| Objective:Immunohistochemical detection rHSG (rat HSG) protein in normal and COPD airway fibroblast cells with different expression; by in vitro transfection hHSG (human HSG) gene to the COPD airway fibroblasts, observed pEGFP-hHSG on fibroblast proliferation, in order to lay the theoretical basis for further gene therapy.Method:COPD rat model by creating, organizing block culture airway fibroblasts, using immunohistochemistry rHSG gene in normal rats and COPD airway fibroblast cells; competent cells transformed into plasmid pEGFP-n1, pEGFP- hHSG, alkaline extraction plasmid by gel electrophoresis and DNA sequencing, and then liposome transfected pEGFP-hHSG COPD patients with airway fibroblasts, transfection by fluorescence microscopy after transfection with immunohistochemistry Detection of the control group (COPD untransfected group), turn natural pEGFP-n1 group, turn natural pEGFP-hHSG group hHSG protein, MTT, cell counting of transfected cells in growth status. Application SPSSll.O software for x±s said.?statistical analysis, experimental data The comparative analysis of variance with one between groups q test, P <0.05 for the difference was significant statistically significant.Results:COPD After the modeling, after cell culture, immunohistochemical studies showed rHSG protein in COPD group and normal rats rats within each expression, the gray value of 113.796±13.173,184.327±3.238, rHSG gene in a large COPD rat airway fibroblasts expressed significantly lower airway fibroblast cells, the difference between the two was statistically significant (P <0.05). Primary culture of normal subjects and patients with COPD airway fibroblasts, anti-vimentin identified the experimental fibroblast cells, purified by time difference of pure airway fibroblast; calcium chloride prepared by the high conversion rate of competent cells (DH5a ), pEGFP-n1, pEGFP-hHSG plasmid transformation, amplification, EcoI and SnaBI restriction enzyme digestion enzyme electrophoresis identification by fragment length about 2274bp gene fragment sequencing confirmed that the plasmid containing the gene coding region hHSG, the fragment length is 2274bp . The GeneBank Blast analysis confirmed that fragment of DNA insertion sequences and completely identical DNA sequences. The pEGFP-n1, pEGFP-hHSG plasmid into COPD airway fibroblasts, can be seen in the silver microscope transfected (pEGFP-n1, pEGFP-hHSG) cells have the sound of green fluorescent protein (GFP) expression The main is located around the nucleus. Detected by immunohistochemistry in the control group were hHSG control, pEGFP-n1, in the pEGFP-hHSG three groups of cells in the nucleus and the nucleus showed a positive reaction to varying degrees, in which the control group (non-transfection group) and transfected pEGFP-n1 group of weak positive reaction, while transfection of pEGFP-hHSG strong positive reaction cells, the control group transfected with pEGFP-n1 group and the group transfected with pEGFP-hHSG gray value measured by the ratio was statistically significant ( P <0.05), shows hHSG in these cells have different levels of expression. Cell counting, MTT and describe cell growth curve, the results show transfected pEGFP-hHSG cell proliferation was inhibited, while the control group transfected with pEGFP-n1 cell proliferation was not changed. Transfected with pEGFP-hHSG group in 1,2,3,4,5 days of the COPD airway fibroblast proliferation inhibition rates were 24.6%, 28.7%, 43.6%, 52.3% and 62%, and the inhibition rate was positively correlated with the time. Transfected with pEGFP-hHSG group and the control group transfected pEGFP-n1 significant difference between groups (P <0.05), and transfected pEGFP-n1 group and control group no significant difference between (P> 0.05).Conclusion:rHSG gene in COPD airway fibroblasts expressed significantly lower airway fibroblast cells. In vitro transfection hHSG to COPD patients with airway fibroblasts hHSG stable expression in the nucleus periphery, cell proliferation was inhibited. |
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