经CM-Dil荧光染料标记后的生物活性及移植后在损伤脊髓中的迁移和分布.方法 取Wistar大鼠的第3代BMSCs消化后分两组,实验组和对照组,实验组以CM.DiI标记,另一组正常细胞不标记,测定生长曲线.以改良Allen法制备脊髓损伤模型,将CM.DiI标记的BMSCs通过蛛网膜下腔注射移植,分别于移植后24 h、1、2、3、4周取损伤脊髓段作冰冻切片,倒置荧光显微镜下观测移植的BMSCs在损伤脊髓中的迁移和分布规律.结果 BMSCs经CM-Dil荧光染料标记成功率100%,实验组和对照组的生长曲线基本吻合;移植后24 h,脊髓损伤区未见标记细胞,移植后1周,标记的BMSCs开始迁移到损伤区硬脊膜下,移植后2～3周.BMSCs迁移、聚集在损伤脊髓的中心,移植后4周,仍有标记细胞聚集在脊髓损伤中心.结论 (1)BMSCs经CM-Dil荧光染料标记后的生物活性保持正常;(2)CM-Dil标记的BMSCs移植后能迁移聚集在损伤脊髓的中心,CM-Dil在活体内至少能维持4周     

脱细胞软骨细胞外基质取向支架复合软骨细胞构建组织工程软骨的实验研究

目的探讨脱细胞软骨细胞外基质(acellular cartilage extracellular matrix,ACECM)取向支架复合软骨细胞构建组织工程软骨的可行性。方法取市售猪关节软骨组织,分离培养关节软骨细胞并传代。取第3代软骨细胞行PKH26荧光标记,MTT检测标记对细胞增殖无影响后,分别取标记及未标记的软骨细胞复合ACECM取向支架并体外培养后,大体观察支架形态,倒置显微镜、荧光显微镜观察软骨细胞在支架中的黏附、生长和分布情况,扫描电镜观察支架中细胞形态,Ⅱ型胶原免疫荧光染色观察软骨细胞外基质分泌情况。将PKH26标记的软骨细胞-支架复合物植入裸鼠背部皮下腔隙,术后观察裸鼠一般情况,4周后分子荧光活体成像系统无创伤性评估细胞-支架复合物生长情况,取材行大体观察以及番红O、甲苯胺蓝、Ⅱ型胶原免疫组织化学染色观察,评价形成软骨组织的能力。结果细胞-支架复合物体外培养7 d,大体观察呈半透明并具有一定硬度;倒置显微镜和荧光显微镜观察软骨细胞在支架上能良好黏附生长,并沿支架管道方向生长,分泌Ⅱ型胶原。细胞-支架复合物植入裸鼠皮下后,分子荧光活体成像系统观察示细胞均存活;术后4周,大体观察见复合物呈类软骨样组织,组织学染色及Ⅱ型胶原免疫组织化学染色示细胞周围软骨细胞外基质分泌,可见"陷窝"样结构形成。结论 ACECM取向支架有利于软骨细胞的黏附、增殖及取向性分布类似于正常软骨结构,并在裸鼠皮下成功异位构建组织工程软骨。     

脱细胞软骨细胞外基质取向支架复合软骨细胞构建组织工程软骨的实验研究北大核心CSCD

目的探讨脱细胞软骨细胞外基质(acellular cartilage extracellular matrix,ACECM)取向支架复合软骨细胞构建组织工程软骨的可行性。方法取市售猪关节软骨组织,分离培养关节软骨细胞并传代。取第3代软骨细胞行PKH26荧光标记,MTT检测标记对细胞增殖无影响后,分别取标记及未标记的软骨细胞复合ACECM取向支架并体外培养后,大体观察支架形态,倒置显微镜、荧光显微镜观察软骨细胞在支架中的黏附、生长和分布情况,扫描电镜观察支架中细胞形态,Ⅱ型胶原免疫荧光染色观察软骨细胞外基质分泌情况。将PKH26标记的软骨细胞-支架复合物植入裸鼠背部皮下腔隙,术后观察裸鼠一般情况,4周后分子荧光活体成像系统无创伤性评估细胞-支架复合物生长情况,取材行大体观察以及番红O、甲苯胺蓝、Ⅱ型胶原免疫组织化学染色观察,评价形成软骨组织的能力。结果细胞-支架复合物体外培养7 d,大体观察呈半透明并具有一定硬度;倒置显微镜和荧光显微镜观察软骨细胞在支架上能良好黏附生长,并沿支架管道方向生长,分泌Ⅱ型胶原。细胞-支架复合物植入裸鼠皮下后,分子荧光活体成像系统观察示细胞均存活;术后4周,大体观察见复合物呈类软骨样组织,组织学染色及Ⅱ型胶原免疫组织化学染色示细胞周围软骨细胞外基质分泌,可见"陷窝"样结构形成。结论 ACECM取向支架有利于软骨细胞的黏附、增殖及取向性分布类似于正常软骨结构,并在裸鼠皮下成功异位构建组织工程软骨。     

脱细胞软骨基质来源的多孔支架复合山羊髓核细胞体内初步构建组织工程髓核的实验研究

[目的]探讨脱细胞软骨基质多孔支架复合PKH26标记的山羊髓核细胞体内异位构建组织工程髓核的可行性.[方法]制备脱细胞软骨基质来源的多孔支架,扫描电镜(scanning electron microscope,SEM)观察、天狼星红染色、HE染色观察、MTT毒性检测;分离山羊髓核细胞,通过倒置显微镜观察、番红O染色、Ⅱ型胶原免疫组化染色进行鉴定;将PKH26标记的山羊髓核细胞接种支架上,体外培养3d后进行LIVE/DEAD活性染色,将细胞支架复合物置入裸鼠皮下,培养6周,病理切片,荧光显微镜下观察,进行番红O、Ⅰ、Ⅱ型胶原免疫组化染色.[结果]扫描电镜观察支架孔隙相连通且分布均匀,天狼星红染色支架呈黄绿相间色,HE支架淡染,MTT检测细胞增殖曲线无统计学差异(P＞0.05);P1代髓核细胞呈软骨样细胞形态,番红O染色、Ⅱ型胶原免疫组化染色均阳性,PKH26标记后的细胞呈红色荧光;体外LIVE/DEAD染色细胞呈绿色荧光,体内培养6周后,带红色荧光的细胞填满支架孔隙,番红O、Ⅱ型胶原免疫组化染色阳性,Ⅰ型胶原免疫组化染色弱阳性.[结论]以脱细胞软骨基质多孔支架复合山羊髓核细胞在体内能够形成组织工程髓核样组织.     

类许旺细胞种植入去细胞神经桥接物后体内外标记示踪及神经功能恢复检测

目的 将类许旺细胞种值入去细胞神经桥接物内后观察体外培养下细胞存活情况及体内桥接坐骨抻终缺损后细胞存活情况及对神经功能恢复的影响。方法 通过Dezawa改良法诱导的类许旺细胞经Hochest 33342标记以10^7／ml显微注入经化学萃取的神经桥接物内，分别体外培养0、5、10、15、20、30d，苏木精-伊红染色及荧光硅微镜观察细胞在侨接物内的存活情况。同时将该神经桥接物桥接2cm长的坐骨神经缺损模型，分别于5、10、15、20、30、40d取材，观察细胞在桥接物内的存活情况、再生轴突的生长速度及再生轴突有髓纤维密度；于第30、50、70d进行神经电生理检测；于第40、60、80d进行坐骨神经功能指数测定，同时将无细胞的单纯神经桥接物桥接坐骨神经缺损及自体坐骨神经切断后原位缝合分别作为对照。结果 类许旺细胞在体内外条件下均能在化学萃取后的去细胞神经桥接物中存活。体内环境可能更有利于类许旺细胞的增殖。种植类许旺细胞的实验组各测定指标明显优于无细胞桥接对照组（P〈0．05或P〈0．01），实验组与自体神经对照组各指标近似（P〉0．05）。结论 类许旺细胞种植入去细胞神经桥接物后在体内外环境均能存话；种入类许旺细胞的去细胞神经桥接物桥接神经缺损可达到近似自体神经移植桥接的效果。     

骨髓基质干细胞治疗脊髓损伤的体外诱导分化及相关蛋白表达

目的 探讨模拟脊髓细胞环境下骨髓基质干细胞(BMSCs)的分化及蛋白差异表达.方法 分离培养Wistar大鼠骨髓的BMSCs和脊髓的神经细胞,设立BMSCs自然分化组、与神经细胞共培养组和双层培养组,8 d后对各组BMSCs进行神经特异性烯醇化酶(NSE)和胶质纤维酸性蛋白(GFAP)免疫荧光检测.应用表面增强激光解析离子化-飞行时间质谱技术(SELDI-TOF-MS)筛选双层培养组BMSCs变化明显的蛋白进行分析.结果 BMSCs与神经细胞共培养和双层培养8 d后,BMSCs呈神经细胞形态.NSE和GFAP检测结果 ,BMSCs与神经细胞共培养组明显高于BMSCs与神经细胞双层培养组(P＜0.05),而BMSCs和神经细胞双层培养组又明显高于BMSCs自然分化组(P＜0.05).SELDI-TOF-MS检测到TIP39_RAT和CALC-RAT增加到原来的5.360和2.807倍,INSL6-RAT、PNOC_RAT和PCSK1_RAT减少到原来的38.0%、49.9%和43.8%.结论 在脊髓神经细胞微环境下体外培养BMSCs能诱导其分化成神经细胞,而且接触培养分化率高;BMSCs在向神经细胞分化机制中与TIP39_RAT、CALC_RAT、INSL6_RAT、PNOC_RAT和PCSK1_RAT密切相关.     

Tracking chondrocytes and assessing their proliferation with PKH26: effects on secretion of proteoglycan 4 (PRG4).

Distinguishing between implanted and host-derived cells, as well as between distinct cell phenotypes, would be useful in assessing the mechanisms of cell-based repair of cartilage. The fluorescent tracker dye, PKH26, was previously applied to several cell types to assess proliferation in vitro and to track cells in vivo. The objectives of this study were to assess the utility of PKH26 for tracking chondrocytes from superficial and middle zones and their proliferation, and determine the effects of PKH26 on chondrocyte functions, in particular, proliferation and secretion of Proteoglycan 4 (PRG4). PKH26-labeled and unlabeled superficial and middle zone chondrocytes were plated in either low- or high-density monolayer culture and analyzed for retention of PKH26 by flow cytometry and fluorescence microscopy at days 0 and 7. Cell suspensions and conditioned media were analyzed for DNA and secretion of PRG4, respectively. Flow cytometric histograms were deconvolved so that the number of cells in each doubling generation contributing to the final cell population could be estimated. Chondrocytes were consistently and intensely labeled with PKH26 through 7 cycles of division. At day 7 of culture, >97% of superficial zone cells seeded at low or high density could be distinguished as fluorescent, as could middle zone cells seeded at high density. Retention of cell fluorescence after PKH26 labeling and lack of adverse effects on cell proliferation and synthesis of PRG4 suggest that PKH26 can be useful in determining the fate and function of implanted chondrocytes in vivo, as well as monitoring proliferation in vitro.     

化学去细胞异体神经添加不同组织来源雪旺细胞对周围神经损伤修复的功能评价

目的构建不同组织来源雪旺细胞(Schwann cells,SCs)及化学去细胞异体神经(chemically extracted acellular nerve allograft,CEANA)移植物,比较其修复周围神经缺损的效果。方法 4周龄SD大鼠3只,体重80～120g,体外培养、扩增和鉴定BMSCs及脂肪来源干细胞(adipose-derived stem cells,ADSCs)。体外诱导BMSCs和ADSCs分化为类雪旺细胞(dMSC,dADSC),并采用胶质细胞标记物p75和抗胶原纤维酸性蛋白(glial fi brillary acidic protein,GFAP)进行鉴定。取出生3d SD大鼠10只,体重6～8g,分离培养SCs。20只成年Wistar大鼠,体重200～250g,取双侧约20mm坐骨神经制备CEANA。40只成年雄性SD大鼠,制备左侧15mm坐骨神经缺损模型,根据神经缺损修复方法不同随机分为5组(n=8):A组,取自体坐骨神经翻转后吻合;B组,取15mm CEANA吻合后补充5×105个SCs;C组,取15mm CEANA吻合后补充5×105个dMSC;D组,取15mm CEANA吻合后补充5×105个dADSC;E组,取15mm CEANA吻合。术后12周行感觉和运动功能恢复评价及组织学评价。结果 BMSCs和ADSCs表面抗原标志为CD34-、CD45-、CD90+。经诱导后BMSCs和ADSCs形态变化与SCs相似,呈双极、星形结构,SCs标记物p75和GFAP均表达阳性。术后12周,A、B、C、D、E组肢体50%回缩阈值分别为(13.8±2.3)、(15.4±6.5)、(16.9±5.3)、(16.3±3.5)和(20.0±5.3)g,A组与E组比较差异有统计学意义(P0.01),B、C、D组间比较差异无统计学意义(P0.05)。A、B、C、D、E组小腿三头肌收缩力恢复率分别为87.0%±9.7%、70.0%±6.6%、69.0%±6.7%、65.0%±9.8%和45.0%±12.1%,A、B、C、D组与E组比较差异有统计学意义(P0.05)。各组远端吻合口神经坚牢蓝染色显示神经纤维排列整齐,无炎性反应。甲苯胺蓝染色和透射电镜观察显示,B、C、D组有髓神经纤维计数及有髓神经纤维髓鞘厚度均大于E组,差异均有统计学意义(P0.01);B组轴突直径大于C、D组,差异有统计学意义(P0.05)。结论 CEANA补充dADSC修复周围神经缺损与补充dMSC和SCs具有类似的修复效果。dADSC来源广泛,可作为组织工程神经理想的种子细胞,在复合CEANA修复周围神经缺损发挥重要作用。     

Skin epithelial cells in mice from umbilical cord blood mesenchymal stem cells

The purpose of this study was investigation of the potential to isolate mesenchymal stem cells (MSCs) from human umbilical cord blood (UCB) and differentiate them into epithelial cells in mouse skin tissues. Mononuclear cells (MNCs) from UCB (UCB-MNCs) were isolated and induced to MSCs in culture. UCB-MSCs were transfected with pEGFP and labeled with PKH26 dye. eGFP-transfected and PKH26-labeled UCB-derived MSCs were purified by flow cytometry to gate purified eGFP(+) PKH26(+) cells and transplanted at a single clone level into injured nude Balb/C mice by tail vein injection. The phenotype of cultured UCB-MSCs, the expression of human cytokeratins and the expression of eGFP(+) PKH26(+) cells in mouse skin tissues were examined by flow cytometry. Human HLA-1 antigen and cytokeratin 10 (CK10) were detected by direct immunofluorescence on mouse skin tissue sections and flow cytometry. Sry gene (sex-determining region of Y chromosome) was detected by PCR reaction. The results showed that MSCs were isolated from UCB and had heterogeneous morphology and growth potential. Moreover, UCB-derived MSCs localized into mouse skin tissues and differentiated into skin epithelial cells confirmed by in vivo cell tracking and human antigen detection. At two weeks after transplant, a number of eGFP(+) PKH26(+) cells were detected in recipient mouse skin tissues. The detection of sry gene and HLA-1 antigen further confirmed that the human UCB-derived cells were present in recipient mouse skin tissues. Human cells localizing to mouse skin and differentiating into skin epithelial cells were demonstrated by cytokeratins (CK) 8 and 10 expression during flow cytometry, and CK10 expression on injured skin tissue section by direct immunofluorescence. CONCLUSION: UCB-derived MSCs localized to injured skin in vivo and differentiated into epithelial phenotypes. The results demonstrate that UCB-derived MSCs contribute to skin tissue regeneration in vivo and may be an ideal cell source for therapy of skin epithelial tissue injury, including burns.     

Study of in vivo differentiation of rat bone marrow stromal cells into schwann cell-like cells

In order to raise an abundant and accessible reservoir for Schwann cells (SCs), which are used as seed cells for constructing tissue-engineered nerve grafts, we investigated the feasibilty of in vivo differentiation of bone marrow stromal cells (MSCs) into SC-like cells. In this study, MSCs were harvested from adult rats' bone marrow, culture-expanded, and characterized. Subcultured MSCs were then labeled with Hoechst 33342, followed by transplantation into the nerve regeneration chamber, which was made of a silicone tube bridging the sciatic nerve defect of the rats. Four weeks after surgery, some of the differentiated MSCs turned into SC-like cells immunopositive to S-100 protein, accompanied by myelination of the regenerated nerve fibers. Walking-track analyses provided evidence that transplantation of MSCs contributed to reconstruction of the sciatic nerve and reinnervation of target tissues. The experimental results suggest that MSCs are capable of differentiating into SC-like cells in vivo, making them a promising candidate for cell transplantation in peripheral nerve repair.     

骨髓间充质干细胞向雪旺细胞分化的体内外实验研究

目的研究骨髓间充质干细胞在体内、外条件下向周围神经雪旺细胞分化的可行性。方法利用SD大鼠间充质干细胞(取自股骨和胫骨)贴壁生长的特性，培养纯化，传代扩增。用复合诱导因子(Beta-mercaptoethanol，retinoic acid，forskolin，basic—FGF，PDGF，heregulin-β)在体外诱导间充质干细胞分化。用免疫细胞化学(P75，S-100，GFAP)鉴定体外的诱导结果。将PKH67标记的间充质干细胞移植人损伤的周围神经动物模型内，术后2周行组织切片免疫荧光染色，激光共聚焦定位移植细胞的去向。结果诱导后的间充质干细胞形态类似雪旺细胞，免疫荧光鉴定骨髓间充质干细胞具有雪旺细胞性质，表达雪旺细胞的表面标志(GFAP，S-100和P75)。周围神经组织切片免疫荧光染色后共聚焦定位的间充质干细胞表达雪旺细胞的表面标志(GFAP，S-100和P75)。结论骨髓间充质干细胞在体内、外都可以分化为具有雪旺细胞性质的细胞，表达GFAP，S-100和P75。骨髓间充质干细胞有可能替代雪旺细胞促进周围神经的再生。     

Study in vitro of populating autogenous Schwann cells into chemical extracted allogenous nerve

Objective:To look for an ideal substance to repair large gap of nerve difect after injury by culture of Schwann cells(Scs)and preparation of acellular allogenous nerve grafts(ANG) with chemical extraction.Methods:The double adhesion culture and Arab-c to prohibit the fibroblast growth were used to achieve high-purified Scs.Triton-x-100 and sodium deoxycholate were used to achieve ANG.Finally the Scs were microinjected into the acellural nerve grafts and cultured in vitro.The consequence was analysed.Results:High purified Scs and ANG were acquired, which could integrate each other well.Scs could survive and transfer to aline in vitro.Conclusions:Populating Scs into chemical extracted ANG may be an ideal substance to repair the large gap of nerve defect after injury.