Prolonged gene expression in primary porcine pancreatic cells using an Epstein-Barr virus-based episomal vector

Epstein-Barr virus (EBV)-based plasmids containing the origin of replication (oriP) and EBV nuclear antigen 1 (EBNA-1) are well known for the stable episomal maintenance in human cells. In order to clarify whether an EBV-based plasmid can be maintained stably in the porcine pancreatic cells which are the primary candidate sources of islet xenotransplantation, we constructed pEBVGFP encoding the green fluorescent protein (GFP). Monolayer culture of the porcine neonatal pancreatic cells was lipofected with pEBVGFP or pGFP which was derived from pEBVGFP by deleting out oriP and EBNA-1. pEBVGFP significantly prolonged GFP expression not only in human cell lines but also in the primary porcine pancreatic cells compared with pGFP. Interestingly, the duct cells that are believed as the pancreatic precursor cells were preferentially transfected and conveniently enriched among the mixed primary cell populations using a hygromycin B selection. To our knowledge, this is the first report suggesting the potential application of an EBV-based plasmid for the extended gene expression in the primary porcine pancreatic duct cells.     

Polyethylenimine-mediated suicide gene transfer induces a therapeutic effect for hepatocellular carcinoma in vivo by using an Epstein-Barr virus-based plasmid vector

The present study aimed to establish a novel efficient nonviral strategy for suicide gene transfer in hepatocellular carcinoma (HCC) in vivo. We employed branched polyethylenimine (PEI) and combined it with Epstein-Barr virus (EBV)-based plasmid vectors. The HCC cells transfected with an EBV-based plasmid carrying the herpes simplex virus-1 thymidine kinase (HSV-1 Tk) gene (pSES.Tk) showed up to 30-fold higher susceptibilities to ganciclovir (GCV) than those transfected with a conventional plasmid vector carrying the HSV-1 Tk gene (pS.Tk). The therapeutic effect in vivo was tested by intratumoral injection of the plasmids into HuH-7 hepatomas transplanted into C.B-17 scid/scid mutant (SCID) mice and subsequent GCV administrations. Treatment with pSES.Tk, but not pS.Tk, markedly suppressed growth of hepatomas in vivo, resulting in a significantly prolonged survival period of the mice. These findings suggest that PEI-mediated gene transfer system can confer efficient expression of the suicide gene in HCC cells in vivo by using EBV-based plasmid vectors.     

HGF基因真核表达质粒的构建及其细胞表达

目的:构建真核表达质粒p EGFP-N1-HGF并观察干细胞生长因子(hepatocyte growth factor,HGF)在人皮肤成纤维中的表达。方法:从扩增人的间充质干细胞中提取全长RNA为模板,设计合成引物,用RT-PCR法扩增出HGF基因片段,然后将HGF基因片段连接于真核表达质粒p EGFP-N1中。双酶切鉴定及测序分析后,用脂质体包裹转染体外培养的人的皮肤成纤维细胞,通过荧光显微镜观察其转染效果,酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA)检测HGF蛋白的表达。结果:经酶切鉴定及测序验证p EGFP-N1-HGF构建正确,经转染的人皮肤纤维细胞中观察到较强的绿色荧光并且可以表达一定量的HGF。结论:成功构建了真核表达质粒p EGFP-N1-HGF,为基因治疗脱发疾病提供实验基础。     

稳定表达ALB启动子及荧光素酶报告基因的肝干细胞株的构建

目的构建稳定表达ALB启动子及荧光素酶报告基因的肝干细胞株。方法PCR扩增获得ALB启动子,并与pBGLuc连接获得携带ALB启动子及荧光素酶报告基因的pBGLuc—ALB质粒,脂质体转染质粒到不同细胞,ALB—GLuc活性检测功能。构建逆转录病毒,感染HP14．5肝干细胞株获得携带ALB启动子及荧光素酶报告基因的稳定细胞株,经Dex、HGF体外诱导后第3、6、9、12天ALB—GLuc检测荧光素酶活性,免疫荧光检测ALB的表达。结果PCR、酶切及测序结果显示ALB启动子正确插入至荧光素酶GLuc基因上游,HEK293、HP14．5、LC14d及Hepa1-6细胞中ALB—GLuc活性与免疫荧光结果一致。HP14．5ALB—Gluc稳定细胞株在高浓度的稻瘟菌素中存活,免疫荧光结果显示Dex、HGF诱导后细胞中ALB的表达逐渐增强,并与ALB—Gluc活性升高一致。结论成功构建了稳定表达ALB启动子及荧光素酶报告基因的肝干细胞株,为研究肝干细胞的体外成熟分化提供了重要的细胞手段。     

Instability of extrachromosomal cosmid DNA in SV40-transformed human (ataxia-telangiectasia) cells

The ability of SV40-transformed human (ataxia-telangiectasia) fibroblasts to maintain Epstein-Barr virus (EBV)-based plasmids and cosmids extrachromosomally has been investigated. Transfection of a culture of cells with two different plasmids gave rise to cell clones which were able to maintain both plasmids extrachromosomally. When an EBV-based cosmid library was transfected into the cells and an individual cell clone was isolated, the extrachromosomal DNA derived from the cosmid contained numerous deletions and rearrangements. When individual cosmids were transfected into the culture, and several cell clones were isolated, the intracellular cosmid-derived DNA again showed the presence of multiple deletions and rearrangements. We conclude that although SV40-transformed cells are able to maintain more than one different EBV-based plasmid extrachromosomally, large EBV-derived molecules are extensively rearranged. SV40-transformed human fibroblasts cannot therefore be usefully used in attempting to clone genes from EBV-based cosmid libraries.     

The Use of Epstein–Barr Virus-Based Shuttle Vectors in Rat PC12 Cells

1. The rat pheochromocytoma PC12 cell line has been a commonly used model for studies of neuronal development, function, and death. Thus the abilityto transfect PC12 cells in an efficient manner and to manipulate their gene expression would enhance the usefulness of these cells.2. We demonstrate that EBV-based vectors provide a useful expression system for gene manipulation in rat PC12 cells.3. The EBV-based vectors replicate episomally in PC12 cells for at least 2months, as evidence by their recovery from the transfected cells and by the digestion of the episomal plasmid with the isoschizomer MboI and DpnI restriction enzymes.3. PC12 cells are efficiently transfected by EBV-based vectors both transiently and stably.4. Transfection of PC12 cells with an EBV-based vector containing tau cDNA in the antisense orientation resulted in a decrease in the level of tau protein in the transfected cells.5. The results demonstrate that EBV-based vectors can be a useful expression system for gene manipulation in PC12 cells.     

Improvement of survival of skin flaps by combined gene transfer of hepatocyte growth factor and prostacyclin synthase

BACKGROUND: Increasing the local blood flow is a critical factor for long-term survival of skin flaps. Thus, a molecular therapy to increase the blood flow by means of an angiogenic factor is considered to be a useful strategy to improve skin flap survival. We focused on a combined strategy to stimulate not only angiogenesis, but also vasodilation of local microvessels, using co-transfection of the hepatocyte growth factor (HGF) and prostacyclin synthase (PGIS) genes to enhance the survival of random-pattern skin flaps. METHODS AND RESULTS: A 2 x 8 cm full thickness cranial pedicled random-pattern flap was made on the back of each 12-week-old male rat. At 3 days before operation, 400 microg of human HGF and PGIS naked plasmid DNA or control plasmid was transfected into the flaps by needle-less injection using a Shima Jet, resulting in successful expression of human HGF and PGIS in the skin flaps. Transfection of both genes into the distal half of skin flaps at 3 days prior to operation significantly increased the survival rate of skin flaps, while transfection all over the flaps did not. In addition, transfection prior to operation was more effective than simultaneous treatment. Moreover, co-transfection of these genes improved the survival area of skin flaps, accompanied by an increase in blood flow of skin flaps, even in a diabetic model. CONCLUSIONS: Overall, these results indicate that combination treatment with HGF and PGIS genes by Shima Jet could be an effective strategy to improve skin flap survival.     

EBV-based plasmid DNA rearrangements after transfection of eukaryotic cells

The cDNA encoding influenza virus (A/Udorn/307/72 strain) M2 protein was subcloned into the EBV-based vector pREP9. Three continuous kidney cellular lines of different origin were transfected with recombinant plasmid pREP9-M2. One and 5 months after transfection plasmid DNA rearrangements were detected by means of restriction analysis of recovered plasmids and their hybridization with an influenza-virus-specific radioactive probe. Deletions were the most frequent type of pREP9-M2 mutations. PCR with primers corresponding to cellular genome and plasmid DNA followed by Southern blot analysis with the [(32)P]-labeled M2-fragment allowed host DNA rearrangements to be revealed in transfected cells.     

Non-replicating Epstein-Barr virus-based plasmids extend gene expression and can improve gene therapy in vivo

To date, no gene transfer vector has produced prolonged gene expression following a single intravenous injection and then efficiently re-expressed the delivered gene following repeated systemic injection into immunocompetent hosts. To overcome these limitations, a gene therapy regimen using non-replicating Epstein-Barr virus (EBV)-based expression plasmids was developed. One plasmid contains the FR (EBV family of repeats) sequence and the expressed gene. The other encodes Epstein-Barr nuclear antigen 1 (EBNA-1), but lacks FR. Although unable to replicate in mice, intravenous co-injection of EBV-based plasmids in cationic liposome-DNA complexes (CLDCs) substantially prolonged luciferase gene expression. The use of a two-vector system limited host exposure to the EBNA-1 gene product. Furthermore, this EBV-based vector system could be intravenously re-injected multiple times into immunocompetent mice without loss of transfection efficiency. Use of this vector system significantly improved the therapeutic efficacy of the biologically important human granulocyte colony-stimulating factor gene. Delivery of the human granulocyte colony-stimulating factor gene in EBV-based plasmids increased circulating white blood counts for at least 2 months following a single CLDC-based intravenous co-injection. Conversely, white blood counts were never elevated following injection of CLDCs lacking EBV-derived elements. Thus, this EBV-based plasmid vector system both markedly prolongs gene expression at therapeutic levels and efficiently and repeatedly re-transfects immunocompetent hosts. These properties of EBV-based plasmid vectors appear to be due, at least in part, to the documented abilities of the EBNA-1 protein both to retain FR-containing DNA intracellularly and within the nucleus and to block anti-EBNA-1 cytotoxic T cell responses.     

腺病毒载体介导PDX-1在骨髓间充质干细胞中的表达

为研究PDX-1基因在骨髓间充质干细胞中的表达情况及生物学功能的发挥,构建了含PDX-1基因的重组腺病毒载体. 酶切PDX-1基因并连入穿梭质粒pAdTrack-CMV.用电穿孔法使穿梭质粒pAdTrack-CMV-PDX-1与病毒骨架质粒pAdEasy-1在大肠杆菌BJ5183中同源重组.利用脂质体介导重组腺病毒载体转染293细胞,包装出完整的腺病毒.分离、培养、扩增骨髓间充质干细胞.用重组腺病毒感染间充质干细胞.用荧光显微镜、RT-PCR、免疫荧光染色等方法检测PDX-1、胰岛素基因及蛋白质的表达,用放射免疫分析法检测转基因细胞分泌胰岛素情况.结果表明:通过测序、PCR、酶切等鉴定PDX-1基因已正确插入穿梭质粒中,并与病毒骨架质粒重组.重组腺病毒滴度为6.3×10⁷ PFU/ml.通过荧光显微镜观察证实重组腺病毒可高效感染骨髓间充质干细胞,经RT-PCR、免疫荧光染色证实转染pAd-PDX-1后培养7天的细胞中有PDX-1及胰岛素基因的表达.这些转基因的细胞向胞外分泌的胰岛素量为(15.21±3.50) mIU/L.     

VEGF165 expressing bone marrow mesenchymal stem cells differentiate into hepatocytes under HGF and EGF induction in vitro

A short half-life and low levels of growth factors in an injured microenvironment necessitates the sustainable delivery of growth factors and stem cells to augment the regeneration of injured tissues. Our aim was to investigate the ability of VEGF₁₆₅ expressing bone marrow mesenchymal stem cells (BMMSCs) to differentiate into hepatocytes when cultured with hepatocyte growth factor (HGF) and epidermal growth factor (EGF) in vitro. We isolated, cultured and identified rabbit BMMSCs, then electroporated the BMMSCs with VEGF₁₆₅-pCMV6-AC-GFP plasmid. G418 was used to select transfected cells and the efficiency was up to 70%. The groups were then divided as follows: Group A was electroporated with pCMV6-AC-GFP plasmid + HGF + EGF and Group B was electroporated with VEGF₁₆₅-pCMV6-AC-GFP plasmid +HGF + EGF. After 14 days, BMMSCs were induced into short spindle and polygonal cells. Alpha-fetoprotein (AFP) was positive and albumin (ALB) was negative in Group A, while both AFP and ALB were positive in group B on day 10. AFP and ALB in both groups were positive on day 20, but the quantity of AFP in group B decreased with prolonged time and was about 43.5% less than group A. The quantity of the ALB gene was increased with prolonged time in both groups. However, there was no significant difference between group A and B on day 10 and 20. Our results demonstrated that VEGF₁₆₅-pCMV6-AC-GFP plasmid modified BMMSCs still had the ability to differentiate into hepatocytes. The VEGF₁₆₅ gene promoted BMMSCs to differentiate into hepatocyte-like cells under the induction of HGF and EGF, and reduced the differentiation time. These results have implications for cell therapies.     

博尔纳病病毒核蛋白调控神经干细胞存活增殖及ERK1／2信号通路的实验研究

目的观察博尔纳病病毒核蛋白（Borna disease virus p40,BDV p40）对大鼠海马源性神经干细胞（Neural stem cells,NSCs）增殖、存活、分化及ERK1／2信号通路的影响,揭示BDV引起神经精神疾病的部分发病机制。方法（1）分别用pEGFP—N1—p40及pEGFP—N1质粒转染NSCs,观察转染效率并鉴定BDVp40在NSCs中的表达。（2）实验设置3组：未转染组、pEGFP—N1空转对照组及pEGFP—N1—p40转染组,用CCK-8试剂盒、Brdu摄入实验及免疫组化分别检测细胞存活、增殖及分化为神经元、星型胶质细胞、少突胶质细胞的比例的变化,并经Western Blot检测ERK1／2磷酸化的改变。结果（1）成功建立表达BDVp40的NSCs模型;PCR结果显示只有pEGFP—N1-p40转染组细胞有BDVp40基因表达。（2）BDVp40抑制NSCs的存活、增殖,但对于转染后贴壁分化14d时3组细胞分化为神经元、星型胶质细胞、少突胶质细胞的比例未见显著差异。WesternBlot结果显示BDVp40下调了磷酸化ERK1／2在蛋白水平的表达。结论BDVp40抑制NSCs的存活、增殖,但是对NSCs的分化方向没有明显的影响。BDVp40有可能通过下调磷酸化ERK1／2活性对NSCs的存活、增殖起抑制作用。     

Effects of extracellular matrixes and growth factors on the hepatic differentiation of human embryonic stem cells

Hepatocytes derived from human embryonic stem cells (hESCs) are a potential cell source for regenerative medicine. However, the definitive factors that are responsible for hepatic differentiation of hESCs remain unclear. We aimed to evaluate the effects of various extracellular matrixes and growth factors on endodermal differentiation and to optimize the culture conditions to induce hepatic differentiation of hESCs. The transgene vector that contained enhanced green fluorescent protein (EGFP) under the control of human alpha-fetoprotein (AFP) enhancer/promoter was transfected into hESC lines. The transgenic hESCs were cultured on extracellular matrixes (collagen type I, laminin, and Matrigel) in the presence or absence of growth factors including hepatocyte growth factor (HGF), bone morphogenetic protein 4, fibroblast growth factor 4, all-trans-retinoic acid, and activin A. The expression of AFP-EGFP was measured by flow cytometry. The culture on Matrigel-coated dishes with 100 ng/ml activin A showed 19.5% of EGFP-positive proportions. Moreover, the sequential addition of 100 ng/ml activin A and 20 ng/ml HGF resulted in 21.7% and produced a higher yield of EGFP-positive cells than the group stimulated by activin A alone. RT-PCR and immunocytochemical staining revealed these EGFP-positive cells to differentiate into mesendoderm-like cells by use of activin A and then into hepatic endoderm cells by use of HGF. Two other hESC lines also differentiated into endoderm on the hepatic lineage by our method. In conclusion, we therefore found this protocol to effectively differentiate multiple hESC lines to early hepatocytes using activin A and HGF on Matrigel.     

Induction of insulin‐producing cells from umbilical cord blood‐derived stromal cells by activation of the c‐Met/HGF axis

Efficient and effective therapies are required for diabetes mellitus. The use of adult stem cells for treating diabetes represents a major focus of current research. We have attempted to differentiate adult stem cells produced from umbilical cord blood‐derived stromal cells into insulin‐producing cells (IPCs). By activating the c‐Met/HGF axis through temporal hypoxia treatment and hepatocyte growth factor (HGF) supplementation, our protocol resulted in the differentiation of cells into functional pancreatic endocrine cells with increased viability. Glucose stimulation test results showed that significantly greater amounts of C‐peptide and insulin were released from the differentiated cells than from undifferentiated cells. These IPCs were capable of reversing the hyperglycemia of diabetic mice. In conclusion, targeting the c‐Met/HGF axis can be considered an effective and efficient means of obtaining IPCs from adult stem cells.     

软骨寡聚基质蛋白过表达对BMP-2诱导骨髓间充质干细胞分化的影响

目的：研究软骨寡聚基质蛋白（cartilage oligomeric matrix protein,COMP）过表达对BMP-2诱导骨髓间充质干细胞成骨及成软骨分化的影响。方法：BMP-2诱导骨髓间充质干细胞分化,通过脂质体转染含人COMP基因的质粒使骨髓间充质干细胞过表达COMP,采用实时定量PCR和Western blotting分析COMP基因过表达、成骨相关基因Ⅰ型胶原、RUNX2、骨钙蛋白以及成软骨相关基因Ⅱ型胶原、SOX9、蛋白聚糖、X型胶原的表达变化;通过茜素红染色观察成骨终末阶段矿化结节的生成情况,阿利新蓝染色观察细胞基质蛋白多糖的合成情况。结果：质粒转染后骨髓间充质干细胞COMP基因蛋白和mRNA表达水平显著提高（P<0.05）。COMP基因过表达后,成骨标记基因RUNX2、Ⅰ型胶原（Col1a1）mRNA水平均显著低于对照组（P<0.05）,RUNX2、骨钙蛋白（Osteocalcin）蛋白表达水平明显低于对照组（P<0.05）,而成软骨标记基因SOX9、蛋白聚糖（Aggrecan）mRNA水平均显著高于对照组（P<0.05）,SOX9、Ⅱ型胶原（Col2a1）蛋白表达均明显多于对照组（P<0.05）。细胞成骨茜素红染色弱于对照组,而阿利新蓝染色强于对照组。过表达组细胞X型胶原（Col10a1）基因表达显著低于对照组（P<0.05）,结论：骨髓间充质干细胞COMP基因过表达可抑制BMP-2诱导其成骨分化,促进骨髓间充质干细胞成软骨分化,并抑制软骨细胞的成熟肥大,为软骨组织工程研究提供新的方向。