Effects of anti-leukemia by antisense c-myc by retrovirus vector

AIM: To study the role of c-myc oncogene in L6565 leukemia oncogenesis and the effects of therapy by inhibition of its expression with antisense c-myc. METHODS: A recombinant retroviral vector containing antisense c-myc of the murine (pGNCas) was constructed and then transfected into PA317 cells by the method of calcium phosphate precipitation. L6565 clone cells were infected with retrovirus particles. Stable integretion of antisense c-myc was shown by PCR. The change of the malignance and phenotype of L6565as were detected by the examination of the growth, morphology, cells cycle, agar assay and expression of c-myc. RESULTS: The shape of most L6565as cells became spherical. The growth of L6565as was inhibited compared to control cells. The analysis of cells cycle: L6565as cells were arrest in G₀/G₁ phase, decreased in S phase. The ability of L6565as cells to form colony in soft agarose was significantly suppressed. c-myc in L6565as cells was lowly expressed. CONCLUSION: (1)c-myc plays a critical role in L6565 leukemia oncogenesis; (2)Inhibition of expression of c-myc makes partly reversion of malignant phenotype of L6565 murine leukemia clone cells.     

Effects of c-myb antisense RNA on the proliferation and collagen Ⅰ gene expression in cultured hepatic stellate cells

AIM:To investigate the effects of c-myb antisense RNA on the proriferation and collagen Ⅰ gene expression in cultured hepatic stellate cells(HSC) in rats.METHODS:The c-myb antisense gene recombinant retroviral vector(pDOR-myb) was constructed, and then was transfected into retroviral package cell line PA 317 by means of N-[1-(2,3-Dioleoyloxy) propyl]-N, N, N-trimethylammonium methyl-sulfate(DOTAP) liposomal transfection reagent. The pseudoviruses produced from the resistant PA317 cells selected with G418 were collected, with which HSCs isolated from rat liver were infected. The cell proliferation was measured by MTT method, c-myb, α₁-Ⅰ collagen mRNA expression and c-myb protein in HSCs were detected with semi-quantitative RT-PCR and western-blot, respectively.RESULTS:HSCs from rats were isolated successfully with the viability >98%. In the pDOR-myb infected HSCs, c-myb expression levels, the cell proliferation, and α₁-Ⅰ collagen mRNA expression were repressed significantly.CONCLUSIONS:c-myb plays a key role in the activation and proliferation of HSC. c-myb antisense RNA can inhibit cell proliferation and α₁-Ⅰ collagen mRNA expression in the infected HSC. These data suggest that inhibition of c-myb gene expression would be a potential way for the treatment of liver fibrosis.     

Effects of human growth arrest-specific homeobox gene delivery on proliferation,migration and cell cycle distribution of vascular smooth muscle cells

AIM: To explore a new gene therapeutic strategy for vein graft restenosis by investigating the effects of adenovirus-mediated human growth arrest-specific homeobox (Ad5-hGax) gene delivery on the proliferation, migration and cell cycle distribution of serum-induced rabbit vascular smooth muscle cells (VSMCs). METHODS: The recombinant adenovirus vector containing hGax gene was constructed and transfected into rabbit VSMCs. The expression of hGax in VSMCs was detected by RT-PCR and immunofluorescent staining. Methyl thiazolyl tetrazolium (MTT) assay was used to assess the effect of hGax over-expression on serum-induced proliferation of VSMCs. Wound healing method was applied to examine the distance of serum-induce VSMCs migration. Flow cytometry was employed to analyze the cell cycle distribution. RESULTS: The recombinant adenovirus vector Ad5-hGax was successfully constructed. The results of RT-PCR and immunofluorescent staining revealed that the hGax -transfected cells contained a 174 bp specific fragment of hGax gene and target protein 48 h after transfection. The proliferation of serum-induced VSMCs was significantly inhibited by overexpression of hGax gene as compared with control group. The migration of serum-induced VSMCs was inhibited after hGax gene delivery. Flow cytometry showed that 72 h after serum induction, the cells in G₀/G₁ phase in Ad5-hGax group were significantly increased, whereas the cells in G₂/M+S phase were significantly decreased. CONCLUSION: Overexpression of hGax gene inhibits the proliferation and migration of serum-induced rabbit VSMCs, and arrests the cells in G₀/G₁ phase. It is likely that hGax gene is a potential target for the gene therapy of vein graft restenosis.     

Effects of hepatitis C virus core protein on cell cycle of HepG2 cells

AIM: To investigate the molecular mechanism of hepatitis C virus (HCV) chronic infection by studying the effect of its core protein on cell growth and the expression of cell cycle regulators such as cyclin D1 and pRb/p130 in HepG2 cells. METHODS: A eukaryotic expression vector that carried a gene encoding HCV-core-1b was constructed. The cDNA of HCV core protein was subcloned into pBabe-Flag-puro vector to generate pBabe-Flag-HCV-core-1b. The plasmid was transfected into Pheonix 293T packaging cells to produce retroviruses. The virus-containing supernatant collected from the cell culture was used to infect HepG2 cells and subsequently the cell line that stably expressed the core protein of HCV was obtained.The cell cycle was analyzed by flow cytometry. The expression of cyclin D1 and pRb/p130 was examined by Western blotting. RESULTS: The pBabe-Flag-HCV-core-1b vector was confirmed by DNA sequencing. The expression of HCV gene type 1b core protein was verified by Western blotting. The overexpression of HCV gene type 1b core protein impaired the cell cycle progression in G₀/G₁ phase and significantly reduced the levels of cyclin D1 and pRb/p130 in the cells. CONCLUSION: A eukaryotic expression plasmid that contains the cDNA of HCV core protein is successfully constructed, and a HepG2 cell line which stably expressed the core protein of HCV is also established. HCV gene type 1b core protein inhibits the cell cycle possibly through down-regulation of cyclin D1 and pRb/p130 proteins in the cells.     

The killing effect of herpes simplex virus thymidine kinase/ganciclovior system on osteosarcoma cells

AIM:To investigate killing effect of herpes simplex virus thymidine kinase/ganciclovior system(HSV-TK/GCV) on osteosarcoma cell and its mechanisms. METHODS: Recombinant retroviral vector (DORHyTK) containing hygromycin phosphotransferase-thymidine kinase fusion gene(HyTK) was constructed and introduced into human osteosarcoma cell line OS732 with DOTAP. DNA and total RNA extracted from HyTK expressing cells (OS732TK) were tested by PCR and RNA dot blot analysis. A chemosensitivity of OS732TK cells to GCV and "bystander effect" were measured by means of MTT colorimetric assay. Hoeschst 332258 staining and flow cytometric analysis were performed for mechanism of HSV-TK/GCV system gene therapy. RESULTS:DORHyTK was constructed successfully; the HyTK gene existed and expressed in OS732TK cells; All OS732TK cells were killed after 5 days of exposure to 5 mg/L GCV. The "bystander effect" was observed in both a high population and a low population, but the former was stronger than the latter. Hoeschst 332258 staining revealed the characteristic hallmarks of apoptosis, however necrosis also existed. Flow cytometric analysis of DNA content showed a G₀-G₁ phase blockade. CONCLUSION:HSV-TK/GCV gene therapy system showed its strong killing effects on osteosarcoma cells OS732; The phenomenon of "bystander effect" was also very apparent. GCV exposure induced both necrotic and apoptotic death in HSV-TK expressing cells, and perturbed the cell cycle. HSV-TK/GCV gene therapy system may provide a new therapeutic approach for treatment of osteosarcoma.     

Construction and in vitro analysis of recombinant adenovirus vector carrying red fluorescent protein reporter gene

AIM: To provide important tools for gene therapy and gene vaccine research by constructing an adenovirus vector containing red fluorescent protein ( RFP ) reporter gene with the approach of in vitro recombinant ligation. METHODS: The RFP gene fragment of pTurboRFP-N was digested and ligated into pShuttle transfer vector to construct recombinant vector pShuttle-TurboRFP-N. I- Ceu I/PI- Sce I were used to double digest recombinant vector pShuttle-TurboRFP-N and backbone of vector pH5'040.pkGFP-II. The target fragment was collected and ligated, and recombinant adenovirus vector AdH5'.040.CMV.RFP-N was obtained. After linearization, the vector was transfected into AD293 cells by liposome for virus packaging. The efficiency of virus packaging and RFP expression level in AD293 cells were examined using fluorescent microscope. In addition, the biological activity and titer of the virus were tested. Human lung cancer cell line A549 and breast cancer cell line MDA-MB-231 were infected with recombinant adenovirus vector AdH5'.040.CMV.RFP-N and control adenovirus vector AdH5.CMV.EGFP respectively. The infection efficiencies of the 2 vectors to different cell lines were compared by evaluating the expression levels of RFP and enhanced green fluorescent protein (EGFP). RESULTS: The recombinant adenovirus vector AdH5'.040.CMV.RFP-N was correctly constructed and confirmed by enzyme digestion. The virus was packaged by the vector in AD293 cells and had the ability to infect the target cells. The target gene in eukaryotic cells was also expressed. The number of recombinant adenoviruses and the titer of the virus after amplification and purification were 3.6×10¹⁵ vp/L and 1×10¹³ pfu/L,respectively. The infection efficiencies of recombinant adenovirus vector Ad5'.040.CMV.RFP-N to human lung cancer cell line A549 and breast cancer cell line MDA-MB-231 were higher than those in control adenovirus vector AdH5.CMV.EGFP (P<0.05). CONCLUSION: We have constructed recombinant virus vector carrying RFP reporter gene and provide an important tool for gene therapy and gene vaccine research. The reporter gene can be highly expressed in AD293 cells and has high infection efficiency to cancer cells. RFP is a good substitution and supplement to green fluorescent protein.     

Establishment of T-lymphocytes that express CD20scFv-IgGFc-CD28-ζ and CD20scFv-IgGFc and their killing activity of B-lymphoma cells

AIM: To investigate the target killing effect of T lymphocytes with chimeric CD20scFv gene on Daudi cells and the activation of T lymphocytes. METHODS: Two kinds of plasmids were transfected into retrovirus-packed PA317 cell lines. The supernatant was collected from successfully transfected PA317 culture and was used to infect peripheral blood T lymphocytes. After one-week screening with G418, the cells were used to kill Daudi and K562 cells. The positive rates of AnnexinⅤ in Daudi cells were measured at different times points respectively by flow cytometry. Meanwhile, the level of IL-2 and IFN-γ were determined by ELISA. RESULTS: The Annexin V positive rate was significant higher in Daudi cells compared to control K562 cell lines at 24 h. No difference of AnnexinV in Daudi cells was observed in CD20 modification T lymphocyte groups. The secretions of IL-2 and IFN-γ in CD20scFv-CD80-IgGFc-CD28-ζ gene modified T cells co-cultured with Daudi cells were dramatically higher than that in CD20scFv-IgGFc group at 72 h. CONCLUSION: ① The two kinds of genetic modified specific T cells have no significant difference in inducing early apoptosis of Daudi cells. CD28-ζ cant affect Daudi cell early apoptosis at the CD20scFv target killing. ② The increase in the secretions of IL-2 and IFN-γ is more obvious in CD20scFv-IgGFc-CD28-ζ group, indicating that the self-activation takes place in CD3ζ and CD28 modified T cells without MHC restriction and then increases the activation and killing function of T cells.     

Construction of human prostate-specific membrane antigen expression vector and identification of tissue specificity

AIM: To construct a prostate-specific expression vector of promoter and enhancer of human prostate-specific membrane antigen (PSMA).METHODS: The promoter and enhancer of PSMA were amplified by PCR separately. The two segments were cloned into the expression vector pGL3-Basic, a prostate-specific expression vector pGL3-PSMP-PSME was constructed. The vector was transfected into prostatic carcinoma cell PC-3M and four kinds of non-prostatic carcinoma cells by lipofectamine. The activity of luciferase of transfected cells and tissue specificity of vector was examined at 48 hours after transfection. RESULTS: With DNA sequence of the vector pGL3-PSMP-PSME, the segment of clone was proved correct. The activity of luciferase of the pGL3-PSMP-PSME was expressed distinctly in PC-3M and was not expressed in other nonprostate cell lines. CONCLUSION: The prostate-specific expression vector was constructed successfully. It lays foundation for studying target gene therapy of the prostate cancer.     

Effects of lentivirus-mediated RWDD3 silencing on proliferation and invasion of human glioma U251 cells

AIM: To investigate the effect of RWDD3 gene silencing on the biological characteristics of human glioma U251 cells.METHODS: A lentiviral vector expressing RWDD3 shRNA was constructed and transfeeted into the U251 cells. The expression of RWDD3 at mRNA and protein levels was detected by real-time PCR and Western blot, respectively. The cell activity was determined by MTT assay. The colony formation ability was detected by the colony formation assay. The cell proliferation ability was detected by BrdU incorporation assay. The cell invasion and migration were evaluated by Transwell assay. Flow cytometry was used to monitor the changes of cell cycle distribution and apoptosis.RESULTS: Recombinant lentivirus was successfully transfected into U251 cells. Compared with the cells transfected with the scrambled shRNA and control cells, the cell activity, colony formation ability, and the invasive and migratory activities were inhibited, the cell cycle was arrested in G₀/G₁ phase, and the apoptosis was increased in the U251 cells transfected with RWDD3 shRNA(P<0.05).CONCLUSION: RWDD3 plays a vital role in proliferation and invasion of glioma cells. It may serve as a potential target of gene therapy for glioma.     

Effect of Bmi-1 gene overexpression on proliferation of human gastric mucosal epithelial cell line GES-1

AIM: To investigate the effect of B lymphoma Moloney murine leukemia virus insertion region 1 ( Bmi-1 ) gene overexpression on the proliferation of a human normal gastric epithelial cell line GES-1. METHODS: The plasmid containing Bmi-1 gene or empty plasmid was transfected into GES-1 cells by retroviral mediation. The expression of Bmi-1 at mRNA and protein levels was detected by quantitative real-time PCR (qRT-PCR) analysis and Western blotting, respectively. The effect of Bmi-1 gene overexpression on the cell cycle of GES-1 cells was evaluated by flow cytometry. The proliferation of the stably transfected cells was measured by Cell Counting Kit-8. RESULTS: The results of qRT-PCR analysis and Western blotting demonstrated that stably transfected cell line was successfully established. The results of flow cytometry analysis showed that overexpression of Bmi-1 reduced the G₀/G₁ phase, arrested the cells in G₂/M phase and S phase. The growth curve showed that overexpression of Bmi-1 resulted in increased growth speed. CONCLUSION: Increase in Bmi-1 gene expression regulates the cell cycle and promotes the proliferation of GES-1 cells.     

Effects of WNT5B over-expression on viability and apoptosis of human breast cancer cell line MCF-7

AIM: To detect the expression of WNT5B in normal breast epithelial cells and different breast cancer cell lines, and to investigate the effects of WNT5B over-expression on the viability and apoptosis of human breast cell line MCF-7.METHODS: The mRNA expression of WNT5B was detected by RT-PCR in different breast cancer cells. MCF-7 cells were transfected with plasmid pcDNA3.1/WNT5B or pcDNA3.1, and the expression of WNT5B at mRNA and protein levels was examined in the 2 groups by real-time PCR and Western blotting, respectively. Subsequently, the changes of cell viability and cell apoptosis were analyzed by CCK-8 assay and flow cytometry, respectively. RESULTS: The expression of WNT5B in the breast cancer cell lines was lower than that in MCF10A cells. The WNT5B expression in the MCF-7 cells in experimental group was significantly higher than that in vector group (P<0.05). However, the cell viability in experimental group decreased significantly as compared with vector group (P<0.05). The number of the cells in S-phase obviously increased, while the percentage of the cells in G₁-phase and G₂/M-phase decreased compared with vector group. The number of apoptotic cells in WNT5B group was significantly higher than that in vector group.CONCLUSION: The expression of WNT5B is decreased in breast cancer cells. WNT5B over-expression significantly inhibits the cell growth and promotes the cell apoptosis in breast cancer MCF7 cells.     

Preparation of gfp-bcl-XL-contained recombinant adenovirus vector by the homologous recombination in bacteria

AIM: To prepare gfp-bcl-X_L-contained recombinant adenovirus(rAd-gfp-bcl-X_L).METHODS: Bcl-X_L gene was amplified from pEGFP-C₃-bcl-X_L, subcloned into shuttle plasmid and formed transfer plasmid of pAdTrack-CMV-bcl-X_L. Then pAdTrack-CMV-bcl-X_L was linealinzed with PmeI and co-transformed into BJ5183 bacteria with adenovirus genomic plasmid of pAdEasy-1. The identified recombinant adenovirus plasmid was digested with PacI and transfected into 293 cells to package recombinant adenovirus particles. The target gene was detected by PCR.RESULTS: There were about 35% positive recombinant bacterial clones after the co-transformation of pAdTrack-CMV-bcl-X_L and pAdEasy-1 into BJ5183. Recombinant adenovirus particle were produced and further amplified after the transfection of pAdEasy-1-gfp-bcl-X_L into 293 cells. PCR test indicated that the recombinant Ad contained bcl-X_L gene. The titer of the purified rAd-gfp-bcl-X_L was 6.5×10¹² PFU/L. CONCLUSIONS: The homologous recombination in bacteria is a convenient and high efficient method to prepare rAd-gfp-bcl-X_L. This affords a good gene transfer vector for the gene therapy in human’s diseases.     

Construction of FAK-shRNA retroviral vector and screening of stable HCC-LM3 cell line with persistent knockdown of FAK

AIM: To construct a recombinant retroviral vector of short interfering RNA targeting focal adhesion kinase (FAK) gene and to establish a cell line with stable knockdown of FAK.METHODS: The oligonucleotides that transcribed to short hairpin RNA (shRNA) targeting FAK gene were synthesized in vitro, cloned into retroviral vector pSuper.retro and transfected into Phoenix cell line. The stable clones were screened and high-titer virus was produced. The human hepatocellular carcinoma cell line HCC-LM3 was infected with the virus-rich supernatant. The stable LM3 cell line, which showed significantly to silence FAK and associated proteins, was selected by puromycin.RESULTS: The recombinant retroviral vector was successfully constructed. Persistent knockdown of FAK in the LM3 cell line infected with the supernatant containing the retrovirus was confirmed by Western blotting. Down-regulation of FAK resulted in the inhibition of p-Akt and p-MAPK1/2 expression and led to decreased migration and invasion of the cells. The cell cycle was blocked at G₀/G₁ phase, and apoptosis was increased. The proliferation rate also decreased significantly.CONCLUSION: FAK-shRNA virus generated by recombinant retroviral vector pSuper-FAK can inhibit the protein expression of FAK and phosphorylation of Akt and MAPK1/2 in HCC-LM3 cells. Down-regulation of FAK shows a significant impact on biological behaviors of tumor cells.     

Inhibitory effects of tumor associated mitochondrial protein 12 on HepG2 cell apoptosis

AIM: To explore the inhibitory effects of tumor associated mitochondrial protein 12 (TAMP12) on tumor cell apoptosis. METHODS: (1) A retrovirus expression vector was recombinated and transfected into the packaging cell line PA317. The virus particles were obtained to infect the target cell line HepG2 low expressing of TAMP12. The expression of TAMP12 mRNA was detected by RT-PCR. The subcellular localization and quantification of TAMP12 protein labeled with double fluorescein were observed under confocal laser scanning microscope (CLSM). (2) Hoechst33258 staining and flow cytometry (FACS) were used to analysis the apoptosis of HepG2 cells treated with 5-fluorouracil (5-FU). RESULTS: (1) The CLSM observation showed that TAMP12 protein was mainly expressed in mitochondria of HepG2 cells. The expressions of TAMP12 gene and protein were stable and high in transfected HepG2 cells. (2) Upon treatment with 5-FU, the transfected HepG2 cells showed a fairly integrated nucelus while the control HepG2 cells exhibited chromatin condensation, marginalization and karyorhexix. Moreover, the apoptosis rate of transfeced HepG2 cells was significantly lower than that in control HepG2 cells (P<0.05 or P<0.01) by FACS. CONCLUSION: TAMP 12 is an anti-apoptotic element in 5-FU induced HepG2 cell apoptosis.     

Expression of human inducible nitric oxide synthase in fibroblast cell line V79 and effects of H4Bon iNOS activity

AIM: To explore the feasibility of human iNOS transfected into V₇₉ cells by gene transfer and investigate the effects of H₄B on iNOS activity. METHODS: Human iNOS was transfected into V₇₉ cells with the karyocyte expressive vector. The cloned cells were selected by G₄₁₈. The expression of iNOS mRNA was quantified by RT-PCR and iNOS expression was observed by immunofluorescence. NO product in cells was determined by measuring nitrite (NO^-₂) release using the Griess reaction. RESULTS: V₇₉ cells infected human iNOS was proved to have iNOS mRNA at 462 bp by RT-PCR, and iNOS protein in the cytochylema by immunofluorescence. When the cells were incubated without H₄B, the content of NO in pcDNA₃ cells was minimal, with NO^-₂ production (82.32±13.08) just above the normal group (74 38±9 80, P>0.05, n=6) There was no significant difference between pcDNA₃ cells incubated with or without H₄B, (P>0.05, n=6) NO^-₂ production by pcDNA₃-iNOS cells without H₄B was higher (105 58±13 33) (n=6, P<0.01vs the normal cells or pcDNA cells). However, in pcDNA₃-iNOS cells incubated with H₄B, NO^-₂ production was much higher (236 57±3183) (n=6, P<0.01vs the all former groups). CONCLUSION: iNOS activity was increased by adding H₄B in pcDNA₃-iNOS cells, and the fibroblast can be a target cell of iNOS gene transfer.     

Effects of xeroderma pigmentosum group D and p53 on replication of HBV

AIM:To investigate the effects of xeroderma pigmentosum D (XPD) and p53 on the replication of hepatitis B virus (HBV). METHODS:Recombinant plasmid pEGFP-N2/XPD and vacant vector plasmid pEGFP-N2 were transfected into HepG2.2.15 cells by liposome. On the next day, these cells were incubated with pifithrin-α, a p53 inhibitor, at a concentration of 20 μmol/L for 24 h. The cells were divided into 5 groups: blank control group, pEGFP-N2 group, pEGFP-N2/XPD group, pEGFP-N2/XPD+pifithrin-α group and pifithrin-α group. The mRNA expression of XPD, hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg) and hepatitis B virus X protein (HBx) was detected by RT-PCR. The content of HBsAg and HBeAg in the supernatants of culture medium was measured by ELISA. The content of HBV-DNA in the supernatants of culture medium was examined by fluorescence quantitative PCR. Using the method of bDNA, the content of HBV-DNA in the core particles was assessed. RESULTS:The expression of XPD mRNA was elevated by the transfection of recombinant plasmid pEGFP-N2/XPD. The increase in XPD expression significantly down-regulated the mRNA expression of HBsAg, HBeAg and HBx. The content of HBsAg and HBeAg in the supernatants of culture medium was significantly decreased by the increase in XPD expression. The results of fluorescence quantitative PCR showed that the content of HBV-DNA in the supernatants of culture medium was significantly down-regulated by the increase in XPD expression. bDNA results showed that the content of HBV-DNA in the core particles was significantly decreased by the increase in XPD expression.Pifithrin-α abolished the above-mentioned effects of XPD (all P<0.01). CONCLUSION: XPD inhibits the replication of HBV through p53 pathway. Therefore, XPD and p53 may be the targets for antiviral therapy of hepatitis B.     

Construction and expression of retroviral vector containing mIL-12 gene

AIM: To construct retroviral expression vector pLXmIL-12SN containing mouse IL-12 (mIL-12) gene and detect the gene expression in B16F10 cells. METHODS: Vector pGCp35-IRES-p40SN was digested to obtain p35-IRES-p40 (mIL-12 gene fragment) and retroviral vector pLXSN was digested by restriction enzyme, and then they were linked by ligase. The recombinant vector that mIL-12 gene fragment had correctly been inserted into pLXSN was identified by sequencing. The recombination vector with mIL-12 gene was transfected into B16F10 cell and was detected by RT-PCR. RESULTS: Expression vector pLXmIL-12SN was successfully constructed. mIL-12 gene was confirmed to express in B16F10 cells at the level of mRNA. CONCLUSION: Acquired vector pLXmIL-12SN and confirmed mIL-12 gene expression lays a foundation for mIL-12 gene therapy to eye diseases.     

High-efficient genetic transfection of CD41+,UT7, U937 and MDA-MB-435 cells with a recombined murine stem cell retroviral vector

AIM: Gene transduction with a recombined murine stem cell retroviral vector has been investigated to find an effective way of gene transduction and to offer theory and experimental basis for the recombined murine stem cell retroviral vector used for gene transduction. METHODS: 1. Construction of retrovirus vector: EC1-4 gene (repeats 1-4 of cadher in-5 extracellular domain) and mutant (Ser 222A) MEK1 gene were cloned into retrovirus vector pMSCV after cut by Bgl Ⅱ and EcoR 1 restriction endonuclease. 2. Obtaining CD41⁺ cells and cell culture: Cells expressing CD34⁺ from cord blood were isolated. The inducement of cells expressing CD41 from CD34⁺ cells was performed by using TPO and cells CD41⁺ were selected by FACS. NIH 3T3 cells were cultured in high sugar DMEM medium and U937 in RPMI 1640 medium. UT7 cells which is cytokine-dependent cell line were grown in Iscove's modified Dulbeco's medium supplemented by GM-CSF. 3. Determination of viral titers: Retroviral vectors were transferred to packaging cell line 293. Retroviral containing supenatant was collected after transfection. The viral titers was tested on infection of NIH 3T3 cells by FACS analysis. 4. Western blot: Transduced CD41⁺, UT7, U937 and MDA-MB-435 cells were analyzed by western blot to examine expression of transduced genes. RESULTS: A packaging cell line 293 produces high-titer MEK1 pMSCV retroviruses (3.1×10⁷) and EC1-4 pMSCV retroviruses (1.0×10⁸). With 8-folds dilution retroviruses, 60.73% GFP positive cells have been obtained in MEK1 pMSCV transduced UT7 cells, 72.56% in U937 cells and 30.57% in CD41⁺ cells, respectively. GFP positive cells have reached up 97.54 % in EC1-4 pMSCV transducted MDA-MB-435 cells. Phosphorylated MEK1 has been decreased in experiment group when TPO has stimulated CD41⁺ and UT7 cells or serum has stimulated U973 cells. This indicates that is a dominant negative effect of mutation MEK gene. EC1-4 gene transduced MDA-MB-435 cells have expressed EC1-4. CONCLUSION:The recombined murine stem cell retrovirus can effectively mediate gene transduction of CD41⁺,UT7,U937 and MDA-MB-435 cells,and transduced genes can be stably expressed.     

Inhibitory effect of 1,25-(OH)2D3 on proliferation of airway smooth muscle cells from passively-sensitized patients

AIM: To investigate the effects of 1,25-dihydroxyvitamin D₃ on the proliferation of passively-sensitized human airway smooth muscle cells (HASMCs), and to explore its potential role in asthmatic airway remodeling.METHODS: HASMCs were passively sensitized with 10% serum from asthmatic patients.1,25-(OH)₂D₃ was used as the interventor.The effect of 1,25-(OH)₂D₃ on the cell proliferation and its optimal concentration were determined by MTT colorimetric assay.The cell cycle analysis was performed by flow cytometry.The expression of proliferating cell nuclear antigen (PCNA) was measured by the method of immunocytochemical staining.RESULTS: 1,25-(OH)₂D₃ at the concentrations of 10^-9-10^-7 mol/L markedly inhibited the cell proliferation and the maximum effect was observed at the concentration of 10^-7 mol/L.This concentration of 1,25-(OH)₂D₃ markedly suppressed the PCNA-positive rate and hampered the G₁/S transition in HASMCs passively-sensitized by asthmatic serum.CONCLUSION: 1,25-(OH)₂D₃ has direct inhibitory effects on the proliferation of passively-sensitized HASMCs in vitro, which may be concerned with the beneficial role of 1,25-(OH)₂D₃ on the prevention and therapy of asthmatic airway remodeling.