Non-transfected Group Research Articles

Potential of nucleofected human MSCs for insulin secretion

The goal of this experiment was to generate insulin-producing human mesenchymal stem cells (hMSCs) as a therapeutic source for type I diabetes mellitus, which is caused by insulin deficiency due to the destruction of islet β cells. In various trials for the treatment of type I diabetes, cell-based therapy using adult stem cells is considered to be one of the most useful candidates for the treatment. In this experiment, a non-viral method called nucleofection was used to transfect hMSCs with pEGFP-C2 and furin-cleavable human preproinsulin gene (hPPI) to produce insulin-secreting cells as surrogate β cells. Transfection efficiency was determined using flow cytometry analysis. Expression and production of insulin were tested using RT-PCR and ELISA. The expression, production and maturation of insulin from the genetically engineered hMSCs showed an increase when compared with a non-transfected control group. Insulin expression from hMSCs using nucleofection in this study has shown the potential for type I diabetes therapy. For further study, an evaluation for in vivo experiments and clinical applications must be supplemented.

Effect of hypoxia-inducible VEGF gene expression on revascularization and graft function in mouse islet transplantation

For gene transfer strategies to improve islet engraftment, vascular endothelial growth factor (VEGF) expression should be regulated in a way that matches the transient nature of revascularization with simultaneously avoiding undesirable effects of overexpression. The aim of this study was to investigate the effects of hypoxia-inducible VEGF gene transfer using the RTP801 promoter on islet grafts. We implanted pSV-hVEGF transfected, pRTP801-hVEGF transfected or nontransfected mouse islets under the kidney capsule of streptozotocin-induced diabetic syngeneic mice. Human VEGF immunostaining of day 3 grafts revealed that the pRTP801-hVEGF transfected group had higher hVEGF expression compared with the pSV-hVEGF transfected group. BS-1 staining of day 3 grafts from the pRTP801-hVEGF transfected group showed the highest vascular density, which was comparable with day 6 grafts from the nontransfected group. In 360 islet equivalent (IEQ)-transplantation which reverted hyperglycemia in all mice, the area under the curve of glucose levels during intraperitoneal glucose tolerance test 7 weeks post-transplant was lower in mice transplanted with pRTP801-hVEGF transfected grafts compared with mice transplanted with nontransfected grafts. In 220 IEQ-transplantations, diabetic mice transplanted with pRTP801-hVEGF islets became normoglycemic more rapidly compared with mice transplanted with pSV-hVEGF or nontransfected islets, and diabetes reversal rate after 50 days was 90%, 68%, and 50%, respectively. In conclusion, our results indicate that regulated overexpression of hVEGF in a hypoxia-inducible manner enhances islet vascular engraftment and preserves islet function overtime in transplants.

Influences of steroidogenic factor-1 gene silencing on angiotensin II -mediated aldosterone secretion

Objective To observe the changes of CYP11B2 mRNA and aldosterone secretion in human adrenocortical carcinoma H295R cells after angiotensin Ⅱ (AT Ⅱ ) stimulation, and investigate the effects of steroidogenic factor-1 ( SF-1 ) gene silencing. Methods The plasmids pGenesil1-SF-1-shRNA containing U6 promoter and SF-1 -specific short hairpin RNA (shRNA) or pGenesil1 -negative-shRNA containing unspecific shRNA were transfected into the H295R cells. The expression of SF-1 was detected by Western blotting and real-time polymerase chain reaction (RT-PCR). CYP11B2 mRNA was detected at 3,6, 9, 12, 15, 18 h after ATⅡ stimulation ( 100 nmol/L), and the maximal amplitude was compared between two groups. Aldosterone was measured by radioimmunoassay at 3, 6, 9, 12, 15, 18 h after AT Ⅱ stimulation ( 100 nmol/L), and the amplitude of aldosterone was compared between two groups. Results As compared with those in control group, the protein and mRNA levels in SF-1-transfected group were reduced by 69.07% and 71.20% respectively. The highest CYP11B2 mRNA level was detected at 12 h after ATⅡ stimulation, and the amplitude in SF-1-transfected group and non-transfection group was 50. 21 fold and 800.09 fold respectively compared to that of cells without ATⅡ stimulation ( P ＜ 0.01 ). Similarly, aldosterone secretion in control group was (0. 061 ± 0. 007 ) and (0. 256 ± 0.014) μg/L before and after ATⅡ intervention (P＜0. 01), and that in SF-1-transfected group was (0. 101 ±0.010) and (0.252 ±0.016) μg/L (P ＜ 0. 01 ) respectively. Conclusion Down-regulation of SF-1 decreased the sensitivity and response of CYP11B2 mRNA and aldosterone to ATⅡ stimulation. Key words: Steroidogenic factor-1; H295R cell; AngiotensinⅡ; Aldosterone

Urokinase Receptor Gene Expression Inhibited by siRNA Cocktails in Co-culture of Spermatogenic Cells with Sertoli Cells from 3-Week-Old Rat

Objective To study the functions of urokinase receptor (uPAR) during spermatogenesis by adding targeted siRNA cocktails to the co-culture of Sertoli cell with spermatogenic cells. Methods The seminiferous tubule samples from SD rats aged 20-23 d were digested with collagenase for 15-20 min, then were cut into small fragments. Tubular fragments were digested with collagenase again for 5-10 min, then gently resuspended in F12/DMEM supplemented with vitamins and hormones. Cell samples were seeded in plate and cultured at 32°C for 2 weeks. After 48 h, siRNA cocktails at a concentration of 15 nmol/L or 30 nmol/L were introduced into this cocultured Sertoli/spermatogenic cells. After then for 48 h, expression of uPAR mRNA was analyzed by real-time RT-PCR. Results Some spermatogenic cells took place morphologic changes and generated an obvious flagellum. The expression levels of uPAR mRNA silencing with siRNA cocktails at a concentration of 15 nmol/L or 30 nmol/L were significantly lower than those in nontransfected group (P Conclusion The meiosis and spermiogenesis could take place in this culture system, and the siRNA cocktails can effectively inhibit the gene expression of uPAR in co-cultured Sertoli/spermatogenic cells.

Effect of siRNA-mediated beta-catenin gene on Wnt signal pathway in lung adenocarcinoma A549 cell

To study the effects of siRNA-mediated beta-catenin down-regulation on Wnt/beta-catenin signal transduction pathway in lung adenocarcinoma A549 cells. A549 cell was divided into three groups: PGCsil-CTNNB1-siRNA group (transfection with beta-catenin interference plasmids), negative control group (transfection with negative control plasmids) and non-transfection group (blank control). For each group, real-time PCR was employed to detect the expression of beta-catenin and cyclin D1 (a target gene of Wnt/beta-catenin pathway). A MTT cell proliferation assay was performed to study the proliferating capacity. Flow cytometry was used for cell cycle analysis. Clone formation and Millicell chamber experiment were performed to evaluate the clone formation and migration capacities of cells. The expression of beta-catenin and cyclin D1 in PGCsil-CTNNB1-siRNA cell decreased at the mRNA level. It was lower than negative control (0.002+/-0.001 vs 0.023+/-0.002, P<0.01; 0.005+/-0.002 vs 0.040+/-0.020, P<0.05) and blank control groups (beta-catenin mRNA: 0.021+/-0.003, P<0.01; cyclin D1 mRNA: 0.042+/-0.004, P<0.05). Also the proliferating capacity at Days 5-7 was inhibited in comparison with negative control and blank control groups (P<0.05, P<0.01). The cell-doubling time (58.1 h) was markedly longer than those of negative control group (37.9 h, P<0.05) and blank control group (34.2 h, P<0.05). The number of cells in G0-G1 phrase increased in PGCsil-CTNNB1-siRNA group (86.4%+/-2.6%) in comparison with negative control (73.8%+/-0.9%, P<0.01) and blank control groups (75.8%+/-1.5%, P<0.01). In PGCsil-CTNNB1-siRNA group, the clone formation ratio (31.6%+/-7.7%) was lower than negative control (46.9%+/-7.3%, P<0.05) and blank control groups (44.2%+/-2.5%, P<0.05). And the number of cells (16.0+/-3.8) passing through the membrane of Millicell chamber was smaller than negative control (32.7+/-3.1, P<0.01) and blank control groups (33.0+/-2.7, P<0.01). Knocking down beta-catenin not only inhibits the Wnt/beta-catenin signal transduction pathway in lung adenocarcinoma A549 cells but also decreases cell proliferation, clone formation and migration capacity. Thus it may become a novel target for lung cancer therapy.

Effect of metastasis suppressor gene Kai1/CD82 on proliferation of laryngo-carcinoma Hep-2 cell line

Objective To study the effect of metastasis suppressor gene Kai1/CD82 on the cell proliferation of Hep-2 cell line.Methods Recombinant adenovirus rAd-Kai1 was amplified,purified,and was used to transfect laryngo-carcinoma Hep-2 cell line.Cells transfected with blank vector and untransfected cells served as controls.MTT assay was used to assess the influence of Kai1 gene on proliferation of Hep-2 cells;RT-PCR was used to examine the expression of Kai1 gene in cells of different groups.Results The titer of rAd-Kai1 reached 6×1010PFU/ml after expansion and purification.When the concentration of the adenovirus was 30 MOI,the transfection rate of Hep-2 cells could be higher than 90%.MTT assay showed that the cell proliferation abilities were similar in the two control groups,and the proliferation ability of cells in the Kai1 transfection group was significantly lower than that in the non-transfected group(P0.05),with the inhibitory rate in the Kai1 group being 29%.Conclusion The Kai1/CD82 gene can inhibit the proliferation of Hep-2 cells.

Over-expression of the Beclin1 gene upregulates chemosensitivity to anti-cancer drugs by enhancing therapy-induced apoptosis in cervix squamous carcinoma CaSki cells

The purpose of this study was to investigate whether the autophagy-related gene, Beclin1, plays a role in the regulation of chemosensitivity to anti-cancer drugs in cervical cancer CaSki cells. Expression of the Beclin1 protein was up-regulated in pcDNA3.1-Bec transfectants and led to cell arrest in the G 0/ G 1 phase of the cell cycle. The MTT assay indicated that over-expression of Beclin1 sensitized CaSki cells to chemotherapeutic drugs (cisplatin, paclitaxel, 5-fluorouracil, and epirubicin) and induced greater degrees of cytotoxicity than vector-only controls. After treatment with anti-cancer drugs, flow cytometric analysis indicated that the Beclin1-transfected group showed a greater increase in apoptosis than did the non-transfected group. Furthermore, pSUPER-Bec transfectants did not lead to a significant increase of resistance to each of these anti-cancer drugs. These results suggest that Beclin1 plays an important role in the regulation of potent anti-tumor activity, and over-expression of Beclin1 in CaSki cells may enhance apoptosis signaling induced by anti-cancer drugs.

Effect of RNA interference targeting brain-derived neurotrophic factor gene on HeLa cell proliferation and apoptosis

To screen effective sequences of short hairpin RNA on brain-derived neurotrophic factor (BDNF) gene and the effect of RNA interference on the proliferation and apoptosis of HeLa cells, a cervix carcinoma cell line with high expression of BDNF. Two recombinant eukaryotic human-BDNF siRNA expression vectors were designed and constructed. Sequences were confirmed by restrictive endonuclease digestion and DNA sequencing. The empty vector pGenesil-1 and two recombinant plasmids, pGenesil-shRNA-BDNF1 and pGenesil-shRNA-BDNF2, were transfected into HeLa cells using Lipofectamine 2000 (groups: P(0), P(1) and P(2), respectively). The mRNA and protein levels of BDNF in HeLa cells were detected by RT-PCR and Western blot, respectively. The cellular proliferation rates were determined by MTT assay and the apoptotic rates were measured by flow cytometry and Hoechest 33258. The recombinant eukaryotic BDNF siRNA expression vectors were successfully constructed. The expression of mRNA and protein of BDNF in P(1) group were significantly decreased, comparing with non-transfected group, P(0) and P(2) groups (F = 48.19, P < 0.01). P(2) group failed to meet the expected results (P > 0.05). In addition, the proliferation activity was reduced in P(1) group and the peak point of proliferation curve was prolonged. Moreover, the early cell apoptotic rates were statistically increased in P(1)[(53.4 +/- 4.2)%] VS. non-transfected [(0.8 +/- 0.4)%], P(0) [(5.1 +/- 1.8)%] and P(2)[(7.9 +/- 2.4)%] groups (F = 269.77, P < 0.01). HeLa cells express a high level of BDNF. BDNF gene silencing by RNA interference increases the apoptosis of HeLa cells and inhibits cell proliferation, offering a possible target for efficient tumor therapy.

Beclin1 Induces Autophagy and its Potential Contributions to Sensitizes SiHa Cells to Carboplatin Therapy

To investigate the role of Beclin1 in the relationship between autophagy and apoptosis after carboplatin treatment. After overexpression or partial silencing of Beclin1 in cervical cancer SiHa cells, the transfected group and the control group were treated with carboplatin for 48 hours. The expression of Beclin1 and LC3 was measured using a western blot. The ultrastructural analysis was conducted with an electron microscope. Fluorescent changes in autophagic cells were observed using reverse fluorescent microscopy. The percentage of apoptotic cells and autophagic cells and cell proliferation were assessed by flow cytometry and MTT assay. Expression of LC3 and Beclin1 protein was up-regulated in overexpressed transfectants of SiHa cells. After treatment with carboplatin for 48 hours, the flow cytometric analysis indicated that the Beclin1 transfected group showed a greater increase in apoptosis and autophagic cells than did the nontransfected group. MTT assay showed that carboplatin inhibited proliferation of SiHa cells in Beclin1-overexpressed transfectant cells more than in nontransfectant cells. These results suggest that autophagy and apoptosis had differential contributions to carboplatin-induced death of cervical cancer SiHa cells. Overexpression of Beclin1 in SiHa cells may enhance apoptosis signaling induced by carboplatin.

Apoptosis of HepG2 cells after transfection with LIGHT gene and interferon-γ.

Objective To investigate apoptosis of HepG2 ceils after transfecfion with LIGHT gene and interferon-γ. Methods LIGHT gene and interferon-γ were transfected into HepG2 cells by liposome mediated method. The HepG2 cells were divided into group A (transfected with LIGHT gene or interferon-γ), group B (transfeeted with LIGHT gene and interferon-γ) and group C (non-transfection group). The apoptosis rate of the HepG2 cells and the expression of Bcl-2 and Caspase-8 were detected 12, 24, 48 hours after transfeetion. Results (1) The apoptosis rates of HepG2 cells at hour 12, 24 and 48 after transfeetion were 18.8% ± 3.5%, 25.7%± 2.8% and 36.4% ±3.6% in group A, 23.8% ±2.4%, 31.1% ±2.1% and42.5% ±4.5% in group B, and 8.7% ± 2.1%, 9.3% ± 1.6% and 10.9% ± 1.2% in group C. There was a significant difference in apoptosis rate among the 3 groups (F = 15.69, 53.33, 48.28, P < 0.01). (2) The expression of Bcl-2 in HepG2 cells at hour 12, 24 and 48 after transfection was 16.4% ± 5.0%, 13.4% ± 3.5% and 8.6% ± 2.3% in group A, 14.7%±3.8%, 9.1% ±2.0% and 4.6% ±2.0% in group B, and 25.3% ±6. 3%, 19.8% ±4.4% and 10.1% ±3.8% in group C. There was a significant difference in the expression of Bcl-2 among the 3 groups (F = 6.19, 12.29, 5.81, P <0.05). (3) The expression of Caspase-8 at hour 12, 24 and48 after transfection were 19.3% ±2.4%, 27.2% ±1.9% and 33.7% ±3.0% in group A, 22.7% ±2.2%, 30.9% ±3.1% and 38.2% ±3.2% in group B, and 1.2% ±0.8%, 1.8% ±0.6% and 3.2% ±1.5% in group C. There was a significant difference in the expression of Caspase-8 among the 3 groups (F =71.54, 112. 78, I01.61, P < 0.01). Condusions LIGHT gene can signiticanfly promote cell apoptosis through regulating the expression of Bcl-2 and Caspase-8. Interferon-γ enhanced the effect of LIGHT gene on the apoptosis of HepG2 cells. Key words: Carcinoma,hepatocellulur; HepG2 cells; LIGHT gene; Intederon-γ; Apoptosis

Proliferation and differentiation of precartilaginous stem cells in response to stable expression of hTGF-β3

Objective To study the effect of stable expression of reconstructed human transforming growth factor-β3 ( hTGF-β3) on proliferation of precartilaginous stem cells ( PSCs) and their differentiation into cartilage chondrocytes.Methods After isolated and purified by immunological microbeads,PSCs of rats were transfected with pcDNA3.1 ( + )-hTGF-β3.MTT reduction assay ( MTT) and flow cytometry were performed to investigate the effect of transduction on proliferation and DNA synthesis.Biosynthesis of hTGF-β3 and expressions of cartilage associated genes and proteins were examined by qRT-PCR,immunohistology and Western blot.Results hTGF-β3 was expressed in PSCs stably.Compared with non-transfection group,PSCs' DNA synthesis level and proliferation rate were significantly increased after transfection.Quantitative real-time PCR and immunological investigation suggested up-regulated expression of specific genes and proteins of chondrocyte and increase of deposition of chondrocyte typical extracellular matrices proteoglycan and collagen type Ⅱ .Conclusions Gene enhanced PSCs can stably express hTGF-β3 protein to promote proliferation of PSCs and induce differentiation of PSCs in to chondrocytes,which provides a fresh approach to cartilage tissue engineering. Key words: Transforming growth factor β; Stem cells; Transfection; Cell differentiation

The effect of mitofusin-2 gene on the neoplasia,proliferation and metastasis of human breast carci-noma cells in vivo

Objective To investigate the effects of Mitofusin-2 gene (mfn2) on the neoplasia, proliferation and metastasis of Human breast carcinoma ceils in vivo. Methods The cells were divided in-to three groups:non-transfection group as negative control group, pEGFP-C2 transfection group as experi-mental control group, and pEGFPmfn2 transfection group as experimental group. Fifteen nude mice were randomly divided into 3 pups:the experimental group, the experimental control group, and the negative groups that were heterografted with three groups' cells respectively. The expression of mfn2 was detected by RT-PCR in tumor tissues. The expression of Ki-67 and VEGF was detected by immunohistochemistry. Results The tumor weight in negative control, experimental control and experimental groups was (2.77± 0.12), (2.84±0.16) and (1.78±0.13) g, respectively. The tumor weight in experimental group was significantly lighter than in two control groups (P<0.05). The tumor volume in experimental group was markedly less than in experimental control and negative control groups (P<0.05). The expression of mfn2 in experimental group was higher than in experimental control and negative control groups (P< 0.05). The expression of Ki-67 in experimental group was higher than in experimental control and negative control groups (P<0.05),but the expression of VEGF in experimental group was lower than in experi-mental control and negative control groups (P<0.05). Conclusion Exogenous mfn2 can strongly inhibit proliferation and metastatic progression of breast carcinoma MCF-7 in xenograft implantation model. Key words: Mitofusin-2 gane; Neoplasia; Gene therapy

Effect of ROR γt on regulation of IFN-γ secretion of T cell subpopulations (90.15)

Abstract It has been known that retinoid-related orphan receptor γt (RORγt) is a key transcription factor for development of IL-17 producing CD4 T cells (Th17), which are negatively regulated by interferon-γ (IFN-γ) produced by Th1 cells, however, whether the RORγt regulates IFN-γ production in T cell subsets has not been elucidated. In our study the peripheral blood mononuclear cells isolated from peripheral blood of healthy human donors were stimulated with CD3 mAB and cultured in IL-2 containing medium for 12 days. The activated T cells were transfected with three chemical synthesized small interfering RNA (siRNA) sequences for RORγt gene using liposomes. By using semi-quantity RT-PCR assay, the RORγt gene expression was inhibited in activated T cells that transfected with RORγt-siRNA-1026 and -1197, but was not inhibited in that with RORγt-siRNA-982. By using flowcytometry, the percentages of IFN-γ producing cells in total T cells, CD4+ T cells and CD8+ T cells that transfected with the sequence of RORγt-siRNA-1026, significantly decreased to 35.19, 32.78, and 41.84, respectively, in comparison of nontransfected group (43.39, 44.54, and 50.04, respectively) (p&amp;lt; 0.05). In conclusions, the transcription factor RORγt may also be involved in the positive regulation for IFN-γ production in human T cells, especially in CD4+ T cells.

Influence of constitutively active Akt1 gene in combination with CTLA-4Ig on xeno-islet transplantation in diabetic mice

Objective To explore the effects of transiection of constitutively acUve Aktl into rat islets on xeno-islet transplantation. Methods Rat islets were separated and purificated, and then transfected with Adv-CA-Aktl. AO/EB stain was used to examine the survival,and TUNEL test to detect the apoptosis of islets. And 300 IEQs islets were transplanted under the kidney capsule of BALB/C diabetic mice. The mice were divided into 4 groups in random. In experimental group, transfection of Aktl gene into islets in combination with intraperitoneal injection of CTLA4Ig;in Aktl group：transfection of Aktl gene into islets only ; in CTLA4 Ig group ： intraperitoneal injection of CFLA4Ig only ; in nontransfected group ： simple islet transplantation. Blood glucose level and graft survive time were determined. Results The islets with Aktl gene gained more ability to survive in vitro, and apoptosis ratio was decreased by 25%. In experimental group, the glucose level was decreased and normal in longer term （ 32.5±6.6） days, and the life splan （ 39.6 ± 5.9 ） days was longer obviously than the other groups [ Aktl group ： （ 15.4 ± 3.5 ）, （ 22.5 ± 1.7 ） days ; CTLA-4Ig group ： （ 18.7 ±4.5 ）, （ 21.7 ±3.5 ） days ; nontransfected group ： （ 4.5 ± 2.8 ）, （ 6.8± 3.1 ） days,P 〈 0.05 ]. Conclusion Aktl gene in conbination with CTLA-4Ig is an attractive target for future strategies aimed at decreasing islet apoptosis and prolonging the life span effectively in xeno-genic islet transplantation. Key words: Islets transplantation ; Genes ; Transfection

Effect of p27Kip1 inhibition on proliferation of bovine corneal endothelial cells by RNA interference

Three plasmids (pGenesil-P1, pGenesil-P2, pGenesil-P3) with different p27Kip1-shRNA sequences were designed and synthesized. Their effects on the proliferation of bovine corneal endothelial cells (bCEC) were investigated. Plasmid expressing irrelevant shRNA with a random combination was used as negative control (pGenesil-HK). The recombination of four plamids was confirmed by restrictive enzyme digestion and sequence analysis. The expression of mRNA and protein of p27Kip1 was detected by RT-PCR and Western blotting after stable transfection. The expressions of p27Kip1 mRNA and p27Kip1 protein of pGenesil-P1 group, pGenesil-P2 group and pGenesil-P3 group were all lower than those in the pGenesil-HK group and the blank group (non-transfected group). pGenesil-P3 had the strongest inhibitory effect and was selected for the next steps. The proliferation rates of the pGenesil-P3 group, the pGenesil-HK group and the blank group were assessed by MTT. The influence of shRNA-p27Kip1 on bCEC cell cycle was detected by flow cytometry (FCM). Compared with the control groups, the proliferation rate of the pGenesil-P3 group was increased significantly, and the ratio of S-phase also increased. It is concluded that shRNA-p27Kip1 could down-regulate the expression of p27Kip1 effectively and increase the proliferation of bCEC. RNA interference (RNAi) may be an effective means to promote the proliferation of CEC.

Effects of Par-4 gene silencing induced by siRNA on the expression of Smac gene, activity of caspase-3, and apoptosis of human bone marrow mesenchymal stem cells

To investigate the effects of Par-4 gene silencing induced by siRNA on the expression of Smac gene, activity of caspase-3, and apoptosis of human bone marrow mesenchymal stem cells (hBMSCs). Bone marrow was obtained from a healthy young man and hBMSCs were isolated and cultured. Two siRNAs (Par-4-siRNA-1 and -2) targeting Par-4 gene were chemically synthesized. Eukaryocytic expression vectors containing these Par-4 siRNA sequences were established and transfected into the hBMSCs. The hBMSCs were divided into 4 groups: non-transfected hBMSCs (normal control group), blank Pae-4 plasmid transfected hBMSCs (Par4 control group), Par4-siRNA-1 transfected hBMSCs, and Par-4-siRNA-2 transfected hBMSCs. The expression of Par-4 mRNA was detected by real-time PCR. Another hBMSCs were inoculated in DMEM and divided into 4 groups: non-transfected normal hBMSCs, glutamate (an apoptosis inducer) + non-transfected hBMSC group, glutamine + Par-4-siRNA-1 hBMSC group, and glutamate + Par4-SiRNS-2 hBMSC group. Flow cytometry was used to detect the apoptotic rate. The relative activity of caspase-3 was determined by colorimetric assay. Western blotting was used to detect the Smac protein expression. The relative mRNA expression levels of Par-4 gene of the Par-4-siRNA-1 hBMSCs, Par-4 SiENA-2 hBMSCs, and Par-4 control hBMSCs were 0.12 +/- 0.03, 0.33 +/- 0.09, and 0.97 +/- 0.02 respectively, decreased by 88%, 67%, and 3% respectively compared with that of the normal control. The percentages of apoptotic cells of the glutamate + Par-4-siRNA-1 hBMSCs was (37.2 +/- 6.3)%, significantly lower than that of the glutamate + non-transfected hBMSC group [(58.9 +/- 8. 9)%, F = 58.26, P < 0.01). The Smac protein expression level of the glutamate + non-transfected hBMSC group was significantly higher than that of the normal control group (P < 0.01); however, the Smac protein expression level of the Par-4-siRNA-1 hBMSC group was significantly lower than that of the glutamate + non-transfected hBMSC group (P < 0.01). The caspase-3 activity of the glutamate + Par-4-siRNA-1 hBMSC group was significantly lower than that of the glutamate + non-transfected BMSC group (P < 0.01). Par-4-siRNA-1 inhibits markedly the apoptosis of the hBMSCs induced by glutamate. Par-4 gene silencing induced by siRNA inhibits the apoptosis of hBMSCs. The mechanism of the inhibition may be closely related to suppression of the up-regulation of Smac gene expression and caspase-3 activity.

Effect of Ad-Gax transfection on apoptosis of human lung adenocarcinoma A549 cells and its mechanism

Apoptosis is closely related to development of lung cancer. It is a strategy of lung cancer therapy to induce apoptosis. The aim of this study is to explore the effects of growth arrest-specific homeobox (Gax) transfection on apoptosis and expression of Bcl-2 and Bax proteins of human lung adenocarcinoma A549 cells. A549 cells were transfected with Gax gene by a replication-deficient adenovirus expressing the hemagglutinin-tagged Gax cDNA (Ad-Gax). Apoptosis of A549 cells was observed by transmission electronic microscope and terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) positive staining. Apoptotic rate of A549 cells was evaluated by flow cytometry. Expressions of Bcl-2 and Bax proteins in A549 cells were detected by immunocytochemistry. Before Ad-Gax transfection, none or few of TUNEL-positive A549 cells were detected. After Ad-Gax transfection, a marked increase in TUNEL-positive staining occurred, especially at 24 h later. The ratio of apoptosis of A549 cellsin non-transfection group and transfection groups at 12 h, 24 h, 48 h were 0.25%, 12.57%, 17.29%, 15.03%, respectively. Compared with non-transfection group, the apoptotic rates of transfection groups increased significantly (Chi-square value was 7.357, 11.126 and 9.943 respectively, P < 0.01). The average optical density (AOD) of Bcl-2 protein in A549 cells in non-transfection group and transfection groups at 12 h, 24 h, 48 h were 2.02±0.07, 1.79±0.02, 1.25±0.51 and 1.21±0.24 respectively. Compared with non-transfection group, AOD of Bcl-2 protein in A549 cells in transfection groups decreased significantly (t value was 6.651, 7.089 and 7.438 respectively, P < 0.01). On the other hand, Bax protein expression in transfection groups increased, the AODs of Bax were 4.49±0.61, 4.24±0.37 and 3.95±0.43, respectively. Compared with non-transfection group (3.12±0.42), AOD of Bax protein in A549 celle in transfection groups increased significantly (t value was 7.469, 7.287 and 6.473 respectively, P < 0.01). In the Ad-Gax transfection groups the lower Bcl-2/Bax ratio was, the higher the apoptotic rate of A549 cells was (r=-0.49, P < 0.01). Ad-Gax transfection can induce A549 cells apoptosis. Possible mechanism is that Gax can downregulate Bcl-2 protein expression and upregulate Bax protein expression, and A549 cells apoptosis is related to the Bcl-2/Bax ratio.

Expression and Characterization of TWIK‐2 in CHO cells

We have recently demonstrated that TWIK-2, a two-pore domain potassium channel, is highly expressed in rat middle cerebral artery (MCA) (AJP 291:, 2006). In this study, we cloned TWIK-2 from rat MCA and expressed it heterologously as a fusion protein to GFP for characterization. Direct fluorescence analysis by deconvolution microscopy in transiently transfected COS cells revealed that GFP-rTWIK-2 localized to the plasma membrane in a punctate pattern by 48 hours after transfection. Stable transfectants of GFP-rTWIK-2 were selected in CHO cells and analyzed by whole-cell electrophysiology. Whole cell currents were 16-fold greater than the nontransfected control group, were non-rectifying in physiological K+, but were inwardly rectifying in symmetrical K+. The observed reversal potentials in different concentrations of extracellular K+ were similar to the theoretical reversal potentials. In physiological K+, membrane potential was -81 ± 2 mv (n=13) for transfected and −64 ± 4 mv (n=5) for control cells. 1 mM BaCl2 inhibited currents by 89 ± 1% with a calculated IC50 of 88 μM. The TWIK-2 currents were minimally affected by 10 mM TEA. Arachidonic acid (10 μM) increased the currents 27 ± 7% (n=4). Since vascular smooth muscle of the rat MCA expresses a functional TWIK-2 channel, TWIK-2 has the potential of being an important regulator of vascular tone. (RO1 NS 46666, NS 038660, and AHA Bugher Award 027011N)

In vitro anti-tumor immune response induced by dendritic cells transfected with recombinant adenovirus carrying mutant k-ras genes

The specific anti-tumor immune response induced by mouse bone marrow dendritic cells (DCs) transfected with recombinant adenovirus carrying mutant k-ras genes was investigated. DCs were generated from mouse bone marrow in the presence of rmGM-CSF (3.3 ng/mL) and rmIL-4 (1.3 ng/mL) and detected by FACS, and then transfected with the recombinant adenovirus encoding mutant k-ras gene. The efficacy of transfection and T cell stimulating activity of DCs were detected. CTL activity of the mice vaccinated with DCs was observed. The results showed that DCs had dendritic veiled morphology. BmDCs highly expressed B7-1 (80%), B7-2 (77%), MHC II (70%), CD11c (65%), CD40 (70%) and CD54 (96%) with FACS, and no significant difference in the expression was observed before and after the transfection (P > 0.05). The DCs transfected by mutant k-ras gene could significantly stimulate lymphocytes proliferation as compared with those transfected by Ad-c or non-modified DCs (P < 0.05). DC vaccine transfected by mutant k-ras gene could induce CTL activity against Lewis lung cancer, but not against B16. The specific cytotoxicity against Lewis lung cancer in Ad-k-ras/12-transduced DC group was significantly higher than those in the control, vector and non-transfected DCs groups (P < 0.05). It was concluded that special antitumor response could be induced by DCs transfected with recombinant adenovirus carrying mutant k-ras genes.

Inhibitory effect of coxsackie adenovirus receptor on invasion and metastasis phenotype of ovarian cancer cell line SKOV3.

Full-length coxsackie adenovirus receptor (CAR) eukaryotic expression plasmid was transfected into an ovarian cell line, SKOV3, and its effect on the change of malignant metastasis phenotype was explored. CAR mRNA and protein expression levels among 4 ovarian cancer cell lines (A2780, SKOV3, SW626, CAOV3) and the positive control 293 (a transformed human embryo kidney cell line) was detected by using semi-quantitative RT-RCR and Western blot and compared. CAR-negative SKOV3 was transfected with the eukaryotic expression plasmid containing a full-length CAR cDNA and mock-vector respectively. The positive clones were screened by G418. The biological behavior changes of positive transfected cells were gauged by colony formation in soft agar assay and cell adhesion assay. Among the cell lines, there were obviously different CAR expression levels. CAR could not be detected in SKOV3. In transfected cell group, CAR expression was enhanced obviously as compared with non-transfected or mock-transfected groups. Cell adhesion in the transfected group was promoted. The number of colony formation was reduced significantly in transfected groups (25.32 +/- 8.91) as compared with that in non-transfected group (88.75 +/- 13. 98) and mock-transfected group (82.53 +/- 19.37). Among the 4 ovarian cancer cell lines, CAR expression level was variable. Exogenous CAR expression had a potential role in inhibiting the malignant metastasis phenotype of ovary cancer cells.