【作者】 杨建虹；

【导师】 刘兢；

【作者基本信息】 中国科学技术大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 糖尿病影响骨代谢,导致骨量减少,诱发骨质疏松,但是糖尿病性骨质疏松的发病机制仍不十分清楚。胰岛素是骨形成促进因子,对成骨细胞的增殖、分化和成熟具有促进作用,但是具体的作用机制也不清楚。本文通过体内和体外实验,探讨了胰岛素缺乏导致骨质疏松的发病机制以及胰岛素促进骨生长和分化的分子作用机制,为预防骨质疏松提供一定的理论依据。营养指导,饮食补充等非药物预防策略干预骨质疏松已受到人们的广泛重视,从中药中筛选抗骨质疏松药物具有广阔的应用前景。本文探讨了鹿茸血缓解去势大鼠骨质疏松的生物学效应机制。主要结果如下：发现糖尿病引起钙(Ca)、磷(P)、镁(Mg)、锌(Zn)、锶(Sr)等骨矿元素流失以及血清睾酮(T)、胰岛素样生长因子-1(IGF-1)和骨钙素(OC)等水平的降低是糖尿病骨密度(BMD)降低的重要原因,而胰岛素处理能够提高Ca、P、Mg等骨矿元素含量,恢复血清T、IGF-1和OC的水平,增加骨密度,缓解骨质疏松。接着,通过细胞分子生物学的研究,进一步阐明了胰岛素通过激活MAPK通路上调Osx和IGF-1的表达,与激活PI3K通路下调Runx2的表达促进成骨细胞生长和分化的分子作用机制。该结果进一步阐明了糖尿病性骨质疏松的发病机制,为胰岛素预防和治疗糖尿病性骨质疏松提供一定的理论指导并在临床应用方面具有一定的现实意义。此外,首次报道了鹿茸血通过提高Ca、P和Zn等骨矿元素水平以及通过提高血清T和IGF-1的水平,缓解去势大鼠骨质疏松的生物学效应机制。为鹿茸血抗骨质疏松的应用提供一定的理论依据。绪论中,从影响成骨细胞生长、分化的调节因子、信号通路以及转录因子等方面,简要综述了骨质疏松的发病机制。首次运用同步辐射X射线荧光探针分析技术(SRXRF),发现糖尿病引起钙Ca、P、Zn、Sr等骨矿元素流失是导致骨密度降低的重要原因之一,从骨矿元素变化阐明了糖尿病性骨质疏松的发病机制。研究表明：糖尿病组骨密度显著低于对照组(P<0.01),同时Ca、P和Zn元素的相对含量明显低于对照组(P<0.01)；与对照组相比,糖尿病组中Sr元素的相对含量降低11%；而糖尿病大鼠硫(S)元素的相对含量显著高于对照组(P<0.01)。元素铬(Cr),铜(Cu),铅(Pb)的相对含量在两组间无显著差异。统计分析显示Ca分别和P(R=0.85,P<0.001),Sr(R=0.38,P<0.05),Zn(R=0.37,P<0.05)呈正相关性；而Zn和S呈负相关性(R=-0.40,P<0.05)。首次利用电感耦合等离子体原子发射光谱法(ICP-AES),发现胰岛素治疗可以通过提高Ca、P、Mg等元素的含量使骨密度得到恢复,缓解骨质疏松,表明胰岛素具有骨形成促进作用,同时也进一步证明糖尿病大鼠股骨中Ca、P、Mg、Sr、K等元素的丢失是导致BMD降低的原因之一。研究表明：与正常组相比,糖尿病组大鼠股骨骨密度显著降低(P<0.01),胰岛素治疗后显著升高(P<0.05)。ICP-AES元素分析结果显示：与正常组相比,糖尿病组大鼠股骨中Ca、P、Mg、Sr、K等5种元素含量显著降低(P<0.05),而胰岛素治疗后,元素Ca、P、Mg含量得到了恢复(P<0.05)。相关性分析显示：正常组大鼠股骨的Ca、P、Mg元素含量间具有很高的相关性。接着,报道了糖尿病引起血清睾酮、胰岛素样生长因子-1和骨钙素等的降低也是导致BMD降低的原因之一,而胰岛素治疗可以恢复血清睾酮、胰岛素样生长因子-1和骨钙素水平,预防骨流失和BMD降低,进一步证明了胰岛素的骨形成促进作用。实验结果表明：(1)、糖尿病组大鼠的骨密度显著降低(P<0.01),小梁骨间距明显增加,骨小梁流失显著(P<0.01),而胰岛素治疗可以降低骨流失,恢复骨密度；(2)、糖尿病组血清睾酮水平显著降低(P<0.01),胰岛素治疗后,睾酮水平升高；(3)、糖尿病组血清中骨保护素、胰岛素样生长因子-1和骨钙素也明显降低(P<0.01),经胰岛素治疗后其相应水平提高；(4)、而糖尿病组血清总碱性磷酸酶水平显著升高,胰岛素治疗后其值降低；(5)、血清钙、磷水平在三组间没有显著差异。通过细胞分子水平的研究,进一步阐明胰岛素通过激活MAPK通路上调Osx和IGF-1的表达以及激活PI3K通路下调Runx2的表达促进成骨细胞生长、生存和分化成熟的分子机制。采用人成骨样MG-63细胞,胰岛素刺激或者胰岛素刺激的同时分别加入特异性的阻断剂阻断PI3K通路和MAPK通路,结果显示：胰岛素上调成骨细胞表面胰岛素受体的表达,并呈时间依赖性；胰岛素能促进成骨细胞的增殖、生存；增加碱性磷酸酶的活性和1型胶原蛋白的分泌；促进钙化结节的产生；上调Osx、IGF-1、OC和BMP-2的表达,下调Runx2的表达。阻断PI3K通路后,成骨细胞的增殖受到抑制并发生明显的凋亡,碱性磷酸酶的活性和1型胶原蛋白的分泌、钙化结节的产生、OC的表达都受到明显抑制,但Runx2的表达明显增加。阻断MAPK通路也能抑制成骨细胞的增殖和生存,碱性磷酸酶的活性和1型胶原蛋白的分泌、钙化结节的产生、OC的表达受到明显抑制,同时,Osx、IGF-1的表达明显降低,BMP-2的表达也有所降低。因此,胰岛素通过上调成骨细胞表面胰岛素受体的表达,激活PI3K通路下调Runx2的表达,而激活MAPK通路上调Osx和IGF-1的表达,从而促进成骨细胞生长、生存和分化成熟。此外,首次运用同步辐射X射线荧光探针技术(SRXRF)报道了鹿茸血通过提高Ca、P和Zn等骨矿元素水平,缓解去势大鼠骨质疏松的作用机制。结果表明：当大鼠去势后导致骨质疏松时,其股骨的BMD明显降低,则股骨元素Ca、P、Zn和Sr的相对强度也都降低,说明由于Ca和P等元素的大量丢失,引起骨质疏松。当鹿茸血治疗后,其骨组织中Ca、P及微量元素Zn的相对强度都有不同程度的升高,因而,BMD也显著升高,骨质疏松得到显著恢复。因此,鹿茸血可以通过改善骨组织Ca、P和Zn元素的含量达到抗骨质疏松的作用。接着,报道了鹿茸血通过提高血清睾酮和IGF-1的水平,使BMD得到增加,缓解骨质疏松的生物学效应机制。研究表明：去势导致E2的缺乏,伴随血清睾酮、IGF-1水平的降低,致使股骨和椎骨的BMD降低,引发骨质疏松。然而,鹿茸血治疗后,通过提高血清睾酮和IGF-1的水平,使股骨和椎骨的BMD得到增加,具有抗骨质疏松效应。但是,鹿茸血缓解骨质疏松的分子机制仍需进一步的研究。更多还原

【Abstract】 Diabetes mellitus affects bone metabolism and leads to osteopenia and osteoporosis, but its pathogenic mechanism remains unknown. Insulin has positive effects on bone growth and differentiation, but the underlying mechanism is not clear. To address these problems, here we investigate the relation between insulin deficiency and osteoporosis in vivo animal model, and the effect of insulin on osteoblast proliferation, differentiation and maturation in vitro cell culture system.Nonpharmacologic management of osteoporosis includes nutrition guidelines, dietary supplementation and so on and is very important to improve bone heath for a patient at risk of osteoporosis. Screening anti-osteoporosis drugs from Chinese herbal medicine hold a very broad appliance future. Here, we investigate the biological mechanism of anti-osteoporosis of antler blood in ovariectomized rats. The thesis consists of eight chapters.First, we present a brief introduction to the mechanism of osteoporosis involved in the regulator, signaling pathway as well as transcription factor which effect on osteoblast growth and differentiation.Second, we revealed for the first time that loss of calcium (Ca), phosphorus (P), zinc (Zn) and strontium (Sr) element contents accounted for the bone mineral density (BMD) reduction in diabetic animal models. With synchrotron radiation X-ray fluorescence (SRXRF) microprobe analysis technique, we found that relative mineral content of Ca, P and Zn in diabetic femurs decreased significantly compared to controls. And Sr in diabetics reduced 11%(P= 0.09). Relative content of sulfur (S) in average was statistically higher (P< 0.01) in diabetics than that in controls. But no obvious difference was observed in relative content of chromium (Cr), iron (Fe), copper (Cu), and lead (Pb) between the two groups. Statistical analysis revealed that Ca correlated positively with P (R= 0.85, P< 0.001), with Sr (R= 0.38, P< 0.05) and with Zn (R= 0.37, P< 0.05). Whereas Zn correlated negatively with S (R=-0.40, P< 0.05).Third, we demonstrated for the first time that insulin treatment could restore BMD by increasing the Ca, P and magnesium (Mg) element contents which was determined by inductively coupled plasma atomic emission spectrometry (ICP-AES) in type1 diabetic rat models. The results showed that the femoral BMD in diabetic group was significantly lower than that in normal group (P< 0.01) but reserved by insulin treatment (P< 0.05). ICP-AES analysis revealed that the element content of Ca, P, Mg, Sr, potassium (K) in diabetic group were remarkably lower than those in normal group (P<0.05) but only Ca, P and Mg content were significantly increased compared with diabetic group (P< 0.05) after insulin treatment. However, no significant differences were observed in element zinc (Zn) content among three groups.Fourth, we also showed that the decrease in serum levels of testosterone, insulin-like growth factor-1 (IGF-1) and osteocacin(OC) caused by insulin deficiency accounted for the bone mineral density (BMD) reduction in diabetic animal models. However, insulin treatment increased serum levels of testosterone, IGF-1 and OC and is capable of preserving bone loss. Results of BMD and histomorphometry revealed serious.bone loss in diabetes. Circling testosterone was significantly reduced in diabetic rats (P< 0.01). osteoprotegerin(OPG), IGF-1 and OC in diabetics also decreased noticeably (P< 0.01). A marked increase was observed in serum concentration of total alkaline phosphatase(ALP), bone ALP of diabetics (P< 0.01) compared to the controls. However insulin treatment induced a marked increase in the levels of testosterone, OPG, IGF-1, OC; and a decrease of total ALP, bone ALP. No significant differences were observed in serum Ca and P among three groups. Our results suggest that bone metabolism is seriously affected by diabetes. Insulin deficiency combining with testosterone depression are associated with alterations in bone turnover, resulting in the development of osteoporosis. And insulin treatment is capable of preserving bone loss and testosterone depression.Fifth, we reported the mechanism of the action of insulin on osteoblast growth and differentiation. Human osteoblastic cell line-MG63 was used and stimulated by insulin in the presence or absence of extracellular signal-regulated kinase (ERK) inhibitor PD98059, PI3-K inhibitor LY294002 or inhibitor PD98059+LY294002. Insulin receptor expression was determined by Confocal laser scanning microscopy (CLSM). Cell proliferation was detected by MTT assay. Cell apoptosis was determined by FACS. ALP activity was determined by ALP kit. Collagen 1 synthesis was determined by ELISA. The mineralized nodules were stained by Alizarin red stain. The mRNA expression of OC, Osx, Runx2, IGF-1, BMP-2 were quantified by real-time RT-PCR. Our results showed that insulin positively regulated the expression of its receptor. Insulin could promote cell proliferation, and blockage of each of the two pathways could decrease the proliferation and cause remarkable cell apoptosis. ALP activity and collagenl synthesis, OC expression, and mineralized nodule formation were increased in insulin treated group, whereas these indicators were decreased in both blockage groups. Down-regulation of Runx2 expression was reversed significantly in PI3K blocked group, but up-regulation of Osx expression was decreased significantly in MAPK blocked group, and so were IGF-1 and BMP-2 after insulin treatment. Therefore, insulin promoted osteoblast differentiation through MAPK signaling pathway to up-regulate Osx expression and PI3K signaling pathway to down-regulate Runx2 expression.In addition, it’s the first time that we reported the antiosteoporotic effects of antler blood. The femoral BMD was analyzed by DXA and the element relative content was determined by SRXRF microprobe in ovariectomized rats. The results showed that the femoral BMD was significantly lower than that of sham-operated rats (p< 0.05) but reversed by antler blood (Cervus nippon Temminck) treatment (p< 0.05). A further study demonstrated that the relative contents of P, Ca, Zn and Sr were obviously lower in ovariectomized rats compared to sham-operated rats but only the relative contents of P, Ca and Zn were normalized by antler blood treatment (p< 0.05). Our experiments revealed that loss of element Ca, P, Zn and Sr was closely related to the BMD reduction in ovariectomized rats and the anti-osteoporotic effect of antler blood was mediated by increasing the contents of P, Ca and Zn.Forthermore, the antiosteoporotic effects of antler blood were evaluated in ovariectomized Wistar rats. Compared with SHAM group, serum 17β-estradiol (E2) level decreased significantly (p<0.05) and OC level increased significantly (p<0.05) in OVX group, indicating successful model of osteoporosis. However, serum levels of IGF-1 and testosterone (T) were lower obviously in OVX group than those in SHAM group (p<0.05) but reserved by antler blood treatment (p<0.05) and the BMD of the lumbar spine and left femur in OVX group decreased remarkably (p<0.05) compared to SHAM group but normalized by treated with the antler blood (p<0.05). No obvious changes in serum levels of Ca, P, total ALP and OPG were observed among three groups. These results showed that bone growth promoting effects of antler blood were mediated by high serum concentration of T and IGF-1.Finally, we summarize the contents of seven chapters above.更多还原