| Part I The preparation and test of nanometer vector HGPAEsObjective:To prepare the nanometer vector HGPAEs and observe its safety and efficiency in vivo. Methods:The nanometer vector HGPAEs was prepared and tested. Normal saline group, nanometer group and nanometer vector pHK-shRNA control group were established for comparison and direct portal vein injection was performed to transfect liver. Liver specimens and blood were taken on the first day and the third day after transfection. Spectrofluorometer was used to detect the green fluorescent protein (EGFP) intensity and biochemical indicators like ALT, AST, TBIL. Crea and Urea were also detected. Results:The nanometer vector HGPAEs was successfully prepared and it was proved to be a nice vector for gene transfection. The EGFP intensity in liver of nanometer vector pHK-shRNA control group was significantly higher than normal saline group and nanometer vector control group both on the two experimental time points (P<0.01),and it showed that the intensity on the third day was higher than that on the first day in the plasmid group(P<0.01). Compared with normal value, the ALT and AST of all the three groups significantly increased on the first day after transfection (P<0.01). while there were no significant difference among the three groups themselves (P>0.05), and all those biochemical indicators became normal on the third day after transfection (P>0.05). Conclusions.-The nanometer vector HGPAEs was easy to be prepared and it was a promising gene transfer vector because it was effective and safe. Highly efficient liver transfection can be achieved through direct portal vein injection of the nanometer vector and plasmid DNA.Part Ⅱ In vivo study of inhibiting genes CⅡTA and MyD88with the help of nanometer vectorObjective:To observe the inhibition effects of RNAi on immune recognition genes CIITA, MHC-Ⅱand MyD88. Methods:Totally6groups, including normal saline control group, nanometer vector control group, nanometer vector pHK-shRNA control group and nanometer vector pC Ⅱ TA-shRNA group, nanometer vector pMyD88-shRNA group and nanometer vector pC Ⅱ TA-pMyD88-shRNA group, received liver transfection through portal vein injection. Real time PCR and western blot were performed to detect the expression of C ⅡTA, MHC-Ⅱ and MyD88in liver3days after transfection. Results:Realtime PCR and western blot showed that, compared with normal saline control group, nanometer vector control group and nanometer vector pHK-shRNA control group, the expression of C Ⅱ TA and MHC-Ⅱ were obviously inhibited both in nanometer vector pC Ⅱ TA-shRNA group and nanometer vector pC Ⅱ TA-pMyD88-shRNA group(P<0.01), and the expression of MyD88was also significantly inhibited in both nanometer vector pMyD88-shRNA group and nanometer vector pCⅡTA-pMyD88-shRNA group(P<0.01), while there was no obvious expression difference of C Ⅱ TA, MHC-Ⅱ and MyD88among the three control groups (P>0.05)). Conclusions:Highly efficient liver transfection can be achieved through direct portal vein injection of the nanometer vector and plasmid DNA. The expression of C Ⅱ TA, MHC-Ⅱ and MyD88in rat liver can be obviously inhibited by plasmids containing shRNAs targeting C Ⅱ TA and MyD88.Part Ⅲ The study of reducing transplantation rejection of rat liver by interferencing donor liverObjective:To observe the effect of inhibiting the expression of CⅡ TA and MyD88of graft on transplantation rejection in high responder model of rat orthotopic liver transplantation and investigate its anti-rejection mechanism. Methods:The high responder model of rat orthotopic liver transplantation was established. There were7groups:normal saline control group, nanometer vector control group, nanometer vector pHK-shRNA control group, nanometer vector pC Ⅱ TA-shRNA group, nanometer vector pMyD88-shRNA group, nanometer vector pC Ⅱ TA-pMyD88-shRNA group and CsA treating group. The donors in the first6groups received portal vein injection of normal saline or nanometer vector or nanometer vector and shRNA plasmid3days before transplantation.6recipients in each group were randomly chosen and sacrificed for their liver and blood as specimen5days after transplantation and the remaining6recipients were monitored for survival. The data of pathological rejection grading were collected by pathological examination, real-time PCR and western blot were used to test the mRNA and protein expression of CⅡTA, MHC-Ⅱ and MyD88in graft respectively, Flow Cytometry was used to test the ratio of CD4/CD8in blood, ELISA was used to observe the concentration of IL-2and IFN-y in serum, and liver function was also detected. Results:The high responder model of rat orthotopic liver transplantation was stably established. Compared to normal saline control group, nanometer vector control group and nanometer pHK-shRNA control group, the survival time of nanometer vector pC Ⅱ TA-shRNA group, nanometer vector pMyD88-shRNA group and nanometer vector pC Ⅱ TA-pMyD88-shRNA group extended significantly (median survival time16days,14days and21days vs1Odays,9days and8days, P<0.01), pathological rejection grading significantly depressed(P<0.01), the expressions of CⅡTA, MHC-Ⅱ and MyD88decreased significantly both on mRNA and protein level(P<0.01), the ratio of CD4/CD8in blood decreased obviously, the concentration of IL-2and IFN-γ in serum as well as liver function decreased significantly(P<0.01). Conclusion:High responder model of rat orthotopic liver transplantation is an ideal animal modelfor the study of liver transplantation rejection. Acute rejection reaction can be significantly relieved and survival time is extended by pretreatment of graft with portal vein injection of nanometer and shRNA plasmid targeting C Ⅱ TA and MyD88genes, which shows new way for both transplantation rejection research and clinical therapy. |
PDF Full Text Request