新生大鼠前软骨干细胞株的体外培养分选、鉴定及永生化研究

背景：前软骨干细胞虽然具有较强的自我增殖能力和多向分化潜能，但生物学性状不稳定，易分化。导入外源性基因能使其永生化，且不改变细胞的表型和性状。 目的：建立永生化大鼠前软骨干细胞株，为以后精确调控前软骨干细胞的增殖与分化打下基础。 设计、时间及地点：单一样本观察，于2005-10/2006-09在华中科技大学同济医学院附属同济医院骨科实验室完成。 材料：出生< 24 h的新生SD大白鼠，雌雄不限，用于取前软骨干细胞。 方法：用LipofectamineTM2000介导基因转染，将含有SV40T抗原基因的真核表达载体pCMVSV40T/PUR导入经免疫磁珠分选出的原代前软骨干细胞进行稳定表达，用嘌呤霉素筛选出阳性克隆并扩大培养。 主要观察指标：①前软骨干细胞的生物学性状。②质粒鉴定。③用免疫细胞化学方法和反转录聚合酶链反应鉴定SV40T抗原基因在转染细胞中的表达。④反转录聚合酶链反应结果。⑤细胞生长曲线。 结果：①免疫磁珠分离获得细胞阳性克隆，用免疫组织化学证实FGFR-3表达阳性。②双酶切质粒，电泳证实pCMV为3 kb，SV40T基因为2.3 kb。③嘌呤霉素分离获得转化后细胞阳性克隆，用免疫组织化学证实FGFR-3表达阳性。④提取RNA后用反转录-聚合酶链反应法成功扩增出588 bp的片段。转染细胞经扩大培养，命名为永生化前软骨干细胞。⑤贴壁培养的转染细胞群体倍增时间为 (22.98±2.77) h，传代、冻存和复苏对细胞形态及生长无明显影响。 结论：在体外培养条件下，可以从新生大鼠干骺端中分离、培养出前软骨干细胞，pCMVSV40T/PUR转染能使其永生化。     

人转化生长因子β3基因转染KLD-12自组装纳米肽纤维支架三维培养前软骨干细胞

背景：作者前期试验证实了外源性细胞因子人转化生长因子β3具有很强的诱导前软骨干细胞向成熟软骨分化并促进其增殖的能力。国外的研究均证实了在自组装肽纳米纤维支架中三维培养有利于软骨源性干细胞的增殖与分化。 目的：将人转化生长因子β3基因转染于三维培养于KLD-12肽纳米纤维支架内的前软骨干细胞中，并比较其与二维培养前软骨干细胞转染效率的差异。 方法：以免疫磁珠分选法获取并纯化前软骨干细胞，将其种植于KLD-12肽纳米纤维支架内，使用xfectTM stem纳米干细胞转染试剂将人转化生长因子β3基因分别转染入KLD-12三维支架内培养的前软骨干细胞和普通二维培养基培养的前软骨干细胞，通过免疫荧光和反转录-聚合酶链反应法测定转染后48 h不同组别转化生长因子β3基因的表达情况，并利用酶联免疫吸附法测定转染后不同时间细胞培养上清液中转化生长因子β3的质量浓度。 结果与结论：免疫荧光结果显示，KLD-12三维支架内培养的前软骨干细胞转染后转化生长因子β3阳性细胞率明显高于普通二维培养基内前软骨干细胞(P < 0.05)，与反转录-聚合酶链反应法检测结果相同。酶联免疫吸附法测定结果显示，转染24 h前软骨干细胞上清液中转化生长因子β3蛋白水平呈上升趋势，72 h后达到峰值，随后稍下降进入平台期。提示人转化生长因子β3基因在三维培养于KLD-12肽纳米纤维支架内前软骨干细胞中的转染效率高于普通二维培养基培养的前软骨干细胞。 关键词：前软骨干细胞；转化生长因子β3；KLD-12肽纳米纤维支架；转染效率；纳米转染试剂 doi:10.3969/j.issn.1673-8225.2010.29.005     

SV40 LT基因诱导人骨髓间充质干细胞的实验研究

目的 利用猿猴病毒40大T抗原(SV40 LT)基因异位表达建立永生化人骨髓间充质干细胞(hM-SCs)系.方法 使用含有SV40大T片段的重组质粒载体转染人原代骨髓间充质干细胞,经G418筛选,连续传代培养,通过形态学、免疫组织化学及PCR技术等方法对细胞系进行鉴定.结果 转染后骨髓间充质干细胞基本保持了原代细胞的表型特征、形态均一、多为梭形,呈漩涡状或放射状排列,有特征性表型.该系已培养5个月,超过36代.细胞免疫组化染色显示有SV40 LT的表达.结论 成功地构建了SV40 LT基因永生化的人骨髓间充质干细胞系,为其在神经系统疾病治疗中的应用奠定了实验基础.     

转染SV40永生化基因对正常成人肝细胞生长特性的影响

背景：生物型人工肝采用猪肝细胞或肝癌细胞作为移植物来源存在动物源性疾病和致瘤性的担心,而正常成人肝细胞也具有一定的局限性。 目的：通过检测转染前、后正常成人肝细胞的活率、生长曲线和细胞周期的变化,了解转染SV40永生化基因对正常成人肝细胞生长特性的影响。 方法：培养不同时间的正常人肝细胞和永生化正常成人肝细胞,采用胎盘蓝染色和AO-PI染色,于培养后1~8 d采用MTT染色法计数细胞活率;用MTT比色法测定细胞的A值并绘制生长曲线;采用流式细胞术检测细胞的生长周期。 结果与结论：3种染色法检测显示两种细胞的活率为95%~99%,存活率无明显差异。转染前、后的两种正常成人肝细胞的生长曲线无明显差异,均在培养 3~5 d时呈指数型生长,但转染后正常成人肝细胞较转染前增殖略快。采用流式细胞术测得的两种肝细胞的细胞周期：转染后肝细胞S期细胞占65.64%,G0~G1期细胞占34.36%,G2期细胞占0%;转染前肝细胞S期占21.27%,G0~G1期细胞占62.64%,G2期细胞占12.09%,提示转染后肝细胞的S期细胞明显增多,增殖能力增强。转染SV40永生化基因的正常成人肝细胞较转染前细胞的增殖活力增高,存活率无明显差异,提示转染后细胞增殖能力更强。     

hTERT介导脐带间充质干细胞实现永生化的研究

目的 通过转染人端粒酶逆转录酶催化亚单位(hTERT)建立永生化脐带间充质干细胞(UCMSCs),用以获得足够多的UCMSCs满足体外研究与临床需要. 方法 原代分离培养人UCMSCs,取第4代UCMSCs行流式细胞术检测UCMSCs表面特异标志物表达:利用脂质体介导pLXSN-hTERT正义表达质粒转染UCMSCs,建立永生化UCMSCs:RT-PCR检测转染前后hTERTmRNA表达水平,细胞免疫荧光方法检测hTERT蛋白表达,流式细胞术观察hTERT转染后细胞周期改变. 结果 流式细胞术结果显示UCMSCs强表达CD29、CD44、CDl05,极低表达CD31、CD34、CD45,检测结果符合人UCMSCs特征;RT-PCR检测单纯UCMSCs低表达hTERT mRNA,pLXSN-hTERT正义表达质粒转染后hTERT mRNA表达明显增高;细胞免疫荧光显示hTERT全部表达于细胞核,转染后胞核荧光强度明显增强;细胞周期检测结果显示转染后处于S期UCMSCs数目明显增多,细胞可获得长期稳定传代(＞35代). 结论 以hTERT转染UCMSCs在体外可获得长期稳定传代培养细胞,可基本满足体外研究与临床需要.     

转化生长因子β3基因转染兔骨髓间充质干细胞诱导其向软骨表型的分化

背景：内源性诱导软骨分为就是通过一定的载体将目的基因整合入干细胞内,使其自行分泌诱导因子诱导自身进行分化。 目的：观察将转化生长因子β3通过腺相关病毒载体转染诱导兔骨髓间充质干细胞向软骨表型转化的能力。 方法：取体外培养的第3代骨髓间充质干细胞进行重组腺相关病毒转染,将转染后3,6,9,12 d细胞裂解提取蛋白进行酶联免疫检测目的蛋白转化生长因子β3的体外表达。RT-PCR,免疫印迹western blot分别从基因和蛋白水平上检测1,2周Ⅱ型胶原的表达,甲苯胺蓝染色检测1,2周蛋白多糖的表达。 结果与结论：重组腺相关病毒转染后,骨髓间充质干细胞可以较稳定的表达目的蛋白转化生长因子β3,并且转染成功的骨髓间充质干细胞较阴性对照组能够更好的向软骨表型转化。证实转化生长因子β3可以腺相关病毒为载体转染骨髓间充质干细胞并诱导其向软骨表型分化。     

软骨源形态发生蛋白基因转染自体骨髓间充质细胞修复关节软骨缺损

背景：以支架或干细胞在一定程度上能够修复软骨缺损,但是研究中发现较大块的缺损修复效果还达不到令人满意的程度。基因转染后的干细胞能够不断的释放生长因子有可能够为软骨缺损研究带来突破。 目的：进一步验证软骨源形态发生蛋白转染的骨髓间充质干细胞是否会促进体内兔软骨修复。 方法：从兔骨髓内分离获得骨髓间充质干细胞,脂质体感染的方法将软骨源形态发生蛋白基因转染到骨髓间充质干细胞中。实验组移植转染后的细胞;单纯骨髓间充质干细胞组移植未转染的骨髓间充质干细胞;空白对照组缺损区不移植细胞。术后行组织学检测及组织学评分分析修复情况。 结果与结论：体内修复过程中,软骨源形态发生蛋白转染的骨髓间充质干细胞促进了软骨再生。透明软骨填充了软骨缺损区,并且深部区域显示了软骨下成骨。透明软骨的重建区域优于单纯骨髓间充质干细胞移植组。组织学评分实验组高于骨髓间充质干细胞对照组和空白组。提示转染软骨源形态发生蛋白基因的骨髓间充质干细胞能够促进和提高关节软骨的改建和修复。     

兔骨髓间充质干细胞体外培养定向诱导分化为软骨细胞

背景：骨髓间充质干细胞具有分化为骨、软骨、肌腱、脂肪等组织的多分化潜能。 目的：体外培养兔骨髓间充质干细胞并定向诱导分化为软骨细胞,探讨体外诱导分化为软骨的方法和条件。 方法：取兔股骨骨髓,全骨髓贴壁法分离培养骨髓间充质干细胞,碱性成纤维生长因子体外培养后,取第3代细胞在培养基中添加不同质量浓度转化生长因子β1的软骨分化诱导剂,2周后倒置显微镜观察细胞形态,甲苯胺蓝染色,RT-PCR技术检测Ⅱ型胶原和聚集蛋白聚糖的表达。 结果与结论：可以从兔骨髓中培养出骨髓间充质干细胞,碱性成纤维生长因子能明显的促进骨髓间充质干细胞增殖。经软骨诱导分化后骨髓间充质干细胞呈软骨细胞形态,甲苯胺蓝染色阳性,Ⅱ型胶原和聚集蛋白聚糖的表达阳性。10 μg/L的转化生长因子β1诱导成软骨分化能力最强。     

腺病毒介导人转化生长因子β2基因转染诱导骨髓间充质干细胞向软骨细胞的定向分化

背景：前人的工作已证实转化生长因子β1能促进骨髓间充质细胞的增殖,诱导其向软骨细胞分化。 目的：进一步验证人转化生长因子β2(human transforming growth factor-β2,hTGFβ2)基因转染诱导骨髓间充质干细胞向成骨细胞定向分化作用。 方法：取清洁级3周龄雄性Lewis大鼠16只,用于分离培养骨髓间充质干细胞。通过腺病毒重组载体Ad-hTGFβ2将外源性hTGFβ2基因转染到体外培养的第2代大鼠骨髓间充质干细胞中,48 h后采用免疫组织化学染色、RT-PCR和Western blotting的方法检测转染细胞中目的基因与软骨特异性蛋白——Ⅱ型胶原和蛋白多糖表达的情况。 结果与结论：从成体大鼠骨髓组织中培养出骨髓间充质干细胞,体外培养呈成纤维细胞样,能大量稳定增殖传代。Ad-hTGFβ2转染骨髓间充质干细胞获得稳定表达,免疫组织化学染色和Western blotting检测到基因转染细胞中Ⅱ型胶原蛋白的合成表达,RT-PCR检测到Ⅱ型胶原和蛋白多糖aggrecan mRNA的表达。结果提示,从骨髓组织中可获得具有多分化潜能的间充质干细胞,并能在体外稳定增殖传代;Ad-hTGFβ2成功转染骨髓间充质干细胞,诱导其向软骨细胞分化。     

hTERT基因转染人神经干细胞向永生化细胞转变实验研究

目的明确外源性人端粒酶催化亚单位(hTERT)在人神经干细胞永生化过程中的作用。方法利用构建了hTERT基因的逆转录病毒载体PLXSN转染人NSCs,G418筛选后,扩大增殖,稳定后观察其生物学特性,检测端粒酶活性,通过核型分析、裸鼠移植致瘤性观察来了解转染细胞特性。结果hTERT基因转入培养的NSC后,端粒酶活性增强,细胞增殖加速,传代时间缩短,传代50代仍稳定增殖,核型分析正常,无致瘤性。结论hTERT基因转染NSCs,能获得永生化细胞,其表型能得以正常维持,可以作为进一步研究的种子细胞。     

Gene expression by simian virus 40 large T antigen-induced medulloblastomas in mice

Signaling pathways known to have components with mutations in human medulloblastoma include sonic hedgehog,Wnt/beta-catenin and insulin-like growth factor.Microarray analysis was applied to examine the gene expression changes in medulloblastomas of pTet-on/pTRE-SV40Tag transgenic mice.Altogether,14 112 genes were detectable,including 152 genes with significantly different expression levels.These genes were associated with immunity,the cell cycle,signal transduction,cytoskeleton and metabolism.To further confirm the microarray data,real-time polymerase chain reactions were used to examine the expression changes of genes related to sonic hedgehog,Wnt/beta-catenin and insulin-like growth factor signal pathways.Immunohistochemistry detected insulin receptor substrate-1 in the nuclei of brain tumor tissue cells from pTet-on/pTRE-SV40Tag transgenic mice,suggesting that SV40 large T antigen may activate the insulin-like growth factor signal pathway to promote tumorigenesis.     

Immortalization of immature and mature mouse astrocytes with SV40 T antigen

The ability of neonatal astrocytes to promote neurite outgrowth in vitro and in vivo diminishes as astrocytes mature. This property correlates with the developmental loss of the central nervous system's ability to regenerate after injury. Cell lines representative of immature and mature astrocytes would be useful for studies to determine differences between these two populations. Previous work on immortalization of bipotential neural/glial precursors and fully differentiated glial cells suggests that immortalization of astrocytes at timed intervals of culture may yield cell lines trapped in different maturation states. To test this, neonatal mouse cortical astrocytes were immortalized by retrovirus-mediated transfer of the SV40 T antigen (Tag) gene at 2, 6 and 17 days of culture. The clonal cell lines express Tag and are contact-inhibited. Three phenotypes that change as a function of astrocyte maturation were examined to determine the fidelity with which the cell lines represent immature and mature astrocytes. These were: (1) cell morphology, growth pattern and size, (2) level of glial fibrillary acidic protein (GFAP) expression, and (3) neurite outgrowth promotion. First, immature and mature lines resemble mortal type 1 astrocytes of corresponding ages with respect to morphology and growth pattern, and retain a quantitative difference in cell size (mature cells are larger). Second, the pattern of GFAP expression is preserved, with immature lines expressing lower levels than mature cell lines, but the overall GFAP levels are significantly lower in immortalized cell lines compared to mortal cells. Finally, promotion of neurite outgrowth from embryonic chick retinal ganglion cells on monolayers of the cell lines was examined. While all neurite outgrowth measures are significantly greater for the immortalized lines than for control 3T3 cells, they are attenuated relative to mortal astrocytes. The age-related pattern of stronger outgrowth support on immature astrocytes is retained for neurite initiation, but not retained for mean neurite length. Thus, SV40 Tag-immortalized astrocytes have a complex phenotype characterized by retention of age-related differences in morphology, growth pattern and cell size, and by a marked attenuation of some astrocyte-specific characteristics but retention of age-related differences in the expression level of these same characteristics, and finally, loss of the ability to support neurite extension at levels characteristic of immature astrocytes. © 1994 Wiley-Liss, Inc.     

人脑胶质瘤组织中SV40 DNA及大T抗原研究

目的：观察猴病毒40（SV40）DNA及大T抗原（Tag)在人脑胶质瘤的定位表达情况，探讨其在胶质瘤发生发展中的生物学意义。方法：采用原位杂交和免疫组化技术，检测256例人脑胶质瘤和11例正常脑组织标本。结果：SV40 DNA定位于胶质瘤细胞核，阳性细胞呈弥漫或片灶状分布。胶质瘤SV40 DNA阳性率40．6％（104／256），正常脑组织均未检测出SV40 DNA，SV40阳性率与病理分级无关；Tag表达分布与SV40 DNA一致，Tag阳性率28．5％（73／256）较SV40 DNA感染率为低，但Tag表达与病理分级呈显著正相关（P＜0．01）。结论：SV40感染与人脑胶质瘤病因学密切相关，Tag可能是SV40在胶质瘤发生发展中起作用的重要因素，其机制及生物学意义需进一步研究。     

Simian virus 40 persistent infection in long-term immortalized human fibroblast cell lines

Episomal simian virus 40 (SV40) DNA was detected in various SV40-immortalized human fibroblast cell lines, without rearrangements or mutations. In these cells, SV40 established a persistent infection with the release of a viral progeny. However, electron microscopy analysis showed that virions are morphologically altered, whereas infectivity assay indicated that viral production was hampered. The data suggest that in SV40-infected human fibroblasts, some cells support a complete SV40 productive cycle, whereas other cells resist to the SV40 infection. This sort of "balance" observed within the same human fibroblast population may be responsible for the semipermissiveness of these cells to SV40 infection.     

Distribution and localization of fibroblast growth factor-8 in rat brain and nerve cells during neural stem/progenitor cell differentiation

The present study explored the distribution and localization of fibroblast growth factor-8 and its potential receptor,fibroblast growth factor receptor-3,in adult rat brain in vivo and in nerve cells during differentiation of neural stem/progenitor cells in vitro.Immunohistochemistry was used to examine the distribution of fibroblast growth factor-8 in adult rat brain in vivo.Localization of fibroblast growth factor-8 and fibroblast growth factor receptor-3 in cells during neural stem/progenitor cell differentiation in vitro was detected by immunofluorescence.Flow cytometry and immunofluorescence were used to evaluate the effect of an anti-fibroblast growth factor-8 antibody on neural stem/progenitor cell differentiation and expansion in vitro.Results from this study confirmed that fibroblast growth factor-8 was mainly distributed in adult midbrain,namely the substantia nigra,compact part,dorsal tier,substantia nigra and reticular part,but was not detected in the forebrain comprising the caudate putamen and striatum.Unusual results were obtained in retrosplenial locations of adult rat brain.We found that fibroblast growth factor-8 and fibroblast growth factor receptor-3 were distributed on the cell membrane and in the cytoplasm of nerve cells using immunohistochemistry and immunofluorescence analyses.We considered that the distribution of fibroblast growth factor-8 and fibroblast growth factor receptor-3 in neural cells corresponded to the characteristics of fibroblast growth factor-8,a secretory factor.Addition of an anti-fibroblast growth factor-8 antibody to cultures significantly affected the rate of expansion and differentiation of neural stem/progenitor cells.In contrast,addition of recombinant fibroblast growth factor-8 to differentiation medium promoted neural stem/progenitor cell differentiation and increased the final yields of dopaminergic neurons and total neurons.Our study may help delineate the important roles of fibroblast growth factor-8 in brain activities and neural stem/progenitor cell differentiation.     

免疫磁珠法分离、培养人脑胶质瘤干细胞

目的建立免疫磁珠法分离，并培养人脑胶质瘤干细胞的方法。方法将术中取得的脑胶质瘤标本，通过剪切、消化和吹打成单细胞悬液，筛网过滤，免疫磁珠分选试剂盒分选出CD133^＋细胞，用神经干细胞无血清培养法培养出具有单细胞克隆能力的细胞球，取第3代进行诱导分化，分化前后用免疫细胞荧光化学方法鉴定肿瘤干细胞及分化后细胞。结果免疫磁珠分选出的CD133^＋细胞，可悬浮生长并形成神经干细胞样细胞球，有较强的增殖能力，干细胞标志物巢蛋白（nestin）阳性，分化后细胞表达神经元小管相关蛋白β-3（β-tubulin3）和星形胶质细胞胶质纤维酸性蛋白质（GFAP）特异性抗原，而巢蛋白、CD133^＋阴性，并具有肿瘤的核型。结论免疫磁珠分选法可避免原代培养中众多细胞混杂生长的发生，能够从大量肿瘤细胞中分离出只占极少比例的肿瘤干细胞，细胞结合磁珠后在体外可以长期培养和传代，进一步证实了肿瘤干细胞的存在，并为胶质瘤干细胞的研究奠定基础。     

Evidence for a novel neurotrophic factor for dopaminergic neurons secreted from mesencephalic glial cell lines

Our previous studies have shown that primary mesencephalic glia secrete factors that promote dopaminergic cell survival and differentiation in vitro. To obtain enough starting material to identify the neurotrophic activity, embryonic day (E)14.5 rat mesencephalic glia were stimulated with acidic fibroblast growth factor to increase number of cells. These cells were replated in the absence of neurons and immortalized by transfection with the SV 40 large T-antigen. Clonal cell lines were established and characterized for immunoreactivity (IR) to various glial and non-glial markers. Media conditioned by these cell lines were tested for survival-promoting effects on dopaminergic neurons in serum-free cultures of the dissociated E14.5 rat mesencephalon. All cell lines expressed IR for the astrocytic marker, GFAP, the oligodendroglial marker, CNP, and for A2B5, a marker for O-2A progenitor cells, but were negative for the neuronal marker, microtubule associated protein-2, and the fibroblast marker, fibronectin. Moreover, treatment of serum-free cultures of the dissociated E14.5 mesencephalon with glial cell line-conditioned medium (CM) delayed dopaminergic cell death in a dose-dependent manner, resulting in a maximal twofold to sixfold increase in the number of surviving tyrosine hydroxylase-IR neurons at various days in vitro. This increase in dopaminergic cell survival was not mimicked by GDNF, BDNF or NT-3 within the initial 3 days of cultivation. Moreover, initial biochemical characterization demonstrated that the neurotrophic activity is restricted to the high MW fraction of >50 kD of glial cell line-CM. Since the apparent MW of this factor exceeds the size of most known growth factors, it may represent a novel dopaminergic neurotrophic factor. © 1996 Wiley-Liss, Inc.     

Increase in the proliferative capacity of human myoblasts by using the T antigen under the vimentin promoter control

Normal myoblasts have a strictly limited growth potential and senesce after a defined number of population doubling. The objective of this study was to determine whether the proliferative capacity of human myoblasts could be extended without inhibiting myogenic differentiation. We have established a stable transfected human myoblast cell line that expresses the SV 40 large T antigen under the control of the human vimentin promoter. We show that these cells have an increased proliferative capacity compared with that of normal myoblasts. Indeed, the final proliferative capacity was increased to 19 passages (5 for normal myoblasts). Moreover, they retained their capacity to differentiate fully, as indicated by their morphology and electrophysiological properties as well as by the expression of different markers of differentiation. The generation of human myogenic cell lines with the ability to proliferate for a longer period of time than primary myoblasts and while retaining the capacity to differentiate into myotubes could provide a valuable tool for the derivation of cell lines from human diseased muscle cells. © 1997 John Wiley & Sons, Inc. Muscle Nerve, 20, 437–445, 1997