Effects Of Knock Down Of TGFβ1 Gene By RNA Interference On Human Temporal Arteritis And Its Mechanisms

Objective:To construct a plasmid vector of RNA interference(RNAi) of TGF-β1 gene and lay a foundation for the construction of lentiviral vector.Methods:Three target sequences were selected according to TGFβ1 mRNA sequence firstly,then three pairs of oligonucleotide sequences according to these target sequences and one pair of negative control oligonucleotide sequence were designed and synthesized.The annealed oligonucleotide fragments were subcloned into pRNA-U6.2/Lenti plasmid and was named pRNA-U6.2/Lenti-1, pRNA-U6.2/Lenti-2,pRNA-U6.2/Lenti-3 and pRNA-U6.2/Lenti-4,respectively. After being identified by restriction enzyme digestion and sequencing,the recombinant plasmids were transfected into Hela cells.TGF-β1 expression in the transfected cells was assayed by RT-PCR and ELISA.Results:Enzyme digestion and DNA sequencing showed that the oligonucleotide fragments were correctly inserted into pRNA-U6.2/Lenti plasmid,and TGF-β1 expression in the transfected cells was knocked down significantly by pRNA-U6.2/Lenti-1 and pRNA-U6.2/Lenti-2 at both the protein and mRNA level compared with the control group.TGF-β1 protein was reduced by a percent of 79.2% and 53.7%in pRNA-U6.2/Lenti-1 and pRNA-U6.2/Lenti-2 group,respectively, statistically different from that of control group(p＜0.01).Conclusion:The RNAi plasmid vector of TGF-β1 was constructed successfully. Chapterâ…¡:Construction and identification of lentiviral vector of RNA interference of TGFβ1 geneObjective:To construct a lentiviral vector of RNA interference(RNAi) of TGF-β1 gene and lay a foundation for gene therapy.Methods:The most effective plasmid vector of RNA interference in the chapterI-pRNA -U6.2/Lenti-1 and the negative control plasmid vector--pRNA-U6.2/Lenti-4 were cotransfected along with ViraPower^TM Lentiviral Packaging Mix into 293FT cells for the package of lentiviral particles.Then the lentiviral vector particles were transfected into Hela cells and TGF-β1 expression in the transfected cells was assayed by Real-Time PCR and ELISA.Results:It was confirmed by digestion and sequencing that lentiviral vectors had the correct structure and could express high titer of virus.After transfected into Hela cells,TGF-β1 expression was decreased significantly by pRNA-U6.2/Lenti-1 lentiviral vectors at both the protein and mRNA level.TGF-β1 protein was reduced by a percent of 90.7%,statistically different from that of control group(p<0.01). Meanwhile.the negative control lentiviral vector--pRNA-U6.2/Lenti-4 show no effects on the expressions of TGF-β1 at both the protein and mRNA level.Conclusion:The lentivirus RNAi vector of TGF-β1 was constructed successfully. Objective:To investigate the effects of knock down of TGFβ1 gene by lentiviral vector--ptLNA-U6.2/Lenti-1 on temporal arteritis and its potential mechanisms.Methods:Each temporal arteritis specimen(confirmed by histopathology) was divided in four fragments,and were implanted into NOD-SCID mice.After transplantation,mices were divided into control group,pRNA-U6.2/Lenti-1 group, dexamethasone group and therapeutic alliance group(treat with pRNA-U6.2/Lenti-1 and dexamethasone).Mice was received NS(100ul) in control group,pRNA-U6.2/Lenti-1(100ul,3.1×10⁷TU/ml) in pRNA-U6.2/Lenti-1 group,dexamethasone(4mg/kg) in dexamethasone group,pRNA-U6.2/Lenti-1(100ul, 3.1×10⁷ TU/ml) and dexamethasone(4mg/kg) in therapeutic alliance group subcutaneous for 3 week.After the interference,inflammatory cell infiltration and tunica intima hyperplasia level of the temporal artery in each group were observed by light microscope,mRNA level of TGFβ1,PCNA,NF-KB,IL-2,MCP-1 and ICAM-1 were assayed by RT-PCR,protein level of TGFβ1,PCNA,NF-KB,IL-2,MCP-1 and ICAM-1 were measured by western-blotting or immunohistochemisty.Results:1.After treated with lentiviral vector--pRNA-U6.2/Lenti-1,the tunica intima hyperplasia level of temporal artery were decreased compared with the control group (p＜0.05);The protein and mRNA level of TGFβ1,PCNA,NF-KB and IL-2 were reduced significantly compared with the control group(p＜0.05).2.After treated with dexamethasone,the inflammatory cell infiltration level of temporal artery were decreased compared with the control group(p＜0.05);the protein and mRNA level of MCP-1,ICAM-1,NF-KB and IL-2 were reduced significantly compared with the control group(p＜0.05).3.After treated with lentiviral vector--pRNA-U6.2/Lenti-1 and dexamethasone together,the inflammatory cell infiltration level of temporal artery were decreased compared with the control group(p＜0.05),the tunica intima hyperplasia level of temporal artery were decreased compared with the control and pRNA-U6.2/Lenti-1 group(p＜0.05);Compared with the control group,the protein and mRNA level of TGFβ1,PCNA,NF-KB,IL-2,MCP-1 and ICAM-1 were reduced significantly (p＜0.05),Compared with pRNA-U6.2/Lenti-1 group,the protein and mRNA level of PCNA were reduced significantly(p＜0.05).Conclusions:1.Lentiviral vector--pRNA-U6.2/Lenti-1 can reduce the tunica intima hyperplasia level of temporal artery by inhibit the expression of TGFβ1,maybe due to the inhibition of smooth muscle cell proliferation;when administered with dexamethasone,the tunica intima hyperplasia will be reduced more significantly, which may due to the coordination inhibition effects of the smooth muscle cell proliferation by them.2.TGFβ1 is not the main reason for the level of inflammatory infiltration,and so it is not enough to treat the temporal arteritis with pRNA-U6.2/Lenti-1 alone.4.Lentiviral vector--pRNA-U6.2/Lenti-1 can reduce the functions of CD4+Cells by inhibit the expression TGFβ1,partly due to the reduction of NF-KB.