抗CD20单克隆抗体及IL-10基因联合免疫治疗对胰岛β细胞保护作用及机制研究
发布时间：2018-07-20 16:22
【摘要】：目的观察抗CD20单克隆抗体(Anti-CD20)及腺病毒介导的鼠白细胞介素10(Adenovirus vector-mediated murine interleukin, Ad-mIL-10)联合干预对未发病非肥胖型糖尿病(Nonobese diabetic, NOD)小鼠胰岛炎、胰岛β细胞凋亡的影响和胰岛β细胞的预防保护作用。 方法3～5周龄未发病雌性NOD小鼠24只,随机分为①Anti-CD20组、②Anti-CD20+IL-10组、③IL-10组和④生理盐水对照(Normal control, NC)组。分别于第1d、第8d、第15d和第21d静脉给予Anti-CD20、Anti-CD20+IL-10、IL-10和生理盐水治疗。定期监测小鼠体质量、尿糖及血糖,治疗12w后麻醉下取小鼠血清测定Insulin、IL-10、CD20和CD19水平。分离胰腺行病理切片HE染色及免疫组化观察胰岛炎症浸润程度、TUNEL法观察胰岛β细胞凋亡情况和免疫组化检测胰腺Insulin、Caspase3、IL-10和CD20表达情况。Western Blot和RT-PCR方法检测胰腺中Bcl-2、Caspase3和PDX-1蛋白和RNA表达情况。 结果1.各组小鼠体质量终值和治疗后第4w、8w和12w小鼠随机血糖组间比较差异均无统计学意义(P0.05)。观察结束时各组小鼠均未发病,且血糖维持在正常水平； 2. Anti-CD20和IL-10联合治疗可使血清中Insulin和IL-10水平升高(vsAnti-CD20组、NC组,P=0.0002;vs IL-10组,P=0.01)；各组血清CD20和CD19水平比较,差异均无统计学意义。Anti-CD20、IL-10单独治疗组胰腺中Insulin、IL-10蛋白高表达,联合治疗组较单独治疗组Insulin、IL-10蛋白胰腺局部表达水平更高(P0.0001)： 3. Anti-CD20组、IL-10组胰岛炎积分(Anti-CD20组vs NC组,P=0.0029：IL-10组vs NC组,P=0.0066)、β细胞凋亡率(NC组vs Anti-CD20,p=0.0001;NC组vs IL-10组,P=0.001)均明显低于NC组；而Anti-CD20+IL-10组胰岛炎积分(P0.0001)、β细胞凋亡率(vs IL-10组、NC组,P0.0001；vs Anti-CD20组,P=0.001)显著低于单独治疗组和NC组； 4.免疫组化结果显示：Anti-CD20组、IL-10组Caspase3蛋白低表达,Caspase3蛋白表达水平联合治疗组较IL-10组更低(P=0.0055)；联合治疗组与Anti-CD20组、IL-10组和NC组比较,CD20表达量显著升高(vs IL-10组、NC组,P0.0001；vs Anti-CD20组,P=0.0004)。Western Blot和RT-PCR结果显示：小鼠Anti-CD20+IL-10治疗后,小鼠胰腺中Caspase3凋亡蛋白局部表达、蛋白定量和RNA定量均显著降低：胰腺中Bcl-2、PDX-1蛋白和RNA表达量均明显高于单独治疗组和生理盐水组,P0.01。 结论1Anti-CD20+IL-10联合干预维持动态血糖和体质量变化在正常范围； 2.Anti-CD20、IL-10单独干预可以减轻胰岛炎症程度,Anti-CD20+IL-10联合治疗使以上作用更加显著； 3.Anti-CD20+IL-10联合干预增加血清胰岛素、IL-10水平和其在胰腺局部表达水平； 4.Anti-CD20和/或IL-10干预可减轻胰岛B细胞凋亡率,使Caspase3蛋白和RNA低水平表达,联合干预起到协同作用。同时增强抗凋亡蛋白、PDX-1蛋白和RNA表达。有效发挥细胞免疫调节和β细胞保护作用,减少胰岛B细胞凋亡和损伤,从而更好的保护残存胰岛B细胞的数量和功能。
[Abstract]:Objective to observe the effects of anti-CD20 monoclonal antibody (Anti-CD20) and adenovirus-mediated Adenovirus vector-mediated murine interleukin-10 (Ad-mIL-10) on islet inflammation in nonobese diabetic mice. Effect of islet 尾 cell apoptosis and prevention and protection of islet 尾 cell. Methods Twenty-four female nod mice at the age of 5 weeks were randomly divided into 1 Anti-CD20 group and 2 Anti-CD20 IL-10 group and 4 normal control group (NC). Anti-CD20 IL-10IL-10 and normal saline were administered intravenously on day 1, day 8, day 15 and day 21 respectively. The body weight, urine sugar and blood sugar were monitored regularly. After 12 weeks of treatment, the serum levels of insulinine IL-10 CD20 and CD19 were measured. Detection of apoptosis of 尾 -cell in pancreatic islets by Tunel method and expression of IL-10 and CD20 by immunohistochemistry in pancreatic insulin.Western Blot and RT-PCR for detection of Bcl-2Caspase3 and PDX-1 in pancreas Protein and RNA expression. Result 1. There was no significant difference in the final body weight of each group and the random blood glucose group at the 4th week and 12th week after treatment (P0.05). At the end of the observation, the mice in each group did not develop the disease, and the blood sugar was maintained at the normal level. 2. Anti-CD20 and IL-10 combined therapy could increase insulin and IL-10 levels in serum (vs Anti-CD20 group vs IL-10 group P < 0.01), but there was no significant difference in serum CD20 and CD19 levels between each group. Anti-CD20 / IL-10 alone treatment group had high insulin IL-10 protein expression. The local expression level of insulin IL-10 protein in pancreas in combination group was higher than that in control group (P 0.0001): 3. In Anti-CD20 group (Anti-CD20 group vs NC group, P < 0.0066), 尾 cell apoptosis rate (NC group vs Anti-CD20 / IL-10 group vs IL-10 group) was significantly lower than that in NC group (P < 0.001), while in Anti-CD20 IL-10 group (P 0.0001), 尾 cell apoptosis rate (vs IL-10 group NC group P 0.0001) was significantly lower than that in NC group (P 0.0001), and 尾 cell apoptosis rate (vs IL-10 group NC group P 0.0001) was significantly lower than that in anti-CD20 IL-10 group (P 0.0001). Vs Anti-CD20 group (P < 0.001) was significantly lower than that of single treatment group and NC group. Immunohistochemical results showed that the low expression of Caspase3 protein and the expression of Caspase3 protein in the combined treatment group were lower than those in the IL-10 group (P0. 0055), the expression of CD20 protein in the combined treatment group was significantly higher than that in the anti CD20 group and the control group (vs IL-10 group, P 0. 0001, P 0. 0001), the expression of caspase3 protein in the combined treatment group was significantly higher than that in the anti CD20 group (P 0. 0001). The results of Western blot and RT-PCR showed that after treatment with Anti-CD20 IL-10, the local expression of Caspase3, the quantitative expression of Caspase3 and the quantity of RNA in pancreas were significantly decreased, and the expression of Bcl-2PDX-1 protein and RNA in pancreas were significantly higher than those in control group and saline group (P0.01). Conclusion (1) Anti-CD20 / IL-10 combined with anti-CD20 / IL-10 can reduce the degree of islet inflammation and the combined treatment of Anti-CD20 / IL-10 can make the changes of blood glucose and body mass in normal range, and the combined therapy of Anti-CD20 / IL-10 can make the above effects more significant. 3. Anti-CD20 / IL-10 combined intervention increased serum insulin level and its local expression level, 4. Anti-CD20 and / or IL-10 intervention reduced the apoptosis rate of islet B cells and reduced the expression of Caspase3 protein and RNA. Joint intervention plays a synergistic role. At the same time, the expression of PDX-1 protein and RNA was enhanced. In order to protect the number and function of residual islet B cells, the apoptosis and injury of islet B cells can be reduced by exerting the effect of cell immune regulation and 尾 cell protection.
【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R587.1
本文编号：2134087
[Abstract]:Objective to observe the effects of anti-CD20 monoclonal antibody (Anti-CD20) and adenovirus-mediated Adenovirus vector-mediated murine interleukin-10 (Ad-mIL-10) on islet inflammation in nonobese diabetic mice. Effect of islet 尾 cell apoptosis and prevention and protection of islet 尾 cell. Methods Twenty-four female nod mice at the age of 5 weeks were randomly divided into 1 Anti-CD20 group and 2 Anti-CD20 IL-10 group and 4 normal control group (NC). Anti-CD20 IL-10IL-10 and normal saline were administered intravenously on day 1, day 8, day 15 and day 21 respectively. The body weight, urine sugar and blood sugar were monitored regularly. After 12 weeks of treatment, the serum levels of insulinine IL-10 CD20 and CD19 were measured. Detection of apoptosis of 尾 -cell in pancreatic islets by Tunel method and expression of IL-10 and CD20 by immunohistochemistry in pancreatic insulin.Western Blot and RT-PCR for detection of Bcl-2Caspase3 and PDX-1 in pancreas Protein and RNA expression. Result 1. There was no significant difference in the final body weight of each group and the random blood glucose group at the 4th week and 12th week after treatment (P0.05). At the end of the observation, the mice in each group did not develop the disease, and the blood sugar was maintained at the normal level. 2. Anti-CD20 and IL-10 combined therapy could increase insulin and IL-10 levels in serum (vs Anti-CD20 group vs IL-10 group P < 0.01), but there was no significant difference in serum CD20 and CD19 levels between each group. Anti-CD20 / IL-10 alone treatment group had high insulin IL-10 protein expression. The local expression level of insulin IL-10 protein in pancreas in combination group was higher than that in control group (P 0.0001): 3. In Anti-CD20 group (Anti-CD20 group vs NC group, P < 0.0066), 尾 cell apoptosis rate (NC group vs Anti-CD20 / IL-10 group vs IL-10 group) was significantly lower than that in NC group (P < 0.001), while in Anti-CD20 IL-10 group (P 0.0001), 尾 cell apoptosis rate (vs IL-10 group NC group P 0.0001) was significantly lower than that in NC group (P 0.0001), and 尾 cell apoptosis rate (vs IL-10 group NC group P 0.0001) was significantly lower than that in anti-CD20 IL-10 group (P 0.0001). Vs Anti-CD20 group (P < 0.001) was significantly lower than that of single treatment group and NC group. Immunohistochemical results showed that the low expression of Caspase3 protein and the expression of Caspase3 protein in the combined treatment group were lower than those in the IL-10 group (P0. 0055), the expression of CD20 protein in the combined treatment group was significantly higher than that in the anti CD20 group and the control group (vs IL-10 group, P 0. 0001, P 0. 0001), the expression of caspase3 protein in the combined treatment group was significantly higher than that in the anti CD20 group (P 0. 0001). The results of Western blot and RT-PCR showed that after treatment with Anti-CD20 IL-10, the local expression of Caspase3, the quantitative expression of Caspase3 and the quantity of RNA in pancreas were significantly decreased, and the expression of Bcl-2PDX-1 protein and RNA in pancreas were significantly higher than those in control group and saline group (P0.01). Conclusion (1) Anti-CD20 / IL-10 combined with anti-CD20 / IL-10 can reduce the degree of islet inflammation and the combined treatment of Anti-CD20 / IL-10 can make the changes of blood glucose and body mass in normal range, and the combined therapy of Anti-CD20 / IL-10 can make the above effects more significant. 3. Anti-CD20 / IL-10 combined intervention increased serum insulin level and its local expression level, 4. Anti-CD20 and / or IL-10 intervention reduced the apoptosis rate of islet B cells and reduced the expression of Caspase3 protein and RNA. Joint intervention plays a synergistic role. At the same time, the expression of PDX-1 protein and RNA was enhanced. In order to protect the number and function of residual islet B cells, the apoptosis and injury of islet B cells can be reduced by exerting the effect of cell immune regulation and 尾 cell protection.
【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R587.1
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