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星形胶质细胞株的构建

目的 构建永生化大鼠星形胶质细胞，为转基因细胞移植镇痛提供细胞载体。方法 采用差速粘附法体外分离和培养大鼠大脑皮层星形胶质细胞。利用脂质体将含有猿肾病毒40大T抗原(SV40Tag)基因的质粒pCMVSV40T／PUR转染培养的大鼠星形胶质细胞。经嘌呤霉素1．5μg／ml筛选后，挑选阳性细胞克隆扩大培养并连续传代。PCR、RT-PCR及免疫组化检测阳性细胞克隆中SV40Tag基因的整合情况及其表达，并对传代细胞的胶质原纤维酸性蛋白(GFAP)进行检测。结果 成功获得体外培养的大鼠星形胶质细胞，GFAP阳性表达；筛选获得阳性细胞克隆，连续传代培养近50代；PCR、RT-PCR产物经1．5％琼脂糖凝胶电泳分析显示558bp处有一特异性扩增条带，与阳性对照条带相同，而未转染pCMVSV40T／PUR的细胞无扩增条带，回收片段经测序、比对与SV40Tag的基因序列一致(100％)；同时转染的阳性细胞克隆SV40Tag和GFAP免疫染色阳性。结论 成功地构建了SV40Tag基因永生化的大鼠星形胶质细胞株。     

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.     

Insertion of a suicide gene into an immortalized human hepatocyte cell line

For developing a bioartificial liver (BAL) device, an attractive alternative to the primary human hepatocytes would be the use of highly differentiated immortalized human hepatocytes with a safeguard. To test the feasibility, the primary human hepatocytes were immortalized by a plasmid SV3neo encoding simian virus 40 large T antigen (SV40Tag) gene. A highly differentiated hepatocyte line OUMS-29 was established. A suicide gene of herpes simplex virus-thymidine kinase (HSV-TK) was retrovirally introduced into OUMS-29 cells as a safeguard for clinical application. One of the resulting HSV-TK-positive cell lines, OUMS-29/tk, grew in chemically defined serum-free medium with the gene expression of differentiated liver functions. OUMS-29/tk cells were 100 times more sensitive to ganciclovir compared with unmodified OUMS-29 cells in in vitro experiments. We have established a tightly regulated immortalized human hepatocyte cell line. Essentially unlimited availability of OUMS-29/tk cells may be clinically useful for BAL therapy.     

Establishment of cementoblast cell lines from rat cementum lining cells by transfection with temperature-sensitive simian virus-40 T-antigen gene

Defining the regulatory mechanisms promoting differentiation and proliferation of cementoblasts has not been well understood, because of the lack of cell models in vitro. To establish an in vitro cell model for the cementoblasts, extracted rat molars obtained from 8-week-old rats were used. Cells lining the root surface (cemetoblasts) were obtained by an enzymatic digestion method, and immediately immortalized by transfection of thermolabile SV40 T-antigen gene. The transfected cementum lining cell clones, RCM-C3 and -C4, were maintained for more than 200 population doublings (PD), while the original cells stopped their growth at 60 PD. Thus, immortalized cell lines decreased expression of SV40 T-antigen and subsequently cell proliferation at non-permissive temperature (39 degrees C). Reverse-transcribed-polymerase chain reaction indicated expression of gene for type I collagen, alkaline phosphatase (ALP), osteopontin, and osteocalcin mRNA at both permissive (33 degrees C) and non-permissive (39 degrees C) temperatures. RCM-C4 expressed higher bone siaploprotein (BSP) mRNA than RCM-C3, and further RCM-C4 showed higher BSP mRNA at 39 degrees C than 33 degrees C. High ALP activity and mineralized nodule formation were observed at 39 degrees C in both cell lines. These findings suggested that the cell lines, RCM-C3 and -C4, are useful model for studying the regulatory mechanisms of differentiation and proliferation of cementoblasts.     

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。     

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.     

pcDNA3-hBMP2转染对成纤维细胞 生物学性状的影响

目的 探讨人BMP2基因转染对成纤维细胞NIH3T3生物学性状的影响。方法 构建重组真核表达载体pcDNA3-hBMP2,并在脂质体介导下,将其导入NIH3T3成纤维细胞,通过G418筛选获得阳性克隆,用细胞原位杂交和免疫组织化学方法检测hBMP2基因在NIH3T3成纤维细胞内的表达情况;MTT法和FCM检测pcDNA3-hBMP2转染pcDNA3-hBMP2后成纤维细胞超微结构的改变,碱性磷酸酶的检测观察转染pcDNA3-hBMP2后成纤维细胞向成骨细胞分化情况。结果 转染pcvDNA3-hBMP2后的NIH3T3细胞内有大量hBMP2mRNA的转录及其蛋白的表达;转染pcDNA3-hBMP2对成纤维细胞增殖和细胞周期无影响;转染pcDNA3-hBMP2后的成纤维细胞超微结构可见粗面内质网丰富,囊腔扩张明显其内充满中等电子密度的蛋白分泌物;碱性磷酸酶活性显著上升。结论 pcDNA3-hBMP2转染对成纤维细胞NIH3T3增殖和细胞周期无影响。转染后的成纤维细胞不仅可形成BMP2,而且具有向成骨细胞系分化的特性。     

强力霉素调控表达脑啡肽的永生化大鼠星形胶质细胞株的构建

目的构建受强力霉素（Dox）调控表达脑啡肽的永生化大鼠星形胶质细胞株（IAST）。方法采用脂质体介导法将重组质粒pRevTREhPPE和调节质粒pRevTet-On分别转染逆转录病毒包装细胞PT67；将含有RevTet—On和RevTRE／hPPE病毒上清感染IAST，得到稳定表达脑啡肽的IAST／Tet-On／hPPE细胞株，实时定量PCR检测Dox定量调控该细胞株前脑啡肽原（hPPE）基因的表达，免疫细胞化学及放射免疫分析法检测Dox定量调控该细胞株中脑啡肽的表达。结果IAST／Tet-On／hPPE细胞株中，hPPE基因的表达和脑啡肽的分泌受Dox调控，Dox浓度为100～5000nedml时，随着Dox浓度增高，hPPE基因的表达和脑啡肽的分泌增加，Dox浓度为5000ng／ml时达峰值。结论成功构建了受四环素及其衍生物强力霉素定量调控表达脑啡肽的IAST。     

转导人端粒酶逆转录酶基因致人骨髓间充质干细胞永生化并向软骨细胞诱导分化的研究

目的 建立永生化人骨髓间充质干细胞系并向软骨细胞诱导分化,以供软骨组织工程基础研究及临床应用.方法 原代培养人骨髓间充质干细胞(hMSC),用含有人端粒酶逆转录酶(hTERT)基因的逆转录病毒转染hMSC,G418筛选得到阳性克隆,体外连续培养,检测端粒酶的表达及活性.TGF-β1和地塞米松对转化后的hMSC-hTERT细胞诱导,使其向软骨细胞分化,并用原位杂交和免疫组化检测II型胶原.结果 外源性hTERT在转染细胞中稳定表达并传至第50代,永生化的hMSC细胞经TGF-β1和地塞米松诱导在体外分化为软骨细胞.结论 外源性hTERT基因可以有效地在体外使hMSC永生化,永生化的hMSC细胞经诱导可在体外分化为软骨细胞,从而作为软骨组织工程研究的细胞来源.     

增强型绿色荧光蛋白转染活细胞效率的研究

目的 研究影响增强型绿色荧光蛋白(EGFP)逆转录病毒表达系统转染活细胞效率的主要相关参数,建立一种稳定高效的活细胞EGFP标记技术.方法 培养、扩增携带EGFP逆转录病毒载体的包装细胞PGl3,分别于12h、24h、36 h和48 h收集病毒液,转染骨髓基质细胞(BMSCs),通过流式细胞仪和荧光显微镜检测不同时间点病毒转染效率.选择转染率最高时间点的病毒液,连续转染BMSCs两次,小剂量嘌呤霉素筛选6h,观察病毒转染率及细胞活力.再以上述方法转染脂肪基质细胞(ADSCs)和成纤维细胞(FBs),观察这种方法标记其他干细胞或成熟分化细胞的可行性.结果 根据流式细胞仪及荧光显微镜观察结果,24h收集的病毒液转染BMSCs效率最高(64.56±2.53)％,36 h次之(56.98±3.22)％,12 h(47.39+1.82)％与48 h(37.84±1.77)％相对较低.连续转染两次并经短暂筛选后,BMSCs转染率可达80％以上,且细胞活力不受影响.采用相同方法转染脂肪基质细胞(ADSCs)和成纤维细胞(FBs),转染率均能达到80％,且细胞形态、增殖能力均未见明显改变.结论 本实验建立了一种稳定高效的活细胞EGFP转染技术,为研究组织工程种子细胞的体内、外转归及细胞间相互作用提供了稳定且直观的检测手段.     

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。     

Retinoic acid modulation of mRNA levels in malignant, nontransformed, and immortalized osteoblasts.

Clonal cell lines presumably "arrested" at a particular stage of differentiation are useful models to study the processes of differentiation in osteoblasts. UMR-201 is a presumptive preosteoblastic nontransformed rat clonal cell line with a limited life span in culture. Two immortalized cell lines, UMR-201-10A (10A) and UMR-201-10B (10B), were derived from UMR-201 by stable transfection with simian virus (SV) 40 large T antigen. This study compares the growth and profile of gene expression of the immortalized cell lines with those of UMR-201 and UMR-106-06, a rat clonal cell line with well-defined osteoblast-like phenotypic characteristics. All four cell lines constitutively expressed the mRNA for the gamma, alpha, and beta receptors for retinoic acid (RA), the growth hormone receptor, pro-alpha 1(I) collagen, osteonectin, bone proteoglycan I, and bone morphogenetic proteins (BMP) 1 and 2A. Alkaline phosphatase mRNA was absent in the preosteoblast cell lines but was induced by treatment with 10(-6) M RA, which also increased the steady-state levels of mRNA for osteopontin and BMP1. mRNA for matrix gla protein was constitutively present and further induced by RA in UMR-201 and 10B only. Messenger RNA for bone sialoprotein and bone morphogenetic protein 3 were constitutively expressed in UMR-106-06 and UMR-201 but absent in the immortalized cell lines. None of the cell lines expressed measurable mRNA for bone gla protein or bone proteoglycan II. 10B grew more rapidly than UMR-201, but unlike UMR-201, it was also able to proliferate in serum-free medium and exhibit anchorage-independent growth. In summary, this study identifies novel retinoic acid effects on gene expression in these cells. Differences noted in the expression of mRNAs between UMR-106-06 and the other cell lines may provide some insight into the sequence of expression of these phenotypic characteristics as osteoblasts differentiate.     

Repopulation of human origin hepatocyte progenitor-like cell line, THLE-5b, in the SCID mouse liver under p21-mediated cell growth-arresting conditions

The in vivo repopulation of hepatocytes depends on donor cell growth potential and recipient conditioning. We herein demonstrate the successful cell transplantation of a human hepatocyte cell line, THLE-5b, into the SCID mouse liver by means of a rather mild conditioning using a 55% hepatectomy and p21 transfection. Adult human liver-derived cells, THLE-5b, are SV40 T antigen-immortalized epithelial cells. A phenotypic examination of THLE-5b showed they expressed hepatic stem cell markers such as EpCAM, OCT3/4, and Thy-1, thus indicating the immature nature of the cells. A three-dimensional aggregate culture of THLE-5b showed a higher expression level of liver-specific genes such as albumin, α1-antitrypsin, and CYP3A4, thus suggesting that THLE-5b possess the capability to differentiate into hepatocytes. In a cell transplantation experiment, the cell cycle regulator p21 was transfected with adenoviral vector into the SCID mouse liver. On the next day, 8 × 10(5) cells of GFP-transfected THLE-5b were injected intrasplenically, together with the intraperitoneal administration of anti-asialo GM1 antibodies. The following day, a partial hepatectomy was performed. The GFP-THLE-5b cells were observed to have migrated and become integrated into the liver parenchyma 14 days after transplantation. The present protocol is thus considered to be a novel experimental model to elucidate the mechanism of hepatocyte repopulation and to develop efficient stem cell therapy in the liver.     

PEC-A: An immortalized porcine aortic endothelial cell

Abstract: Cultured porcine aortic endothelium was transfected with sequences that encode the SV40 T antigen, resulting in an immortalized cell line that retains the differentiated properties of normal aortic endothelial cells. Specifically, these cells form cobblestone monolayers, synthesize von Willebrand factor, and endocytose acetylated LDL. These cells can be readily propagated in culture and have been passaged over a year in culture. The cells form tubular structures when grown on Matrigel. The cells in culture express class I but do not express MHC class II antigens. Both class I and class II expression can be induced by treatment with recombinant swine interferon-γ. Expression of CD44 and several other cell surface antigens have been observed. Co-culture of these cells with purified human CD4+ T cells resulted in a significant T-cell proliferative response similar to that observed for primary porcine EC. Finally, the cells are readily susceptible to transfection and express exogenous genes. These cells should be valuable for study of human anti-porcine endothelial responses.     

Making stem cell lines suitable for transplantation

Human stem cells, progenitor cells, and cell lines have been derived from embryonic, fetal, and adult sources in the search for graft tissue suitable for the treatment of CNS disorders. An increasing number of experimental studies have shown that grafts from several sources survive, differentiate into distinct cell types, and exert positive functional effects in experimental animal models, but little attention has been given to developing cells under conditions of good manufacturing practice (GMP) that can be scaled up for mass treatment. The capacity for continued division of stem cells in culture offers the opportunity to expand their production to meet the widespread clinical demands posed by neurodegenerative diseases. However, maintaining stem cell division in culture long term, while ensuring differentiation after transplantation, requires genetic and/ or oncogenetic manipulations, which may affect the genetic stability and in vivo survival of cells. This review outlines the stages, selection criteria, problems, and ultimately the successes arising in the development of conditionally immortal clinical grade stem cell lines, which divide in vitro, differentiate in vivo, and exert positive functional effects. These processes are specifically exemplified by the murine MHP36 cell line, conditionally immortalized by a temperature-sensitive mutant of the SV40 large T antigen, and cell lines transfected with the c-myc protein fused with a mutated estrogen receptor (c-mycERTAM), regulated by a tamoxifen metabolite, but the issues raised are common to all routes for the development of effective clinical grade cells.