【作者】 王慎田；

【导师】 陈丽；

【作者基本信息】 山东大学 ， 内科学， 2009， 博士

【摘要】 研究背景:2型糖尿病的发病率逐年增加,已经成为严重危害人类健康的常见病和多发病。2型糖尿病的主要危害是糖尿病所致的各种严重慢性并发症,包括大血管并发症,如冠心病、脑梗塞、外周动脉硬化等,以及微血管并发症,如糖尿病视网膜病变、糖尿病肾病和神经病变等。防治糖尿病所致的大血管及微血管并发症是糖尿病领域的主要任务,但糖尿病慢性并发症的发病机制目前仍不清楚。非酶蛋白糖化、多元醇通道途径激活、氧化应激、炎性反应及免疫因素等是糖尿病慢性并发症的可能发病机制之一,但上述因素并不能解释糖尿病慢性并发症的全部机制。是否还存在其它机制仍是目前糖尿病研究领域的重要课题之一。护骨素（osteoprotegerin,OPG）是1997年发现的肿瘤坏死因子（tumor necrosis factor,TNF）受体家族新成员,它的主要生物学作用是抑制破骨细胞形成、分化、存活并诱导其凋亡,对于骨代谢有重要意义。近来研究发现,OPG亦是一重要的血管调节因子,与动脉粥样硬化和血管钙化相关联,可能在血管疾病的发生、发展中起重要的调节作用。国外许多学者研究了血清OPG水平与冠心病及外周动脉粥样硬化的关系,结果发现血清OPG水平不仅与冠心病、动脉粥样硬化的存在显著相关,而且与其严重程度有明显关系,高血清OPG水平是发生冠心病和心血管疾病的独立危险因素。糖尿病的慢性并发症主要是大血管和微血管并发症,是否OPG与糖尿病的慢性并发症有关?关于此方面的研究国内未见报道,国外的报道亦较少,部分研究结果表明,糖尿病并冠心病患者或糖尿病并微血管病变患者的血清OPG水平升高,因此OPG可能与糖尿病的慢性血管并发症有关。本课题主要系统研究OPG与2型糖尿病血管并发症的关系,为糖尿病并发症的研究提供新的思路。目的:第一部分:应用ELISA法检测2型糖尿病并大血管病变患者的血清OPG水平,同时应用PCR-RELP测定OPG 950T-C基因多态性。以其探讨（1）血清OPG水平与2型糖尿病并大血管病变的关系。（2）2型糖尿病并大血管病变患者血清OPG水平与FPG、2hPG、HbA1c、血脂、胰岛素抵抗及hsCRP等指标的关系,进一步研究糖尿病并大血管病变的相关危险因素。（3）OPG 950T-C基因多态性是否与糖尿病大血管病变有关。通过以上研究了解OPG在糖尿病大血管并发症中的作用及意义。第二部分:应用ELISA法测定2型糖尿病并肾病患者的血清OPG水平,同时对部分糖尿病肾病患者行肾活检,应用免疫组化技术检测OPG在糖尿病肾组织中的表达。以其探讨（1）血清OPG与2型糖尿病肾病及其严重程度的关系。（2）OPG在肾组织中的表达与糖尿病肾病及其严重程度的关系。（3）糖尿病肾病患者血清OPG水平与血糖控制、炎性指标、血脂、胰岛素抵抗等指标的相关性,进一步研究糖尿病肾病的相关危险因素。第三部分:应用ELISA法测定2型糖尿病并视网膜病变患者的血清OPG水平。以其探讨:（1）血清OPG水平与糖尿病视网膜病变及其严重程度的关系。（2）糖尿病视网膜病变患者的相关危险因素以及与血清OPG水平的关系。材料与方法:第一部分:1.病例选择:2006年3月至2008年9月在山东省潍坊医学院附属青州医院内分泌科、心内科和神经内科住院和门诊的2型糖尿病患者168例。根据有无大血管病变分为大血管病变组86例和无大血管病变组82例。另选择年龄、性别相匹配的40例健康者为对照组。2.标本留取:所有个体空腹12h后次晨抽静脉血,进行生化指标的测定。采取静脉血,分离血清后-70℃保存,集中测定血OPG水平。抽取静脉血肝素抗凝,-70℃保存,集中行OPG基因多态性的测定。3.试验方法（1）血清OPG水平应用ELISA法测定。血糖采用葡萄糖氧化酶法测定。利用化学发光法测定空腹胰岛素（Fins）。胰岛素抵抗指数采用稳态模式评估法（HOMA-IR）计算。HbA1c采用NycoCard ReaderⅡ金标定量检测仪测定。hsCRP采用免疫比浊法测定。血脂指标应用全自动生化分析仪测定。（2）OPG 950T-C基因多态性的测定:①DNA的提取:引物的设计:上游序列:5’-CCCAGGGGCCAGACACCAC-3’,下游序列:5’-GCGCGCAGCACAGCAACTT-3’,目的片段长度330bp。②PCR扩增:PCR反应体系:模板2ul;10×PCR Buffer5ul;25mM MgCL₂ 4ul;4×dNTP 1ul;上游引物P1 0.5ul;下游引物P2 0.5ul;Taq酶2U;超纯水36ul;液体石蜡30ul。PCR反应结束后,取10μl反应产物于1.2%琼脂糖凝胶电泳。电泳结束后,紫外线透射仪上观察结果,拍照或扫描保存。③PCR产物的酶切:应用HincⅡ限制性内切酶对PCR产物进行酶切,HincⅡ限制性内切酶酶切反应体系:PCR产物15μl:10×buffer 2μl;HincⅡ1.5μl;超纯水1.5μl。37℃水浴反应4h,取10μl于1.0%琼脂糖凝胶电泳鉴定。4.统计学处理:结果以（?）±s表示,组间均数比较采用方差分析,比较有差异者进行两两比较。各变量之间的关系用spearman相关分析,多因素相关分析采用Logistic回归分析。统计软件为SPSS12.0。第二部分:1.病例选择:2006年3月至2008年9月在山东省潍坊医学院附属青州医院内分泌科、肾内科住院和门诊的2型糖尿病患者110例。根据24h尿白蛋白排泄率（UAER）,分为正常白蛋白尿组38例,微量白蛋白尿组36例,大量白蛋白尿组36例。另选择年龄、性别相匹配的40例健康者为对照组。2.标本留取:所有个体空腹12h后次晨抽静脉血,进行生化指标的测定。采取静脉血,分离血清后-70℃保存,集中测定血OPG水平。所有患者留取24小时尿,记录尿量,混匀后取2 ml送检测定尿微量白蛋白。肾活检标本的采集:微量蛋白尿及大量蛋白尿组的部分患者经病人及家属同意,签订肾活检协议书。局麻、B超引导下,应用肾活检针,采集肾组织2-4条,石蜡包埋,切片行HE染色和免疫组化检查。3.试验方法（1）血清OPG水平应用ELISA法测定。血糖采用葡萄糖氧化酶法测定。利用化学发光法测定空腹胰岛素。胰岛素抵抗指数采用HOMA-IR计算。HbA1c和尿微量白蛋白采用NycoCard ReaderⅡ金标定量检测仪测定。hsCRP采用免疫比浊法测定。血脂指标应用全自动生化分析仪测定。（2）肾活检组织OPG免疫组化染色:采用即用型免疫组化染色试剂盒,一抗:兔抗人OPG多克隆抗体;二抗:亲和纯化抗体,山羊抗兔IgG。结果判定以染色呈棕黄色颗粒为阳性表达,染色结果根据阳性细胞显色深浅及阳性细胞百分率分级分别进行评分,根据相加得分判定表达强弱。4.统计学处理:结果以（?）±s表示,组间均数比较采用方差分析,比较有差异者进行两两比较。各变量之间的关系用spearman相关分析,多因素相关分析采用多元回归分析。统计软件为SPSS12.0。第三部分:1.病例选择:2006年3月至2008年9月在山东省潍坊医学院附属青州医院内分泌科、眼科住院和门诊的2型糖尿病患者98例。根据有无眼底视网膜病变分为无眼底病变组38例,背景期视网膜病变组30例和增殖期视网膜病变组30例。2.标本的留取与检测:所有个体空腹12h后次晨抽静脉血,进行生化指标的测定。采取静脉血,分离血清后-70℃保存,集中测定血OPG水平。血清OPG水平应用ELISA法测定。其它血清指标的检测同第一部分。3.统计学处理:结果以（?）±s表示,组间均数比较采用方差分析,比较有差异者进行两两比较。各变量之间的关系用spearman相关分析,多因素相关分析采用Logistic回归分析。结果:第一部分:1.2型糖尿病大血管病变组血清OPG水平（4.16±0.65）ng/L显著高于无大血管病变组（2.75±0.59）ng/L与对照组（2.29±0.37）ng/L,差异有显著统计学意义（P＜0.01）,无大血管病变组与对照组差异无显著性。2.2型糖尿病患者血OPG与FPG（r=0.243,p＜0.01）、2hPG（r=0.237,p＜0.01）、HbA1C（r=0.361,p＜0.001）、HOMA-IR（r=0.252,p＜0.01）、hsCRP（r=0.585,p＜0.001）呈明显正相关,与年龄、BMI、腰围、腰臀比、SBP、DBP、TG、TC、LDL-C、HDL-C和Fins无明显相关性。3.2型糖尿病并大血管病变患者OPG基因型CC频率（24.4%）,显著高于无大血管病变组（11.0%）（P＜0.05）。等位基因频率分布也有显著性差异,大血管病变组C等位基因频率（40.7%）显著高于无大血管病变组患者（29.9%）（P＜0.05）。4.2型糖尿病患者不同OPG基因型TT、TC和CC的年龄、BMI、腰围、腰臀比、SBP、DDP、FPG、2hPG、HbA1c、Fins、HOMA-IR、TG、TC、LDL-C、HDL-C和hsCRP无明显统计学差异（均P＞0.05）。但CC基因型的血浆OPG水平（3.96±0.85）显著高于TT基因型（3.36±0.93）和TC基因型者（3.37±0.94）（P＜0.05）。5.Logistic回归分析结果显示血清OPG水平和OPG基因型是糖尿病大血管病变的独立危险因素。第二部分:1.2型糖尿病各组血清OPG水平均显著高于正常对照组（2.29±0.37）ng/L,而且大量白蛋白尿组（4.45±0.76）ng/L高于微量白蛋白尿组（3.62±0.70）ng/L与正常白蛋白尿组（2.77±0.78）ng/L（P＜0.01）,微量白蛋白尿组显著高于正常白蛋白尿组（P＜0.01）。2.糖尿病肾病患者血清OPG与FPG（r=0.223,p＜0.05）、2hPG（r=0.298,p＜0.01）、HbA1C（r=0.520,p＜0.001）、hsCRP（r=0.360,p＜0.001）、HOMA-IR（r=0.257,p＜0.01）及UAER（r=0.639,p＜0.001）呈明显正相关,与年龄、BMI、腰围、腰臀比、SBP、DBP、TG、TC、LDL-C、HDL-C和Fins无明显相关性。3.多元逐步回归分析显示血清OPG水平是2型糖尿病UAER的独立影响因素。4.肾活检组织的OPG免疫组化染色结果:微量白蛋白尿及大量白蛋白尿组患者所有活检病例的肾小球组织,包括轻微病理改变、弥漫性硬化和局灶节段性硬化的肾小球均未见OPG的阳性表达。OPG的阳性表达主要位于肾小管组织,主要表达部位是肾小管细胞的胞浆内。大量白蛋白尿组患者肾小管OPG阳性表达水平显著高于微量白蛋白尿组患者,差异有统计学意义（P＜0.01）。第三部分:1.2型糖尿病无眼底病变患者眼底检查无明显异常,背景期视网膜病变组眼底检查主要表现为微血管瘤伴有硬性或软性渗出,增殖期视网膜病变组眼底检查主要表现为新生血管的形成,或伴有纤维增殖和玻璃体出血。2.2型糖尿病各组血清OPG水平均显著高于正常对照组（2.29±0.37）ng/L。增殖期视网膜病变组（4.07±1.08）ng/L高于背景期视网膜病变组（3.57±0.96）ng/L与无病变组（2.77±0.78）ng/L（P＜0.01）,而且背景期视网膜病变组显著高于无病变组（P＜0.01）。3.糖尿病视网膜病变患者血清护骨素与FPG（r=0.263,p＜0.01）、2hPG（r=0.305,p＜0.01）、HbA1C（r=0.377,p＜0.001）、hsCRP（r=0.453,p＜0.001）及HOMA-IR（r=0.324,p＜0.001）呈明显正相关,与年龄、BMI、腰围、腰臀比、SBP、DBP、TG、TC、LDL-C、HDL-C和Fins无明显相关性。4.Logistic回归分析结果表明血清护骨素水平是2型糖尿病视网膜病变的独立危险因素。结论:1.2型糖尿病并大血管病变、并肾病、并眼底视网膜病变患者血清护骨素水平显著高于无并发症者,且与严重程度有关;血清OPG水平是糖尿病血管病变的独立影响因素,说明血清OPG水平与糖尿病血管并发症有关,OPG在糖尿病血管并发症的发生、发展中可能起一定作用,血清OPG水平升高可作为糖尿病血管病变的标志物。2.2型糖尿病并血管并发症患者血清护骨素水平与FPG、2hPG、HbA1C、HOMA-IR、hsCRP呈明显正相关,表明血清护骨素水平的升高与血糖控制、胰岛素抵抗和炎性反应有关。3.OPG 950T-C基因多态性与2型糖尿病并大血管病变相关联,是2型糖尿病患者并发大血管病变的危险因素之一,对大血管病变的影响可能通过升高血清OPG水平来实现。4.糖尿病肾病患者的肾小球组织无OPG的阳性表达,但肾小管细胞OPG表达明显增加,可能是糖尿病肾病血清OPG升高的原因之一。更多还原

【Abstract】 Backgrounds:Chronic complications in type 2 diabetes are macrovascular complications including coronary heart disease,cerebral infarction as well as peripheral artery atherosclerosis and microvascular complications including nephropathy,retinopathy as well as neuropathy,which lead to a high incidence of death and disability in diabetic patients.The mechanism of diabetic vascular complications is unknown.Osteoprotegerin,a recently identified glycoprotein belonging to the tumor necrosis factor receptor superfamily,was originally discovered as an inhibitor of bone resorption.It has been proposed that osteoprotegerin may be involved in the development of calcifications in the arterial wall,where the molecule is present in concentrations comparable with those in bone.Supporting this idea, studies have shown that expression of osteoprotegerin and production of the protein are altered in arterial areas with calcification.Recently,some studies have indicated that osteoprotegerin also acts as an important regulatory molecule in the vasculature, and increased plasma osteoprotegerin concentrations are associated with coronary artery disease in nondiabetic patients.In some more recent studies in diabetic subjects, a strong association between plasma levels of osteoprotegerin and coronary artery disease was observed.Furthermore osteoprotegerin gene polymorphism(T-to-C at 950) was reported to be associated with coronary heart disease,patients with C/C genotype have higher coronary heart disease risk and higher serum osteoprotegerin levels.Whether serum osteoprotegerin level and osteoprotegerin gene polymorphism are associated with macroangiopathy in Chinese type 2 diabetes is unknown,so we investigated the serum osteoprotegerin level and osteoprotegerin gene polymorphism in type 2 diabetes,whether serum osteoprotegerin level is associated with microvascular complications,only one article reported the serum osteoprotegerin level was increased in diabetic patients with microalbuminuria and retinopathy.At present,no research on the relationships between the degree of diabetic nephropathy, retinopathy and osteoprotegerin was reported.So in this study,we also investigated the relationships between serum osteoprotegerin level and diabetic nephropathy, retinopathy as well as their severity.Objective:1.To investigate the relationships between serum osteoprotegerin level and osteoprotegerin gene polymorphisms with macroangiopathy in type 2 diabetes patients.2.To investigate the serum osteoprotegerin level and osteoprotegerin expression in kidney biopsy tissue in type 2 diabetes with nephropathy.3.To investigate the serum osteoprotegerin level and diabetic retinopathy in type 2 diabetes.Methods:1.Serum osteoprotegerin level was measured by enzyme-linked immunoassay(ELISA) and osteoprotegerin gene polymorphisms(T-to-C at 950) was examined by PCR and restriction enzyme technique in 86 type 2 diabetes with macrovascular complications,82 patients without macrovascular complications and 40 healthy persons.2.Serum osteoprotegerin level was measured by ELISA in 38 type 2 diabetes with normoalbuminuria,36 patients with microalbuminuria,36 patients with macroalbuminuria and 40 healthy persons.Part of diabetes patients with nephropathy were performed kidney biopsy by ultrasound guide.The osteoprotegerin expression in kidney biopsy tissue is examined by immunohistochemistry.3.30 type2 diabetes patients with proliferative retinopathy,30 patients with background retinopathy and 36 patients without retinopathy were collected for the research on the relationship between serum osteoprotegerin and diabetic retinopathy.Results:Part 1:1.The serum osteoprotegerin level in diabetic patients with macroangiopathy was 4.16±0.65 ng/l,which was significantly higher than that in patients without macroangiopathy(2.75±0.59) and control subjects(2.29±0.37 ng/l,p＜0.01).2.Correlation analysis showed that the serum osteoprotegerin has significant positive correlation with the FPG(r=0.243,p＜0.01),2hPG(r=0.237,p＜0.01),HbAlC (r=0.361,p＜0.001),HOMA-IR(r=0.252,p＜0.01),hsCRP(r=0.585,p＜0.001),and no correlation with age,BMI,blood pressure,blood lipid profiles and fasting insulin.3.Gene polymorphisms results showed that C/C genotype was more prevalent in patients with maeroangiopathy than without macroangiopathy(24.4%versus 11%, p＜0.05) and C allele prevalence was also higher than that in patients without macroangiopathy(40.7%versus 29.9%,p＜0.05).Subjects with C/C genotype(3.96±0.85 ng/l) have higher serum osteoprotegerin level than those with T/T(3.36±0.93 ng/l ) and T/C(3.37±0.94) ng/l genotype.(p＜0.05).4.Multivariate logistic regression analysis revealed that serum osteoprotegerin levels and C/C genotype were independently associated with the presence of macroangiopathy in patients with type 2 diabetes.Part 2:1.The serum osteoprotegerin level in macroalbuminuria group was 4.45±0.76 ng/l, which was significantly higher than that in microalbuminuria group(3.62±0.70) and normoalbuminuria group(2.77±0.78 ng/l)(p＜0.01).And the serum osteoprotegerin level in microalbuminuria group was also higher than that in normoaibuminuria group(p＜0.01).2.Correlation analysis showed that the serum osteoprotegerin has significant positive correlation with the FPG(r=0.223,p＜0.05),2hPG(r=0.298,p＜0.01),HbAlC (r=0.520,p＜0.001),HOMA-IR(r=0.257,p＜0.01),hsCRP(r=0.360,p＜0.001) and UAER(r=0.639,p＜0.001).3.Multivariate regression analysis revealed that serum osteoprotegerin level was independently effective factor for UAER in type 2 diabetes.4.The immunohistochemistry results showed that positive immunostaining for osteoprotegerin was observed in the renal tubule cells and not in glomerulus,and the osteoprotegerin expressed level was higher in macroalbuminuria group than that in microalbuminuria group. Part 3:1.The serum osteoprotegerin level in proliferative diabetic retinopathy group was 4.07±1.08 ng/l,which was significantly higher than that in background retinopathy group(3.57±0.96) and no retinopathy group(2.77±0.78 ng/l)(p＜0.01).And the serum osteoprotegerin level in background retinopathy group was also higher than that in no retinopathy group(p＜0.01).2.Correlation analysis showed that the serum osteoprotegerin has significant positive correlation with the FPG(r=0.263,p＜0.01),2hPG(r=0.305,p＜0.01),HbAlC (r=0.377,p＜0.001),hsCRP(r=0.453,p＜0.001) and HOMA-IR(r=0.324,p＜0.001).and no correlation with age,BMI,blood pressure,blood lipid profiles and fasting insulin.3.Multivariate logistic regression analysis revealed that serum osteoprotegerin level was independently associated with diabetic retinopathy in patients with type 2 diabetes.Conclusions:1.Serum osteoprotegerin levels were significantly increased in type 2 diabetes with macroangiopathy and microangiopathy,and there is a independently significant association between serum osteoprotegerin levels and the presence and severity of diabetic vascular complications,which supports the growing concept that osteoprotegerin acts as an important regulatory molecule in the vasculature of diabetes.2.The serum osteoprotegerin level has significant positive correlation with the FPG,2hPG,HbAlC,hsCRPand HOMA-IR.,indicating that the high serum osteoprotegerin levels in diabetic vascular complications are associated with diabetic control,insulin resistance and inflammation.3.Osteoprotegerin gene polymorphism at 950 was associated with macrovascular complications in chinese type 2 diabetes.This polymorphism may play a role in the development of macroangiopathy in type 2 diabetes.4.Positive immunostaining for osteoprotegerin was observed in the renal tubule cells and not in glomerulus,and the osteoprotegerin expressed level was higher in macroalbuminuria group than that in microalbuminuria group,indicates that renal tubule cells may be the important source of elevated serum osteoprotegerin level in type 2 diabetes with nephropathy.更多还原