抗CD20单克隆抗体联合IL-10基因治疗对发病早期非肥胖型糖尿病小鼠肝脏Sox9、Ngn3、Hes1表达的影响

发布时间：2018-03-03 04:02

本文选题：抗CD20单克隆抗体　切入点：白介素10　出处：《青岛大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:观察抗CD20单克隆抗体联合腺病毒介导的IL-10基因对发病早期非肥胖性糖尿病鼠(NOD)肝脏细胞Sox9、Ngn3、Hes1基因表达的情况。方法:选择17-19周龄新诊断1型糖尿病(T1D)NOD鼠24只,随机分为A、B、C、D四组,分别代表抗CD20单抗单药治疗组、抗CD20+IL-10联合治疗组、Ad-IL-10单药治疗组及生理盐水对照(NS)组。分别于发病后第1,3,6,9天尾静脉注射抗CD20单抗250ug,抗CD20单抗250ug+Ad-IL-10 100ul,Ad-IL-10 100ul,NS 100ul,每3天1次,首剂加倍,共注射4次。期间每天同一时间监测小鼠血糖水平,若血糖≥25mmol/L,给予20U/kg皮下注射来得时;每周同一时间测量2次体重,并记录。自最后1次给药后,连续观察9周,处死小鼠。摘眼球后取血,静置离心取血清,行ELISA检测。解剖分离肝脏,部分行Western Blot和RT-PCR检测;部分用4%甲醛固定,制作石蜡切片,行免疫组化检测。结果:动态血糖监测示联合治疗可明显降低NOD鼠血糖水平。动态体质量监测示各组干预对NOD鼠体质量影响不大,差异无统计学意义(P0.05)。RT-PCR结果示联合组小鼠肝脏Sox9、Ngn3基因表达明显高于单药治疗组和生理盐水组,差异有统计学意义(P0.05),Hes1基因表达量在各组间差别不大,差异无统计学意义(P0.05);Western Blot示联合治疗组IL-10蛋白表达水平明显高于其它组,差异有统计学意义(P0.05),Hes1蛋白表达量在各组间差别不大,差异无统计学意义(P0.05);免疫荧光结果示胰岛素表达细胞在联合治疗组明显增多,差异有统计学意义(P0.05);ELISA结果示联合治疗组较单药治疗组血清IL-17、IL-1β明显减低,联合治疗组血清TGF-β、Insulin水平较其他三组明显较高,差异具有统计学意义(P0.05)。结论:腺病毒介导的IL-10基因在肝脏组织内高表达。抗CD20单克隆抗体与腺病毒介导的IL-10联合应用可促进NOD鼠胰岛素的分泌,更好的维持血糖稳定。联合干预可以减低IL-17、IL-1β等炎性因子水平,促进TGF-β合成,维持免疫稳态。同时,联合干预可以通过激活Notch信号通路,促进Sox9、Ngn3基因表达等方式促进肝脏合成IPC(胰岛内分泌细胞),分泌肝源性胰岛素,对残存胰岛β细胞功能具有一定的保护作用。
[Abstract]:Objective: to observe the expression of CD20 monoclonal antibody combined with adenovirus mediated IL-10 gene in liver cells of nonobese diabetic rats. Methods: Twenty-four newly diagnosed type 1 diabetic T1D nod mice were selected from 17 to 19 weeks old. They were randomly divided into four groups: group A, B, C, D, representing the treatment group of anti CD20 McAb, respectively. Anti CD20 IL-10 combined with Ad-IL-10 monotherapy group and normal saline control group were injected intravenously with anti-#en2# McAb 250ugand anti-#en3# McAb 250ug Ad-IL-10 100uln NS 100U every 3 days at the end of 9 days after the onset of the disease, and the first dose was doubled. The blood glucose level of mice was monitored at the same time every day. If the blood glucose was more than 25 mmol / L, 20 U / kg subcutaneous injection was given to the mice, and the weight was measured twice a week at the same time, and recorded. The mice were sacrificed, blood was taken from eyeball, serum was collected by static centrifugation, ELISA was detected, liver was dissected, Western Blot and RT-PCR were detected partly, paraffin sections were made by fixed with 4% formaldehyde. Results: dynamic blood glucose monitoring showed that combined treatment could significantly reduce the blood glucose level of NOD mice. Dynamic body mass monitoring showed that the intervention of each group had little effect on the body mass of NOD mice. The results of RT-PCR showed that the expression of Ngn3 gene in liver of combined group was significantly higher than that of single drug treatment group and normal saline group. The expression of IL-10 protein in the combined treatment group was significantly higher than that in the other groups, and there was no significant difference in the expression of Hes1 protein between the two groups. The results of immunofluorescence showed that the expression of insulin cells in the combined treatment group was significantly increased, and the difference was statistically significant. The results of Elisa showed that the serum levels of IL-17 and IL-1 尾 in the combined treatment group were significantly lower than those in the single drug treatment group. The serum TGF- 尾 insulin level in the combined treatment group was significantly higher than that in the other three groups. Conclusion: adenovirus-mediated IL-10 gene is highly expressed in liver tissue. The combination of anti-adenovirus monoclonal antibody and adenovirus-mediated IL-10 can promote insulin secretion in NOD mice. The combined intervention can reduce the level of inflammatory factors such as IL-17 and IL-1 尾, promote the synthesis of TGF- 尾 and maintain the immune homeostasis. At the same time, the combined intervention can activate the Notch signaling pathway. Promoting the expression of Sox9 Ngn3 gene and promoting the synthesis of IPCs (islet endocrine cells, secreting hepatogenic insulin, etc.) has a certain protective effect on the function of residual islet 尾 cells.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1

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