Cotransfected human chondrocytes: over-expression of IGF-I and SOX9 enhances the synthesis of cartilage matrix components collagen-II and glycosaminoglycans

Damage to cartilage causes a loss of type II collagen (Col-II) and glycosaminoglycans (GAG). To restore the original cartilage architecture, cell factors that stimulate Col-II and GAG production are needed. Insulin-like growth factor I (IGF-I) and transcription factor SOX9are essential for the synthesis of cartilage matrix, chondrocyte proliferation, and phenotype maintenance. We evaluated the combined effect of IGF-I and SOX9 transgene expression on Col-II and GAG production by cultured human articular chondrocytes. Transient transfection and cotransfection were performed using two mammalian expression plasmids (pCMV-SPORT6), one for each transgene. At day 9 post-transfection, the chondrocytes that were over-expressing IGF-I/SOX9 showed 2-fold increased mRNA expression of the Col-II gene, as well as a 57% increase in Col-II protein, whereas type I collagen expression (Col-I) was decreased by 59.3% compared with controls. The production of GAG by these cells increased significantly compared with the controls at day 9 (3.3- vs 1.8-times, an increase of almost 83%). Thus, IGF-I/SOX9 cotransfected chondrocytes may be useful for cell-based articular cartilage therapies.

Establishing HEK-293T cell model carrying human sodium channel and PRKAG2 gene / 第二军医大学学报

Objective: To establish a HEK-293T cell model with sodium channel function similar to that in PRKAG2 syndrome. Methods: Vectors ofPRKAG2 gene and SCN5A gene were constructed and were used to co-transfect HEK-293T cells. Immunofluorescence, real-time PCR and Western blotting analysis were used to examine the transfection results and the expression of interested mRNA and protein. Results: Imunofluorescence findings showed red fluorescence and green fluorescence in HEK-293T cells 48 hours after co-transfection. Real-time PCR and Western blotting analysis showed SCN5A expression in pure SCN5A group, SCN5A + wild-type group, SCN5A + R302Q mutant group, and SCN5A + G100S mutant group, but not in the blank control group, showing significant significance (P<0.05). Expression of PRKAG2 gene and protein in SCN5A+PRKAG2 group was significantly higher than those in the blank control group, pure SCN5A group, SCN5A+R302Q, and SCN5A+G100S groups (P<0.05). Conclusion: We have successfully established a HEK-293T cell model with sodium channel function similar to that in PRKAG2 syndrome, paving a way for future study.

Establishment of a lung cancer cell line A549 highly expressing DNA oxidative damage related repair gene hOGG1 / 第二军医大学学报

Objective: To establish a human lung cancer cell line A549 highly expressing human 8-oxoguanine-DNA glycosylase (hOGG1) by co-transfecting pcDNA 3.1 (+)/Myc-HisA-hOGG1 and PGL3 promoter, and to observe the biological behavior of the transfected cells. Methods: PcDNA3.1 (+)/Myc-His A-hOGG1 and PGL3 promoter were steadily co-transfected into A549 cells via mediation of Fu GENE 6 (transfected group); untransfected cells served as blank control and cells transfected with PGL3 + pcDNA3.1 (+)/Myc-HisA served as negative control. The hOGG1 mRNA expression in A549 cells was detected by Bio-luciferase activity and the hOGG1 protein expression by Western blotting analysis. Cells in the three groups were exposed to hyperoxia condition, and the morphological changes were observed by phase-contrast microscope. Comet cell rate and olive tail moment of cells were tested after different exposure periods. After exposure, the cells were incubated for 0, 60, 120, and 180 min, and the same indices were determined by modified comet assay; changes of DNA oxidative damage markers 8-hydroxy-desoxoguanosine (8-OHdG) was tested at the same time. Results: The bio-luciferase activity was stable in the co-transfected cells. Western blotting analysis showed that the expression of hOGG1 protein in co-transfected A549-T cells was significantly higher than those in the other two groups, indicating the successful establishment of hOGG1 highly expressing cells. Under the same hyperoxia condition, the morphological changes of transfected cells were greatly alleviated, and the Comet cell rate and olive tail moment of cells were obviously lower than those of the other two groups(P< 0.05). Transfected A549-T cells had significantly increased ability of DNA repair and shorter repair time compared with cells in the other two groups (P<0.05). Furthermore, the level of 8-OHdG in transfected A549-T cells was significantly lower than those of the other two groups under the same hyperoxia condition, indicating a significantly higher ability of DNA damage resisting and repair (P<0.05). Conclusion: High hOGG1 expression can decrease the cell sensitivity to DNA damage and improve the repair ability of cells.

Construction of Recombinant Adeno-Associated Virus Vector (AAVCMVp53) for Human Cervical Cancer Gene Therapy / 대한산부인과학회잡지

OBJECTIVE: To eliminate the potential problem of adenovirus contamination during recombinant adeno-associated virus (rAAV) vector production, we investigated new rAAV production method by a triple transfection of vector plasmid, packaging plasmid, and adenovirus helper plasmid. METHODS: This study was carried by triple transfection for the production of recombinant adeno-associated virus vector. This new production system was conducted with a plasmid construct which contained a mini-adenovirus genome capable of propagation of rAAV in the presence of adeno-assoceated viral rep and cap genes. To examine the helper functions of adenoviral plasmid on the production of rAAV vector, we carried out cotransfection with three plasmids, AAV vector, packaging construct, and adenovirus helper plasmids. The optimized plasmid quantity of transfection by calcium phosphate precipitation method was 25 micro gram of total plasmid DNA per 10 cm diameter plate of 293 cell. RESULTS: We found that rAAV yields peaked at 48 hr after Ad infection. The titer of rAAV was measured by the dot blot analysis to measure the number of particles/mL based on the quantification of viral DNA. CONCLUSION: We constructed recombinant AAVp53 without adenovirus contamination. We thought that we construct high titer rAAVp53 particle through HPLC column in the near future.

Inhibitory Effect of Co-Transfection of tPA Gene and PCNA-ASODN on Restenosis of Autograft Artery in Rabbits / 中国普外基础与临床杂志

Objective To observe the inhibitory effects of local co-transfection of tissue-type plasminogen activator(tPA) gene and proliferating cell nuclear antigen antisense oligodeoxynucleotides(PCNA-ASODN) on the intima proliferation and restenosis of autograft artery in rabbits. Methods One hundred and twenty male Zelanian rabbits were randomly divided into four groups(n=30, in each group): control group, PCNA-ASODN group, tPA group and tPA+PCNA-ASODN group. The left and right external iliac arteries (length 1.0 cm) were transplanted reciprocally. The transplanted arteries were respectively soaked in lipofection, PCNA-ASODN, pBudCE4.1/tPA and pBudCE4.1/tPA+PCNA-ASODN solution about 15 minutes. The transplanted arteries were sutured with 9-0 sutures soaked in PCNA-ASODN and pBudCE4.1/tPA solution. Each group were divided into five subgroups(n=6, in each subgroup) according to the sacrifice time (3 d, 7 d, 14 d, 28 d and 56 d after operation). On every sacrifice time point, the vascular specimens were harvested. The thrombocyte assembling and thrombus forming lining vessel wall were observed by scanning electron microscope. The pathological morphology of transplanted arteries were observed under microscope(HE). The intimal areas and stenosis ratio(%) of transplanted arteries were calculate and analyzed statistically among groups by computer system. The mRNA expression of tPA gene in transplanted ressel wall was detected with vevere transcription-PCR(RT-PCR). The number of PCNA positive cells in transplanted vessel wall was counted by SP immunochemisty. Results The mRNA expression of tPA gene in the transplan-ted vessel wall in tPA and tPA+PCNA-ASODN groups was higher than that of the other two groups (P