本文選題：2型糖尿病　切入點(diǎn)：骨質(zhì)疏松　出處：《山西醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:本實(shí)驗(yàn)旨在探討2型糖尿病(DM)大鼠骨微環(huán)境氧化應(yīng)激(OS)水平與成骨細(xì)胞(OB)分化調(diào)節(jié)因子Runx2的關(guān)系,同時(shí)觀察脂聯(lián)素(APN)干預(yù)是否可以通過改善骨微環(huán)境氧化應(yīng)激影響成骨細(xì)胞分化,進(jìn)一步探討糖尿病性骨質(zhì)疏松癥(DOP)的發(fā)生機(jī)制及早期干預(yù)措施,為臨床預(yù)防及治療糖尿病性骨質(zhì)疏松癥提供新思路。方法:選擇4周齡雄性SD大鼠隨機(jī)分為正常對(duì)照組(n=18只)、糖尿病模型組(n=42只)。對(duì)照組予普通飼料喂養(yǎng),模型組高膽固醇高糖喂養(yǎng)2個(gè)月,并一次性腹腔注射鏈脲佐菌素(STZ)30mg/kg建2型DM大鼠模型。剔除造模失敗、死亡大鼠,建模成功的大鼠分為糖尿病組(DM組=18只)和脂聯(lián)素干預(yù)組(APN組=18只),APN組予腹腔注射球形脂聯(lián)素10μg/kg*d干預(yù),分別于第4、8、12周末處死動(dòng)物,迅速取出雙側(cè)股骨及脛骨,提取一側(cè)股骨及脛骨骨髓液,經(jīng)離心分別留取骨髓細(xì)胞及骨髓上清液,另一側(cè)股骨經(jīng)測(cè)定骨密度后脫鈣包埋切片。ELISA、RT-PCR等方法檢測(cè)Runx-2、BALP、AOPPs,骨組織免疫組化染色觀察AGEs表達(dá),HE染色觀察骨質(zhì)量(骨小梁結(jié)構(gòu)、密度)及脂肪細(xì)胞量。結(jié)果:1.各組大鼠血糖水平比較隨著喂養(yǎng)時(shí)間的延長(zhǎng),可見DM組大鼠血糖水平明顯升高。APN干預(yù)后,血糖有所改善。2.三組大鼠骨髓上清液AOPP水平比較隨著喂養(yǎng)時(shí)間延長(zhǎng),正常大鼠組骨髓上清液AOPP無明顯變化,在DM組、APN干預(yù)組均升高(P0.05),但APN干預(yù)組升高幅度較DM組小。在4周時(shí),DM組較正常對(duì)照組AOPP水平升高(P0.05),APN干預(yù)后較DM組無明顯差異;8周及12周時(shí)DM組AOPP水平升高更顯著(P0.05),APN干預(yù)后,8周時(shí)有輕微下降趨勢(shì),12周時(shí)較DM組下降且有統(tǒng)計(jì)學(xué)意義(P0.05)。3.三組大鼠骨組織免疫組化AGEs表達(dá)比較隨著時(shí)間變化,正常大鼠組骨組織免疫組化表達(dá)AGEs無明顯差異,在DM組、APN干預(yù)組表達(dá)均升高,(P0.05),但APN干預(yù)組升高幅度較DM組小。在4周時(shí),DM組較正常對(duì)照組AGEs表達(dá)水平升高(P0.05),APN干預(yù)后較DM組降低(P0.05),8周及12周時(shí)DM組AGEs表達(dá)升高更顯著(P0.05),APN干預(yù)后較DM組降低(P0.05),且12周較8周AGEs下降幅度顯著增大。4.三組大鼠骨髓細(xì)胞Runx2 mRNA表達(dá)水平及骨髓上清液BALP水平比較隨著飼養(yǎng)時(shí)間增加,正常對(duì)照組、DM組、APN干預(yù)組Runx2 mRNA、BALP水平均逐漸降低,且DM組下降幅度更顯著(P0.05)。在4周時(shí),兩兩組間比較均無明顯統(tǒng)計(jì)學(xué)差異;8周時(shí),DM組較正常對(duì)照組Runx2 mRNA、BALP水平開始降低,APN干預(yù)后較DM組,上述指標(biāo)下降幅度減低,水平升高(P0.05);12周時(shí),DM組較對(duì)照組明顯降低,APN干預(yù)后較DM組上述指標(biāo)升高(P0.05),且APN組與DM組間各指標(biāo)的差異較8周時(shí)顯著。5.三組大鼠股骨骨密度(BMD)比較4周與12周隨時(shí)間變化,正常對(duì)照組、DM組、APN干預(yù)組三組BMD值降低;4周時(shí)三組兩兩比較BMD差異性較小無統(tǒng)計(jì)學(xué)意義,但是12周時(shí),DM組較對(duì)照組BMD下降,APN干預(yù)后較DM組有所升高且有統(tǒng)計(jì)學(xué)意義(P0.05)。6.骨組織切片HE染色觀察骨小梁結(jié)構(gòu)、脂肪細(xì)胞含量比較成模后培養(yǎng)4周時(shí),三組間骨小梁結(jié)構(gòu)尚可,脂肪細(xì)胞量少;8周及12周時(shí),三組骨小梁結(jié)構(gòu)出現(xiàn)不同程度破壞,以DM組明顯,骨小梁變細(xì)、變稀疏,部分骨小梁結(jié)構(gòu)消失,髓腔擴(kuò)大,出現(xiàn)較多脂肪細(xì)胞,APN干預(yù)后骨小梁結(jié)構(gòu)有所恢復(fù),脂肪細(xì)胞含量減少,且12周時(shí)上述變化更明顯。結(jié)論:1.2型糖尿病大鼠骨髓微環(huán)境中氧化應(yīng)激水平明顯升高,可能對(duì)成骨分化的調(diào)節(jié)因子Runx2有直接抑制作用,可能引起成骨細(xì)胞分化減少,骨形成降低,結(jié)合AGEs水平偏高,骨膠原蛋白非酶糖化影響骨質(zhì)及骨密度,均可能參與糖尿病性骨質(zhì)疏松癥的發(fā)生。2.脂聯(lián)素可改善糖代謝,減少AGEs生成,并通過降低糖尿病大鼠骨微環(huán)境氧化應(yīng)激水平,對(duì)骨骼可能起保護(hù)作用。具體機(jī)能尚需深入研究。
[Abstract]:Objective: To investigate the type 2 diabetes mellitus (DM) rat bone microenvironment of oxidative stress (OS) and the level of osteoblast (OB) differentiation regulator Runx2, adiponectin (APN) and observe whether intervention can improve the bone microenvironment effect of oxidative stress in osteoblast differentiation, further study of diabetic osteoporosis osteoporosis (DOP) in the pathogenesis and early intervention, to provide new ideas for clinical prevention and treatment of diabetic osteoporosis. Methods: 4 week old male SD rats were randomly divided into normal control group (n=18), diabetic model group (n=42). Control group was given normal diet, model group of high cholesterol and sugar for 2 months, and intraperitoneal injection of streptozotocin (STZ) 30mg/kg type 2 DM rat model. To eliminate the failure model, the death of rats of successful model rats were divided into diabetic group (DM group =18) and intervention group (APN group, adiponectin =18, APN) group were given intraperitoneal injection of globular adiponectin 10 g/kg*d were killed in the intervention, animal 4,8,12 weekend respectively, quickly remove the bilateral femur and tibia, femur and tibia bone marrow extraction side, bone marrow cells were collected by centrifugation and the supernatant of bone marrow, the other side of the femoral bone density after decalcified paraffin embedded.ELISA. RT-PCR method for detection of Runx-2, BALP, AOPPs, bone tissue immunohistochemical staining to observe the expression of AGEs, HE staining to observe the bone mass (trabecular structure, density) and fat cells. Results: the blood glucose level of 1. rats in each group were compared with the feeding time extension, the blood glucose level in DM group rats increased significantly.APN the prognosis, improve blood glucose level of AOPP bone marrow supernatant.2. three rats, compared with feeding time, normal rats bone marrow supernatant AOPP no significant changes in the DM group, APN intervention group were higher (P0.05), but the APN intervention group L 楂樺箙搴﹁緝DM緇勫皬.鍦，

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