【作者】 张丽萍；

【导师】 荣海钦； 陈丽；

【作者基本信息】 山东大学 ， 内科学， 2008， 博士

【摘要】 第一部分糖尿病大鼠骨组织形态计量学和生物力学的特点目的:采用高糖高脂肪饮食结合小剂量链脲佐菌素和单次大剂量链脲佐菌素的方法,用雄性Wistar大鼠制备2型和1型糖尿病模型,以正常血糖大鼠和病程匹配的自发性2型糖尿病模型GK大鼠分别作为阴性和阳性对照,测定离体骨密度(BMD)、骨组织形态计量学和生物力学等指标、骨转换血清生化标志物及调节骨代谢的细胞因子,观察糖尿病状态下大鼠骨代谢的特点;并与大鼠胰岛素水平及代谢控制等因素进行相关分析,寻找导致其骨代谢紊乱的可能机制。方法:实验动物分为四组:正常对照组(NC组)、2型糖尿病组(T2D组)、1型糖尿病组(T1D组)和自发性2型糖尿病模型GK大鼠(GK组)。T2D组大鼠在给予高脂饮食8周后,空腹状态下一次性腹腔注射小剂量STZ(30mg/kg),1周后空腹状态下行腹腔葡萄糖耐量试验,空腹血糖≥7.0mmol/L或2小时血糖≥11.1 mmol/L确认为造模成功。T1D组大鼠空腹状态下给予一次性腹腔注射大剂量STZ(60mg/kg),72 h后测随机血糖≥16.7 mmol/L者,确定糖尿病模型建立。建模成功后继续喂养20周,同时纳入病程相近的GK大鼠作为阳性对照。所有动物在处死前第14天开始进行四环素标记。试验结束时,留取24小时尿检测尿Ca、肌酐和羟脯氨酸,计算羟脯氨酸(HOP)排出率;所有大鼠禁食12小时后测空腹体重,麻醉状态下取血样测定空腹血糖、胰岛素、糖化血红蛋白(GHb)、Ca、P、肿瘤坏死因子(TNF-α)、白介素(IL)-1β、IL-6、胰岛素样生长因子(IGF)-1、骨钙素(OCN)水平和抗酒石酸酸性磷酸酶(TRAP)活性;留取腰椎、股骨、胫骨等骨标本,测定第五腰椎及股骨BMD,做腰椎压缩试验及股骨三点弯曲试验,取右侧胫骨上端以甲基丙烯酸甲酯包埋,制作不脱钙骨切片。应用多媒体病理图像分析软件对Von Kossa染色的5μm切片和未染色的7μm切片进行骨组织形态计量学分析。结果:1、糖代谢状态的检测:T2D组空腹血糖、GHb、胰岛素水平及HOMA-IR均明显高于NC组(P＜0.01),血清TG、TC和LDL水平也明显升高(P＜0.05或P＜0.01)。2、BMD和骨量的变化:T2D组、T1D组和GK组股骨、腰椎BMD和股骨灰干重比均明显低于NC组(P＜0.05);各指标在糖尿病大鼠各组间无明显差异(P＞0.05)。3、生物力学结果显示:股骨三点弯曲实验中T2D组、T1D组和GK组的最大载荷、能量吸收、最大应变、截面惯性矩和弯曲刚性系数均明显降低(P＜0.05或P＜0.01);T1D组生物力学指标较T2D组更显著(P＜0.05),而GK大鼠股骨生物力学指标无明显差异。腰椎压缩试验中各组糖尿病大鼠的弹性模量和最大载荷均较对照组显著降低(P＜0.01);以T1D组的变化更明显(P＜0.05或P＜0.01),而GK大鼠各项生物力学指标变化与T2D组大鼠相似(P＞0.05)。4、骨组织形态学结果:各组糖尿病大鼠骨体积显著低于对照组(P＜0.01),Tb.Th、OS/BS和O.Th均明显降低(P＜0.01或P＜0.05);T1D组和GK大鼠的破骨细胞标记ES/BS也有明显下降(P＜0.05)。各组糖尿病大鼠骨组织动力学参数MS/BS、骨矿化沉积率和BFR均明显降低(P＜0.01),T1D组和T2D组的类骨质成熟时间Omt明显延长(P＜0.05);但骨矿化延迟时间Mlt没有明显变化。糖尿病大鼠各组间没有明显差异(P＞0.05)。5、血尿骨代谢指标的变化:各组大鼠血钙、磷水平无明显差异(P＞0.05)。与NC组相比,各组糖尿病大鼠血清24h尿钙、TRAP活性和羟脯氨酸排除率HOP/Cr显著升高(P＜0.01),血清OCN水平明显降低(P＜0.01);同时,血清IGF-1水平明显下降(P＜0.01),且T1D大鼠IGF-1水平明显低于T2D大鼠(P＜0.01)。6、血清炎性细胞因子:各组糖尿病大鼠血清IL-1β、IL-6和TNF-α水平均明显高于对照组(P＜0.01),糖尿病大鼠各组间无明显差异(P＞0.05)。7、相关分析:GHb与骨密度、骨生物力学指标及BV/TV均显著负相关,IL-1β、IL-6和TNF-α与骨密度也显著负相关(P＜0.05)。结论:1、高糖高脂饮食和小剂量STZ诱导的Wistar大鼠呈现明显的高血糖、高胰岛素血症和胰岛素抵抗,并伴有血脂代谢紊乱,可用来成功制造2型糖尿病动物模型;2、该模型大鼠无明显肥胖,可摒除肥胖对骨代谢的影响,可作为研究2型糖尿病骨代谢紊乱的理想动物模型。3、糖尿病状态下大鼠表现为骨密度和骨量减少,骨力学强度降低;4、糖尿病状态下大鼠不仅出现血、尿骨代谢标记的异常,且可出现明显的骨组织形态结构异常,成骨细胞功能障碍可能是糖尿病骨代谢失衡的主要机制,但破骨细胞也可能参与其中。5、糖尿病大鼠骨密度、骨生物力学和骨形态学指标与糖化血红蛋白明显负相关,提示高血糖及其糖基化终产物可能在糖尿病大鼠骨代谢紊乱中具有重要作用。第二部分糖尿病大鼠骨组织RAGE、OPG、RANKL和IGF-1基因表达的研究目的:采用单次注射大剂量链脲佐菌素的方法,用Wistar大鼠制备胰岛素缺乏的糖尿病模型,免疫组化方法检测骨组织中AGEs受体RAGE的表达,并用RT-PCR方法测定骨组织RAGE、RANKL、OPG、IGF-1等基因表达,初步探讨糖尿病状态下高血糖和糖基化终产物对大鼠骨代谢相关基因表达的影响。方法:雄性Wistar大鼠随机分为正常对照组(NC)、糖尿病组(T1D组)和糖尿病胰岛素治疗组(DI组)。T1D糖尿病的建模方法同前,成模后治疗组给予胰岛素0.6～1u/d以控制随机血糖在10mmol/L以下,T1D组大鼠隔日皮下注射长效胰岛素0.3～0.6u,以防止酮症发生,但不明显影响血糖水平,随机血糖在16mmol/l以上。糖尿病建模成功后继续喂养20周处死。试验结束时,留取24小时尿检测尿肌酐和羟脯氨酸,计算羟脯氨酸(HOP)排出率;麻醉状态下取血样测定空腹血糖、糖化血红蛋白(GHb)、骨钙素(OCN)水平和抗酒石酸酸性磷酸酶(TRAP)活性;留取股骨、胫骨等骨标本,测定左侧股骨BMD,用手术刀片刮除右侧股骨骨膜后装于冻存管中迅速投入液氮中,再转入-70℃冰箱保存。胫骨经甲基丙烯酸甲酯包埋,制作不脱钙骨切片。采用免疫组化方法检测胫骨组织RAGE蛋白表达,应用多媒体病理图像分析软件测定RAGE阳性细胞积分光密度值。提取大鼠骨组织的总mRNA,利用实时荧光定量RT-PCR方法检测大鼠股骨RAGE、RANKL、OPG和IGF-1基因表达。结果:1、与NC组相比,T1D组骨组织RAGE免疫组化平均光密度值明显升高(P＜0.01)。RT-PCR显示T1D组大鼠股骨IGF-1 mRNA表达明显下降(P＜0.01),RAGE、RANKL mRNA水平显著升高(P＜0.01或P＜0.05),OPG mRNA水平无明显变化(P＞0.05),而OPG/RANKL比值明显低于对照组(P＜0.01)。胰岛素治疗可显著改善上述糖尿病大鼠的变化(P＜0.01)。2、相关分析分析显示,糖尿病大鼠RAGE蛋白免疫组化染色平均光密度值和mRNA水平与糖化血红蛋白水平显著正相关(r=0.656,P＜0.01;r=0.535,P＜0.05);RAGE mRNA与股骨BMD和血清骨钙素水平均显著负相关(r=-0.583,P＜0.01;r=-0.519,P＜0.05),与血清TRAP和羟脯氨酸排泄率无明显相关性。糖尿病大鼠骨组织RAGE mRNA与RANKL mRNA和RANKL/OPG比值均显著正相关(r=0.555,P＜0.05;r=0.651,P＜0.01);与IGF-1 mRNA显著负相关(r=-0.459,P＜0.05);与OPG mRNA没有显著相关性。IGF-1 mRNA与RANKL mRNA和RANKL/OPG比值均显著负相关(r=-0.672,P＜0.01;r=-0.567,P＜0.05)。结论:1、高血糖状态下累积的AGEs诱导其受体RAGE mRNA和蛋白表达上调;2、糖尿病大鼠骨组织RAGE表达增加,可能通过上调RANKL表达作用于偶联骨重建的OPG/RANKL/RANK轴,使破骨细胞活性增加,而成骨细胞功能受抑,参与糖尿病大鼠骨丢失的发生。3、RAGE表达上调可能与骨组织局部IGF-1表达降低有关,后者可直接或间接通过对OPG/RANKL/RANK轴的作用而影响糖尿病大鼠骨重建过程。更多还原

【Abstract】 Objective:To characterize the bone metabolism in type 2 and type 1 diabetic rats induced by high-fat feed combined with low dose of streptozotocin and high dose of streptozotocin.Non-diabetic Wistar rats and Goto-Kakizaki(GK) rats,a spontaneous type 2 diabetic model,were enrolled as negative and positive controls respectively. Bone mineral density(BMD),bone histomorphometric and biomechanical properties, as well as the bone biochemical indices and cytokines were observed,and correlations between bone metabolism and insulin level,metabolic control were analyzed to seek the possible mechanism for bony metabolic disturbance.Methods:Male Wistar rats were randomly served as non-diabetic controls(NC), inducible type 2 diabetic group(T2D) and type 1 diabetic group(T1D),with sex-and course of disease- matched GK rats served as positive controls.Type 2 diabetes was established by high fat and high-sugar feeding for eight weeks combined with one injection of small-dose(30 mg/kg body weight) of streptozotocin(STZ).The diabetes was confirmed by an intraperitoneal glucose tolerance test(IPGTT) one week after the injection of STZ.Type 1 diabetes was established by one injection of large-dose (60 mg/kg body weight) of STZ.The diabetes was confirmed with random blood glucose levels≥16.7 mmol/L detected 72 hours later.Double labeling for bone histomorphometry was conducted by an intraperitoneal injection of tetracycline 14 days before the sacrifice.The rats were killed 20 weeks after the onset of diabetes. Urine was collected from the rats for calcium,creatinine and hydroxyproline analyses. Blood specimens were collected from carotid arteries for fasting blood glucose, glycosylated hemoglobin,calcium,phosphate,osteocalcin and tartrate-resistant acid phosphatase activity analyses.The femur,tibia and the fifth lumbar vertebra were harvested and freed of soft tissue attachments carefully.Analyses for bone mineral density was performed by dual energy X-ray absorptiometry on femur and the fifth vertebral body respectively followed by the biomechanical studies of three-point bending test and compressive test.The metaphyseal tibiae were embedded in methylmethacrylate to obtain the undecalcified sections.Histomorphometry analysis was performed on 5μm(stained with Von Kossa) and 7μm bone sections with an analyze software for multimedia pathological image.Results:①Fasting plasma glucose,glycosylated hemoglobin,insulin concentrations, and HOMA-IR of the T2D rats were higher than the NC group significantly(P＜0.01), companied by significantly increased serum levels of TG,TC and LDL.②All the diabetic rats showed significantly deceased ratio of bone ash and dry weight,as well as the bone density determined on femur and lumber vertebra(P＜0.05).There was no significant difference among the diabetic groups(P＞0.05).③The maximal load, maximal energy,maximal strain,cross-sectional moment of inertia and coefficient of stiffness in the three-point bending test of the femora decreased significantly(P＜0.05或P＜0.01) in the three groups of diabetic rats,among which the T1D group had the most evident changes(P＜0.05).The maximal load and elastic modulus of vertebral bodies significantly decreased in the diabetic rats(P＜0.01),but there was no significant difference in the elastic modulus of femur among the groups.④Histomorphometrical study showed decreased trabecular bone volume,trabecular thickness,osteoid surface,osteoid thickness and osteoid volume in all the diabetic rats (P＜0.05或P＜0.01),as well as erosion surface in the T1D group and GK rats(P＜0.05).Mineralizing surface,mineral apposition rate and bone formation rate also decreased in all the diabetic rat(P＜0.01)s,along with an increase in osteoid maturation time in the T1D group and GK rats(P＜0.05).There was no significant difference among the diabetic groups(P＞0.05).⑤There was no significant difference in the serum calcium and phosphate levels among all the groups(P＞0.05). Compared with the healthy controls,the T2D rats had significantly lower serum osteocalcin and insulin-like growth factor-1 concentrations,and higher 24h urinary calcium,hydroxyproline exclusion and serum tartrate-resistant acid phosphatase activity(P＜0.01).The serum levels of IGF-1 were significantly lower in the T1D group than in the T2D groups(P＜0.01).⑥Compared to healthy controls,diabetic rats had significantly increased interleukin 1βand 6,and tumor necrosis factorαconcentrations in the serum(P＜0.05 or 0.01).There was no significant difference among the diabetic groups(P＞0.05).⑦Partial correlation analysis showed that the levels of GHb were significantly correlated with the BMD,biomechanical properties and BV/TV,and the serum levels of IL-1β,IL-6 and TNF-αwere negatively correlated with the BMD in the control and diabetic rats.Conclusions:1.Wistar rats induced by high fat and high-sugar feeding combined with one injection of small-dose streptozotocin can be the type 2 diabetes model successfully,which were charactered by high blood glucose,hypefinsulinemia, insulin resistant and metabolic disturbance of blood fat.2.This rat model without obesity excludes the interference of obesity on bone and can be the ideal animal model for study on abnormality of bone metabolism under type 2 diabetes.3.The diabetic rats had decreased bone density,bone mass and biomechanical forces.4.the diabetic rats had disturbance of bone metabolic markers in the blood and urine and abnormal bone histomorphometry.Dysfunction of osteoblasts might be the major mechanism for bone metabolic disturbance under diabetic status,in which osteoclasts may involve.5.The bone mineral density,bone biomechanical and histomorphometric indices were inversely correlated with the glycosylated hemoglobin significantly in the diabetic rats,indicating that hyperglycemia and advanced glycation endproducts might involve in the bone metabolic disturbance in diabetic rats. Objective:To observe the expression of RAGE,OPG,RANKL and IGF-1 in bone from the STZ-treated diabetic rats in order to study the possible mechanisms of hyperglycemia and advanced glycation endproducts on bone metabolism.Methods:Male Wistar rats were randomly divided into control group(NC),untreated diabetic group(T1D) and insulin-treated diabetic group(DI).Diabetes was established as previously detailed in part one.Rats in the DI group were treated with insulin to control the blood glucose level under 10 mmol/L.All the rats were killed 20 weeks after the onset of diabetes.Urine was collected from the rats for creatinine and hydroxyproline analyses.Blood specimens were collected for fasting blood glucose, glycosylated hemoglobin,osteocalcin and tartrate-resistant acid phosphatase activity analyses.Analyses for bone mineral density was performed by dual energy X-ray absorptiometry on the left femora.RNA samples were extracted directly from the right femora.The expression of RAGE,OPG,RANKL and IGF-1 mRNA were detected by real-time reverse transcription polymerase chain reaction(RT-QPCR). The expression of RAGE protein was detected by immunohistochemieal analysis on the tibiae sections embedded in methylmethacrylate with an analyze software for multimedia pathological image.Results:1.Compared with the healthy controls,the untreated T1D diabetic rats had significantly higher optical density value of RAGE protein in the tibiae(P＜0.01).The expression of IGF-1 mRNA significantly decreased(P＜0.01),and both RAGE and RANKL mRNA significantly increased(P＜0.05 or 0.01) in the untreated T1D diabetic rats.The change in the expression of OPG mRNA was undetectable.Then the ratio of OPG/RANKL decreased significantly in the untreated diabetic rats(P＜0.01).Insulin improved all the above changes in the diabetic rats(P＜0.01).2.The optical density value of RAGE protein and expression of RAGE mRNA were positively correlated with the glycosylated hemoglobin in the diabetic rats(r=0.656, P＜0.01;r=0.535,P＜0.05).The expression of RAGE mRNA was negatively correlated with the femoral BMD,serum OCN and BV/TV(r=-0.583,P＜0.01;r= -0.519,P＜0.05;r=-0.564,P＜0.05),while positively correlated with ES/BS(r =0.535,P＜0.05).There was no correlation between the expression of RAGE and hydroxyproline exclusion and serum tartrate-resistant acid phosphatase activity.The expression of RAGE mRNA was positively correlated with the expression of RANKL mRNA and RANKL/OPG(r=0.555,P＜0.05;r=0.651,P＜0.01),and negatively correlated with the expression of IGF-1 mRNA(r=-0.459,P＜0.05).There were negative correlations between the expression of IGF-1 mRNA and RANKL mRNA and RANKL/OPG(r=-0.672,P＜0.01;r=-0.567,P＜0.05).Conclusions:1.AGEs accumulated in the diabetes induce both the protein and mRNA expression of its receptor,RAGE.2.The increased RAGE in bones might play a role in the bone loss of the diabetic rats through stimulating the osteoclasts activities and inhibit the osteoblasts by increasing the expression of RANKL.3.The increased expression of RAGE may be associated with the decreased expression of IGF-1.The latter impacts the bone remodeling directly or indirectly through the OPG/RANKL/RANK axis in diabetic rats.更多还原