The Protective Effects Of Adenovirus-mediated IL-10 Gene And Anti-CD20 Monoclonal Antibody On The Pancreatic β Cells Of NOD Mice In The Early Stage Of Natural T1D Onset

Type 1 diabetes mellitus(T1D)is an autoimmune disease characterized by an absolute lack of insulin secretion and progressive destruction of pancreatic β cells,with lifelong subcutaneous injection of insulin remaining its major treatment means at present.However,lifelong subcutaneous injection of insulin has brought tremendous sufferings for children patients.In addition,fewer functions of the residual pancreatic β cells in the children patients will lead to greater dependence on insulin,more unstable blood glucose,earlier occurrence of acute and chronic complications of T1 D,and a poorer prognosis for the children patients.Therefore,actively protecting functions of T1 D residual pancreatic β cell is of great clinical significance.T1 D is an autoimmune disease,which involves cellular and humoral immune dysfunction during its pathogenesis.This project was thereby carried out proposing to recover cellular immune homeostasis using adenovirus-mediated IL-10 gene and recover humoral immune homeostasis with anti-CD20 monoclonal antibody,with an aim to observe the protective effects of drug combination on pancreatic β cells early in the natural incidence of T1 D in NOD rats and to investigate its relevant mechanism.This project mainly contained two parts:Part Ⅰ Purify,Enrich and Proliferate The Recombinant Adeno virus –Mediated IL-10 GeneObjective To purify,enrich and proliferate recombinant adenovirus-mediated IL-10 gene(Ad-IL-10).Methods Total RNA was extracted from rat splenocytes cultured with lipopolysaccharide(LPS),and IL-10 cDNA was amplified with reverse transcription polymerase chain reaction(qRT-PCR),which was then directionally subcloned into pAdtrack-CMV to construct the adenovirus shuttle plasmid pAdtrack-CMV-IL-10.It was transformed into adenovirus framework plasmid pAdEasy-containing BJ5183 Escherichia coli after enzyme digestion linearization,so as to obtain the recombinant adenovirus plasmid pAd-IL-10.Lipofectamin was adopted to package and transfect 293 cell,so as to obtain the recombinant adenovirus Ad-IL-10.Expression of green fluorescence was observed under fluorescence microscope.Similarly,null adenovirus vector without target gene(Ad-GFP)was packaged and amplified according to the same method,which was served as the control virus vector.Virus titer was determined through rapid adenovirus infectious titer detection kit.Results Expression quantity of recombinant adenovirus fluorescence carrying green fluorescent protein and rat IL-10 gene was high in HEK293 cell.Virus titer was determined through rapid adenovirus infectious titer detection kit after enrichment and amplification of adenovirus,which was 1.0×1011pfu/ml.Similarly,expression quantity of adenovirus vector(Ad-GFP)carrying green fluorescent protein only was high in HEK293 cell.Virus titer was determined through rapid adenovirus infectious titer detection kit after enrichment and amplification of adenovirus,which was 1.0×1011pfu/ml.Conclusion Recombinant adenovirus-mediated IL-10 gene(Ad-IL-10)and null vector(Ad-GFP)are successfully enriched and amplified,which has established foundation for subsequent animal experiments.Part Ⅱ The Protective Effects of Adenovirus-mediated IL-10 Gene and Anti-CD20 Monoclonal Antibody on the Pancreatic β Cells of NOD Mice in the Early Stage of Natural T1 D OnsetObjective The experiment blocked the proliferation and activation of β cells through anti-CD20 monoclonal antibody,and restored the ratio of Th cell subset through adenovirus-mediated IL-10 gene(Ad-IL-10),with an aim to observe the protective effects of combined intervention on the pancreatic β cells of NOD mice in the early stage of T1 D onset.Methods 35 NOD mice that were aged between 17 and 20 weeks and were diagnosed with T1 D within recent 3 days were selected and divided randomly into the anti-CD20 monoclonal antibody alone group,the anti-CD20 combined with IL-10 group(the combined group),the IL-10 alone group(the Ad-IL-10 group),the null vector group(the Ad-GFP group),and the normal saline group(the NS group).250 ug of anti-CD20 monoclonal antibody,250 ug of anti-CD20 monoclonal antibody combined with 100 ul of Ad-IL-10,100 ul of Ad-IL-10,100 ul of Ad-GFP,as well as 100 ul of NS were injected to the above-mentioned groups through the caudal vein,respectively,for four times with once every three days and the first-dosage doubled.During this period,the blood glucose level of the mice should be closely monitored.Observed for 9 weeks successively from the last time of administration,then the mice were sacrificed.Observed the inflammatory infiltration of the pancreatic islets with HE staining;detected the quantity of pancreatic β cells by TUNEL assay;assessed the levels of IL-14,IL-10,insulin,C-peptide,TNF-α and INF-γ in mice serum through ELISA method;evaluated the in situ expression of IL-10 and CD20 through immunohistochemistry;detected the expression of IL-10,CD20,insulin,Bcl-2 and Bax genes of mice pancreas with qRT-PCR;and detected the expression of related proteins,such as IL-10,CD20,pro-caspase 3,cleaved-caspase 3,cleaved-caspase 8,cleaved-caspase 9,Bcl-2 and Bax of the pancreas through western blot.Results The blood glucose level of mice in the combined group was reduced.A majority of the insulitis was grade 0-1 in the combined group,and grade 2-3 in the Ad-GFP group and the NS group,with the insulitis grading of NOD mice being remarkably decreased in the combined group.The apoptosis rate of pancreatic β cells was significantly lower in the combined group than in the IL-10 alone group,the Ad-GFP group and the NS group,with all p<0.05.Immunohistochemistry indicated that IL-10 could be highly expressed locally in the pancreatic islets,while the expression of CD20 was reduced in the combined group.The results of qRT-PCR showed that high expression of IL-10 could be found in the IL-10 alone group and the combined group,but the expression of CD20 showed no difference among groups.The insulin gene was highly expressed in the combined group with all p<0.01 when compared with the other groups.The expression of Bcl-2 gene was notably increased in the combined group,while that of Bax gene was distinctly decreased,and the ratio of Bcl-2/Bax was apparently higher than that of other groups with p<0.01.The results of western blot revealed that the expression of pro-caspase 3,cleaved-caspase3,cleaved-caspase 8 and cleaved-caspase 9 was lower in the combined group than in the Ad-GFP group and the NS group,with p<0.05.The expression of Bcl-2 protein was increased in the combined group,while that of Bax protein was reduced,and the ratio of Bcl-2/Bax was higher than that in the IL-10 alone group,the Ad-GFP group,and the NS group,with p<0.05.Conclusion Adenovirus-mediated IL-10 gene showed high expression in local pancreatic islets,which when combined with anti-CD20 monoclonal antibody could accelerate the secretion of insulin in NOD mice,control blood glucose,and alleviate insulitis.Combined intervention exerted protective effects on pancreatic β cells through activating Bcl-2 anti-apoptosis pathway,inhibiting the expression of TNF-α and INF-γ,and blocking caspase-8--caspase-3 as well as caspase-9--caspase-3 apoptosis pathways.In sum,combined intervention of anti-CD20 monoclonal antibody and IL-10 gene could reduce the apoptosis of pancreatic β cells of NOD mice in the early stage of onset,and had certain protective effects on the residual pancreatic β cell function.