小鼠胚胎干细胞分化为血管内皮细胞的永生化研究

本文探讨了小鼠胚胎干细胞（ES细胞）、诱导分化的血管内皮细胞永生化。在体外培养系统中,以维甲酸（RA）和转化生长因子－β1（TGF－β1）诱导小鼠胚胎干细胞（ES细胞）的拟胚体（EB）分化为“圆形细胞”和由这些“圆形细胞”组成的血管样结构。经光学和扫描电镜及免疫荧光等法分析检测,证明组成血管样结构的细胞具有专一性vWF荧光染色,表明是血管内皮样细胞。利用脂质体将人端粒酶催化亚基逆转录酶（hTERT)基因转染诱导分化中的“圆形细胞”。应用Dot-blot,RT－PCR,Western blot及免疫组织化学等方法分析、观察和证明了诱导分化的组成血管样结构的园形细胞和被hTERT基因转染的“圆形”细胞的形态和生物学特性。结果表明,携带hTERT基因的从ES细胞分化来的圆形细胞在体外可大量增殖,持续传代,95％具有血管内皮细胞的一些特有标志和管道化生长特征。因此,通过人端粒酶基因的转染途径可解决由ES细胞诱导分化而来的内皮细胞扩增和永生化问题,为构建组织工程化血管及其它人工血管的内皮化提供种子细胞来源打下基础。     

永生化后细胞的生物学特性

近年来,永生化细胞技术快速发展,对细胞生物学特性的研究及其临床应用具有深远的意义。目前通过导入永生化基因SV40抗原和hTERT基因成为细胞永生化的常用方法,但存在安全性隐患。利用可逆性永生化技术,使细胞在获得永生化能力的同时去除永生化基因,使细胞恢复到原始状态。文章综述了永生化后细胞生物学特性的变化,并讨论了临床应用可能面临的问题。     

小鼠胚胎干细胞分化为血管内皮细胞的永生化研究

本文探讨了小鼠胚胎干细胞(ES细胞)诱导分化的血管内皮细胞永生化。在体外培养系统中,以维甲酸(RA)和转化生长因子-β1(TGF-β1)诱导小鼠胚胎干细胞(ES细胞)的拟胚体(EB)分化为“圆形细胞”和由这些“圆形细胞”组成的血管样结构。经光学和扫描电镜及免疫荧光等法分析检测,证明组成血管样结构的细胞具有专一性vWF荧光染色,表明是血管内皮样细胞。利用脂质体将人端粒酶催化亚基逆转录酶(hTERT)基因转染诱导分化中的“圆形细胞”。应用Dot-blot,RT-PCR,Western blot及免疫组织化学等方法分析、观察和证明了诱导分化的组成血管样结构的园形细胞和被hTERT基因转染的“圆形”细胞的形态和生物学特性。结果表明,携带hTERT基因的从ES细胞分化来的圆形细胞在体外可大量增殖,持续传代,95%具有血管内皮细胞的一些特有标志和管道化生长特性。因此,通过人端粒酶基因的转染途径可解决由ES细胞诱导分化而来的内皮细胞扩增和永生化问题,为构建组织工程化血管及其它人工血管的内皮化提供种子细胞来源打下基础。     

细胞永生化技术及其应用研究进展

细胞永生化是目前细胞生物学研究的热点之一.引起细胞永生化的因素很多,其中端粒酶在这一过程中发挥重要作用.细胞永生化技术在未来的成功应用,不仅为生物制品的研究与开发提供了有力工具,而且也为疾病的诊断与治疗提出了新的思路.就细胞永生化技术的原理、方法、途径及其应用等方面研究进展作一综述.     

肝细胞永生化的研究与应用

正常体外培养的细胞在体外有一定的寿命,一般传代不超过50代,被称为有限细胞系,但有些体外培养的细胞可经过自发或外界因素的影响从凋亡危机中逃离出来,从而形成具有无限增殖能力的细胞系,成为永生化细胞系.由于肝细胞在体外增殖能力极弱,培养困难,其应用受到较大限制,而肝细胞经永生化后具有了无限增殖优势,使其在肝细胞移植,生物人工肝以及药理、毒理学等研究方面有广阔的应用前景.就近年来肝细胞永生化方法及其应用作简要的综述.     

端粒酶在细胞永生化和癌变中的作用

重点讨论了端粒酶在肿瘤细胞和永生化细胞中的作用和功能,以及它与细胞衰老和永生化的关系.多数真核细胞的端粒酶能将单一重复序列加到端粒DNA的3′末端.端粒酶主要由模板RNA和端粒酶蛋白催化亚基组成,后者以前者为模板起逆转录酶的作用.端粒酶活性存在于肿瘤细胞中,而在良性肿瘤、体细胞中未发现端粒酶.     

肝细胞永生化策略研究进展

正常体细胞在细胞分裂过程中由于无法维持端粒的长度和染色体的稳定而衰老、死亡。建立基因稳定、无致瘤性的永生化细胞是永生化研究的目的所在。利用Cre-loxP系统构建的可恢复性永生化细胞可用于以细胞为基础的临床治疗。分化良好并具有肝脏正常合成代谢功能的永生化肝细胞在生物医学研究,尤其是在肝细胞移植、生物人工肝支持治疗以及药理学和毒理学研究方面具有广泛的应用前景。     

人卵巢浆液性囊腺癌永生化细胞系BUPH∶OVCA-3的建立及其生物学特性

介绍人卵巢浆液性囊腺癌永生化细胞系的建立 ,研究其生物学特性 .以卵巢浆液性乳头状囊腺癌的腹水细胞为材料 ,进行体外培养 .将永生化基因———SV4 0T抗原基因转染第 2代细胞 ,得到永生化细胞系 .通过光学显微镜、生长曲线测定、染色体分析、双层软琼脂培养、裸鼠接种、免疫组化等 ,研究其生物学特性 ,并与其来源细胞的生物学特性进行比较 .建立了一株人卵巢浆液性囊腺癌永生化细胞系 ,命名为BUPH∶OVCA 3,现已传至 6 0余代 .其生物学特性为 ,细胞生长旺盛 ;具有人体恶性细胞的核型特征 ;细胞恶性度较低 ,不具有集落形成能力及裸鼠接种致瘤性 ;除较未永生化细胞生长速率增快 ,饱和密度增加外 ,仍保留上皮细胞的分化表型 .结果表明 ,BUPH∶OVCA 3为一株恶性度较低的人卵巢浆液性囊腺癌永生化细胞系 ,保留其来源细胞的生物学特性 ,可作为研究恶性度较低的卵巢上皮癌的体外模型     

人神经干细胞的体外生物学特性

本实验利用有丝分裂因子,体外诱导生成人神 经干细胞(NSCs),观察其生长特性并进行鉴定。取胎龄10-22周的大脑半球,分散细胞后种于添加表皮生长因子(EGF,20ng/ml)和/或碱性成纤维生长因子(bFGF,20ng/ml)的培养基中。利用免疫组织化学方法鉴定分化后的细胞类型。同时,进行细胞克隆分析、传代培养及端粒酶活性检测。结果显示:NSCs呈悬浮生长的干细胞球,其特异性抗原nestin阳性。NSCs具有增殖能力,可连续传代而不丢失其增殖和多分化潜能的干细胞特性。撤除EGF和bFGF的作用,细胞停止分裂,并分化为神经元、星形胶质细胞和少突胶质细胞。克隆分析显示NSCs生长呈密度依赖性。人NSCs表达较低的端粒酶水平,并随培养时间延长而下调。研究表明,利用有丝分裂因子,可在体外成功诱导生成人NSCs,其生长,分化受内外源因素的调节,相关的机制还有待阐明。     

Subsenescent telomere lengths in fibroblasts immortalized by limiting amounts of telomerase

Human fibroblasts expressing the catalytic component of human telomerase (hTERT) have been followed for 250-400 population doublings. As expected, telomerase activity declined in long term culture of stable transfectants. Surprisingly, however, clones with average telomere lengths several kilobases shorter than those of senescent parental cells continued to proliferate. Although the longest telomeres shortened, the size of the shortest telomeres was maintained. Cells with subsenescent telomere lengths proliferated for an additional 20 doublings after inhibiting telomerase activity with a dominant-negative hTERT mutant. These results indicate that, under conditions of limiting telomerase activity, cis-acting signals may recruit telomerase to act on the shortest telomeres, argue against the hypothesis that the mortality stage 1 mechanism of cellular senescence is regulated by telomere positional effects (in which subtelomeric loci silenced by long telomeres are expressed when telomeres become short), and suggest that catalytically active telomerase is not required to provide a protein-capping role at the end of very short telomeres.     

Identification and characterization of various differentiative growth plate chondrocytes from porcine by countercurrent centrifugal elutriation

Countercurrent centrifugal elutriation was used to separate growth plate chondrocytes from porcine basing on their differences in sizes and densities. Eighteen fractions of cells with different sizes and densities were obtained. The mean cellular volumes increased progressively in each of successive fractions, and that increase was associated with specific phenotypic changes, such as biochemical differences in DNA synthesis, proteoglycan synthesis, and activities of alkaline phosphatase. Three distinct chondrocyte subpopulations with their unique characteristics were identified among the elutriated fractions. The resting chondrocytes were found to be small in size and quiescent. The hypertrophic chondrocytes were found to be large in size and metabolically active both in alkaline phosphatase and in proteoglycan productions. The proliferative chondrocytes exhibited a high DNA synthesis rate, and their sizes were found to be between those of the resting and hypertrophic chondrocytes. © 1996 Wiley-Liss, Inc.     

Metalloproteinase expression by control and telomerase immortalized human endometrial endothelial cells

Vascular endothelial cells play a critical role in the maintenance of endometrial homeostasis. Indeed many pathological conditions causing abnormal endometrial bleeding including progestin only contraception, hormone replacement therapy, endometrial polyps, myomas, hyperplasia and cancer are associated with aberrant angiogenesis. Critical to the process of angiogenesis is the breakdown of the surrounding tissues by matrix metalloproteases (MMPs). In addition to the cells surrounding the endometrial endothelial cells, the endothelial cells themselves produce their own panel of MMPs. We now characterize the specific MMPs that are expressed by endothelial cells derived from human endometrium. These include MMP-1, MMP-2 and MMP-10 but not MMP-3. In addition, in order to successfully carry out consistent, homogeneous and sufficient numbers of studies we investigated the in vitro expression of the MMPs with both freshly isolated, early passaged endometrial endothelial cells (HEECs) as well as with newly telomerase immortalized HEECs (T-HEECs). The latter were karyotypically normal and expressed classic endothelial cell endpoints such as tubulogenesis on matrigel and expression of the endothelial cell markers CD-31 (PECAM), von Willebrand's factor, and the Tie-2 receptors. The levels of MMP expression as well as that of the metalloprotease inhibitors TIMP-1 and TIMP-2 were similar in parent and immortalized endothelial cells.     

Functional characterization of human T cells immortalized by oncogene transfection

We have succeeded in immortalizing human lymphocytes derived from the peripheral blood of a healthy donor and of an atopic patient, and from the lymph node of a cancer patient by oncogene transfection (Alam et al., 1996). All immortalized human lymphocytes were shown to be CD3+ and CD19–, indicating that these immortalized human lymphocytes were all T cells. We established 317, 154 and 692 individual immortalized human T cell lines derived from the healthy donor, the atopic patient and the cancer patient, respectively. The ratios of CD4+ and CD8+ subpopulations within the set containing immortalized T cells derived from the healthy donor were shown to be varied depending on the combinations of transfected oncogenes used. However, CD8+ cells were found to be the dominant subpopulation of immortalized T cells derived from the atopic patient and the cancer patient. These immortalized T cells showed different proliferative responses in the presence of exogenous human IL–2 depending on their origin, and was consistent with the surface expression of the IL–2 receptor. Furthermore, the cytokine secretion patterns of these immortalized T cells stimulated with mitogen were investigated. The results showed that the immortalized T cells from the healthy donor is able to secrete various kinds of cytokines such as IL–2, IL–10, -IFN and GM-CSF. However, immortalized T cells from the cancer patient was shown to only secrete IL–2 and GM-CSF. These results suggest that depending on the origin, the immortalized T cells came from different subsets or from cells in different activated states. Mixed lymphocytes reactions demonstrated that these immortalized T cells are able to proliferate in the presence of allogenic or xenogenic stimulator cells, suggesting that they maintain the ability to recognize specific antigens on the stimulator cells and can proliferate even after the immortalization. Furthermore, immortalized T cells derived from the healthy donor and the cancer patient strongly responded to K562 cells, suggesting that MHC-nonrestricted killer T cells were also immortalized.Abbreviations IL–2R – interleukin 2 receptor; MLR – mixed lymphocyte reaction     

Odontoblast cells immortalized by telomerase produce mineralized dentin-like tissue both in vitro and in vivo

The formation of dentin provides one well accepted paradigm for studying mineralized tissue formation. For the assembly of dentin, several cellular signaling pathways cooperate to provide neural crest-derived mesenchymal cells with positional information. Further, "cross-talk" between signaling pathways from the mesenchymal derived odontoblast cells and the epithelially derived ameloblasts during development is responsible for the formation of functional odontoblasts. These intercellular signals are tightly regulated, both temporally and spatially. When isolated from the developing tooth germ, odontoblasts quickly lose their potential to maintain the odontoblast-specific phenotype. Therefore, generation of an odontoblast cell line would be a valuable reproducible tool for studying the modulatory effects involved in odontoblast differentiation as well as the molecular events involved in mineralized dentin formation. In this study an immortalized odontoblast cell line, which has the required biochemical machinery to produce mineralized tissue in vitro, has been generated. These cells were implanted into animal models to determine their in vivo effects on dentin formation. After implantation, we observed a multistep, programmed cascade of gene expression in the exogenous odontoblasts as the dentin formed de novo. Some of the genes expressed include the dentin matrix proteins 1, 2, and 3, which are extracellular matrix molecules responsible for the ultimate formation of mineralized dentin. The biological response was also examined by histology and radiography and confirmed for mineral deposition by von Kossa staining. Thus, a transformed odontoblast cell line was created with high proliferative capacity that might ultimately be used for the regeneration and repair of dentin in vivo.