| Diabetes mellitus(DM)is a chronic disease severely threated public health in global,and type 2 diabetes mellitus(T2DM)accounts for more than 90%of DM.Studies showed that a significant factor impacted the pathogenesis of T2DM was the impaired glucose-dependent insulinotropic polypeptide(GIP)function.GIPR played a key role in the function of GIP.Blood sugar levels and insulin response in body are affected by GIP/GIPR signal which transmission is inhibited by dominant inhibitory GIPR(GIPRdn).In our research,rat insulin 2 promoter was cloned and GIPR and GIPRdn were artificially synthesized for constructing pLV-RIP2-GIPR,pLV-RIP2-GIPRdn vectors,then lentivirus carrying GIPR/GIPRdn gene was packed and used to infect pancreatic ? cell.The influence of GIPRdn on ? cells insulin secretion through GIP-GIPR-PKA signaling pathway was determined,and with the purpose to lay a foundation for developing the T2DM model of Guangxi Bama mini-pig by transgenic technology.The results were as follow:The RIP2 sequence was cloned using the designed primer based on the sequence of rat insulin 2(RIP2)gene published in GenBank.The RIP2 sequence was inserted into EGFP-1 vector without promoter for constructing recombinant vector EGFP-1-RIP2,which was respectively transfected into?TC-6,C2C12 and PK15 cells to identify the promoter function of the sequence.The results showed that the homology between reference sequence and cloned RIP2 sequence of 703bp was 99%,including 3 nucleotide mutations.Two transcription promoter regions were predicted and core element TATA-box and CAAT-box were found in RIP2 sequence.Meanwhile,cell transfection showed that the RIP2 sequence was capable of specifically turning on the expression of green fluorescent protein in ?TC-6 cell.Respectively inserting synthesized human dominant negative GIPR(GIPRdn)and normal GIPR gene into lentivirus vector pLV and substituting the RIP2 sequence to CMV promoter of pLV to construct the recombinant lentivirus vector of pLV-RIP2-GIPRdn,pLV-RIP2-GIPR,which were transfected into 293T cell with packaging plasmid for packing.The lentivirus was infected pTC-6 cell after concentrating and titers detection.The results showed that titers of the lentivirus respectively expressing GIPR and GIPRdn gene was 2×107TU/ml.The bright green fluorescence was observed and the expression of GIPRdn and GIPR was detected in ?TC-6 cell after the vectors of pLV-RIP2-GIPRdn,pLV-RIP2-GIPR and the lentivirus transfected(infected).The lentivirus respectively carrying GIPR and GIPRdn were infected ?TC-6 cell which was cultivated with hormones GIP.qPCR,Western Blot and ELISA respectively were used to detect the different effect of the overexpression of GIPR,GIPRdn gene on ? cells insulin secretion regulation in collected cells and cell culture after 48h and 72h cultivation.The qPCR results showed that the overexpression of GIPR gene promoted the expression of INS compared with the control group in ? TC-6 cells cultivated with GIP,while the overexpression of GIPRdn gene inhibited the expression of Gnabl,PKACa-1,CREB and PDX-1 gene,which did not affect the expression level of INS gene.Western Blot results showed that the overexpression of GIPR increased the PDX-1,total CREB,p-CREB and INS protein expression level,while the overexpression of GIPRdn reduced the PDX-1,total CREB,p-CREB and INS protein expression level.ELISA results showed that insulin concentration in cell cultures had no significant change when lentivirus infected in 48h.The insulin concentrations in cell cultures of overexpression of GIPR gene were significantly higher than the control group in 72h,and the insulin concentrations were significantly lower than the control group after overexpression of GIPRdn gene.Consequently,infection of lentivirus with GIPRdn gene to ? cells would competitively inhibit GIP-GIPR-PKA signaling pathways,and reduced the insulin secretion of P cells. |
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