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。     

Genetically engineered human mesenchymal stem cells produce met-enkephalin at augmented higher levels in vitro

We have reported that transplantation of adrenal medullary chromaffin cells that release endogenous opioid peptides into pain modulatory regions in the CNS produce significant antinociceptive effects in patients with terminal cancer pain. However, the usefulness of this procedure is minimal because the availability of human adrenal tissue is very limited. Alternative xenogeneic materials, such as porcine and bovine adrenal chromaffin cells present problems of immune rejection and possible pathogenic contamination. In an attempt to develop opioid peptide-producing cells of autologous origin, we have transfected human mesenchymal stem cells (hMeSCs) with a mammalian expression vector containing a fusion gene of green fluorescent protein (GFP) and human preproenkephalin (hPPE), a precursor protein for enkephalin opioid peptides. Enkephalins are major neurotransmitters that play an important role in analgesia by activating peripheral opioid receptors. Following the establishment of stable transfection of hMeSCs, the expressions of hPPE and GFP were confirmed and the production of methionine enkephalin (Met-enkephalin) was significantly increased compared to control naive hMeSCs (p < 0.05). Our in vitro data demonstrated that genetically engineered hMeSCs with transfected hPPE gene can constitutively produce opioid peptide Met-enkephalin at an augmented high level. hMeSCs are relatively easy to isolate from a patient's bone marrow aspirates and expand in culture by repeated passages. Autologous hMeSCs would not require immunosuppression when transplanted back into the same patient. Through targeted gene manipulation such as hPPE gene transfection, this may offer a virtually unlimited safe cell supply for the treatment of opioid-sensitive pain in humans.     

EphA1基因可控性双稳转内皮祖细胞系EPCsTet-On-EphA1SiRNA的建立

目的建立可调控EphA1基因表达的内皮祖细胞系EPCsTet-On-EphA1SiRNA。方法将pWHE146质粒转染到内皮祖细胞系中,筛选出稳定表达的细胞克隆;扩增后瞬时转染pTRE-hyg-luc质粒,强力霉素诱导表达后,检测荧光素酶活性,挑选出高表达、低背景的受强力霉素调控的EPCsTet-On细胞株;再将重组质粒pTRE-EphA1SiRNA转染入EPCsTet-On细胞株,筛选出稳定表达细胞克隆EPCsTet-On-EphA1SiRNA;通过强力霉素诱导后,利用RT-PCR和Western blotting法检测EphA1基因mRNA与蛋白的表达。结果成功构建了受强力霉素调控的高表达低背景的EPCsTet-On-EphA1SiRNA细胞株;强力霉素可诱导EPCsTet-On-EphA1SiRNA细胞株中EphA1mRNA表达下调,较之未调控组其差异有统计学意义(P<0.05);强力霉素调控EphA1蛋白表达的能力在一定范围内呈剂量依赖性关系。结论成功建立强力霉素调控EphA1基因表达的大鼠双稳转内皮祖细胞系EPCsTet-On-EphA1SiRNA,为深入研究EphA1基因在内皮祖细胞参与肝癌血管生成过程中的作用提供了有效的实验手段。     

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.     

人前脑啡肽原基因修饰人骨髓间充质干细胞系的构建

目的 构建人前脑啡肽原(PENK)基因修饰的并可稳定分泌脑啡肽蛋白的人骨髓间充质干细胞系(hMSCs).方法 采用脂质体法将PENK基因逆转录病毒载体质粒(pBABE-PENK)转染至Phoenix-293T细胞,收集病毒上清液感染hMSCs细胞,经过嘌呤霉素筛选得到稳定表达PENK基因的hMSCs细胞株(hMSC-PENK细胞).以转染空载体细胞作为对照,即hMSC-pBABE细胞.采用RT-PCR法检测PENK mRNA的表达,免疫荧光法测定亮氨酸脑啡肽(LEK)的表达,ELISA法测定细胞培养上清液LEK浓度.结果 与hMSCs细胞和hMSC-pBABE细胞比较,hMSC-PENK细胞PENK mRNA和LEK表达上调,细胞培养上清液中LEK浓度升高(P＜0.05或0.01).结论 PENK基因修饰的hMSCs可表达PENK基因并分泌脑啡肽蛋白,成功构建了稳定分泌镇痛物质的细胞系.     

Expression of tyrosine hydroxylase in an immortalized human fetal astrocyte cell line: In vitro characterization and engraftment into the rodent striatum

The use of primary human fetal tissue in the treatment of neurodegenerative disorders, while promising, faces several difficult technical and ethical issues. An alternative approach that would obviate these problems would be to use immortalized cell lines of human fetal central nervous system origin. An immortalized human fetal astrocyte cell line (SVG) has been established (45) and herein we describe the in vitro and in vivo characteristics of this cell line which suggest that it may be a useful vehicle for neural transplantation. The SVG cell line is vimentin, GFAP, Thy 1.1 and MHC class I positive, and negative for neurofilament and neuron specific enolase, consistent with its glial origin. To determine whether the cell line could be used as a drug delivery system, a cDNA expression vector for tyrosine hydroxylase was constructed (phTH/Neo) and stably expressed in the SVG cells for over 18 months as demonstrated by immunohistochemistry and Western blotting of the stable transfectants. HPLC analysis of the supernatant from these cells, termed SVG-TH, consistently found 4–6 pmol/ml/min of I-dopa produced with the addition of BH₄ to the media. Furthermore, in cocultivation experiments with hNT neurons, PC-12 cells and primary rat fetal mesencephalic tissue, both the SVG and SVG-TH cells demonstrated neurotrophic potential, suggesting that they constituitively express factors with neuroregenerative potential. To determine the viability of these cells in vivo, SVG-TH cells were grafted into the striatum of Sprague-Dawley rats and followed over time. A panel of antibodies was used to unequivocally differentiate the engrafted cells from the host parenchyma, including antibodies to: SV40 large T antigen (expressed in the SVG-TH cells), human and rat MHC class 1, vimentin, GFAP, and tyrosine hydroxylase. While the graft was easily identified with the first week, over the course of a four week period of time the engrafted cells decreased in number. Concomittantly, rat CD4 and CD8 expression in the vicinity of the graft increased, consistent with xenograft rejection. When the SVG-TH cells were grafted to the lesioned striatum of a 6-hydroxydopamine lesioned rats, rotational behavior of the rat decreased as much as 80% initially, then slowly returned to baseline over the next four weeks, parallelling graft rejection. Thus, the SVG-TH cells can induce a functional recovery in an animal model of Parkinson's disease, however as a xenograft, the SVG cells are recognized by the immune system.     

转人GM—CSF基因人膀胱癌细胞株的建立及体外生物学特性

经逆转录病毒载体将人GM-CSF基因导入人膀胱癌细胞株BIU-87细胞中,建立了转基因细胞株BIU/GM。经流式细胞仪行细胞DNA周期分析表明GM-CSF基因的导人及表达对BIU-87细胞的增长无影响。免疫荧光测定发现转GM-CSF基因及表达不能促进BIU-87细胞表面HLA-ABC、DR、DQ抗原的表达。转基因瘤细胞株经6000rad X射线照射灭活后,丧失增殖能力,逐步死亡,但能维持一定水平的GM-CSF分泌活性达两周以上。从而为制备灭活的转基因瘤苗提供了初步经验。     

Immortalized hepatocytes using human artificial chromosome

The shortage of organ donors has impeded the development of human hepatocyte transplantation. Immortalized hepatocytes could provide an unlimited supply of transplantable cells. To determine whether immortalized hepatocytes could provide global metabolic support in end-stage liver disease, rat hepatocyte clones were developed by transduction with the gene encoding the Simian virus 40 T antigen (SVT) using the human artificial minichromosome (HAC). The SVLT sequence was excised by FRT recombination. Following HAC infusion, the transduced hepatocytes express SVT, blasticidine resistance (BS), and the PGK promoter TK gene. Forty-six cell clones were obtained and at least partially characterized, as previously described, for albumin, alpha-1-antitrypsin, glucose-6-phosphatase (G6Pase), dipeptidylpeptidase 4 (Dpp4), gamma-glutamyltransferase 1 (Ggt), SVT, and beta-actin expression using RT-PCR. Clones were also assessed for albumin secretion into the culture medium using ELISA. All of the cell line secreted approximately 10 mg/dl of albumin, which is equivalent to the amount secreted by primary hepatocytes. In further experiments, this cell line will be used for transplantable cells or artificial organ using HAC. These results represent an important step toward the development of immortalized hepatocytes.     

Isolation of immortalized, INK4a/ARF-deficient cells from the subventricular zone after in utero N-ethyl-N-nitrosourea exposure

OBJECT: Brain tumors, including gliomas, develop several months after rats are exposed in utero to N-ethyl-N-nitroso-urea (ENU). Although pathological changes cannot be detected until these animals are several weeks old, the process that eventually leads to glioma formation must begin soon after exposure given the rapid clearance of the carcinogen and the observation that transformation of brain cells isolated soon after exposure occasionally occurs. This model can therefore potentially provide useful insights about the early events that precede overt glioma formation. The authors hypothesized that future glioma cells arise from stem/progenitor cells residing in or near the subventricular zone (SVZ) of the brain. METHODS: Cells obtained from the SVZ or corpus striatum in ENU-exposed and control rats were cultured in an epidermal growth factor (EGF)-containing, chemically defined medium. Usually, rat SVZ cells cultured in this manner (neurospheres) are nestin-positive, undifferentiated, and EGF-dependent and undergo cell senescence. Consistent with these prior observations, control SVZ cells undergo senescence by the 12th to 15th doubling (20 of 20 cultures). In contrast, three of 15 cultures of cells derived from the SVZs of individual ENU-treated rats continue to proliferate for more than 60 cell passages. Each of these nestin-expressing immortalized cell lines harbored a common homozygous deletion spanning the INK4a/ARF locus and was unable to differentiate into neural lineages after exposure to specific in vitro stimuli. Nevertheless, unlike the rat C6 glioma cell line, these immortalized cell lines demonstrate EGF dependence and low clonogenicity in soft agar and did not form tumors after intracranial transplantation. CONCLUSIONS: Data in this study indicated that immortalized cells may represent glioma precursors that reside in the area of the SVZ after ENU exposure that may serve as a reservoir for further genetic and epigenetic hits that could eventually result in a full glioma phenotype.     

Role of hensin in mediating the adaptation of the cortical collecting duct to metabolic acidosis

PURPOSE OF REVIEW: The cortical collecting duct is able to secrete HCO3-, a state that can be converted to acid secretion during metabolic acidosis. Bicarbonate secretion in this segment is mediated by beta-intercalated cells whereas alpha-intercalated cells perform acid secretion. During metabolic acidosis, the number of beta-intercalated cells is reduced while that of alpha-intercalated cells increases without a change in the total number of intercalated cells, suggesting conversion of one cell type to another. Using an immortalized intercalated cell line we found that this adaptation is mediated by an extracellular protein named hensin. Hensin is secreted as a monomer which is then polymerized in the extracellular environment by a complex process requiring at least three other proteins. RECENT FINDINGS: We describe that a cyclophilin, via its cis/trans prolyl isomerase activity, is required for this polymerization. This may explain the distal renal tubular acidosis observed with cyclosporin A therapy. In addition, galectin-3 is needed to aggregate the protein. Finally, we recently found that activation of integrins is also necessary for the development of the hensin fiber. Hensin is expressed in all epithelia and deletion of its gene is embryonic lethal at an early stage when the first columnar epithelia develop. SUMMARY: These studies suggest that the response of intercalated cells to metabolic acidosis uses a pathway that is involved in terminal differentiation of columnar epithelia.     

miR-126在胃癌中的表达及其靶基因Crk的鉴定

目的:分析miR-126在胃癌中的表达水平并鉴定miR-126的靶基因,以阐明miR-126在胃癌发生机制中的功能。方法:采用qRT-PCR分别检测miR-126在胃癌细胞株、正常胃黏膜组织、永生化胃黏膜上皮细胞、胃癌及配对癌旁组织的表达水平,并与胃癌组织的临床病理指标进行相关性分析。采用生物信息学方法预测出miR-126的靶基因,并通过荧光素酶报告系统加以验证;采用qRT-PCR及Western印迹法检测miR-126对靶基因mRNA及蛋白质表达水平的影响。结果:qRT-PCR检测结果显示,miR-126在胃癌细胞株中的表达水平明显低于其在正常胃黏膜组织及永生化胃黏膜上皮细胞株的表达水平。miR-126在60例病人的胃癌组织中表达的水平显著低于其在配对癌旁组织中表达的水平;且胃癌组织miR-126表达水平低者,肿瘤组织体积较大,胃壁浸润较深,易发生淋巴结转移,病理分期也较晚。生物信息学分析提示Crk mRNA的3′UTR含有miR-126直接作用的靶序列,荧光素酶报告系统检测进一步验证了该靶序列,qRT-PCR及Western印迹法证实miR-126对Crk蛋白表达的调控发生在转录后水平。结论:miR-126有望成为研究胃癌的新型标志物;miR-126通过对其靶基因Crk的调控参与了胃癌的发生、发展过程。     

缺氧诱导因子1α的表达对HepG2细胞增殖与凋亡的影响

目的观察缺氧诱导因子1α（HIF-1α）对HepG2细胞增殖相关CyclinD1、CyclinA基因与凋亡相关Survivin、Bcl-2基因的影响。方法对Tet—on基因表达系统调控的缺氧诱导因子1α转染入HepG2细胞,用不同质量浓度的强力霉素对受强力霉素调控、稳定表达HIF—1α的HepG2^Tet-on进行凋亡诱导,检测肝癌细胞的凋亡和HIF-1α、CyclinD1、CyclinA、Survivin、Bcl-2的基因表达。结果终浓度分别为0、0．1、1、5mg／L的对HIFHepG2^Tet-on进行细胞凋亡诱导,细胞凋亡率分别为56．4％±7．8％、42．7％±4．9％、31．5％±6．1％、19．7％±4．5％;随着强力霉素浓度的增加,HIF-1α、CyclinD1、CyclinA、Survivin、Bcl-2基因表达水平增加,差异有统计学意义（P〈0．01）。结论HIF-1α基因在体外可以促进HepG2细胞的增殖,抑制其凋亡,而且随着HIF—1α基因表达水平的增加作用不断增强,这可能与HIF-1α诱导CyclinD1、CyclinA、Survivin、Bcl-2基因表达有关。     

局部进展期胰腺癌术前介入治疗新方法的应用

目的　通过术前联合化疗药物区域性动脉灌注介入治疗 ,探讨局部进展期胰腺癌介入治疗新方法应用的疗效。方法　 94例局部进展期胰腺癌患者术前进入介入治疗组和未介入治疗组 ,观察介入治疗前后肿瘤大小的改变、疼痛缓解率、血清肿瘤标志物的变化和副反应程度 ,通过免疫组化法检测介入治疗对肿瘤细胞凋亡和凋亡基因bcl 2、bax表达的影响 ,并比较两组患者手术切除率。结果　术前介入组和未介入组的手术切除率分别为 4 6 .8%和 2 3.3% (P <0 .0 5 ) ;术前介入治疗组肿瘤细胞调亡明显增加 ,肿瘤细胞bcl 2基因表达明显减少 ,而bax基因的表达显著增加 (P <0 .0 5 ) ;介入治疗能显著降低血清肿瘤标志物水平 ,并能明显缓解患者疼痛 (缓解率为 6 8.6 % ,P <0 .0 5 )。结论　术前介入治疗对胰腺癌肿瘤细胞有明显的杀伤和诱导凋亡作用 ,有助于提高手术切除率和改善患者疾病相关症状 ,是胰腺癌综合治疗的有效措施之一     

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.     

Expression and role of parathyroid hormone-related protein in human renal proximal tubule cells during recovery from ATP depletion

Parathyroid hormone (PTH)-related protein (PTHrP) is widely expressed in normal fetal and adult tissues and regulates growth and differentiation in a number of organ systems. Although various renal cell types produce PTHrP, and PTHrP expression in rat proximal renal tubules is upregulated in response to ischemic injury in vivo, the role of PTHrP in the kidney is unknown. To study the effects of injury on PTHrP expression and its consequences in more detail, the immortalized human proximal tubule cell line HK-2 was used in an in vitro model of ATP depletion to mimic in vivo renal ischemic injury. These cells secrete PTHrP into conditioned medium and express the type I PTH/PTHrP receptor. Treatment of confluent HK-2 cells for 2 h with substrate-free, glucose-free medium containing the mitochondrial inhibitor antimycin A (1 microM) resulted in 75% depletion of cellular ATP. After an additional 2 h in glucose-containing medium, cellular ATP levels recovered to approximately 75% of baseline levels. PTHrP mRNA levels, as measured in RNase protection assays, peaked at 2 h into the recovery period (at four times baseline expression). The increase in PTHrP mRNA expression was correlated with an increase in PTHrP protein content in HK-2 cells at 2 to 6 h into the recovery period. Heat shock protein-70 mRNA expression was not detectable under baseline conditions but likewise peaked at 2 h into the recovery period. Treatment of HK-2 cells during the recovery period after injury with an anti-PTHrP(1-36) antibody (at a dilution of 1:250) resulted in significant reductions in cell number and uptake of [3H]thymidine, compared with nonimmune serum at the same titer. Similar results were observed in uninjured HK-2 cells. It is concluded that this in vitro model of ATP depletion in a human proximal tubule cell line reproduces the pattern of gene expression previously observed in vivo in rat kidney after ischemic injury and that PTHrP plays a mitogenic role in the proliferative response after energy depletion.     

Tet-on基因表达系统调控HIF-1α表达对肝癌细胞增殖的影响

目的 探讨HIF-1α表达对肝癌细胞增殖和细胞周期的影响.方法 利用Tet-on基因表达系统调控肝癌细胞系HepG2中HIF-1α的表达,检测细胞周期和细胞增殖的变化.结果 酶切和DNA测序证实Tet-on基因表达系统反应质粒PTRE-HIF-1α构建成功.获得了受强力霉素调控、稳定表达HIF-1α的肝癌细胞.随着强力霉素浓度增加,hiF-1α基因表达增加,HIF-1α在体外明显增加HepG2细胞增殖活性;G0/G1期细胞指数减少,细胞增殖指数增多.RT-PCR检测结果表明,随着HIF-1α基因表达增加,Cyclin A mRNA表达增加(P＜0.001),Cyclin D1和Cyclin E mRNA表达水平无明显变化(P＞0.05).结论 HIF-1α基因在体外可以通过HIF-1α表达水平的增加而促进肝癌细胞增殖活性,通过CyclinA表达增加缩短了肝癌细胞增殖周期.     

Promotion of survival and regeneration of nigral dopamine neurons in a rat model of Parkinson's disease after implantation of embryonal carcinoma-derived neurons genetically engineered to produce glial cell line-derived neurotrophic factor

OBJECT: The P19 embryonal carcinoma-derived cell line consists of undifferentiated multipotential cells, which irreversibly differentiate into mature neurons after exposure to retinoic acid (RA). In the present study, the authors genetically engineered P19 cells to produce glial cell line-derived neurotrophic factor (GDNF), and grafted the cells in a rat model that had been rendered parkinsonian. METHODS: Undifferentiated P19 cells were grown in vitro and transduced with GDNF complementary DNA. The level of GDNF released from the transduced cells was measured using an enzyme-linked immunosorbent assay, and its neurotrophic activities were assessed by testing the effects on rat embryonic dopamine (DA) neurons in culture. After having been exposed to RA for 48 hours and allowed to differentiate into postmitotic neurons, the GDNF gene-transduced cells were implanted into the midbrain of immunosuppressed rats. A unilateral nigrostriatal lesion was then induced by intrastriatal infusions of 6-hydroxydopamine. Immunohistochemical analyses performed 4 weeks postgrafting revealed that the GDNF-producing cells expressed several neuronal markers without evidence of overgrowth. The grafts expressed GDNF protein and prevented the death of nigral DA neurons. Furthermore, the GDNF-producing cells implanted 4 weeks after nigrostriatal lesions restored the expression of tyrosine hydroxylase in injured DA neurons and induced their dendritic sprouting. CONCLUSIONS: The results indicate that the P19 cell line transduced with the GDNF gene can stably secrete functional levels of GDNF, even after being converted to postmitotic neurons. Because it is has been established that GDNF exerts trophic effects on DA neurons, the means currently used to deliver GDNF into the brain could be a viable strategy to prevent the death of nigral DA neurons in cases of Parkinson's disease.