發(fā)布時間：2018-09-01 05:47

【摘要】：目的:糖尿病(Diabetes mellitus,DM)是繼腫瘤、心血管疾病之后第三大非傳染性的慢性疾病,嚴重威脅著人類健康。據(jù)報道,全球已有3.82億人患有DM,預(yù)計到2035年將達到5.92億。我國就有近1億人患有DM,約占全球患病人數(shù)的四分之一,患病率位居世界第一位。糖尿病腎病(Diabetic Nephropathy,DN)是1型和2型DM患者的主要并發(fā)癥之一,是終末期腎臟疾病導(dǎo)致患者致死致殘的主要原因。據(jù)統(tǒng)計,大約35%～50%1型、約20%2型DM患者發(fā)展成為DN。DN主要病理改變是基底膜增厚,系膜基質(zhì)增多,細胞外基質(zhì)增加,最終導(dǎo)致彌漫性或結(jié)節(jié)性腎小球硬化。目前,其發(fā)病的內(nèi)在機制尚未完全闡明。近年研究確認,氧化應(yīng)激是公認的DN發(fā)生發(fā)展的中心環(huán)節(jié)。其關(guān)鍵酶誘導(dǎo)型一氧化氮合酶(inducible nitric oxide synthase,iNOS)轉(zhuǎn)基因會引起DN 樣病變。iNOS 基因轉(zhuǎn)錄最重要的調(diào)控因子是核因子-κB(nuclear factor kappa B,NF-κB),當受到相應(yīng)刺激時被激活轉(zhuǎn)位入核,調(diào)控相關(guān)基因轉(zhuǎn)錄。P300是細胞內(nèi)最重要的共激活因子,具有核蛋白乙�；�轉(zhuǎn)移酶活性,參與多種轉(zhuǎn)錄因子的調(diào)控。研究證實DM時,NF-κB的P65亞基與共激活因子P300結(jié)合增多,異常激活基因轉(zhuǎn)錄,產(chǎn)生大量iNOS,在DN的發(fā)生發(fā)展中起關(guān)鍵性作用。C肽是含31個氨基酸的多肽,在胰島素合成過程中產(chǎn)生,并與胰島素等摩爾分泌。一直以來,C肽僅作為評價胰島β細胞功能的指標。近年來,一些研究表明對于實驗動物和DM患者,外源性C肽可以防止或逆轉(zhuǎn)DN。一系列研究發(fā)現(xiàn)生理濃度的C肽能結(jié)合胞膜G蛋白偶聯(lián)受體,或進入胞質(zhì)胞核發(fā)揮細胞保護效應(yīng)。本課題前期研究表明高糖(25 mmol/L)刺激下,0.5 nmol/L的C肽定位于大鼠腎小球系膜細胞核,抑制iNOS基因轉(zhuǎn)錄及其蛋白的表達,亦能抑制高糖引起的NF-κB核轉(zhuǎn)位。然而C肽是否通過調(diào)控NF-κB與P300的相互作用而發(fā)揮防治DN的作用,尚未見相關(guān)報道。本研究從細胞和動物整體兩個方面進行實驗。采用大鼠腎小球系膜細胞株HBZY-1進行細胞培養(yǎng),用激光共聚焦顯微鏡(Confocal)觀察C肽(0.5nmol/L)對高糖(25mmol/L)刺激的細胞中NF-κB的作用及其與P300共定位,用染色質(zhì)免疫沉淀(chromatin immunoprecipitation,ChIP)技術(shù)檢測C肽對NF-κB啟動iNOS的調(diào)控作用,用免疫共沉淀(co-immunoprecipitation,Co-IP)技術(shù)檢測C肽對NF-κB與P300相互作用的影響。用鏈脲佐菌素(streptozotocin,STZ)復(fù)制SD大鼠DM模型,隨機分為正常組、病理組、C肽防治組、C肽治療組和亂碼C肽組,實驗前后測定血糖、體重變化,測定24h尿白蛋白排泄量。取大鼠腎小球皮質(zhì)于光鏡及透射電鏡下進行形態(tài)學(xué)觀察,進一步探討C肽對防治DN的整體功效及作用。方法:1細胞實驗1.1細胞培養(yǎng)大鼠腎小球系膜細胞株(GMCs)HBZY-l,用含10%胎牛血清的低糖DMEM(5.5 mmol/L),0.25%EDTA-胰蛋白酶消化傳代,接種于100 ml培養(yǎng)瓶中,在37℃、5%CO2培養(yǎng)箱中培養(yǎng)。1.2實驗設(shè)計與分組(1)Confocal技術(shù)觀察NF-κB P65核轉(zhuǎn)位及其與P300共定位,同時確定C肽作用的最佳時間。實驗分組:1)正常組(Control):GMCs(HBZY-1)用低糖DMEM(5.5mmol/L)正常培養(yǎng);2)高滲對照組(HO):GMCs用L-葡萄糖 DMEM(25 mmol/L)刺激作用 24 h;3)高糖組(HG):GMCs(HBZY-1)用高糖DMEM(25 mmol/L)刺激作用24 h;4)NF-κB抑制劑(BAY11-7082)加高糖組(IH):正常培養(yǎng)的 GMCs 加入 BAY11-7082(10μM)孵育1 h,換高糖DMEM(25 mmol/L)刺激作用24 h;5)C肽治療組(CP):GMCs用高糖DMEM刺激作用24 h后,換成0.5 nmol/L C肽-高糖DMEM 分別培養(yǎng) 10、20、30、40、50、60 min。(2)ChIP技術(shù)檢測C肽對NF-κB啟動iNOS的調(diào)控作用。實驗分組:1)正常組(Control):GMCs 用低糖 DMEM(5.5 mmol/L)正常培養(yǎng);2)高糖組(HG):GMCs用高糖DMEM(25mmol/L)刺激作用24h;3)C肽治療組(CP):GMCs用高糖DMEM刺激作用24 h后,換成0.5 nmol/L C肽-高糖DMEM培養(yǎng)30 min。(3)Co-IP技術(shù)檢測C肽對NF-κB與P300結(jié)合作用的影響。實驗分組:1)正常組(Control):GMCs 用低糖 DMEM(5.5mmol/L)正常培養(yǎng);2)高糖組(HG):GMCs用高糖DMEM(25mmol/L)刺激作用24h;3)C肽治療組(CP):GMCs用高糖DMEM刺激作用24 h后,換成0.5 nmol/L C肽-高糖DMEM培養(yǎng)30 min。2動物實驗2.1實驗設(shè)計與分組健康雄性SD大鼠80只,隨機分為2組,正常對照組(NC,8只),其余72只注射STZ溶液(STZ溶于濃度為0.1 mol/L的檸檬酸納緩沖液,pH 4.4,新鮮配制,終濃度為10mg/mL,按45mg/kg腹腔注射)制備DM模型。注射3天后,尾靜脈采血測血糖值≥16.7mmol/L,尿糖+++以上,為造模成功。造模成功68只,再隨機分成4組:病理組(DM,17只),C肽防治組(DM+CP-P,17只),C肽治療組(DM+CP-T,17只),亂碼C肽治療組(DM+scCP,17只),以上五組均于20～25℃室溫下正常飲食喂養(yǎng)。DM+CP-P組在造模成功后,開始皮下注射人C肽(130 nmol/kg),每天2次,治療6周后停藥。DM+CP-T組和DM+scCP組在病程6周后,分別開始同劑量皮下注射人C肽或亂碼C肽,每天2次,治療6周。最后股動脈放血處死。2.2檢測血糖和24 h尿液取尾靜脈血,用血糖儀檢測并記錄注射STZ前、后第3天和第12周的血糖變化。收集24h尿液,檢測尿蛋白排泄量。2.3形態(tài)學(xué)觀察取大鼠腎小球皮質(zhì)作病理切片,分別于光鏡和透射電鏡下觀察。結(jié)果:1不同時間點,C肽對CP組GMCs NF-κB P65核轉(zhuǎn)位及其與P300共定位的影響Confocal結(jié)果顯示,高糖刺激GMCs 24 h,會引起NF-κB P65轉(zhuǎn)位入核,且NF-κB P65與P300共定位于細胞核;0.5 nmol/L C肽治療在30 min時效果顯著,即NF-κB P65聚集于胞質(zhì),P300聚集于胞核;40～60 minC肽作用逐漸減弱至基本消失。因此選30 min作為C肽治療高糖長時間刺激細胞最佳的作用時間。2 ChIP技術(shù)檢測C肽治療30 min時,對CP組GMCs NF-κB啟動iNOS的調(diào)控作用高糖刺激GMCs 24 h后,細胞內(nèi)NF-κB在iNOS啟動子區(qū)近端的結(jié)合作用(4.44 ± 0.29,P0.05)與Control組相比明顯升高;0.5 nmol/L C肽治療30 min后,細胞內(nèi)NF-κB在iNOS啟動子區(qū)的近端結(jié)合作用(1.86 ±0.21,P0.01)與HG組相比顯著下降。高糖刺激GMCs 24 h后,細胞內(nèi)NF-κB在iNOS啟動子區(qū)遠端的結(jié)合作用(5.06 ±0.43,P0.05)與 Control 組相比明顯升高;0.5 nmol/L C肽治療30 min后,細胞內(nèi)NF-κB在iNOS啟動子區(qū)的近端結(jié)合作用(0.94 ±0.07,P0.05)與HG組相比明顯下降。3 Co-IP技術(shù)檢測C肽治療30 min時,Control組、HG組和CP組GMCs NF-κB P65與P300的結(jié)合作用Control組、HG組和CP組細胞蛋白裂解液分別加入P300抗體沉淀處理后,Western blot結(jié)果顯示:HG組GMCs P65與P300蛋白結(jié)合量(18.31 ±0.82,P0.05)比 Control 組(5.99 ± 0.07)明顯升高;CP 組 GMCs P65與P300蛋白結(jié)合量(5.220±0.33,P0.01)比HG組顯著下降。Control組、HG組和CP組細胞蛋白裂解液分別加入P65抗體沉淀處理后,Western blot結(jié)果顯示:HG組GMCs P300與P65蛋白結(jié)合量(9.19±0.17,P0.01)比 Control 組(3.54±0.21)顯著升高;CP 組 GMCsP300與P65蛋白結(jié)合量(2.98 ±0.23,P0.01)比HG組顯著下降。4大鼠的一般表現(xiàn),C肽對DM大鼠血糖、體重和尿白蛋白排泄量的影響造模成功的大鼠逐漸出現(xiàn)消瘦,皮毛疏松無光澤,反應(yīng)遲鈍,多飲、多尿、多食,生長發(fā)育遲緩等癥狀。隨著實驗進行,DM組和DM+scCP組病癥更加明顯,而DM+CP-P組和DM+CP-T組病癥與NC組相似。各組大鼠入選時血糖和體重無顯著性差異。整個實驗中,DM+CP-P組平均血糖(29.96 ± 0.52)mmol/L,DM+CP-T 組(28.90 ± 0.74)mmol/L,DM+scCP 組(29.74 ± 0.66)mmol/L,均與 DM 組(29.23 ± 0.66)mmol/L無顯著性差異。以上四組均顯著高于NC組(6.40 ± 0.25)mmol/L(P0.01)。C肽治療結(jié)束時,DM+CP-T組平均體重(271.82 ±8.76)g,DM+scCP組(248.35 ±7.19)g,均與DM組(249.0 ±7.13)g無顯著性差異。以上四組均顯著低于NC組(515.5 ± 14.42)g(P0.01)。C肽治療結(jié)束時,DM組24小時尿蛋白排泄量(327.93±32.58)mg和DM+scCP 組(293.79 ±49.40)mg 均明顯高于 NC 組(15.42 ± 4.06)mg(P0.05);DM+CP-P 組(55.21 ± 4.06)mg 和 DM+CP-T組(70.2 ± 9.46)mg明顯低于DM組(P0.05)。5腎小球形態(tài)學(xué)觀察腎小球光鏡(× 400)下觀察:NC組腎小球未見異常;DM組可見到腎小球結(jié)構(gòu)異常、腎小囊腔增大;DM+scCP組與DM組相似,并且有腎小球壞死的現(xiàn)象;而DM+CP-P組和DM+CP-T組,可見腎小囊腔縮小,與病理組相比有所改善,且DM+CP-P組比DM+CP-T組的病變較輕。腎小球透射電鏡(×20K)下觀察:NC組腎小球未見異常。DM組可見基底膜重度增厚,局部呈丘狀隆起,血管內(nèi)皮細胞重度增生,部分足細胞足突融合;DM+scCP組與DM組相似,基底膜明顯增厚;DM+CP-P組和DM+CP-T組可見基底膜和血管內(nèi)皮細胞接近正常,足細胞足突部分輕度融合,比DM組有明顯改善,且DM+CP-P組較DM+CP-T組的病理改變減輕。結(jié)論:1 C肽調(diào)控NF-κB與P300防治糖尿病腎病的機制是:(1)C肽能抑制NF-κB P65的核轉(zhuǎn)位及其與P300的共定位。(2)C肽能抑制高糖引起的NF-κB核轉(zhuǎn)位與iNOS啟動子區(qū)遠、近端均有相互作用,從而抑制NF-κB啟動iNOS基因。(3)C肽能抑制高糖引起的NF-κB與P300之間的相互作用。2光鏡和透射電鏡下觀察到,C肽能明顯改善DM大鼠腎小球的功能和形態(tài)學(xué)變化。
[Abstract]:Objective: Diabetes mellitus (DM) is the third largest non-infectious chronic disease after tumor and cardiovascular disease, which seriously threatens human health. It is reported that 382 million people worldwide have DM, and it is expected to reach 592 million by 2035. Diabetic nephropathy (DN) is one of the major complications of type 1 and type 2 diabetes mellitus (DM) and the leading cause of death and disability in patients with end-stage renal disease. According to statistics, about 35%-50% of type 1 diabetes mellitus patients and 20% of type 2 diabetes mellitus patients develop into DN. The main pathological changes of DN are thickening of basement membrane, increasing of mesangial matrix and extracellular matrix. The underlying mechanism of diffuse or nodular glomerulosclerosis has not been fully elucidated. Recent studies have confirmed that oxidative stress is the central link in the genesis and development of DN. The most important regulator of gene transcription is nuclear factor kappa B (NF-kappa B), which is activated and transported into the nucleus when stimulated. P300 is the most important co-activating factor in the cell. It has the activity of nuclear protein acetyltransferase and participates in the regulation of many transcription factors. In recent years, C peptide is a polypeptide containing 31 amino acids, produced during insulin synthesis and secreted with insulin and other moles. C peptide has been used only as an index to evaluate the function of islet beta cells. Some studies have shown that exogenous C peptides can prevent or reverse DN in experimental animals and DM patients. A series of studies have found that C peptides at physiological concentrations bind to membrane G protein-coupled receptors, or enter the cytoplasmic nucleus to exert cytoprotective effects. Previous studies have shown that 0.5 nmol/L C peptides are localized in rat kidneys stimulated by high glucose (25 mmol/L). The nucleus of mesangial cells inhibits the transcription of iNOS gene and the expression of iNOS protein, and also inhibits NF-kappa B nuclear translocation induced by high glucose. However, whether C peptide can prevent and treat DN by regulating the interaction between NF-kappa B and P300 has not been reported. Cell line HBZY-1 was cultured. The effect of C-peptide (0.5 nmol/L) on NF-kappa B and its co-localization with P300 in the cells stimulated by high glucose (25 mmol/L) were observed by Confocal microscopy. The regulatory effect of C-peptide on the activation of iNOS by NF-kappa B was detected by chromatin immunoprecipitation (ChIP) and co-immunoprecipitation (co-immunoprecipitation). The effects of C-peptide on the interaction between NF-kappa B and P300 were examined by co-immunoprecipitation, Co-IP. SD rats were randomly divided into normal group, pathological group, C-peptide prevention and treatment group, C-peptide treatment group and disordered C-peptide group. Methods: The glomerular mesangial cells (GMCs) of rats were cultured in 1.1 cell experiment, digested and subcultured with low glucose DMEM (5.5 mmol/L) containing 10% fetal bovine serum and 0.25% EDTA-trypsin, and inoculated in 100 ml. The experiment design and grouping (1) Confocal technique were used to observe the nuclear translocation of NF-kappa B P65 and its co-localization with P300, and to determine the optimal time of C peptide action. High glucose group (HG): GMCs (HBZY-1) stimulated by high glucose DMEM (25 mmol/L) for 24 hours; 4) NF-kappa B inhibitor (BAY11-7082) plus high glucose group (IH): Normal GMCs were incubated with BAY11-7082 (10 mu M) for 1 hour, replaced with high glucose DMEM (25 mmol/L) for 24 hours; 5) C peptide treatment group (CP): GMCs stimulated by high glucose DMEM for 24 hours, and then replaced with high glucose DMEM (25 mmol/L) for 24 hours. (2) ChIP assay was used to detect the regulatory effect of C peptide on the initiation of iNOS by NF-kappa B. Experimental grouping: 1) Normal group (Control): GMCs were cultured with low-glucose DMEM (5.5 mmol/L); 2) High-glucose group (HG): GMCs were stimulated with high-glucose DMEM (25 mmol/L) for 24 hours; 3) C peptide treatment group (CP): GMCs were stimulated with high-glucose DMEM (25 mmol/L); 3) GMCs were treated with high-glucose DMEM (GMCs) for 24 hours. After 24 hours of stimulation, the cells were cultured with 0.5 nmol/L peptide-high glucose DMEM for 30 minutes. (3) Co-IP technique was used to detect the effect of C peptide on the binding of NF-kappa B to P300. After 24 hours of stimulation, 80 healthy male SD rats were randomly divided into 2 groups: normal control group (NC, 8 rats), and the rest 72 rats were injected with STZ solution (STZ dissolved in 0.1 mol/L sodium citrate buffer, pH 4.4, freshly prepared, the final concentration was 10 mg/mL, according to 45 mg/kg). DM model was established by intraperitoneal injection. After 3 days of injection, blood glucose (> 16.7 mmol/L) and urine glucose (+ + +) were collected from the caudal vein. Sixty-eight rats were successfully established. They were randomly divided into 4 groups: pathological group (DM, 17 rats), C peptide prevention and treatment group (DM + CP-P, 17 rats), C peptide treatment group (DM + CP-T, 17 rats) and disorderly coded C peptide treatment group (DM + scCP, 17 rats). DM+CP-T group and DM+scCP group were given subcutaneous injection of human C-peptide (130 nmol/kg) twice a day for 6 weeks. DM+CP-T group and DM+scCP group were given subcutaneous injection of human C-peptide or scrambled C-peptide at the same dose twice a day for 6 weeks. Blood glucose of caudal vein was measured and recorded by blood glucose meter before and after STZ injection. 24 hours urine was collected and urinary protein excretion was measured. Confocal results showed that high glucose stimulated GMCs for 24 hours could induce NF-kappa B P65 translocation into the nucleus, and NF-kappa B P65 and P300 co-localized in the nucleus; 0.5 nmol/L C peptide treatment had a significant effect in 30 minutes, that is, NF-kappa B P65 aggregated in the cytoplasm, P300 aggregated in the nucleus; 40-60 min C peptide effect gradually weakened to disappear. ChIP assay was used to detect the regulatory effect of C peptide on the initiation of iNOS by GMCs NF-kappa B in C P group. After 24 hours of stimulation with high glucose, the binding of NF-kappa B to the proximal end of iNOS promoter (4.44 0.29, P 0.05) was significantly higher than that in control group. After 30 minutes of treatment, the proximal binding of intracellular NF-kappa B to iNOS promoter region (1.86+0.21, P 0.01) was significantly lower than that of HG group. After 24 hours of high glucose stimulation, the binding of intracellular NF-kappa B to distal end of iNOS promoter region (5.06+0.43, P 0.05) was significantly higher than that of C ontrol group; after 30 minutes of 0.5 nmol/L C peptide treatment, intracellular NF-kappa B was significantly higher in iNOS promoter region. The proximal binding of S promoter region (0.94+0.07, P 0.05) was significantly lower than that of HG group. 3 The binding of GMCs NF-kappa B P65 to P300 in control group, HG group and CP group was detected by Co-IP after 30 minutes of treatment with control group, HG group and CP group, respectively. The results of Western blot showed that GMCs P in HG group was precipitated by P300 antibody. The binding capacity of GMCs P65 to P300 protein (18.31+0.82, P 0.05) in control group was significantly higher than that in control group (5.99+0.07), and the binding capacity of GMCs P65 to P300 protein (5.220+0.33, P 0.01) in CP group was significantly lower than that in HG group. The combined dose of GMCsP300 and P65 protein (2.98+0.23, P 0.01) in CP group was significantly lower than that in HG group. The symptoms of DM + CP - P group and DM + CP - T group were similar to those of NC group. There was no significant difference in blood glucose and body weight between DM + CP - P group and DM + CP - T group. There was no significant difference between DM + scCP group and DM group (29.23 + 0.66) mmol / L. All the above four groups were significantly higher than NC group (6.40 + 0.25) mmol / L (P 0.01). At the end of C peptide treatment, the average body weight of DM + CP-T group (271.82 + 8.76) g, DM + scCP group (248.35 + 7.19) g, and DM group (249.0 + 7.13) g had no significant difference. At the end of C-peptide treatment, 24-hour urinary protein excretion in DM group (327.93 (+32.58) mg and DM+scCP group (293.79 (+49.40) mg were significantly higher than those in NC group (15.42 (+4.06) mg (P 0.05), DM+CP-P group (55.21 (+4.06) mg and DM+CP-T group (70.2 (+9.46) mg) significantly lower than those in DM group (P 0.05). The glomeruli of NC group were not abnormal; the glomeruli of DM group were abnormal and the cysts of DM group were enlarged; the glomeruli of DM + scCP group were similar to DM group and glomerular necrosis was observed; while the glomeruli of DM + CP - P group and DM + CP - T group were smaller, and the pathological changes of DM + CP - P group were less than DM + CP - T group. The glomeruli of NC group were normal. In DM group, the basement membrane was thickened severely, the vascular endothelial cells were proliferated severely, and some podocytes were fused. In DM + scCP group, the basement membrane was thickened obviously, and the basement membrane and vascular endothelial cells were close to normal in DM + CP - P group and DM + CP - T group. Conclusion: The mechanism of C peptide regulating NF-kappa B and P300 in preventing and treating diabetic nephropathy is: (1) C peptide can inhibit the nuclear translocation of NF-kappa B P65 and its co-location with P300. (2) C peptide can inhibit the nuclear translocation of NF-kappa B induced by high glucose far from the iNOS promoter region. (3) C peptide could inhibit the interaction between NF-kappa B and P300 induced by high glucose. 2 Light and transmission electron microscopy showed that C peptide could significantly improve the function and morphological changes of glomeruli in DM rats.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R587.2;R692.9

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