体外碱性成纤维细胞生长因子聚乳酸纳米缓释微球对人脂肪干细胞增殖和成脂分化的影响

目的 体外观察碱性成纤维细胞生长因子(basie fibroblast growth factor,bFGF)聚乳酸纳米缓释微球对人脂肪干细胞增殖和成脂分化的影响,为bFGF缓释微球应用于脂肪组织工程的研究提供理论依据.方法 体外分离培养脂肪干细胞,并行多向诱导分化鉴定.配制含有0、1、2、3、4、5 mg/ml bFGF聚乳酸缓释微球的脂肪干细胞培养液及成脂分化诱导液.将脂肪干细胞接种至96孔板,第2天更换含不同浓度hFGF缓释微球的培养液和成脂诱导液,分别用四甲基偶氮噻唑蓝比色法(MTT)和油红O定量检测法定期检测细胞增殖和成脂分化的情况.所得数据均用SPSS13.0软件进行统计学处理.结果 bFGF聚乳酸缓释微球有明显促进脂肪干细胞增殖和成脂分化的作用.增殖实验和成脂诱导实验合适的作用浓度分别为3 mg/ml和4 mg/ml.结论 bFGF聚乳酸纳米缓释微球体外可以明显促进脂肪下细胞的增殖和成脂分化,可作为一种较理想的细胞因子缓释系统应用于脂肪组织工程的研究.     

音猬因子体外诱导人骨髓基质干细胞转化为神经元样细胞的研究

目的：研究体外使用音猬因子（sonic hedgehog，SHH）诱导人骨髓基质干细胞（BMSCs）分化为神经元样细胞（神经干细胞、神经细胞、神经胶质细胞）的可行性。方法：从健康志愿者髂骨抽取骨髓5ml．离心、分离BMSCs。接种于含10％胎牛血清（FBS）的DMEM／F12培养液中，BMSCs体外扩增、纯化到第五代后分别种于含有不同浓度诱导液的24孔板中：A组为空白对照；B组加SHH 700ng/ml，碱性成纤维生长因子8（fibroblast growth factor-8，FGF-8）140ng／ml；C组加SHH500ng/ml，FGF-8100ng／ml；D组加SHH250ng/ml，FGF-8 50ng／ml；E组加SHH125ng／ml，FGF-825ng／ml，诱导BMSCs分化。使用免疫组化及免疫荧光鉴定微管相关蛋白（MAP-2）、胶质纤维酸性蛋白（GFAP）、神经元烯醇化酶（NSE）的表达。结果：诱导7d后，部分BMSCs胞体收缩、突起伸出，表现出神经元样细胞的形态。除A组外其余各组免疫组化及免疫荧光染色NSE、MAP-2均为阳性，各组免疫组化及免疫荧光染色均有GFAP阳性细胞存在，且以B组、C组的MAP-2、GFAP、NSE染色阳性细胞数最多。结论：人BMSCs具有较强的自我更新和多向分化潜能，一定浓度的SHH可以在体外有效诱导人BMSCs转化为神经元样细胞。     

大鼠脂肪干细胞体外定向诱导为血管内皮细胞的研究

目的研究大鼠脂肪干细胞(Adipose-Derived Stem Cells, ADSCs)体外定向诱导分化为血管内皮细胞(vascular endothelial cells, VECs)的可行性.方法取SD大鼠腹股沟区脂肪组织获取ADSCs进行体外培养并检测其多向分化能力,取4代细胞在血管内皮细胞生长因子(vascular endothelial growth factor, VEGF)、碱性成纤维细胞生长因子(basic fibroblast growth factor, bFGF)诱导下向内皮方向分化,检测内皮细胞标志物血管性假血友病因子(Von Willebrand Factor, vWF)表达.结果 ADSCs在体外能迅速扩增,具有多向分化的能力.在 VEGF、bFGF 诱导下,ADSCs能迅速分化为VECs,大多数细胞vWF阳性表达,与阳性对照组相似.结论 ADSCs在特定诱导环境下可以分化为VECs,有望成为组织工程领域内治疗勃起功能障碍(erectile dysfunction, ED)新的种子细胞来源.     

成体神经干细胞培养方法的建立

目的 探讨体外建立培养成体神经干细胞(MSC)的方法.方法 从6周龄大鼠脑组织中分离神经干细胞,用神经干细胞培养液[DMEM/F12培养液添加1%N2、2%B27、20 μg/L表皮生长因子(EGF)和20μg/L碱性成纤维细胞生长因子(bFGF)]使其稳定增殖,10%胎牛血清诱导其贴壁分化.倒置显微镜下观察神经干细胞形态学变化;流式细胞仪检测神经干细胞表面标记物巢蛋白(Nestin)、神经元特异性烯醇酶(NSE,成熟神经元的特异性标志)、半乳糖脑苷脂(Galc-C,成熟少突胶质细胞的标记物)的表达.结果 分离所得细胞能在体外传代培养,流式细胞仪检测显示Nestin阳性细胞为97.6%,细胞经胎牛血清诱导分化后能形成NSE、Galc-C阳性细胞.结论 采用无血清的神经干细胞培养液能培养出具有分化潜能的成体神经干细胞.     

碱性成纤维细胞生长因子和表皮生长因子对骨骼肌源性干细胞的促增殖效应

目的观察碱性成纤维细胞生长因子（basic fibroblast growth factor，bFGF）和表皮生长因子（epidermal growth factor，EGF）单独及联合应用对骨骼肌源性干细胞（muscle derived stem cells，MDSCs）生长的影响。方法取出生24h内的昆明小鼠15只，采用连续预贴壁法从小鼠后肢肌分离培养MDSCs，用含2％胎牛血清的DMEM培养基促进其向骨骼肌细胞分化。取原代MDSCs及MDSCs分化细胞，采用免疫细胞化学染色检测干细胞标志Sca-1和骨骼肌细胞标志α-Sarcomeric肌动蛋白的表达。HE染色观察细胞肌管形成。MTT比色法检测不同浓度（6．25、12．50、25．00、50．00、100．00ng／ml）的bFGF和EGF单独应用96h对MDSCs增殖的影响以及二者（100．00ng／ml）联合作用24、48、72和96h对MDSCs增殖的影响。结果从新生小鼠后肢肌成功分离培养MDSCs，免疫细胞化学染色90％以上的MDSCs呈Sca-1阳性，分化形成的肌管呈α—Sarcomeric肌动蛋白阳性。HE染色可见肌管形成。bFGF、EGF对MDSCs的促增殖效应随浓度的增加而增加。与阴性对照组比较，bFGF于12．50ng／ml出现促增殖效应（P〈0．05）；25．00ng／ml组与12．50ng／ml组比较，作用提高（P〈0．01）；50．00、100．00ng／ml组较25．00ng／ml组无明显提升（P〉0．05）；EGF的作用与bFGF类似，但于50．00ng／ml时趋于饱和。与阴性对照组比较，EGF于72h、bFGF于96h表现促增殖效应（P〈0．01），而二者联合应用于24h即表现促增殖效应（P〈0．01），并于48、72和96h增殖效应均较单独应用显著提高（P〈0．05）。结论bFGF和EGF都能促进MDSCs的增殖，联合作用更快、更强。     

毛囊干细胞体外诱导分化为血管内皮细胞的实验研究

目的探讨体外诱导人毛囊干细胞成血管内皮细胞的可行性。方法采用中性蛋白酶(Dispase)分离人毛囊干细胞,用含10 ng/mL VEGF、2 ng/mL bFGF及10%血清的EGM-2诱导液对其诱导,以无诱导因子的基础培养液为对照组,对每代细胞形态进行观察。诱导4代后,检测vWF(von Willebrand factor)与CD31的表达。结果在诱导液的作用下,细胞形态逐步向内皮细胞的铺路石样形态转变,对照组细胞形态改变不明显。至第4代,实验组已明显表达vWF与CD31,对照组表达不明显;流式细胞仪检测显示,实验组阳性表达率近80%,对照组则低于5%;RT-PCR显示,实验组表达vWF,对照组未见明显表达。结论使用含10 ng/mL VEGF、2 ng/mL bFGF及10%血清的EGM-2诱导液,可在体外诱导人毛囊干细胞分化成血管内皮细胞。     

小鼠胚胎神经干细胞的长期培养和分化

目的：探索小鼠胚胎神经干细胞的体外培养和分化条件,为神经干细胞的深入研究奠定基础。方法：无菌条件下分离小鼠胚胎脑皮质,制成单细胞悬液,种植在N2培养基中培养,培养基中加入20ng/ml的碱性成纤维细胞生长因子（每次实验用1－2只小鼠胚胎,实验重复8次）。采用机械方法传代,常规方法冻存和复苏。用免疫细胞化学方法对培养的细胞进行鉴定。结果：成功培养出小鼠的神经干细胞,神经干细胞呈悬浮状态生长,形成典型的神经球。细胞表达巢蛋白和波形蛋白2种神经干细胞的标志物。细胞在胎牛血清的诱导下,可分化成神经元、星型胶质细胞和少突胶质细胞,它们所占的比例分别为7％、85％－90％和2％－4％。结论:鼠的神经干细胞可以在体外稳定地培养和传代,为细胞治疗提供了一个理想的细胞来源。     

骨形成蛋白-2诱导脊髓神经干细胞分化的实验研究

目的：研究不同浓度的骨形成蛋白-2(bone norphogenetic protein-2，BMP-2)对脊髓源性神经干细胞(neural stem cells，NSCs)诱导分化成为神经细胞(神经元和神经胶质细胞)的影响。方法：取孕14d的胚胎小鼠脊髓，原代培养出脊髓NSCs。培养四代后，分别在碱性成纤维细胞生长因子(bFGF)存在与否的不同条件下，加入不同浓度的BMP-2．观察细胞的增殖、分化情况，用免疫组织化学的方法进行鉴定，并进行统计学分析：结果：低浓度的BMP-2(1～10ng／ml)与bFGF(20ng／ml)联合作用，可促进脊髓NSCs向神经元和星形胶质细胞分化，抑制其向少突胶质细胞分化。较高浓度BMP-2(100ng／ml)可抑制脊髓NSCs的增殖甚至导致细胞死亡。结论：脊髓NSCs在低浓度BMP和bFGF联合作用下能有效地分化成具有多种特性的神经样细胞，并与脊髓具有良好的同源性，可为脊髓损伤的修复研究提供良好的细胞学基础。     

端粒酶在大鼠胚胎神经干细胞的表达及其生物学特性

目的观察Wistar大鼠胚胎神经干细胞及其分化细胞的端粒酶活性，探讨神经干细胞维持其生物学特性的分子生物学基础及神经干细胞分化调控的内在机制。方法显微镜下分离大鼠胚胎室管膜下区细胞，在含生长因子的神经干细胞完全培养液中培养形成神经干细胞，然后在不含生长因子而含10％血清培养液中分化14～16d，应用改良的端粒重复序列扩增法(TRAP)研究神经干细胞及其分化细胞的端粒酶活性。结果Wistar大鼠胚胎神经干细胞表达较强的端粒酶活性，而分化后的细胞则不再表达端粒酶活性。结论端粒酶活性的改变可能是胚胎神经干细胞诱导分化的机制。随着端粒酶活性的逐渐下降，胚胎神经干细胞随之分化为具有一定生物学特性的细胞。     

成纤维细胞生长因子和表皮生长因子及复合因子对兔ACL、MCL体外增殖作用

目的观察酸性成纤维细胞生长因子(acid fibroblast growth factor，aFGF)、碱性成纤维细胞生长因子(basic fibroblast growth factor，bFGF)和表皮生长因子(epidermal growth factor，EGF)以及复合因子对兔前交叉韧带(anterior cruciate ligament，ACL)和内侧副韧带(medial collateral ligament，MCL)的促增殖作用。方法分离传代培养10周龄新西兰大白兔骨关节韧带的ACL和MCL的成纤维细胞，接种96孔板，并加入浓度为0(对照组)、1、5、10、50、100ng／ml的aFGF或bFGF，浓度为0(对照组)、1．56、3．13、6．25、12．50、25、50ng／ml的EGF，单独或aFGF EGF两种因子联用与细胞(n=4)共同培养48h，以XTT方法测定其促细胞增殖作用。结果3种生长因子单独应用对ACL和MCL都有促进作用，aFGF对两种细胞均存在量效关系；bFGF 1ng／ml，EGF 5ng／ml对ACL作用最好，而对MCL则是bFGF和EGF均存在量效关系。浓度为5ng／ml的aFGF与50ng／ml的EGF联合1ng／ml aFGF与3．13ng／mlEGF作用于ACL或MCL均有协同作用。结论3种生长因子对ACL和MCL均有促进作用，单独应用aFGF或联用EGF优于单一因子促进兔ACL和MCL细胞的增殖，并且提示低浓度的aFGF联用EGF优于单一生长因子。     

Differentiation of adult human bone marrow mesenchymal stem cells into Schwann-like cells in vitro

Bonemarrowmesenchymalstemcells, alsocalledbonemarrowstromalcells (BMSCs), areisolatedfrombonemarrow, andtheycanmultiplyinvitroanddifferentiateintoosteogeniccells,chondrocytes, adipocytes, musclecellsandneuralcells.1 5 RecentstudieshavedemonstratedthatBMSCsfromadultratscoulddifferentiateintoSchwann likecellsinspecificconditions.6, 7 BMSCsmayhavepotentialapplicationforautologousneuraltransplantation. Inthisstudy, wetrytoinvestigatethedifferentiativecapabilityofadulthumanBMSCsintoSchwann l…     

Feasibility of using early mesencephalic neural plate for intracerebral grafting

The purpose of this study was to elucidate the biological significance and the possibility of intracerebral grafting of neuroepithelial stem cells derived from the mesencephalic neural plate. Immunohistological studies of embryonic day 10.5 (E10.5) Wister rats revealed strong nestin expression in the mesencephalic part of the neural plate. Mesencephalic neural plates removed from E10.5 rats were processed to either tissue or cell dissociation culture. They were cultured in vitro under various conditions and were analyzed 7 days after the primary culture. When they were cultured as a tissue, cell proliferation and differentiation into neurons extending long neurites were obvious in a serum-free medium, in a medium containing 3% serum, and in a medium containing 20 ng/ml epidermal growth factor. On the other hand, in a medium containing 10 ng/ml basic fibroblast growth factor (bFGF), both vigorous cell proliferation and sphere formation were recognized. Furthermore, marked neurite growth was rarely seen in this culture. When they were plated in a dissociation culture, cell proliferation and neurosphere generation were also recognized only in a medium containing bFGF, depending on the initial cell concentration. The spheres, generated 7 days after the primary cell culture, were positively stained by nestin. These data suggested that bFGF was able to amplify the stem cell population present in the mesencephalic neural plate derived from early embryos. This might make it possible to obtain a large number of stem cells as donor material for neural transplantation on demand.     

新生大鼠脑源性神经干细胞培养方法的优化

目的寻求较理想的新生大鼠脑源性神经干细胞(neuralstemcells,NSCs)分离培养方法。方法用不同的接种密度和多种配方的培养基:细胞种植密度采用1×104、1×105、1×106和1×107个/ml4个浓度;培养基除基础成分DMEM/F12外,按照是否添加2%B27、碱性成纤维细胞生长因子(basicfibroblastgrowthfactor,bFGF)、表皮生长因子(epidermalgrowthfactor,EGF)以及开始培养时胎牛血清浓度不同而分别记为第1～8组。分离培养新生大鼠脑源性NSCs并诱导其分化,应用相差显微镜和免疫细胞化学法对其进行观察和比较。结果分离培养出的细胞聚集成神经球,可在体外大量增殖和长期存活,可诱导分化为神经元和神经胶质,具有神经干细胞特性。开始培养时培养液中加入5%胎牛血清有利于NSCs存活,当血清浓度采用10%时,克隆球迅速分化,干细胞收获量极少;干细胞增生速度在种植密度采用1×106个/ml时优于采用其它种植密度时,适当提高其种植密度可加快增生速度;培养基中加入2%B27、bFGF、EGF是必需的,2%B27、20ng/ml的bFGF和EGF同时加入时NSCs生长最好,bFGF和EGF对加快NSCs的增生具有协同作用。结论成功建立了较理想的新生大鼠脑源性NSCs分离培养方法,为进一步研究和应用奠定了基础。     

碱性成纤维细胞生长因子对成骨细胞粘附特性影响的实验研究

目的 针对骨组织工程中成骨细胞与基质材料间促粘附问题,研究碱性成纤维细胞生长因子（ｂＦＧＦ）对成骨细胞粘附特性的影响。方法 取兔骨髓基质干细胞来源的成骨细胞体外培养,分别用５、１０、５０、１００及２００ｎｇ／ｍｌ浓度的ｂＦＧＦ诱导培养２４小时作为实验组;不含ｂＦＧＦ的培养基作为对照组。观察接种后０．５、１、２、４、及８小时各时间点成骨细胞粘附情况,体现学计量粘附细胞数量。结果 １０ｎｇ／ｍｌｂＦＧ     

不同微环境对骨髓间充质干细胞分化为产胰岛素细胞的影响

目的 观察不同的微环境对大鼠骨髓间充质干细胞(MSCs)体外分化成为产胰岛素细胞(IPCs)的影响.方法 采用贴壁法分离纯化大鼠MSCs,用不同的微环境进行诱导,对照组诱导剂为含有角朊细胞生长因子、ITS、尼克酰胺的无血清DMEM/F12培养基、实验组在对照组基础上添加胰腺条件培养液;对诱导后细胞进行光、电镜观察,双硫腙和免疫细胞化学进行鉴定,并进行体外葡萄糖刺激实验,测定细胞分泌胰岛素及C-肽功能.结果 实验组及对照组均可诱导分化为IPCs,但实验组分化而成的IPCs在数量及功能上均高于对照组.结论 大鼠胰腺提取物模拟的微环境能促进大鼠MSCs体外诱导分化为较高质量的IPCs.     

大鼠骨髓间充质干细胞诱导分化为表皮细胞的实验观察

目的:研究表皮生长因子(Epidermal growth factor,EGF)加条件培养基体外诱导大鼠 MSCs 向表皮细胞定向分化的可行性。方法:采用 Ficoll-Paque 淋巴细胞分离液分离扩增大鼠骨髓 MSCs,免疫细胞化学染色及流式细胞仪进行鉴定。传至第3代的大鼠 MSCs 用表皮生长因子(EGF)、条件培养基等定向诱导 MSCs 分化为表皮细胞;免疫细胞化学对细胞角蛋白 CK5/8、19(Cytokeratin5/8、19)阳性表达细胞进行检测。结果:从大鼠骨髓中分离培养的 MSCs 增殖能力强,细胞表面标志 CD34、CD45阴性,CD29、CD44阳性,流式细胞仪检测显示细胞纯度高,诱导后7d 细胞免疫化学显示角蛋白5/8、19染色阳性,具有表皮细胞特征。结论:从大鼠骨髓中分离培养出的问充质干细胞,具有自我更新和增殖能力强的特点,经诱导可定向分化表达角蛋白。     

Using human plasma supplemented medium to cultivate human bone marrow-derived mesenchymal stem cell and evaluation of its multiple-lineage potential

The objective of this study was to evaluate the proliferation and the multiple-lineage differentiation capacity when bone marrow mesenchymal stem cells (BMSCs) were cultured short-term in autologous serum/plasma instead of fetal calf serum (FCS). The BMSCs from 12 donors were cultivated individually in 10% autogenic plasma or serum, with or without bFGF and EGF growth factors. Cell proliferation was examined by a Tetrazolium assay (MTT) after passages 1, 3, and 5. A medium supplemented with 10% human plasma or serum was sufficient to propagate BMSCs. However, no significant proliferation was shown when bFGF and EGF (20 ng/mL each) were added into the medium with autologous serum/plasma. We examined, inductions of adipogenesis, osteogenesis, and chondrocytogenesis, as capacities of multiple-lineage differentiation of cultivated BMSCs (passages 8). Differentiation was investigated by both RT-PCR and immunohistochemistry staining (IHC). Qualitative evidence demonstrated the differentiation capacity was preserved in cultivated BMSCs with autologous serum/plasma.     

Cranial bone defect healing is accelerated by mesenchymal stem cells induced by coadministration of bone morphogenetic protein-2 and basic fibroblast growth factor.

To facilitate bone healing in difficult circumstances, and to replace conventional therapeutic modalities, highly purified bone marrow-derived human mesenchymal stem cells (hMSCs) were investigated for induction of their osteogenic lineage upon provision of cytokine cues in vitro and in the cranial defect model in vivo. Alkaline phosphatase-expressing cells were most frequently observed when the hMSCs were treated with 2.5 ng/ml of basic fibroblast growth factor (bFGF) and 50 ng/ml of bone morphogenetic protein (BMP)-2 for 4 days in culture after a 6-day incubation in osteogenic medium containing dexamethasone, ascorbic acid-2-phosphate, and beta-glycerophosphate. Four-millimeter full-thickness cranial defect wounds were made in male nude rats (F344/NJCl-rnu), whose deficit in the T cell compartment prevented T-cell-mediated cellular rejection. The animals were treated for 4 weeks with hMSCs and application of 10 microg each of bFGF and BMP-2 that had been soaked into a gelatin sponge carrier. Significant bone mineral density was observed by dual X-ray absorptiometry and this treatment also produced histologically mature osteocytes surrounded by both osteoblasts and osteoclasts expressing alkaline phosphatase and osteocalcin. The bone mineral densities and histological structures were matched at 8 weeks post-transplantation. Therefore, human bone marrow-derived mesenchymal stem cells are able to differentiate into an osteogenic lineage upon cytokine stimulation and accelerate healing in a nude rat cranial bone healing model.     

Can a bone marrow cell contribute to organ regeneration? In vivo analysis using transgenic rats with reporter genes

Although implantation of multipotent bone marrow-derived stem cells represents an attractive new cell therapy to repair damaged tissues, recent reports have raised serious concerns over the feasibility of using stem cells deriving from the bone marrow to promote cell transdifferentiation. We established transgenic (Tg) rats with reporter genes as specific molecular tags to examine the effect of bone marrow cells (BMCs) on transdifferentiation into tissues/organs. To monitor transdifferentiation events of locally transplanted BMCs into hepatocytes or capillary endothelial cells, a liver injury model and an ischemic hind-limb model were developed in rats. To test the ability of circulating bone marrow-derived cells to give rise to myocytes after skeletal muscle injury, we used a bone marrow cell transplantation model from Tg rats, which showed ubiquitous expression of beta-galactosidase (lacZ), into lethally irradiated non-Tg rats. Our results show that there was little transdifferentiation of BMCs into the targeted cells in these tissue injury models. However, in the ischemic hind-limb model, laser Doppler imaging and histologic analysis showed that both implantation of BMCs and treatment with microspheres incorporating basic fibroblast-like growth factor (bFGF), which enables the release of bFGF at the site of action over a period of time, effectively induced angiogenesis. In conclusion, rat BMCs with specific marker genes could be a useful tool for detecting transdifferentiation events in vivo.