抗CD20單克隆抗體及IL-10基因聯(lián)合免疫治療對(duì)胰島β細(xì)胞保護(hù)作用及機(jī)制研究
發(fā)布時(shí)間：2018-07-20 16:22
【摘要】：目的觀察抗CD20單克隆抗體(Anti-CD20)及腺病毒介導(dǎo)的鼠白細(xì)胞介素10(Adenovirus vector-mediated murine interleukin, Ad-mIL-10)聯(lián)合干預(yù)對(duì)未發(fā)病非肥胖型糖尿病(Nonobese diabetic, NOD)小鼠胰島炎、胰島β細(xì)胞凋亡的影響和胰島β細(xì)胞的預(yù)防保護(hù)作用。 方法3～5周齡未發(fā)病雌性NOD小鼠24只,隨機(jī)分為①Anti-CD20組、②Anti-CD20+IL-10組、③IL-10組和④生理鹽水對(duì)照(Normal control, NC)組。分別于第1d、第8d、第15d和第21d靜脈給予Anti-CD20、Anti-CD20+IL-10、IL-10和生理鹽水治療。定期監(jiān)測(cè)小鼠體質(zhì)量、尿糖及血糖,治療12w后麻醉下取小鼠血清測(cè)定Insulin、IL-10、CD20和CD19水平。分離胰腺行病理切片HE染色及免疫組化觀察胰島炎癥浸潤程度、TUNEL法觀察胰島β細(xì)胞凋亡情況和免疫組化檢測(cè)胰腺Insulin、Caspase3、IL-10和CD20表達(dá)情況。Western Blot和RT-PCR方法檢測(cè)胰腺中Bcl-2、Caspase3和PDX-1蛋白和RNA表達(dá)情況。 結(jié)果1.各組小鼠體質(zhì)量終值和治療后第4w、8w和12w小鼠隨機(jī)血糖組間比較差異均無統(tǒng)計(jì)學(xué)意義(P0.05)。觀察結(jié)束時(shí)各組小鼠均未發(fā)病,且血糖維持在正常水平； 2. Anti-CD20和IL-10聯(lián)合治療可使血清中Insulin和IL-10水平升高(vsAnti-CD20組、NC組,P=0.0002;vs IL-10組,P=0.01)；各組血清CD20和CD19水平比較,差異均無統(tǒng)計(jì)學(xué)意義。Anti-CD20、IL-10單獨(dú)治療組胰腺中Insulin、IL-10蛋白高表達(dá),聯(lián)合治療組較單獨(dú)治療組Insulin、IL-10蛋白胰腺局部表達(dá)水平更高(P0.0001)： 3. Anti-CD20組、IL-10組胰島炎積分(Anti-CD20組vs NC組,P=0.0029：IL-10組vs NC組,P=0.0066)、β細(xì)胞凋亡率(NC組vs Anti-CD20,p=0.0001;NC組vs IL-10組,P=0.001)均明顯低于NC組；而Anti-CD20+IL-10組胰島炎積分(P0.0001)、β細(xì)胞凋亡率(vs IL-10組、NC組,P0.0001；vs Anti-CD20組,P=0.001)顯著低于單獨(dú)治療組和NC組； 4.免疫組化結(jié)果顯示：Anti-CD20組、IL-10組Caspase3蛋白低表達(dá),Caspase3蛋白表達(dá)水平聯(lián)合治療組較IL-10組更低(P=0.0055)；聯(lián)合治療組與Anti-CD20組、IL-10組和NC組比較,CD20表達(dá)量顯著升高(vs IL-10組、NC組,P0.0001；vs Anti-CD20組,P=0.0004)。Western Blot和RT-PCR結(jié)果顯示：小鼠Anti-CD20+IL-10治療后,小鼠胰腺中Caspase3凋亡蛋白局部表達(dá)、蛋白定量和RNA定量均顯著降低：胰腺中Bcl-2、PDX-1蛋白和RNA表達(dá)量均明顯高于單獨(dú)治療組和生理鹽水組,P0.01。 結(jié)論1Anti-CD20+IL-10聯(lián)合干預(yù)維持動(dòng)態(tài)血糖和體質(zhì)量變化在正常范圍； 2.Anti-CD20、IL-10單獨(dú)干預(yù)可以減輕胰島炎癥程度,Anti-CD20+IL-10聯(lián)合治療使以上作用更加顯著； 3.Anti-CD20+IL-10聯(lián)合干預(yù)增加血清胰島素、IL-10水平和其在胰腺局部表達(dá)水平； 4.Anti-CD20和/或IL-10干預(yù)可減輕胰島B細(xì)胞凋亡率,使Caspase3蛋白和RNA低水平表達(dá),聯(lián)合干預(yù)起到協(xié)同作用。同時(shí)增強(qiáng)抗凋亡蛋白、PDX-1蛋白和RNA表達(dá)。有效發(fā)揮細(xì)胞免疫調(diào)節(jié)和β細(xì)胞保護(hù)作用,減少胰島B細(xì)胞凋亡和損傷,從而更好的保護(hù)殘存胰島B細(xì)胞的數(shù)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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R587.1
本文編號(hào)：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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R587.1
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