Objective To investigate the effect of constitutively active Akt1 gene on rat engrafted islets in apoptosis and revascularization, and to explore potential method of gene therapy in the islet transplantation. Methods Rat islet which was transfected constitutively actived Akt1 gene via adenovirus vector using MOI=500. Thirty-six streptozotocin induced diabetic Wistar rats were divided into 3 groups complete randomly: Adv-CA-Akt1 group, Adv-LacZ group and simple transplantation group. Blood glucose and insulin were determined after operation. TUNEL was used to detect the apoptotic islet cells. HE and immunohistochemical staining of insulin were used to evaluate the histology of the islet grafts. The microvessel density (MVD) was determined by CD31 immunohistochemical staining. Results The fasting glucose level in Adv-CA-Akt1 group restored to normal 2 days after transplantation. However, in Adv-LacZ group and simple transplantation group, it reduced but still kept being hyperglycemia. And the serum insulin level was higher than other two groups ( P ＜ 0.05). Compared to simple transplantation group and Adv-LacZ group, apoptotic rate decreased 25% in Adv-CA-Akt1 group, a large number of islet grafts were seen under the capsule of the kidney, which were positively stained by insulin antibody. In the other two groups, the islet groups mass were lighter, and few positively stained by insulin antibody. MVD showed lighter positive endothelial cells stained by CD31 antibody in the other two groups than Adv-CA-Akt1 group ( P ＜ 0.05). Conclusion Constitutively activate Akt1 gene can prolong graft survival during early posttransplant period, and can accelerate the revascularization of islet grafts effectively.
Objective To elucidate whether glucose transporters-4 （GLUT-4） takes part in glucose uptake of mesenchymal stem cells （MSCs） and whether Akt gene improves translocation and expression of GLUT-4 in MSCs under hypoxic environment ex vivo. Methods MSCs, transfected by Akt gene and no, were cultured with normoxia (5% CO2) or hypoxia (94%N2, 1%O2 and 5% CO2) at 37 ℃ for 8 h. Glucose uptake was assayed by using radiation isotope 2-［3H］-deoxy-Dglucose (3H-G) and the expression of GLUT-4 protein and mRNA was assayed by immunocytochemistry, Western blot and RT-PCR, respectively. Results ①3 H-G intake of MSCs was significantly increased in hypoxiatransfection group than that in hypoxia-non-transfection 〔（1.39±0.13） fold, P＜0.05〕, but which was lower than that in normoxia-non-transfection group, P＜0.05. ②GLUT-4 was expressed by MSCs under any conditions. Compared with normoxia-non-transfection group, hypoxia decreased the expressions of GLUT-4 mRNA and protein significantly (P＜0.05). ③Compared with hypoxianontransfection group, the expression of GLUT-4 〔mRNA（1.756±0.152） fold, total protein in cell （1.653±0.312） fold, protein in plasma membrane （2.041±0.258） fold〕 was increased in hypoxia-transfection group significantly (P＜0.05), but which was lower than that in normoxianontransfection group (P＜0.05). ④There was significantly positive relation between 3H-G intake and GLUT-4 protein expression in plasma membrane (r=0.415, P=0.001).Conclusion GLUT-4 may take part in glucose uptake of MSCs, and the capability of Akt gene to improve MSCs anti-hypoxia may be finished by its role in increasing the expression and translocation of GLUT-4.
Objective To investigate the effect s of T lymphoma invasion and metastasis inducing factor 1 ( Tiam 1) antisense oligonucleotides (ASODN) on morphological remodeling of gast ric cancer cells. Methods The high-invasive and metastastic subgroup (MH ) was separated f rom human gast ric cancer cell line MKN245 (M0 ) by laminin adhesion method in vi t ro. And they were divided into four group s according to different further t reatment s : no t ransfection group (cont rol group ) , liposome t ransfection group , sense oligonucleotides2liposome t ransfection group ( SODN t ransfection with liposome group ) and antisense oligonucleotides2liposome t ransfection group (ASODN t ransfection with liposome group) . Then the expressions of Tiam 1 mRNA and protein were detected by RT-PCR and flowcytomet ry , respectively. The morphology changes between Tima 1 ASODN t ransfected MH cells and no t ransfected cells were observed by using HE stain , cytoskeletal protein stain and scanning elect ronic microscope (SEM) . Results Compared with the other group s , the expressions of Tiam 1 mRNA and protein in MH cells were significantly decreased af ter the cells were t ransfected with 0. 43 μmol/ L Tiam 1 ASODN ( P lt; 0. 01) . Additionally , it was observed that the t ransfected MH cells had less membrane surface projections , fewer or shortener pseudopodia , less irregular cytoskeletal network and less spotted-like actin bodys than no t ransfected MH cells did. Conclusion ASODN t ransfection could effectively suppress the expression of Tiam 1 and the remodeling in gast ric cancer cells , which may play an important role in the invasion and metastasis of gast ric cancer cells.
Objective To establish hepatocellular carcinoma (HCC) cell lines which olig-expressed IGF1R gene stably． Methods An eukaryotic expressing vector pSUPER-IGF1R-siRNA that could block IGF1R expressing was transferred into hepatocellular carcinoma cell lines SMMC7721 and Hep3B with Lipofectamine 2000 reagents． After transferred, cells were selected with G418 to obtain positive clones. The expressions of IGF1R, cyclin D1 and cyclin B1 were detected by RT-PCR and Western-blot． Cell growth curve were painted. Results Two cell lines clones were screened olig-expressing IGF1R gene stably． The experimental cell lines grew more slowly than control cell lines and the expression of cyclin D1 decreased (P＜0.05). Conclusion The HCC cell lines for olig-expressing IGF1R gene stably are established successfully．The plasmid pSUPER-IGF1R-siRNA can inhibit the growth of SMMC7721 and Hep3B cell lines, and the expression of cyclin D1.
【Abstract】ObjectiveTo study the antitumor effects of DNA electrotransfer in muscle (ETM) by using established animal model for human anaplastic thyroid cancer cell line TA-K.MethodsNude mice with implanted TA-K were divided into five groups including: control group, pcDNA-3 plasmid electrotransfered into muscle (pcDNA ETM group), TIMP-3 plasmid injected into muscle (TIMP-3 IM group), TIMP-3 plasmid electrotransfered into muscle (TIMP-3 ETM group), TIMP-3 plasmid electrotransfered into implanted tumor (TIMP-3 ETT group). Electrical parameters used in electrotransfer were: 200 V/cm, 20 ms/pulse; 8 pulses, 1 Hz in muscle and 600 V/cm, 20 ms/pulse; 1 pulses, 1 Hz in implanted tumor respectively. ResultsThe growth of TA-K was inhibited more significantly in the groups of TIMP-3 plasmid electrotransfered into muscle and TIMP-3 plasmid electrotransfered into implanted tumor than the other groups (P＜0.05), and the content of TIMP-3 protein in tumor tissues was higher in these two groups . ConclusionAnti-oncogene has the antitumor effects by DNA electrotransfer in muscle.
【Abstract】ObjectiveTo study the effect of transfection with antisense DNMT3b gene eukaryotic expression vector on the expression of DNMT3b gene in human cholangiocarcinoma cell line QBC-939. MethodsThe constructed antisense DNMT3b gene eukaryotic expression vector was transfected into the human cholangiocarcinoma cell line QBC-939 by using lipofectamine transfection reagents, and positive cell clones were obtained by using G418 selection after transfection. Whether the constructed recombinant vector was transfected into QBC-939 cells successfully was confirmed by amplifying the exogenous neoR gene with PCR method. The expression of DNMT3b gene mRNA and protein were detected by semi-quantitative RT-PCR and FCM methods respectively. ResultsFollowing the transfection of antisense DNMT3b gene eukaryotic expression vector, the mRNA level of DNMT3b gene in QBC-939 cells of human cholangiocarcinoma decreased from 0.956±0.053 to 0.209±0.023, and the protein level of DNMT3b gene also decreased from (75.38±3.22)% to (29.87±3.46)%. There were very significant differences on the expression levels of DNMT3b gene between non-tranfections group and the antisense DNMT3b gene eukaryotic expression vector transfection group (P＜0.01). ConclusionTransfection with antisense DNMT3b gene eukaryotic expression vector significantly reduces the expression level of DNMT3b gene in human cholangiocarcinoma cell line QBC-939, and this study may provide a valid tool and method to investigate the function of DNMT3b gene and its role in cholangiocarcinoma.
Objective To observe the expression of adenovirus vector coding for mouse endostatin gene（Ad-mES） in lung cancer cells and its antiangiogenic activity in human umbilical vein endothelial cells（ECV304） in vitro.Methods Lewis lung cancer（LLC） cells were transfected with Ad-mES at different multiplicity of infection（MOI）.The expression of mES in LLC cells and supernatant after 48 hours was detected by immunohistochemical staining and Western blot respectively.The inhibitory effect of supernatant at different MOI on ECV304 non-stamulated and stimulated by basic fibroblast growth factor（bFGF） was measured by methyl thiazolyl tetrazolium（MTT） assay.Results After transfected for 48 hours，endostatin was identified in the cell plasma of infected LLC and negative result was founded in non-infected LLC.Western blot revealed band of endostatin in 20 kDa in culture supernatant of infected LLC and negative results in non-infected LLC.The inhibitory effects on ECV304 cell proliferation were ber at higher MOI，and the difference was significant between stimulated and non-stamulated cells by bFGF（Plt;0.05）.Conclusion Ad-mES can transfect and express endostatin effectively in LLC with biological activity
Objective To investigate the transfection and expression of recombinant plasmid human vascular endothelial growth factor 165/pcDNA3. 1 （hVEGF165/pcDNA3. 1） in myocardial cells, and to build foundation for gene therapy and cell therapy of coronary artery disease （CAD）. Methods Myocardial cells were cultured in vitro and transfected by hVEGF165/pcDNA3.1 with liposome; then transient expressed protein was detected by reverse transcriptase-polymerase chain reaction （RT-PCR）, immunochemistry and Western blotting. Results A strap as hVEGF165 was obtained by RT-PCR, the protein of hVEGF165 was found in myocardial cells by immunochemistry and in supernatant by Western blotting. Conclusion The recombinant plasmid hVEGFI65/pcDNA3. 1 can be expressed in myocardial cells, and may be used in studying CAD by gene therapy and cell transplantation.