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1、专 业：指导老师：学 生：中国贵州贵阳 2013 年 6 月中文摘要 IV英文摘要 V 前 言 11. BMP蛋白 21.1 BMP结构和功能 21.2 BMP诱导成骨机制 22. Noggin基因的概况 32.1 Noggin基因的结构 32.1.1 Noggin基因结构特征 4 结 果 25致 谢 51参 考 文 献 52中文摘要目的：研究noggin、骨形态发生蛋白2（bone morphogenetic protein 2，BMP2）、胰岛素样生长因子I（insulin like growth factor I，IGF-I）对成骨细胞增殖和分化的调节作用。方法：1、将人成骨肉瘤细胞MG

2、63于RPMI 1640培养基中培养，添加不同浓度的noggin、BMP2、IGF-I蛋白进行药物干预，用四甲基偶氮唑盐（methylthiazolyl-tetrazolium，MTT）法研究细胞的增殖率。2、选用最适浓度的noggin、BMP2、IGF-I蛋白作用成骨细胞，运用ELISA方法和RT-PCR技术分别检测成骨细胞分化标志物骨碱性磷酸酶（bone alkaline phosphatase, BALP）和骨钙蛋白（osteocalcin, OC）的蛋白含量和mRNA的表达情况。结果：1、各药物干预组OD值与对照组相比有统计学差异（P0.05），对成骨细胞增殖的作用效应呈剂量和时间依赖

3、性，其作用的最佳时间均在72h。各药物组内之间比较，确定了最佳作用浓度，BMP2为210-9 mol/L，对MG63细胞的增殖率达179.59；noggin为810-9mol/L，对其增殖的抑制率达到15.51；IGF-I为110-8mol/L，其增殖率达189.80。结论：BMP2、IGF-I对MG63细胞增殖有促进作用，而noggin则抑制MG63细胞的增殖。Noggin、BMP2和IGF-I促进BALP的分泌。Noggin对OC蛋白的分泌有抑制作用，而BMP2、IGF-I则促进OC蛋白的分泌。关键词：Noggin蛋白，BMP2蛋白，IGF-I蛋白，成骨细胞，增值，分化英文摘要Object

4、: The aims is to study the effects of noggin, bone morphogenetic protein 2 （BMP2） and insulin-like growth factor I （IGF-I） on the proliferation rates and differentiation of osteoblasts.Methods: The human osteosarcoma cell line MG63 were chosen using the RPMI1640 medium with 10 fetal bovine serum （FB

5、S）. Then, the cultured MG63 cells were exposed to BMP2, noggin and IGF-I at different concentrations with zero concentration as control group （CON）. Ability of the proliferation of MG63 cells was detected by methylthiazolyltetrazolium method （MTT）. the concentration of BALP and OC of osteoblasts wer

6、e determined by ELISA method.at different time points （12h, 24h, 48h, 72h） after optimal dosage of noggin BMP2, IGF-I were added into the culture medium.And the expression level of BALP and OC were investigated by reverse transcription PCR at the same time.Results: 1. The proliferation ability of MG

7、63 cells treated with BMP2, noggin and IGF-I groups were significantly different compared with that of CON group（P0.05）. The effects of three factors on MG63 cell was dose-dependently and time-dependently after cultured for 72h. The most effective growth of MG63 cells showed at 210-9 mol/L of BMP2 w

8、ith the proliferation rate of 179.59 and 110-8mol/L of IGF-1 with the rate of 189.80, while 810-9mol/L noggin with the inhibitory proliferation rate of 15.51, respectively. 2. The concentration of BALP treated with BMP2, noggin and IGF-I groups were significantly different compared with that of CON

9、group （P0.05） for 48h, especially, the noggin group was extremely significant （P0.01）, and the BALP mRNA expression were also significantly different （P0.05）. 3. The concentration of OC treated with noggin groups were significantly different compared with that of CON group （P0.01） for 24h, but the O

10、C mRNA expression were not significantly different （P0.05）. The concentration of OC of BMP2 group were significantly different compared with that of CON group significantly different compared with that of CON group （P0.01） , and the OC mRNA expression were also significantly different （P0.05）. The c

11、oncentration of OC of IGF-I group were significantly different compared with that of CON group significantly different compared with that of CON group （P0.01）, and the OC mRNA expression were also significantly different （P0.05）.Conclusion: BMP2 and IGF-I promoted the proliferation of MG63 cells whe

12、reas noggin inhibited. Noggin, BMP2, and IGF-I enhanced the concentration of BALP in osteoblasts. BMP2 and IGF-I improved the concentration of OC in osteoblasts, whereas noggin inhibited.Key words: Noggin; bone morphogenetic protein 2 （BMP2）; insulin-like growth factor I （IGF-I）; MG63 cell; prolifer

13、ation; differentiation. 前 言生长是动物个体发育特有的生理特点，其主要表现在体高体长的增长方面。而体高体长的增长则源于骨骼的纵向生长，从而形成相应的骨骼系统，达到体高体长的增长的目的。骨骼系统也是机体非常重要的一个组成部分，它支撑躯体，保护内脏，并且和骨骼肌一起协调配合完成其运动功能。 在骨骼的生长发育过程中，骨组织不断进行着新骨形成和旧骨吸收的骨重建过程，即骨形成（bone formation）与骨吸收（bone resorption）的动态平衡代谢过程。骨形成由成骨细胞介导来完成。在骨形成过程中，成骨细胞主要经历细胞增殖、细胞外基质成熟、细胞外基质矿化和细胞凋亡四个

14、阶段。在幼年生长时期，骨形成远远大于骨吸收（Krupa B et al, 2005），为骨骼的生长提供了良好的基础，也为体高体长的增长提供了重要的保证。在成年时期，骨的形成较为稳定，骨骼的生长不再发生变化，这主要是受到骨形成与骨吸收相对均衡的一个动态调节作用来完成。在晚年时期，则主要趋向于骨吸收大于骨形成，从而骨形成会受到一定的影响，这将导致一系列骨疾病的发生，比如在老年人群体中常出现的骨质疏松症。 然而骨骼的纵向生长是一种十分复杂的过程，是多种内分泌激素、生长因子在时空上对生长板综合作用的结果。内分泌系统的全身性激素如生长激素、降钙素、甲状旁腺激素、性激素、糖皮质激素等，与生长因子如胰岛素样

15、生长因子-（Iinsulin-like growthfactor-I，IGF-I）、骨形态发生蛋白（bone morphogenetic proteins，BMPs）、转化生长因子（transforming growth factor TGF）、成纤维细胞生长因子（fibroblast growthfactors，FGF）、血管内皮生长因子（vascular endothelial growth factor，VEGF）、甲状旁腺素相关肽（PTH-related peptide，PTHrP）等局部因子，以及外源性物质如维生素D等相结合，共同调节骨骼生长过程。1. BMP蛋白（一级标题）1.1 BMP结构和功能（二级标题）在1965年，Urist首次发现骨形成蛋白（bone morphogenetic protein，BMP） ，BMP属于转化生长因子-（transforming growth factor-） 超家族的一员，具有诱导骨形成的作用，现已发现其家族至少有20多个成员。BMP是一种低分子量酸性多肽，属于二硫健交联的生长和分化因子（Wozney JM等，1998）。是结构类似的高度保守的功能蛋白质族，其C-端有一个高度保守的结构域，内含7个位置稳定的半肤氨酸，这对于形成正确的二聚体结构有重要意义。研究发现，BMP的活性形式为二聚体，它既可以两个相同的链形成的同源二