Inhibitory effect of Tum-5 gene on growth of hepatocellular carcinoma cells and its influence in angiogenesis / 吉林大学学报(医学版)

Objective: To explore the inhibitory effect of Tum-5 gene on the tumor growth and its influence in the angiogenesis of hepatocellular carcinoma (HCC), and to illustrate its mechanism involved in antitumorigenesis. Methods: The human HepG2 cells were selected in in vitro experiment and treated with different multiplicity of infection (MOD (0, 1, 5, 10, 25 and 50) of pLXSN and pLXSN-Tum-5 virus particles for 72 h. The proliferation rates of cells in various groups were detected by MTT method. In vivo, the H22 tumor-bearing mouse models were established. The mice were divided into saline group, pLXSN group and pLXSN-Tum-5 group (n=5). The mice in saline group were intratumorally injected with normal saline, and the mice in pLXSN group and pLXSN-Tum-5 group were intratumorally injected with virus particles (MOI =5), 5 times every other day. The volumes and weights of transplanted tumor and the weights of mice in various groups were measured. The CD31 expressions in transplanted tumor tissue were detected by immunohistochemical method and the microvessel density (MVD) was calculated. Results: Compared with pLXSN group, the proliferation rates of cells in pLXSN-Tum-5 group after infected with different MOI of virus particles were not significantly different (P> 0. 05). The volumes and weights of transplanted tumor of the mice in pLXSN-Tum-5 group were significantly smaller than those in pLXSN group and saline group after intratumoral injection (P

Inhibitory effect of Tum-5 gene on growth of hepatocellular carcinoma cells and its influence in angiogenesis / 吉林大学学报(医学版)

Objective:To explore the inhibitory effect of Tum-5 gene on the tumor growth and its influence in the angiogenesis of hepatocellular carcinoma (HCC),and to illustrate its mechanism involved in antitumorigenesis.Methods:The human HepG2 cells were selected in in vitro experiment and treated with different multiplicity of infection (MOI) (0,1,5,10,25 and 50) of pLXSN and pLXSN-Tum-5 virus particles for 72 h.The proliferation rates of cells in various groups were detected by MTT method.In vivo,the H22 tumor-bearing mouse models were established.The mice were divided into saline group,pLXSN group and pLXSN-Tum-5 group (n=5).The mice in saline group were intratumorally injected with normal saline,and the mice in pLXSN group and pLXSN-Tum-5 group were intratumorally injected with virus particles (MOI =5),5 times every other day.The volumes and weights of transplanted tumor and the weights of mice in various groups were measured.The CD31 expressions in transplanted tumor tissue were detected by immunohistochemical method and the microvessel density (MVD) was calculated.Results:Compared with pLXSN group,the proliferation rates of cells in pLXSN-Tum-5 group after infected with different MOI of virus particles were not significantly different (P＞ 0.05).The volumes and weights of transplanted tumor of the mice in pLXSN-Tum-5 group were significantly smaller than thosein pLXSN group and saline group after intratumoral injection (P＜ 0.05 or P＜ 0.01).The immunohistochemical results showed that there was irregular angiogenesis in each group.Compared with saline group and pLXSN group,the mean value of MVD of the transplanted tumor tissue of the mice in pLXSN-Tum-5 group was significantly decreased (P ＜ 0.05).Conclusion:Tum-5 can exert its antitumor activity by inhibiting the formation of neovascularization in HCC.

Construction and identification of the recombinant retroviral vector to carry out hypoxia-regulated expression of neurotrophin-3 / 西安交通大学学报(医学版)

Objective To construct and identify the recombinant retroviral vector containing five copies of hypoxia responsive elements (5HRE)and neurotrophin-3 (NT-3 ).Methods Using PCR,enzyme digestion and DNA ligase,5HRE and human derived NT-3 were cloned into the retroviral vector plasmid (pLNCX)to construct the recombinant retroviral vector plasmid pLNCX-5HRE-SV40-NT3-IRES-EGFP.The retrovirus RV-5HRE-NT3 was packaged in the PT67 cells,and then it was purified and concentrated by high-speed centrifugation.After infected for 48 h with the concentrated retrovirus,the number of the EGFP positive cells in the NIH 3T3 cells was counted by fluorescence activated cells and sorted to calculate the retrovirus titer.Results The retroviral vector plasmid,pLNCX-5HRE-SV40-NT3-IRES-EGFP,was successfully constructed,and the retrovirus was packaged and defined as RV-5HRE-NT3.After purification and concentration,the retrovirus titer reached 9.1 × 10 6 cfu/mL. Conclusion The recombinant retroviral vector which carried out hypoxia-regulated expression of NT-3 was successfully constructed.It may provide basis for studies on hypoxia-regulated expression of the exogenous genes.

Malignant transformation of mature T cells after gammaretrovirus mediated transfer of nucleophosminanaplastic lymphoma kinase oncogene.

Background: Gene therapy has been in use to cure hereditary and acquired diseases by incorporating the desired gene into the cells with the help of gammaretroviral vectors. Despite the success of this therapy in X-linked severe combined immunodefi ciency syndrome, few patients developed leukemia as a major adverse event due to retroviral insertional mutagenesis within stem cells. In experimental animals also, retroviral-mediated gene transfer technique resulted in the development of leukemia. On the other hand, evidence suggests that mature T cells (TC) are relatively resistant to transformation even after retroviral-mediated transfer of potent oncogenes Tcl1, ΔTrkA and LMO2 with no reported side effects yet. Aims: To further address the safety issue for TC use in gene therapy, this study investigated susceptibility of mature polyclonal TC to malignant transformation by the retroviral-mediated transfer of nucleophosminanaplastic lymphoma kinase (NPM-ALK) oncogene. Materials and Methods: Wild-type mature TC, isolated from C57BL/6 donor mice (genetic background Ly5.1) were transduced with gamma-retroviral vectors encoding the potent TC oncogene NPM-ALK or the control vector enhanced green fl uorescent protein eGFP. The cells were then transplanted into RAG-1 defi cient recipient mice (genetic background Ly5.2). Results: Two out of fi ve mice from NPM-ALK oncogene group developed leukemia/lymphoma after latency periods (153 and 250 days, respectively). None of the mice from the control group developed any malignancy throughout the observational period. Conclusion: Mature polyclonal TC are relatively susceptible to malignant transformation after gamma-retroviral mediated transfer of NPM-ALK oncogene; hence safety of TC use in gene therapy should be further investigated to avoid the possible side-effect of development of leukemia/lymphoma.

Construction and expression of retroviral vector pLEGFP-N1-TERT in preparation of seed cells for skin tissue engineering

OBJECTIVE@#To construct the retroviral vector pLEGFP-N1-telomerase reverse transcriptase (TERT) and to investigate the expression of TERT in neonatal mouse hypodermal cells.@*METHODS@#The polymerase chain reaction (PCR)-amplified TERT gene was inserted into plasmid pLEGFP-N1. The positive clone was identified by restriction enzyme digestion and sequencing, then was transfected into packaging cells to produce retrovirus particles. Neonatal mouse hypodermal cells were infected with the virus to generate a stable cell line. The TERT mRNA expression level, telomerase activity, and enhanced green fluorescent protein (EGFP) expression level were analyzed.@*RESULTS@#Retroviral vector pLEGFP-N1-TERT was constructed successfully, and a stable cell line of neonatal mouse hypodermal cells expressing EGFP was established. Western blot and immunohistochemical assay showed that the expression level of TERT was significantly elevated in the neonatal mouse hypodermal cells.@*CONCLUSIONS@#A high titer of retrovirus pLEGFP-N1-TERT mediates high-level expression of the exogenous TERT gene in the neonatal mouse hypodermal cells. This protocol has potential applications for skin tissue engineering and cell transplantation therapy.

The effects of oncostatin M (OSM) gene on proliferation and apoptosis of human breast cancer cells in vitro / 中华内分泌外科杂志

Objective To study the inhibition effects of oncostatin M (OSM) gene on human breast cancer cell line(michigan cancer foundation-7,MCF-7).Methods MCF-7 cells were infected with the optimized dose of retrovirus expressing OSM.Expression of the target gene was determined by reverse transcriptionpolymerase chain reaction(RT-PCR) and enzyme-linked immunosorbent assay(ELISA).Methyl thiazolyl tetrazolium (MTT) assay was employed to examine growth inhibition effect of OSM gene on MCF-7 cells.Cell apoptosis was determined by flow cytometry.The morphologic changes of nucleus was detected by Hochest staining,in addition,the expression of apoptosis related genes in tumor cells were further detected by RT-PCR.Results OSM gene was well expressed in human breast cancer MCF-7 cell by retrovial vetor and it could significantly inhibit the growth of MCF-7 cell(MTT,P =0.0162,P ＜ 0.05) and induce its apoptosis (Annexin V/PI,P =0.02,P ＜ 0.05).Conclusion OSM gene has significant inhibitory effects on MCF-7 cells,which mechanism might be related to the upregulation of Bax and Caspase3 and the downregulation of Bcl-2.

Overexpression of Human Arginine Decarboxylase Rescues Human Mesenchymal Stem Cells against H2O2 Toxicity through Cell Survival Protein Activation

In this study, we explored the potentiality of human arginine decarboxylase (ADC) to enhance the survival of mesenchymal stem cells (MSCs) against unfavorable milieu of host tissues as the low survival of MSCs is the issue in cell transplantation therapy. To address this, human MSCs overexpressing human ADC were treated with H2O2 and the resultant intracellular events were examined. First, we examined whether human ADC is overexpressed in human MSCs. Then, we investigated cell survival or death related events. We found that the overexpression of human ADC increases formazan production and reduces caspase 3 activation and the numbers of FITC, hoechst, or propidium iodide positive cells in human MSCs exposed to H2O2. To elucidate the factors underlying these phenomena, AKT, CREB, and BDNF were examined. We found that the overexpression of human ADC phosphorylates AKT and CREB and increases BDNF level in human MSCs exposed to H2O2. The changes of these proteins are possibly relevant to the elevation of agmatine. Collectively, our data demonstrate that the overexpression of human ADC stimulates pro-survival factors to protect human MSCs against H2O2 toxicity. In conclusion, the present findings support that ADC can enhance the survival of MSCs against hostile environment of host tissues.

The expression of Foxp3 protein by retroviral vector-mediated gene transfer of Foxp3 in C57BL/6 mice / 대한수의학회지

The maintenance of peripheral immune tolerance and prevention of chronic inflammation and autoimmune disease require CD4+CD25+ T cells (regulatory T cells). The transcription factor Foxp3 is essential for the development of functional, regulatory T cells, which plays a prominent role in self-tolerance. Retroviral vectors can confer high level of gene transfer and transgene expression in a variety of cell types. Here we observed that following retroviral vector-mediated gene transfer of Foxp3, transductional Foxp3 expression was increased in the liver, lung, brain, heart, muscle, spinal cord, kidney and spleen. One day after vector administration, high levels of transgene and gene expression were observed in liver and lung. At 2 days after injection, transductional Foxp3 expression level was increased in brain, heart, muscle and spinal cord, but kidney and spleen exhibited a consistent low level. This finding was inconsistent with the increase in both CD4+CD25+ T cell and CD4+Foxp3+ T cell frequencies observed in peripheral immune cells by fluorescence-activated cell-sorting (FACS) analysis. Retroviral vector-mediated gene transfer of Foxp3 did not lead to increased numbers of CD4+CD25+ T cell and CD4+Foxp3+ T cell. These results demonstrate the level and duration of transductional Foxp3 gene expression in various tissues. A better understanding of Foxp3 regulation can be useful in dissecting the cause of regulatory T cells dysfunction in several autoimmune diseases and raise the possibility of enhancing suppressive functions of regulatory T cells for therapeutic purposes.

Effects of up-regulated gene-4 on the proliferation of colonic cancer cells / 中华消化外科杂志

Objective To study the effects of up-regulated gene-4 (URG-4) on colon cancer cellproliferation.MethodsColon cancer cell line with high expression of URG-4 was selected.The recombinant URG-4 siRNA retroviral vector was constructed and packaged by PT67 cell,then retroviral particles which can express URG-4 siRNA in mammal cell and its negative control were obtained.Expressions of URG-4 in MKN45,SW480,LoVo,HCT116,HT29 were detected by RT-PCR and Western blot,respectively.Recombinant virus (interference group),original virus (negative control group) and the same amount of PBS (blank group) were used to transfect LoVo cells respectively.Stably transfected cell lines were screened.The growth condition of cell lines in each group was assayed by MTT.All data were analyzed by the one-way analysis of variance and the t test.Results Sequencing results confirmed the successful construction of retroviral which expressed siRNA,the relative expression levels of URG-4 mRNA in MKN45,SW480,LoVo,HCT116,HT29 were 0.58 +0.02,0.63 ±0.03,0.81 ± 0.01,1.01 ± 0.02,0.91 ± 0.04 and the expression levels of URG-4 protein in the 5 cell lines were 0.73 ±0.02,0.85 ± 0.03,1.42 ± 0.01,0.80 ± 0.30,0.80 ± 0.04,respectively.High expression of URG-4 was observed in the LoVo cells.The expression of URG-4 mRNA in the LoVo cells in the interference group was 0.55 ±0.03,which was significantly lower than 1.15 ±0.02 of the negative control group and 1.15 ±0.01 of the blank group ( t =- 5.179,- 9.285,P ＜ 0.05 ).The inhibition rate of URG-4 mRNA in the interference group was 52.6％.The expression of URG-4 protein in the interference group was 0.82 ± 0.05,which was significantly lower than 1.46 ± 0.07 of the negative control group and 1.54 ± 0.04 of the blank group (t =-4.239,-3.704,P＜0.05).The inhibition rate of URG-4 protein in the interference group was 43.6％.The LoVo cells in each group grew exponentially.Compared with the negative control group,the cell growth of the interference group was inhibited during day 3 to day 6,which had statistical significant difference ( t =- 6.436,-6.045,-6.434,-4.285,P＜0.05).ConclusionInterference of the expression of URG-4 can inhibit the growth of LoVo cells.

Screening for CHO cell line stably expressing inducible costimulator protein and its biological activity / 第二军医大学学报

Objective: To construct a recombinant retroviral vector carrying human inducible costimulator (ICOS) gene, screen for CHO cell line stably expressing ICOS protein and to study its biological activity. Methods: ICOS cDNA was obtained from human peripheral blood mononuclear cells (PBMC) through RT-PCR and was cloned into retroviral vector to construct retroviral recombinant pMSCV-ICOS; the latter was then packed and the high-titer virus producing cells were screened. Then CHO cell was infected by this high-titer virus and the stable cell line was screened. CHO-ICOS cells were co-cultured with PBMC (the ratio of CHO-ICOS to PBMC being 1:1,1:2,1:5, and 1:10) in presence of substimulating dose of anti-human CD3 antibody. The proliferation of PBMC and the CD25 expression on T cells were examined by 3H-TdR incorporation method and flow cytometry,respectively. CHO-pMSCV cells co-cultured with PBMC (1:1) served as the negative control and PBMC served as blank control. Results: We successfully constructed the retroviral recombinant pMSCV-ICOS and obtained CHO cell line stably expressing ICOS protein. 3H-TdR incorporation method and flow cytometry showed that, compared with the negative control group and the blank control group, co-culture with CHO-ICOS cells significantly inhibited the anti-CD3 antibody-induced activation and proliferation of PBMC(P<0.05);the maximal inhibitory rates of activation and proliferation were obtained when the ratio of CHO-ICOS to PBMC was 1:1, being (68±5.9)% and (44.08±3.26)%, respectively. Conclusion: CHO cell line stably expressing ICOS protein has been successfully established, which lays a foundation for future study.

Construction of MafA recombinant retroviral vector and its stable expression in liver epithelial progenitor cells / 第二军医大学学报

Objective: To construct a recombinant retroviral vector harboring MafA and to establish a liver epithelial progenitor cell (LEPC) line for stable expression of MafA. Methods: MafA was amplified by PCR and suncloned into pBMN-Z-IRES-Neo vector to obtain pBMH-MafA-Neo vector. After introducing the pBMN-MafA-Neo into Phoenix package cells, the cell culture supernatant was used to infect LEPCs. LEPCs stably expressing MafA gene were screened out. RT-PCR was used to detect the influence of MafA on the phenotype of LEPCs. Results: We successfully constructed pBMH-MafA-Neo vector and obtained LEPCs which stably expressed MafA. Expression of GK and GLUT2 genes in LEPCs-MafA cells was higher than that in the LEPCs. Conclusion: We have successfully obtained LEPCs which can stably express MafA,laying a basis for studying the differentiation of LEPCs into pancreas cells.

Effect of DPC4 gene transfection on chemotherapy sensitivity of pancreatic carcinoma cells / 中华肝胆外科杂志

Objective To observe the effect of DPC4 gene transfection on the chemotherapy sensitivity of pancreatic carcinoma cells. Methods The human DPC4 complementary DNA was subcloned to the retroviral vector pLXSN to obtain recombinant pLXSN/DPC4 with direct inserting potential. The daughter cell BxPC-3/DPC4 which had DPC4 stable expression was acquired after the pancreatic carcinoma BxPC-3 cells had been transfected with pLXSN/DPC4. The sensitivity of the carcinoma cells for 5-Fu and gemcitabine was observed. Meanwhile, the mRNA level of Mdr-1 and Chk1was detected by semi-quantity PCR assay. Results The 50% inhibiting concentrations (IC50)of 5-Fuand gemcitabin4e for BxPC-3 (culturing for 72 h) were rather lower than those of BxPC-3/pLXSN and BxPC-3/-cells. Moreover, the semi-quantity PCR assay revealed that the mRNA level of Mdr-1 and Chk1 was down-regulated. These findings indicated that pLXSN/DPC4 vector, 5-Fu and gemcitabine could inhibit the growth of pancreatic cancer cells. The combined therapy with pLXSN/DPC4 vector and chemotherapeutic drugs could further inhibit the growth of cancer cells. Conclusion The DPC4 gene transfection could enhance the sensitivity of pancreatic cells to chemotherapy, which may be realized through the down-regulation of Mdr-1 and Chk1 gene expression.

High-efficiency gene transfer into rabbit smooth muscle cells by pseudotyped retroviral vector / 西安交通大学学报(医学版)

Objective To construct pseudotyped retroviral vector MuLV/VSV-G and transfer it into rabbit smooth muscle cells (SMC) in order to provide a high-efficiency vector for SMC gene transfer. Methods We constructed pseudotyped retroviral vector MuLV/VSV-G containing the previously reported gene lacZ, determined the titer, and determined the efficiency of gene transfer into SMC mediated by pseudotyped retroviral vector MuLV/VSV-G. Finally the transfer efficiency was compared with that by MuLV. Results MuLV/VSV-G vector was constructed. The titer of the vector was 6-7.8×10~6CFU, the transfer efficiency was (92±12)% by using MuLV/VSV-G vector and (24±5)% by MuLV vector. Conclusion Pseudotyped retroviral vector MuLV/VSV-G which was constructed successfully is a kind of high-efficiency gene transfer vector in smooth muscle cells.

Autonomous growth of BALB/MK keratinocytes transfected with a retroviral vector carrying the human epidermal growth factor gene

Epidermal growth factor (EGF), which promotes epidermal regeneration and wound closure, is important for the proliferation and differentiation of epidermal and epithelial tissues in animals. Exogenous EGF is a promising therapeutic agent for wound healing, but its general use is restricted by the limited availability of this protein. In this work, we show that the transfection of mouse BALB/MK keratinocytes, which are totally dependent on EGF for growth and migration, with mature cDNA for human EGF via a retroviral vector abolished the cells requirement for exogenous EGF. The transformed cells had normal morphology and a growth rate that varied according to the source of the retroviral vector used. Keratinocyte transfection with EGF cDNA provides a time- and cost-efficient means of culturing keratinocytes and yields cells that may be useful for skin grafting.

Expression of Major Antigen Domains of Gene of E2 CSFV and Analysis of its Immunological Activity / 中国病毒学·英文版

E2 is an envelope glycoprotein of Classical swine fever virus (CSFV) and contains sequential neutralizing epitopes to induce virus-neutralizing antibodies and mount protective immunity in the natural host. In this study, four antigen domains (ABCD) of the E2 gene was cloned from CSFV Shimen strain into the retroviral vector pBABE puro and expressed in eukaryotic cell (PK15) by an retroviral gene expression system, and the activity of recombinant E2 protein to induce immune responses was evaluated in rabbits. The results indicated that recombinant E2 protein can be recognized by fluorescence antibodies of CSFV and CSFV positive serum (Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China) using Western blot, indirect immunofluorescence antibody test (IFAT) and ELISA, Furthermore, anti-CSFV specific antibodies and lymphocyte proliferation were elicited and increased by recombinant protein after vaccination. In the challenge test, all of rabbits vaccinated with recombinant protein and Chinese vaccine strain (C-strain) were fully protected from a rabbit spleen virus challenge. These results indicated that a retroviral-based epitope-vaccine carrying the major antigen domains of E2 is able to induce high level of epitope-specific antibodies and exhibits similar protective capability with that induced by the C-strain, and encourages further work towards the development of a vaccine against CSFV infection.

Retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP 2 in mesenchymal stem cells / 西安交通大学学报·英文版

Objective: To develop retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2 in mesenchymal stem cells. Methods: Mesenchymal stem cells from New Zealand white rabbits were transduced with retroviral pLEGFP-BMP2 vector by the optimized retroviral transduction protocol. Fluorescent microscopy's examination was to evaluate the results of the transduction, flow cytometers analysis was to evaluate the transduction efficiency and the Fluorescence-activated cell sorting method was to sort the transduced cells. Bioactivity test from C2C12K4 cells was to show the expression and bio-activity of the fusion gene. Results: Fluorescent microscopy showed the success of the transduction. By flow cytometer's analysis, the mean efficiency of the transduction with EGFP was (42. 8 ± 6. 1)% SD. Transduced cells were sorted efficiently by the fluorescence-activated cell sorting method and after sorting, almost of those showed the expression of BMP2. Fluorescently and strongly bioactivity test for C2C12 K4 cells demonstrated that fluorescent materials were located the surface of cells and the activity of luciferase increased compared with the control. Analysis of long-term expression showed there was no difference between 2 week-time point and 3 month-time point of culture post-sorting. Conclusion: Mesenchymal stem cells can be transduced efficiently by retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2, the highly pure transduced cells are obtained by the fluorescence-activated cell sorting technique, the expressed chimeric protein embraced the double bioactivity of EGFP and BMP2, and moreover, the expression had not attenuated over time.

The effect of hnRNP K shRNA on K562 cells / 重庆医科大学学报

Objective:To explore the effect of heterogeneous nuclear ribonucleoprotein K(hnRNP K)short hairpin RNA(shRNA)on K562 cells. Methods:hnRNP K shRNA retrovirus was packaged with pSUPER retro hnRNP K shRNA after its identification by enzyme digestion and sequencing analysis,then FCM,Wright staining,MTT assay,RT-PCR were used to analyze its effects on cell cycle and apoptosis,proliferation,hnRNP K and eukaryotic translation initiation factor-4E(eIF4E) mRNA of K562 cells respectively. Results:The sequence of the inserted fragment in pSUPER retro hnRNP K shRNA was correct. Compared with the control K562 cells,hnRNP K shRNA treated group showed significantly more cells at G2/M phase(P

Retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2 in mesenchymal stem cells / 药物分析学报

Objective To develop retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2 in mesenchymal stem cells. Methods Mesenchymal stem cells from New Zealand white rabbits were transduced with retroviral pLEGFP-BMP2 vector by the optimized retroviral transduction protocol. Fluorescent microscopy's examination was to evaluate the results of the transduction, flow cytometer's analysis was to evaluate the transduction efficiency and the Fluorescence-activated cell sorting method was to sort the transduced cells. Bioactivity test from C2C12K4 cells was to show the expression and bio-activity of the fusion gene. Results Fluorescent microscopy showed the success of the transduction. By flow cytometer's analysis, the mean efficiency of the transduction with EGFP was (42.8±6.1)% SD. Transduced cells were sorted efficiently by the fluorescence-activated cell sorting method and after sorting, almost of those showed the expression of BMP2. Fluorescently and strongly bioactivity test for C2C12K4 cells demonstrated that fluorescent materials were located the surface of cells and the activity of luciferase increased compared with the control. Analysis of long-term expression showed there was no difference between 2 week-time point and 3 month-time point of culture post-sorting. Conclusion Mesenchymal stem cells can be transduced efficiently by retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2, the highly pure transduced cells are obtained by the fluorescence-activated cell sorting technique, the expressed chimeric protein embraced the double bioactivity of EGFP and BMP2, and moreover, the expression had not attenuated over time.

Activation of Notch1 Signaling Inhibits Proliferation of Human Colon Carcinoma Cells via Down-regulation of Wnt / 医学研究杂志

Objective To construct the recombinant retroviruses vector PCLNRX-ICN and to explore over-activation Notch1 on the growth of human colon carcinoma cells HT-29.MethodsICN(intracellular domain of Noctch1) genes were inserted into retrovirus expression vector pCLNRX.The expression vector pCLNRX-ICN and packaging vector pCL-10A1 were co-transfected into 293 package cells.The recombinant retroviruses were used to infect HT-29 cells.After being infected,proliferation of HT-29 cells was observed by MTT assay.The expression of c-Myc and ?-catenin detected by RT-PCR and Western Blot.ResultsRecombinant retrovirus vector pCLNRX-ICN was successfully constructed.Overactivation of Notch1 by over-expressing exogenous ICN significantly inhibited the growth of HT-29 cells,and down-regulated ?-catenin,a key regulator of Wnt signaling,in protein level but not in mRNA levels.However,the mRNA or protein levels of c-Myc were not affected by overactivation of Notch1.ConclusionOver-activated Notch1 signaling could inhibit the growth of HT-29 cells partly through down-regulation of Wnt signaling independent of c-Myc inhibition.

Establishment of IBRS-2 Cell Line Stably Expressing T7 RNA Polymerase and Recovery of SVDV From IBRST7 Cells / 生物化学与生物物理进展

The bacteriophage T7 RNAP gene was amplified via PCR from -lysogen DE3, and the gene was cloned into pBABEpuro retrovial vector, a recombinant plasmid named as pT7BABEpuro was constructed and sequenced. Then the pT7BABEpuro and pVSV-G plasmids were cotransfected into GP2-293 packaging cells by liposomese, some pseudotype viruses were ingathered and transfected into IBRS-2 cell under polybrene. The IBRS-2 cell was propagated in DMEM with puromycin. The genome extraction from the cells transfected different times, the T7 RNAP gene was amplified from the genome by PCR, the mRNA of T7 RNAP protein expressed in IBRST7 cells was analyzed by RT-PCR, respectively, the results showed the T7 RNAP gene had been integrated into the chromosome of IBRS-2 cell and expressed stably at high level. To study whether T7 RNAP is of transcriptional activity in the established IBRST7 cell line, a plasmid pIERS-EGFP-ET with a reporter gene (EGFP) under control of the T7 promoter was constructed. IRES element from FMDV (for CAP-independent translation) was cloned into plasmid pET-43.1a-c(+) downstream of the T7 promoter sequence, then EGFP gene was cloned in frame downstream of the AUG codon of the FMDV IRES, resulting in the plasmid. IBRST7 cells were transfected with plasmid pIERS-EGFP-ET using lipfection, EGFP was expressed, the results showed the T7 RNAP in IBRST7 cells has transcriptional activity. IBRST7 cell line was directly transfected with linearized full-length cDNA of swine vesicular disease virus (SVDV) HK/70, infectious SVDV was efficiently recovered from the cDNA. The reverse genetic procedure is simplified to a faster, one step protocol to recover RNA virus and will be useful to understand the mechanisms of molecular pathology of RNA virus and develop effective vaccines.