猿肾病毒40介导的人前软骨干细胞永生化的实验研究

目的 构建猿肾病毒40大T抗原基因(SV40Tag)介导的永生化人前软骨干细胞株,为下一步基因打靶研究其分化分子机制提供稳定的细胞来源. 方法采用脂质体介导的基因转染技术将含有SVd0Tag的质粒pCMVSV40T/PUR转染人前软骨干细胞(PSCs),经嘌呤霉素筛选,阳性克隆扩大培养并连续传代.用免疫组化、RT-PCR、Southern印迹杂交法对转染细胞进行鉴定,并检测SV40Tag在转染细胞中的表达及其与基因组的整合情况. 结果 筛选获得的阳性克隆扩大培养,命名为永生化前软骨干细胞(IPSCs),能连续传代培养,细胞生长迅速.免疫组化和RT-PCR证实IPSCs成纤维生长因子受体-3阳性,并可检测到SV40Tag mRNA及其蛋白的表达.Southern印迹杂交显示IPSCs基因组中存在SV40Tag cDNA. 结论 成功构建了SV40Tag介导的永生化人前软骨干细胞株.     

猿肾病毒40大T抗原基因永生化大鼠神经前体细胞株的构建

目的建立猿肾病毒40大T抗原基因(SV40Tag)永生化大鼠神经前体细胞株,为细胞移植治疗和转基因治疗提供稳定的细胞来源。方法利用脂质体介导的基因转染技术将含有SV40Tag的质粒pCMVSV40T/PUR转染原代培养的新生大鼠神经前体细胞,经嘌呤霉素筛选,阳性克隆扩大培养并连续传代。应用巢蛋白抗体进行细胞鉴定,5%胎牛血清诱导细胞分化后,应用免疫细胞化学法检测其分化能力,观察细胞的形态及其生长状况,绘制细胞生长曲线。用RT-PCR、Southern印迹杂交和免疫细胞化学法检测SV40Tag在转染细胞中的表达。结果转染细胞经筛选培养后获得1 个阳性细胞克隆,免疫细胞化学结果显示细胞的巢蛋白和微管相关蛋白2染色为阳性,增殖能力较强。5%胎牛血清可诱导转染细胞分化为微管相关蛋白2阳性和胶质纤维酸性蛋白阳性细胞。Southern印迹杂交结果显示转染细胞基因组中存在SV40Tag cDNA,并可检测到SV40Tag mRNA及其蛋白的表达。转染细胞经扩大培养,命名为永生化神经前体细胞。贴壁培养的神经前体细胞,群体倍增时间为(22.9±2.7)h,传代、冻存和复苏对细胞形态及生长无明显影响。结论成功地构建了SV40Tag永生化的大鼠神经前体细胞株。     

大鼠骨骺干细胞的分离鉴定及其永生化细胞株的构建

[目的]分离、鉴定大鼠骨骺干细胞并建立永生化的大鼠骨骺干细胞株,为细胞移植和转基因治疗提供稳定的细胞来源.[方法]采用Percoll不连续密度梯度离心法分离骨骺干细胞,利用电穿孔转染技术将含有猿肾病毒40大T抗原基因（SV40Tag）的质粒pCMVSV40T/PUR转染骨骺干细胞,经嘌呤霉素筛选,抗性克隆扩大培养.应用FGFR-3抗体和PCNA抗体进行免疫细胞化学染色,观察细胞的形态及其生长状况并绘制细胞生长曲线.用免疫细胞化学法和RT-PCR检测SV40Tag在转染细胞中的表达.[结果]转染后获得一个阳性细胞克隆,免疫细胞化学结果显示FGFR-3抗体染色阳性.SV40Tag抗体染色和RT-PCR结果显示SV40Tag已稳定转染入骨骺干细胞.转染细胞经扩大培养,命名为永生化骨骺干细胞.[结论]成功纯化大鼠骨骺干细胞并构建了SV40Tag永生化的骨骺干细胞株.     

免疫分选软骨前体细胞并诱导永生化的研究

目的建立永生化大鼠软骨前体细胞株,为细胞移植和转基因治疗提供稳定的细胞来源。方法利用免疫磁珠技术分离纯化具有特异性表面标志成纤维生长因子受体-3(FGFR-3)的软骨前体细胞,用基因转染技术将含有猿肾病毒40大T抗原基因(SV40Tag)的重组质粒pEGFP- IRES2-SV40Tag转染原代培养的新生大鼠软骨前体细胞,经G418筛选,抗性克隆扩大培养。应用FGFR-3、Ⅱ型胶原和X型胶原抗体进行细胞鉴定,检测其分化能力,观察细胞的形态及其生长状况,绘制生长曲线。用逆转录-聚合酶链反应(RT-PCR)、Southern blot和免疫细胞化学法鉴定SV40Tag在转染细胞中的表达。结果获得1个阳性细胞克隆,免疫细胞化学证实为FGFR-3阳性的具有较强增值能力和多分化潜能的软骨前体细胞。经Southern印迹杂交证实,SV40Tag已稳定转染入软骨前体细胞,表达mRNA及其蛋白。贴壁培养的永生化软骨前体细胞株(IPSC),群体倍增时间为23.62 h,传代、冻存和复苏对细胞形态及生长无明显影响。结论SV40Tag导入可诱导软骨前体细胞永生化,为软骨前体细胞的实验研究及其介导的细胞移植治疗提供了稳定的细胞来源。     

四环素调控系统修饰永生化大鼠星形胶质细胞株的构建及鉴定

目的构建四环素及其衍生物强力霉素诱导表达目的基因的永生化大鼠星形胶质细胞株。方法用脂质体法将逆转录病毒载体四环素调控系统中的质粒pRevTet-On转染病毒包装细胞 PT67,经筛选培养后获得病毒载体RevTet-On,采用RT-PCR进行鉴定,并应用NIH313细胞测定病毒滴度。将RevTet-On感染永生化大鼠星形胶质细胞,用有限稀释法挑选阳性细胞单克隆后扩大培养,每个克隆瞬时转染含萤光素酶报告基因的质粒pRevTRE-Lue,加入强力霉素48h后分别检测其萤光素酶活性值,挑选出强力霉素诱导表达高、背景表达低的细胞株并检测其诱导表达的时效、量效关系。结果 RT-PCR结果显示逆转录病毒包装成功,病毒最高滴度为7．4×105CFU／ml。RevTet-On转染永生化大鼠星形胶质细胞后挑选出48个单克隆,瞬时转染pRevTER-Luc后筛选出1株高表达低背景细胞株, 其诱导表达值为876．1 RLU,背景表达值为42．5 RLU,诱导倍数为20．6。该细胞株在加入诱导因子强力霉素1 h后目的基因即开始表达,在24 h时达到高峰,在强力霉素浓度100-2 000 ng/ml的范围内, 其诱导表达活性与药物浓度呈剂量依赖性。结论含四环素调控系统的永生化大鼠星形胶质细胞株诱导活性可靠,可用于调控表达外源基因的研究。     

哌替啶对大鼠齿状回星形细胞形态和GFAP表达的影响

目的　观察不同剂量哌替啶对SD大鼠齿状回星形细胞形态和GFAP表达的影响。方法　单次注射不同剂量哌替啶2 4h后及以8mg/kg给药后不同时间点经心脏灌注、取脑。GFAP免疫组化,共聚焦显微镜观察,测定齿状回GFAP阳性星形胶质细胞的数量及体视学参数。结果　①小剂量哌替啶对大鼠齿状回星形胶质细胞的形态及其GFAP表达无影响,大剂量(>8mg/kg)时星形胶质细胞GFAP的表达显著增加(P <0 0 5 ) ,单位体积内GFAP阳性细胞数目较对照组明显增多,平均细胞面积有所减小。②大剂量(8mg/kg)哌哌替啶给药1d后GFAP的水平增加,GFAP阳性细胞开始增生和分化,给药3d后GFAP的水平继续增加,伴细胞数目增加、体积肥大、侧枝增多,给药7d后GFAP的水平增加达到最高峰,细胞数目进一步增加、体积更肥大、侧枝更丰富,给药14d后GFAP的水平又恢复到正常水平,细胞数目,大小,形态也恢复到给药前正常水平。结论　单次注射哌替啶对星形胶质细胞GFAP的表达影响呈剂量依赖性;小剂量哌替啶对大鼠齿状回星形胶质细胞的形态及其GFAP表达几乎无影响,大剂量时呈时间依赖性影响。     

猿肾病毒40大T抗原基因永生化大鼠星形胶质细胞株的生物学特性

目的研究猿肾病毒40大T抗原基因永生化大鼠星形胶质细胞株(IAST)的生物学特性,探讨其作为转基因细胞镇痛载体的可行性。方法取大鼠大脑皮层星形胶质细胞(AST)和IAST 进行体外培养,观察细胞的形态学、超微结构以及传代复苏的情况;绘制细胞生长曲线,免疫细胞化学法检测细胞胶质纤维酸性蛋白的表达;5’溴-2-脱氧尿苷掺入法和流式细胞仪检测细胞增殖周期;双层软琼脂克隆形成实验和裸鼠接种实验检测细胞的致瘤性。结果AST体外传代至10代左右即出现复制衰老现象,而IAST可连续传代,未出现衰老迹象;细胞生长呈单层,锚着依赖和接触抑制;传代、冻存和复苏对IAST的存活率无影响,而第6代和第10代AST的存活率降低(P<0.01);与第6代和第10代AST相比,IAST增殖较为旺盛,增殖指数较高,群体倍增时间较短、增殖率较高(P<0.01); IAST胶质纤维酸性蛋白免疫染色阳性,软琼脂培养无集落形成,裸鼠接种无致瘤性。结论大鼠IAST是增殖活跃的非恶性转化细胞,可安全地用于转基因细胞移植镇痛研究。     

SV40LT抗原基因逆转录病毒载体的构建及其在肝细胞中的表达

目的　构建含SV 40LT抗原基因的逆转录病毒载体并转染鼠肝细胞 ,检测SV 40LT抗原基因在肝细胞中的表达情况 ,为肝细胞移植研究打下基础。方法　利用体外基因重组技术构建含SV 40LT抗原基因的逆转录病毒载体并酶切、测序鉴定 ,脂质体介导转染PA3 17细胞 ,G 418抗性筛选阳性克隆 ,通过NIH 3T3细胞测定病毒滴度 ;分离、纯化大鼠肝细胞后 ,将含SV 40LT抗原基因的假病毒颗粒感染肝细胞 ,用PCR及免疫组化法检测转染肝细胞中SV40LT抗原基因的表达情况。结果　( 1)酶切分析、测序证明重组逆转录病毒载体含有SV 40LT抗原基因 ;( 2 )病毒滴度为 1.3× 10 6 cfu/ml ;( 3 )转染后的肝细胞含有SV40LT抗原基因 ,其表达在转染后 2 4h明显高于 96h(P <0 .0 5 )。结论　成功构建含SV40LT抗原基因的逆转录病毒载体 ,转染后的肝细胞表达有目的基因 ,有望作为肝细胞体外培养的可用技术     

人碱性成纤维细胞生长因子基因转染大鼠骨髓间充质干细胞的研究

目的 观察人碱性成纤维细胞生长因子(bFGF)基因体外转染对大鼠骨髓间充质干细胞(MSCs)bFGF表达的影响.方法 密度梯度离心、贴壁法培养分离SD雄性大鼠MSCs,体外扩增,流式细胞仪检测MSCs表面抗原表达.利用慢病毒载体系统介导将具有人源性bFGF基因转染至第2代MSCs,在倒置荧光显微镜下观察转染后细胞形态和生长的变化,应用逆转录-聚合酶链反应(RT-PCR)、Western blot法鉴定bFGF在MSCs中的表达.结果 密度梯度离心、贴壁法培养分离可获得MSCs,P3代大鼠细胞利用流式细胞仪检测CD11b/c阳性细胞表达率为(13.2±0.6)%,CD34阳性细胞表达率为(1.2±0.5)%,CD44阳性细胞表达率(97.8±0.9)%,CD90阳性细胞表达率(96.8±1.4)%.MSCs转染48 h后,绿色荧光蛋白的表达明显增强.RT-PCR证实转基因MSCs表达bFGF mRNA明显增强,Western blot检测证实转基因MSCs在49 KDr出现特异性条带,而空白和空载组的MSCs则未见阳性条带.结论 采用慢病毒介导的基因转染技术可以将bFGF基因转染至MSCs中,并有外源性bFGFmRNA和蛋白的有效表达,MSCs可作为bFGF基因治疗的载体.     

破骨细胞转基因永生化问题的初步探讨

目的：通过转基因技术建立破骨细胞永生化细胞系，方法：经1，25（OH）2D3诱导，获得小鼠骨髓来源的破骨前体细胞，脂质体法（Fugene6）将猿猴病毒40（SV40）和绿色荧光蛋白（GFP）质粒分别转染入破抽前体细胞，G418筛选抗性克隆；同时将GFP转染入逆转录病毒包装细胞（P167）中，作为方法对照，继而，用含有GFP的逆转录病毒感染破骨前体细胞，G418筛选抗性克隆。结果：获得小鼠骨髓来源的破骨前体细胞，Fugene6转染SV40和GFP到破骨前体细胞后，G418筛选未获得阳性克隆，但GFP转染PT67细胞获得阳性克隆和含有GFP的逆转录病毒。将含有GFP的逆转录病毒感染破骨前体细胞，G418筛选未获得阳性克隆，结论：通过破骨细胞转基因永生化，建立破骨或破骨前体系细胞系的方法是一个非常有意义的研究课题，但是难度较大。可以初步认为：脂质体和逆转录病毒载体的方法不是破骨细胞转基因的最佳方法。     

Immunological purification of rat precartilaginous stem cells and construction of the immortalized cell strain

Precartilaginous stem cells (PCSC) are adult stem cells that control limb growth of animals and can differentiate directionally. In a previous study, PCSC was reported to begin differentiating at the fifth passage; therefore, sufficient uni-phenotype PCSC cannot be harvested from primary cell culture. The purpose of this study was to examine whether simian virus 40 large T antigen gene (SV40Tag) could induce rat PCSC to immortalize. Immunomagnetic separation was used to isolate PCSC labeled with fibroblast growth factor receptor-3 (FGFR-3). Plasmid pCMVSV40T/PUR containing SV40Tag was transfected into PCSC by liposome transfection method. One anti-puromycin cell clone was obtained, which was confirmed as FGFR-3 positive, and expanded to immortalized cell strain. Results from RT-PCR and immunocytochemistry demonstrated that SV40Tag was highly expressed at both mRNA and protein levels after stable transfection. The cells transfected with SV40Tag were expanded to immortalized cell strain, which could maintain its characteristics for 30 passages, named immortalized precartilaginous stem cells (IPCSC). IPCSC were short fusiform or triangular cells with two or three short axons. Immunocytochemistry results of FGFR-3 and Collagen II demonstrated that IPCSC retained the characteristics of PCSC and the high proliferation capability of IPCSC was confirmed by methyl thiazolyl tetrazolium assay. Therefore, we concluded that rat precartilaginous stem cells were purified and immortalized precartilaginous stem cell strain was established. It may provide a stable cell resource for basic research and cell transplantation therapies.     

单酶消化复合植块法培养雪旺细胞的实验研究

目的探讨一种快速获取高纯度、高活性雪旺细胞的分离、培养方法,并从转录及翻译水平对获取细胞进行鉴定。方法取4周龄SD大鼠双侧坐骨神经,胶原酶Ⅰ消化15 min后接种于培养瓶中,进行体外培养并按1∶2比例传代,倒置相差显微镜观察细胞生长状态及形态变化,MTT法检测细胞增殖情况并绘制生长曲线,RT-PCR检测S100及胶原纤维酸性蛋白(glial fibrillary acidic protein,GFAP)基因表达情况,并行免疫组织化学染色观察S100及GFAP蛋白表达水平,计算细胞纯度。结果胶原酶Ⅰ消化后组织块培养24 h即可见成纤维样细胞迁出,胞体细长,折光性较弱;48 h后可见大量细胞迁出,细胞多呈双极或三极,突起细长,胞体饱满,折光性强,72 h形成细胞集落。细胞传代后48～72 h即可长满瓶底,并出现螺旋形细胞集落,多次传代后细胞仍生长旺盛,细胞质饱满。MTT结果显示,第3代细胞第3天即进入对数生长期,随时间延长逐渐增殖,第7天进入平台期,生长曲线呈"S"形。RT-PCR结果示培养细胞表达S100、GFAP基因;免疫组织化学染色检测显示大部分细胞呈阳性染色,表达S100及GFAP蛋白,雪旺细胞纯度为98.37%±0.30%。结论单酶消化复合植块法可以快速获得高纯度、高活性的大鼠雪旺细胞。     

端粒酶逆转录酶启动子联合CD自杀基因靶向杀伤人肝癌细胞研究

目的 利用肿瘤特异性人端粒酶逆转录酶(hTERT)启动子介导自杀基因CD表达,检测其促进5-FC对肝癌细胞系的杀伤作用.方法 采用聚合酶链反应(PCR)技术获得hTERT基因的启动子片段,将其连至SV40增强子序列后,并克隆到表达荧光素酶基因的报告质粒上,检测hTERT基因启动子在肝癌细胞系Bel-7402和人成纤维细胞HDF中的转录活性,同时将CD自杀基因连至该调控序列后,转染肝癌细胞Bel-7402,通过逆转录(RT)-PCR、噻唑蓝(MTT)比色法检测其作为肝癌基因治疗中肿瘤特异性靶向杀伤载体的可行性.结果 成功构建pGL3-SV40-hTERT载体和SV40-hTERT-CD-GFP质粒载体,转染ST-CD载体后Bel-7402细胞表达CD基因,转染PGL3-SV40-hTERT质粒后Bel-7402细胞中hTERT启动子高表达,相对活性为354%.与未转染组比较,5-Fc对转染SV40-hTERT-CD-GFP质粒载体的肝癌细胞Bel-7402的杀伤效应明显增强(F=27.831,P<0.01).结论 hTERT启动子在肝癌细胞系中具有较强的转录活性,能有效地诱导CD自杀基因的表达.     

Cre/loxP-based reversible immortalization of human hepatocytes

An ideal alternative to the primary human hepatocytes for hepatocyte transplantation would be to use a clonal cell line that grows economically in culture and exhibits the characteristics of differentiated, nontransformed hepatocytes following transplantation. The purpose of the present studies was to establish a reversibly immortalized human hepatocyte cell line. Human hepatocytes were immortalized with a retroviral vector SSR#69 expressing simian virus 40 large T antigen (SV40Tag) gene flanked by a pair of loxP recombination targets. One of the resulting clones, NKNT-3, showed morphological characteristics of liver parenchymal cells and expressed the genes of differentiated liver functions. NKNT-3 cells offered unlimited availability. After an adenoviral delivery of Cre recombinase and subsequent differential selection, efficient removal of SV40Tag from NKNT-3 cells was performed. Here we represent that elimination of the retrovirally transferred SV40Tag gene can be excised by adenovirus-mediated site-specific recombination.     

Long-term culture and transplantation of murine testicular germ cells

The objectives of this study were to develop an in vitro culture system to optimize germ cell proliferation and to measure the potential of the cultured germ cells to produce mature spermatozoa after transplantation into a recipient. Donor germ cells isolated from ROSA26 male mice were cultured with a STO feeder cell layer in Dulbecco's minimal essential medium (DMEM) supplemented with fetal bovine serum (FBS), stem cell factor, leukemia inhibitory factor, basic fibroblast growth factor, insulin-like growth factor 1, interleukin-11, L-glutamine, sodium pyruvate, 2-mercaptoethanol, murine oncostatin M, and platelet-derived growth factor. Donor germ cells formed colonies in the primary cultures after 8-21 days. These cultured colonies were maintained for 4 weeks or longer without subculture and proliferated for up to 8 passages over a period of 3 months. These colonies had alkaline phosphatase activity and incorporated 5-bromo-2'-deoxyuridine. These colonies were positive partially when screened with antibody for germ cell nuclear antigen and c-kit. Germ cells cultured with this supplemented medium showed enhanced colonization vs controls cultured with DMEM and FBS. Cultured germ cells from Rosa26 donors were transplanted into testes and were identified by X-gal staining and histological screening. The cells cultured in the supplemented medium colonized the tubules and initiated spermatogenesis in the recipient mice. This is an improved method for culturing germ cells and may be useful in gene therapy and the production of transgenic animals.     

Simian virus 40 and human pleural mesothelioma

BACKGROUND: An aetiological role for Simian virus 40 (SV40) in malignant mesothelioma has been suggested from studies in the USA and the UK but results have been conflicting. A study was undertaken to look for evidence of SV40 in stored tissue samples from pleural mesotheliomas. METHODS: DNA was extracted from paraffin embedded tissue. The presence of DNA was established by amplification of a 250 bp product from the betaglobin gene. Primers PYV.F and PYV.R were used in a concentration of 50 per mol each per reaction to amplify a 172 bp fragment of a conserved region of SV40 that codes for a portion of large T antigen that is common to SV40 and other polyoma viruses. RESULTS: Twelve of the 17 samples contained amplifiable betaglobin DNA. None of the samples (0/12, 95% CI 0 to 26.5%) was positive for the polyoma large T antigen. CONCLUSIONS: These results do not lend any support to the hypothesis that SV40 infection may be aetiologically relevant to the increasing incidence of mesothelioma in the UK.     

A new method to immortalize primary cultured rat hepatocytes

BACKGROUND: In conventional methods of establishing hepatocyte cell lines, the immortalizing gene alone is introduced into hepatocytes. We designed a new method in which not only the immortalizing gene, the simian virus-40 large T-antigen (SV-40 Tag) gene, but also a drug-resistant gene, under the control of an albumin enhancer/promoter, were introduced into hepatocytes to efficiently obtain immortalized hepatocyte cell lines. METHODS: The plasmid pAPUR contains the puromycin-resistant gene under the control of an albumin enhancer/promoter, and the pSVTag contains the early region of SV-40 enhancer/promoter and the SV-40 Tag gene. Both pAPUR and pSVTag were transferred into isolated rat hepatocytes by electroporation. After these cells were cultured on a collagen-coated dish for 24 hours, puromycin selection was started. Expression levels of albumin, alpha-fetoprotein (AFP), SV-40 Tag, and cytokeratin 19 (CK 19) in the transformed cells were evaluated by western analysis, immunocytochemical staining, and RT PCR. RESULTS: Approximately 3 weeks after transfection, five or six colonies appeared on the dish. Twenty strains were obtained by cloning these cells. All strains that were similar to immature hepatocytes expressed albumin and SV-40 Tag, although CK 19 was not detected. AFP expression was detected in 33% of these strains. CONCLUSIONS: All clones cotransfected by pAPUR and pSVTag expressed albumin. Our new method may be useful to establish hepatocyte cell lines.     

大鼠蛛网膜下腔移植超顺磁性氧化铁纳米离子标记永生化神经前体细胞后的磁共振成像追踪

目的观察大鼠蛛网膜下腔移植超顺磁性氧化铁纳米粒子（SPIO）标记永生化神经前体细胞后的磁共振成像追踪。方法用SPIO-多聚赖氨酸复合物（SPIO-PLL）标记永生化神经前体细胞。采用普鲁士蓝染色鉴定SPIO-PLL标记永生化神经前体细胞的效率，采用MTT法检测标记前后细胞活力，用免疫细胞化学法对标记后1周的细胞进行抗巢蛋白、微管相关蛋白和胶质纤维酸性蛋白（GFAP）染色，检测标记细胞的分化能力。蛛网膜下腔置管成功的SD大鼠10只，随机分为2组（n=5），标记细胞组和未标记细胞组，蛛网膜下腔分别移植标记后2d的永生化神经前体细胞和未标记细胞，移植后30min及移植后1周用MRI对蛛网膜下腔的细胞进行活体追踪，用组织切片进行普鲁士蓝染色和抗猿肾病毒40大T抗原染色。结果SPIO可以高效率地标记永生化神经前体细胞，普鲁士蓝染色显示SPIO—PLL标记永生化神经前体细胞质内出现细小的天蓝色铁颗粒，SPIO-PLL标记对永生化神经前体细胞的活力没有明显的影响，标记后1周，抗巢蛋白、微管相关蛋白染色阳性，GFAP染色阴性。标记细胞组移植后30min及移植后1周MRI活体检查发现标记细胞在磁共振成像上呈明显的低信号改变，脊髓组织学切片结果普鲁士蓝、抗猿肾病毒40大T抗原染色阳性；未标记细胞组磁共振成像上无明显低信号改变。结论利用MRI技术可以对蛛网膜下腔移植后的标记细胞进行活体追踪。