本文選題：IL-17　切入點：RORγt　出處：《鄭州大學》2017年碩士論文

【摘要】：1研究背景2型糖尿病作為一種慢性代謝性疾病,其發(fā)病機制復雜,涉及環(huán)境與遺傳兩大方面,胰島素分泌不足及胰島素抵抗是其發(fā)病的關鍵因素。相關研究表明,2型糖尿病不僅是一種代謝性疾病,也是一種亞臨床炎癥疾病,其發(fā)病機制與慢性低度炎癥密切相關,因此提出2型糖尿病的炎癥學說。大量研究證明,炎性因子、促炎性因子與T2DM密切相關,但其發(fā)病機制尚未完全闡明。近年來研究發(fā)現(xiàn),IL-17與2型糖尿病、1型糖尿病、高血壓及代謝綜合征均有一定的相關性。IL-17是一種促炎細胞因子,具有強大的招募中性粒細胞、促進多種細胞釋放炎性因子、促進細胞增值及抑制腫瘤生長等多種生物學作用,可導致炎癥反應、自身免疫性疾病等發(fā)生和發(fā)展。而IL-17也是一種重要的炎癥介質(zhì),它可以誘導其他炎癥細胞如IL-6、TNF-α等的表達,產(chǎn)生致炎作用。大量研究證實,無論體外還是體內(nèi),Th17分化IL-17細胞都需要RORγt的參與,證實RORγt是Th17細胞分化的關鍵正向轉(zhuǎn)錄調(diào)節(jié)因子。在活化T細胞中RORγt高表達可產(chǎn)生大量的IL-17,并且RORγt基因敲除小鼠體內(nèi)TH17細胞和IL-17明顯減少。二甲雙胍于20世紀進入臨床應用,大量循癥醫(yī)學證據(jù)顯示其有良好的降糖作用,且安全性及效價比均高,目前許多權威的糖尿病指南把二甲雙胍推薦為治療2型糖尿病的一線首選藥物。近年來一些研究顯示其除了降糖作用外同時具有抗炎作用,然而其抗炎作用機制仍未完全闡明。2材料和方法2.1分組及飼喂方法SPF級SD雄性大鼠50只,體重為89.62±7.42g。適應性喂養(yǎng)7天后,隨機分為2組,一組20只,一組30只,分別飼喂普通飼料、高脂高糖飼料,在第8周末時腹腔注射STZ,使用STZ+高脂高糖飼樣聯(lián)合造模,以30mg/kg STZ腹腔注射造模,3天后測空腹血糖,將空腹血糖大于11mmol/L視作為造模成功。將造模組隨機分為3組,每組10只,一組繼續(xù)飼喂高脂高糖(M組),一組飼喂高脂高糖+二甲雙胍(M+MET組),一組飼喂高脂高糖+胰島素(M+INS);普通飼料組隨機分為2組,每組各10只,一組飼喂普通飼養(yǎng)(C),一組飼喂普通飼料+二甲雙胍(C+MET)。MET按200mg/Kg劑量灌胃,不給予MET的大鼠使用生理鹽水灌胃。整個實驗共持續(xù)12周。2.2觀察指標(1)全自動血標本分析儀檢測血糖濃度及胰島素含量,計算胰島素抵抗指數(shù);(2)ELISA法檢測血清中IL-17的表達水平;(3)Western blotting法檢測RORγt蛋白的表達水平;(4)RT-PCR法檢測RORγt mRNA基因的表達;(5)免疫組化觀察脾臟組織中RORγt及IL-17陽性細胞因子的表達。3結果3.1 HOMA-IR各組大鼠HOMA-IR水平之間的差異有統(tǒng)計學意義(F=163.923,P=0.000)。M組、M+INS組和M+MET組的HOMA-IR均比C組高,差異有統(tǒng)計學意義(P0.05)。M+INS組、M+MET組的HOMA-IR均比M組低,差異有統(tǒng)計學意義(P0.05)。3.2血清IL-17的表達水平各組大鼠血清平均IL-17水平之間的差異有統(tǒng)計學意義(F=59.778,P=0.000)。進一步兩兩比較,M組、M+INS組、M+MET組的IL-17水平均高于C組,差異有統(tǒng)計學意義(P0.05)。M+MET組的IL-17水平低于M組,差異有統(tǒng)計學意義(P0.05)。C+MET組的IL-17水平低于M+MET組,差異有統(tǒng)計學意義(P0.05)。M+INS組的IL-17水平高于M+MET組,差異有統(tǒng)計學意義(P0.05)。C組的IL-17水平與C+MET組相比無統(tǒng)計學意義(P0.05)。M組的IL-17水平與M+INS組相比無統(tǒng)計學意義(P0.05)。3.3大鼠HOMA-IR與IL-17水平的相關性分析M組,HOMA-IR與血清IL-17水平呈正相關,r=0.718,P=0.019;M+MET組,HOMA-IR與血清IL-17水平呈正相關,r=0.706,P=0.022。3.4各組大鼠脾臟組織RORγt mRNA的表達各組大鼠脾臟組織平均RORγt mRNA水平之間的差異有統(tǒng)計學意義(F=910.176,P=0.000)。進一步兩兩比較,M組、M+INS組、M+MET組的RORγt mRNA水平均高于C組,差異有統(tǒng)計學意義(P0.05)。M+MET組的RORγt mRNA水平低于M組,差異有統(tǒng)計學意義(P0.05)。C+MET組的RORγt mRNA水平低于M+MET組,差異有統(tǒng)計學意義(P0.05)。M+INS組的RORγt mRNA水平高于M+MET組,差異有統(tǒng)計學意義(P0.05)。C組的RORγt mRNA水平與C+MET組相比無統(tǒng)計學意義(P0.05)。M組的RORγt mRNA水平與M+INS組相比無統(tǒng)計學意義(P0.05)。3.5各組大鼠脾臟組織RORγt蛋白的表達各組大鼠脾臟組織平均RORγt蛋白表達水平之間的差異有統(tǒng)計學意義(F=505.668,P=0.000)。進一步兩兩比較,M組、M+INS組、M+MET組的RORγt蛋白水平均高于C組,差異有統(tǒng)計學意義(P0.05)。M+MET組的RORγt蛋白水平低于M組,差異有統(tǒng)計學意義(P0.05)。C+MET組的RORγt蛋白水平低于M+MET組,差異有統(tǒng)計學意義(P0.05)。M+INS組的RORγt蛋白水平高于M+MET組,差異有統(tǒng)計學意義(P0.05)。C組的RORγt蛋白水平與C+MET組相比無統(tǒng)計學意義(P0.05)。M組的RORγt蛋白水平與M+INS組相比無統(tǒng)計學意義(P0.05)。3.6各組大鼠脾臟組織IL-17與RORγt的免疫組化表達水平M組較M+INS組、M+MET組、C組、C+MET組的脾臟組織中IL-17及RORγt細胞陽性比均高,差異有統(tǒng)計學意義(P0.05)。4結論(1)2型糖尿病大鼠的血清中IL-17及脾臟組織RORγt的表達水平升高。(2)炎癥因子IL-17表達增高可能加重2型糖尿病大鼠的胰島素抵抗。(3)二甲雙胍可通過抑制脾臟RORγt水平使2型糖尿病大鼠IL-17表達減少。
[Abstract]:1 research background of type 2 diabetes mellitus is a chronic metabolic disease, its pathogenesis is complex, involving environmental and genetic aspects of the two, the lack of insulin secretion and insulin resistance is a key factor in its pathogenesis. Related studies have shown that type 2 diabetes is a metabolic disease, is also a kind of subclinical inflammatory disease, the and the pathogenesis of chronic low-grade inflammation is closely related, therefore proposed the type 2 diabetes inflammation theory. Many studies have demonstrated that inflammatory cytokines, inflammatory cytokines are closely related with T2DM, but its pathogenesis has not been fully elucidated. Recent studies have found that IL-17 patients with type 2 diabetes mellitus, type 1 diabetes, hypertension and metabolic syndrome.IL-17 have a certain correlation between syndrome is a proinflammatory cytokine, has a strong recruitment of neutrophils, promote various cells to release inflammatory cytokines, promote cell proliferation and inhibit tumor growth and other biological The role, can cause inflammation, autoimmune disease and development. IL-17 is also an important medium of inflammation, it can induce other inflammatory cells such as IL-6, expression of TNF-, to produce proinflammatory effects. A number of studies have demonstrated that both in vitro and in vivo, Th17 IL-17 cells to ROR gamma the involvement of T, confirmed that the ROR gamma t is a key positive transcription regulatory factor. The differentiation of Th17 cells in the activation of T cells in ROR gamma t high expression can produce a large number of IL-17, ROR and gamma T gene knockout mice TH17 cells and IL-17 decreased significantly. The clinical application of metformin in twentieth Century, a large number of evidence-based medical evidence it has good hypoglycemic effect, and the safety and potency ratio are high, at present many authoritative diabetes guidelines to metformin recommended as the preferred first-line drugs for the treatment of type 2 diabetes. In recent years, some studies show that in addition to its hypoglycemic effect At the same time have anti-inflammatory effects with its anti-inflammatory effect, however the mechanism is not yet fully elucidated.2 material and method 2.1 grouping and feeding method of SPF 50 SD male rats, weight 89.62 + 7.42g. for adaptive feeding for 7 days, were randomly divided into 2 groups, a group of 20, a group of 30 rats were fed normal diet. High fat and high glucose diet, in eighth weeks after intraperitoneal injection of STZ, use the STZ+ high fat and high sugar feeding combined with 30mg/kg like model, intraperitoneal injection of STZ rats, after 3 days of fasting blood glucose, fasting blood glucose greater than 11mmol/L as the successful model. The rats were randomly divided into 3 groups, 10 rats in each group. Continue feeding high fat and high glucose (M group), a group fed with high fat and high glucose + metformin (M+MET group), a group fed with high fat and high glucose + insulin (M+INS); normal diet group were randomly divided into 2 groups, 10 rats in each group, one group was fed with normal chow (C), a group fed with normal diet metformin + (C+MET).MET by 20 0mg/Kg dose administered, not given MET rats using saline. The whole experiment lasted 12 weeks.2.2 observation index (1) full automatic blood analyzer to detect the concentration of blood glucose and insulin levels, insulin resistance index; (2) IL-17 in serum were measured by ELISA expression level; (3) expression the level of detection of ROR gamma t protein Western blotting method; (4) to detect the expression of ROR gene of t mRNA gamma RT-PCR method; (5) immunohistochemistry to observe the expression of.3 in spleen tissue results ROR gamma T and IL-17 positive cell factor differences in the level of HOMA-IR 3.1 HOMA-IR rats between the statistically significant (F=163.923. P=0.000).M group, M+INS group and M+MET group HOMA-IR were higher than C group, the difference was statistically significant (P0.05) in.M+INS group, M+MET group, HOMA-IR was lower than M group, the difference was statistically significant (P0.05) between the serum expression level of rats in each group the average IL-17 level of serum.3.2 IL-17 There was a significant difference (F=59.778, P=0.000). A further 22 compared with M group, M+INS group, M+MET group, IL-17 levels were higher than C group, the difference was statistically significant (P0.05).M+MET levels of IL-17 were lower than those of M group, the difference was statistically significant (P0.05).C+MET levels of IL-17 were lower than group M+MET, there are statistically significant difference (P0.05) of.M+INS group IL-17 was higher than M+MET group, the difference was statistically significant (P0.05).C group IL-17 levels compared with the C+MET group had no statistical significance (P0.05) in.M group compared with M+INS group IL-17 level had no statistical significance (P0.05) of M group between.3.3 and IL-17 levels in HOMA-IR rats and, HOMA-IR was positively related with the serum level of IL-17, r=0.718, P=0.019; group M+MET, HOMA-IR was positively associated with serum levels of IL-17, r=0.706, P=0.022.3.4 between groups of rat spleen tissue ROR mRNA expression of gamma t spleen tissue of rats in each group were averaged ROR gamma t mRNA level There was a significant difference (F=910.176, P=0.000). A further 22 compared with M group, M+INS group, M+MET group, ROR gamma t mRNA levels were higher than C group, the difference was statistically significant (P0.05) group.M+MET ROR y t mRNA level lower than the M group, the difference was statistically significant (P0.05).C+ MET group ROR y t level of mRNA was lower than that in M+MET group, the difference was statistically significant (P0.05) group.M+INS ROR y t mRNA level is higher than that of M+MET group, the difference was statistically significant (P0.05) group.C ROR y t mRNA level compared with the C+MET group had no statistical significance (P0.05) group.M ROR mRNA y t level and M+INS group no significant difference (P0.05) there was significant difference between the expression level of.3.5 group rat spleen tissue ROR protein expression of gamma t spleen tissue of rats in each group were averaged ROR gamma t protein (F=505.668, P=0.000). A further 22 compared with M group, M+INS group, M+MET group, ROR gamma t protein levels were significantly higher than that of C group, difference There was a significant correlation (P0.05) group.M+MET ROR gamma t protein level is lower than the M group, the difference was statistically significant (P0.05) group.C+MET ROR gamma t protein level is lower than the M+MET group, the difference was statistically significant (P0.05) group.M+INS ROR gamma t protein level is higher than that of M+MET group, the difference was statistically significant (P0.05.C) group of ROR gamma t protein levels compared with the C+MET group had no statistical significance (P0.05) group.M ROR gamma t protein levels compared with the M+INS group had no statistical significance (P0.05) group were immunized.3.6 rat spleen tissue IL-17 and ROR gamma t expression level in M group compared with M+INS group, M+ MET group. Group C, IL-17 and ROR gamma t C+MET cells in the spleen tissue were higher than positive, the difference was statistically significant (P0.05) conclusion.4 (1) increased the expression level of IL-17 in serum and spleen tissue of rats with type 2 diabetes in ROR gamma t. (2) increased the expression of inflammatory factor IL-17 may add weight 2 the insulin resistance in diabetic rats (3) metformin can reduce the expression of IL-17 in type 2 diabetic rats by inhibiting the level of ROR gamma t in the spleen.

【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R587.1
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本文編號：1674356