Retroviral Vector-mediated Il-1ra And Tgf-¦Â1 Complex Transfer Into Rabbit Chondrocytes Expression

PART ONE The construction of recombination gene PLNCX₂-IL-1Ra-GFP and PLNCX₂-TGF-β1-RFP[Objective]To construct recombination gene PLNCX₂-IL-1Ra-GFP and PLNCX₂- TGF-β1-RFP,which provide gene transfection with target gene.[Methods]The vector PLNCX₂ being digested and dephosphorylated by the double enzyme Xhoâ… /Hindâ…¢,,the multiple cloning sites result from pCDNA3.1-GFP(-) being digested by the double enzyme Xhoâ… /Hindâ…¢,and the gene GFP were conjoined by ligase T₄DNA to form PLNCX₂-GFP.After amplifying from human brain tissue,gel extracting and being digested by Xhoâ… /Hindâ…¢,Il-1Ra gene combined with PLNCX₂-GFP being digested by Xhoâ… /Hindâ…¢to form PLNCX₂-Il-1Ra-GFP by ligase T₄DNA.the vector PLNCX₂ being digested and dephosphorylated by the double enzyme Hindâ…¢/Notâ… ,the multiple cloning sites result from the vector pDsRed₂ being digested by Hindâ…¢/Notâ… and RFP gene were conjoined by ligase T₄DNA to form PLNCX₂-RFP.After amplifying from PGEMT-TGF,gel extracting and being digested by Xhoâ… /Hindâ…¢,TGF-β1 combined with PLNCX₂-RFP being digested by Xhoâ… /Hindâ…¢to form PLNCX₂-TGF-β1-RFP by ligase T₄DNA.[Results] Recombination gene PLNCX₂-IL-1Ra-GFP's and PLNCX₂- TGF-β1-RFP's sequence were correct after enzyme digesting and identifying.The gene can be read through,it's pillar location conform to corresponding plasmid profile.[Conclude]The recombination gene PLNCX₂-IL-1Ra-GFP and PLNCX₂- TGF-β1-RFP were constructed correctly.PART TWO The packing and measure of recombination gene PLNCX₂-IL-1Ra -GFP and PLNCX₂-TGF-β1-RFP[Objective]To improve transfecting efficiency ofthe retrovirus vetor PLNCX₂-IL-1Ra-GFP and PLNCX₂- TGF-β1-RFP,and to measure the best virus titer used for transfection.[Methods]The packaging cell PT67 was growing until fusing to 80%.Recombination plasmid PLNCX₂-IL-1Ra-GFP and PLNCX₂- TGF-β1-RFP transfected PT67 respectively with liposome mediated method,screening and culturing 14 days with G418, after the transfected cell forming positive clone,selecting out it ,cultivating and passaging,then collecting the virus supernatant of the first to the forth generation clone cell,packaging respectively after filtering,and then cryopeservting on -70℃.Adding 2×10⁴个NIH3T3 cell lines in every well with 6 well culture plate growing until fusing to 80%,and then adding the diluted positive colne virus supernatant 3ml(1:100),after culturing 4hs,replacing supernatant with 300ug/ml G418 to culture 4 weeks,counting the survival anti-clone cell,calculating virus supernatant titer based on formula. [Results]Having observed green and red fluorescence after transfecting the packaged cell,the virus supernatant titer was 1×10⁶C.F.U after transfecting the NIH3T3 cell.[Conclude]To have got higher transfecting efficient recombination gene and the best transfecting virus titer.PART THREE The study of the exto-transfection of IL-â… Ra and TGF-β1 to knee articular chondrocytes of the rabbit in vitro respectively and commonly[Objective]A study of useing IL-1Ra and TGF-β1 transfect knee articular chondrocytes of the rabbit in vitro respectively and commonly,and observing expression of IL-1Ra and TGF-β1 in the knee articular chondrocytes of the rabbit.[Methods]To use expressed carrier PLNCX₂-IL-1Ra-GFP and PLNCX₂- TGF-β1-RFP with aim gene transfect the knee articular chondrocytes of the rabbit respectively and commonly,to detect the effect of the gene transfection on articular chondrocytes by NO- detecting kit and the exexpressing of hIL-â… Ra and hTGF-β1 in cell culture supemate by Elisa method.[Results]Enzyme digestion identification showed that PLNCX₂-IL -1Ra-GFP and PLNCX₂- TGF-β1-RFP were constructed successfully.After transfecting articular chondrocytes,using immunofluorescence inverted microscope can detect the green fluorescence in the TGF-β1 group,the red fluorescence in the IL-1Ra group and the green and red fluorescence in the same cell of the cotransfected group,which tested the transfection are successful.In the culture medium,the NO content of the single gene were(89.71±5.43)umol/L(TGF-β1 group) and(92.15±5.36) umol/L(IL-1Ra group),the cotransfected gene(94.93±4.88) umol/L,the bland control group(60.19±4.68) umol/L and the nude carrier group(57.23±4.29) umol/L,which showed that there was no statistical significance among transfected group(P＞0.05)and non-transfected group(P＞0.05),there was statistical significance between transfected group and nontransfected group(P＜0.05).Detecting expression of hIL-1Ra and hTGF-β1 in the transfected group by ELISA,the single gene group(43.42±4.07)ng/L(hIL-1Ra) and(28.08±3.73)ng/L(hTGF-β1),the cotransfected gene group(46.55±4.85) ng/L and(30.79±3.14) ng/L.There was no expression in the non-transfected group and the nude carrier group.There was statistical significance between the transfected group and the non-transfected group(P＜0.05),no statistical significance between the single gene group and the cotransfected gene group(P＞0.05).[Conclude]IL-1Ra and TGF-β1 mediated by retrovirus PLNCX₂ can successfully transfect knee articular chondrocytes of the rabbit respectively and commonly and get expression stably,which provide IL-1Ra and TGF-β1 respectively and commonly treating OA with theoretical basis.