糖尿病对口腔种植体骨整合影响的作用机制研究

发布时间：2017-12-26 19:38

本文关键词：糖尿病对口腔种植体骨整合影响的作用机制研究　出处：《山东大学》2015年博士论文　论文类型：学位论文

【摘要】：背景和目的：口腔种植是治疗缺牙患者的最有效途径。B ranemark在1971提出的“骨结合Osseointegration"概念成为种植体成功的最重要标准。稳定的骨结合依赖于成骨细胞在种植体表面的粘附和增殖分化,这一过程受到多种局部和全身因素的影响。糖尿病作为口腔种植治疗的相对禁忌症,一直受到学者们的关注,被公认为是影响种植体骨结合的最重要系统因素之一。本研究拟从体外实验观察高糖对MC3T3-E1细胞增殖、成骨分化的影响,并分析PI3K/Akt信号通路在其中的调节作用机制；从体内实验,通过糖尿病大鼠模型,检测不同血糖水平大鼠种植体周围骨组织变化及整合素α5β1和纤维粘连蛋白(FN)表达水平,综合评价糖尿病对种植体骨结合影响的作用机制。材料与方法：体外实验：PI3K/AKT通路对不同高糖状态下MC3T3-E1细胞成骨分化的影响根据文献选取四种培养基糖浓度：5.5mmol/L,15.5mmol/L,25.5mmol/L, 35.5mmol/L,分别模拟人体内正常生理糖浓度及不同程度高血糖浓度,并根据是否添加PI3K/AKT通路抑制剂LY294002来分为八组：1)5.5mM糖浓度组(5.5mM-)；2)15.5mM糖浓度组(15.5mM-);3)25.5mM糖浓度组(25.5mM-);4)35.5mM糖浓度组(35.5mM-);5)5.5mM糖浓度+LY294002组(5.5mM+);6)1 5.5mM糖浓度+LY294002组(15.5mM+);7)25.5mM糖浓度+LY294002组(25.5mM+);8)35.5mM糖浓度+LY294002组(35.5mM+)；其中,5.5mM糖浓度组(5.5mM)为对照组。甲基噻唑基四唑(MTT)法测定1d,3d的细胞增殖；碱性磷酸酶(ALP)活性检测3d,7d的细胞分化情况；茜素红染色观察7d,14d细胞分泌钙结节的情况；荧光实时定量PCR测定Run mRNA和OCN mRNA基因分别在3d、7d的表达；蛋白免疫印迹(Western blot)检测3d、7d磷酸化蛋白激酶(p—Akt)的表达。体内实验：糖尿病大鼠种植体周围骨结合的机制研究33只3月龄雄性健康ZDF(Zucker diabetic fatty)大鼠随机分为3组,每组11只,每只大鼠植入2颗种植体。A组：对照组,ZDF大鼠皮下注射等量生理盐水,直接植入种植体(11只大鼠,22颗种植体)；B组：ZDF大鼠接受艾塞那肽缓释纳米球治疗(皮下注射,0.74%,0.1m]/100g),并同时植入种植体(11只大鼠,22颗种植体)；C组：ZDF大鼠接受艾塞那肽缓释纳米球治疗(皮下注射,0.74%,0.1ml/100g),至血清葡萄糖维持在恒定水平(≤16mmol/L),然后植入种植体(11只大鼠,22颗种植体)。种植手术后,在第7d、14d、30d、60d分批次处死大鼠,外科手术取出双侧股骨,去净肌肉、粘膜等软组织,随后使用切割钻切取种植体近远中3mm的范围的骨块,从而得到一个包含种植体的长方形骨块标本(第7d,标本数n＝4个/每组,第14d、30d、60d,标本数分别为n=6个/每组)。通过硬组织切片、HE染色观察种植体周围骨代谢状况,并通过免疫组化检测种植体周围整合素α5β1和纤维粘连蛋白的表达。结果：MTT实验结果显示,在第24h,与对照组相比,15.5mM-和25.5mM-组的吸光度值均没有显著变化(PO.05)；然而,35.5mM-组的吸光度值较对照组显著降低,差异具有统计学意义(P0.05)；在第72h,与对照组相比较,15.5mM-组的吸光度值有显著升高,差异具有统计学意义(P0.05)；然而,25.5mM-和35.5mM-组的吸光度值较对照组显著降低,且差异具有统计学意义(P0.05)。此外,在每一时间点,使用LY294002处理过的四组细胞的吸光度值较对照组均显著下降,差异具有统计学意义(P0.05)ALP实验结果显示,在第3d,与对照组相比较,15.5mM-组、25.5mM-组、35.5mM-组的吸光度值均无显著变化,差异没有统计学差异(P0.05),然而,添加LY294002处理的四组细胞的吸光度值较对照组均显著降低,差异具有统计学意义(P0.05)；在第7d,15.5mM-组表现出最高的吸光度值,且与对照组相比具有统计学差异(P0.05),然而,25.5mM-组和35.5 mM-组的吸光度值较对照显著降低,差异具有统计学意义(P0.05)。此外,经LY294002处理的四组细胞的吸光度值较对照组显著降低(P0.05),其中,15.5 mM+组的吸光度值高于5.5 mM+组、25.5 mM+组和35.5 mM+组,且差异具有统计学差异(P0.05)。茜素红染色结果显示,在第7d,15.5mM-组钙结节的积分吸光度值显著高于对照组及其他实验组,差异具有统计学意义(P0.05),同时,25.5mM-组和35.5mM-组钙结节的积分吸光度值也要高于对照组,且差异具有统计学差异(P0.05),此外,经LY294002处理的四组细胞的钙结节积分吸光度值较对照组均显著降低,差异具有统计学意义(P0.05)；在第14d,与对照组相比,15.5mM-组钙结节的积分吸光度值要显著增高,差异具有统计学意义(P0.05),然而,25.5mM-组和35.5mM-组钙结节的积分吸光度值与对照组相比无显著差异(P0.05),同样,经LY294002处理的四组细胞的钙结节积分吸光度值较对照组均显著降低,差异具有统计学意义(P0.05)。成骨相关转录因子(Runt-related transcription factor 2, RunX2)表达结果显示,在第3d,5.5mM+组的基因相对表达量显著低于对照组,且差异具有统计学意义(P0.05),其余各组的基因相对表达量与对照组相比没有明显差异(P0.05)；在第7d,15.5mM-组的基因相对表达量较对照组明显增高,差异具有统计学意义(P0.05),同时,15.5mM+组的基因相对表达量与对照组相比无显著差异(P0.05),其余各组的基因相对表达量均较对照组显著降低(P0.05)。锌指结构转录因子(Osterix)表达结果显示,在第3d,5.5mM+组的基因相对表达量显著低于对照组,且差异具有统计学意义(P0.05),15.5mM-组的基因相对表达量显著高于对照组,且差异具有统计学意义(P0.05),其余各组的基因相对表达量与对照组相比没有明显差异(P0.05)在第7d,15.5mM-组及15.5mM+组的基因相对表达量较对照组均明显增高,差异具有统计学意义(P0.05),同时,5.5mM+组的基因相对表达量与对照组相比明显降低,且有统计学意义(P0.05),其余各组的基因相对表达量与对照组相比没有明显差异(P0.05)。骨桥蛋白(Osteoprotegerin, OPN)结果显示,在第3d与7d两个时间点各组的变化趋势一致,15.5mM-组、25.5mM-组及15.5mM+组的基因相对表达量显著高于照组,且差异具有统计学意义(P0.05),35.5mM+组的基因相对表达量与对照组相比显著降低,且差异具有统计学意义(P0.05),其余各组的基因相对表达量与对照组相比没有明显差异(P0.05)。骨钙蛋白(osteocalcin, OCN)结果显示,在第3d,15.5mM-组的基因相对表达量显著高于对照组,且差异具有统计学意义(P0.05),5.5mM+组的基因相对表达量显著低于对照组,且差异具有统计学意义(P0.05),其余各组的基因相对表达量与对照组相比没有明显差异(P0.05)；在第7d,15.5mM-组、25.5mM-组及25.5mM-组的基因相对表达量较对照组明显增高,差异具有统计学意义(P0.05),同时,5.5mM+组、25.5mM+组及35.5mM+组的的基因相对表达量显著低于对照组,且差异具有统计学意义(P0.05),15.5mM+组的基因相对表达量与对照组相比无显著差异(P0.05)。蛋白免疫印迹(Western blot)检测P-Akt的表达结果显示,在第3d,15.5mM-组的P-AKT表达量显著高于对照组,且差异具有统计学意义(P0.05),25.5mM-组的P-AKT的表达量较对照组无明显变化(P0.05),其余各组的P-AKT表达量均较对照组显著降低(P0.05)；在第7d,15.5mM-组和25.5mM-组的P-AKT表达量较对照组无显著差异(P0.05),其余各组的P-AKT表达量均较对照组显著降低(P0.05)。硬组织切片观察结果显示,术后30天,A组：骨没有填满种植体与骨组织之间的空腔；B组：在种植体与骨组织之间的空腔中,填满了新形成的编织骨,且骨与种植直接接触,没有纤维组织细胞的干扰；C组：和B组相比较,新形成的编织骨更加紧致规则,种植体表面的骨开始重建并出现类骨质沉淀。术后60天,C组的种植体周围包绕着非常致密的骨组织,可见骨细胞沉积在板层骨上,新旧骨组织之间可观察到结合线；A组和B组的骨-种植体结合状况与C组类似。HE染色结果显示：术后7天：三组均未见明显的新生骨,并且有大量的炎性细胞浸润；术后14天,对照组和实验组均未见明显的新生骨,可见较多活跃的破骨细胞,C组的炎性细胞明显少于A组和B组,且C组的松质骨较其他两组更为致密。A组和B组的破骨细胞数目在14天后达到峰值,C组在第7天达到峰值,并随时间逐渐下降；但C组的平均破骨细胞数目较A组、B组没有显著差异,p0.05。免疫组化染色观察种植体周围整合素α5β1及纤维连接蛋白的表达结果显示,纤维连接蛋白及其受体主要在种植体周围骨组织中及破骨细胞表面表达,而整合素α5β1则主要在成骨细胞表面表达。纤维连接蛋白及整合素α5β1在C组中的表达要高于其他两组。整合素α5β1的光密度值表达显示,在整个实验过程中,C组种植体周围骨组织中整合素α5β1的表达要显著高于A组和B组(p=0.003)。在第7天,A组和B组的整合素α5β1表达量相同(p0.05),但是,在第14天,B组的整合素α5β1表达量要显著高于A组(p=0.027),第30天以后,A组和B组的差异没有统计学意义(p0.05)。纤维粘连蛋白的光密度值表达显示,在整个实验过程中,C组种植体周围骨组织中纤维粘连蛋白的光密度值表达显著高于A组和B组(p=0.012)。在第7天、14天、30天,A组和B组的纤维粘连蛋白没有统计学差异(p0.05),但是,在第60天开始,B组的纤维粘连蛋白表达量要显著高于A组(p=0.001)。结论：1.适当的高糖(如15.5mM)能促进MC3T3-E1细胞的增殖及成骨分化,过高的糖浓度(25.5 mM和35.5 mM)则起抑制作用；2.P13K/Akt信号通路在成骨细胞的增殖、成骨分化等生理过程中起到重要作用；3.血糖浓度得到稳定控制的糖尿病大鼠,较对照组更早的开始成骨代谢,其种植体周围能形成良好的骨结合；4.整合素α5 β1及纤维粘连蛋白在种植体-骨结合过程中起到重要调节作用,糖尿病得到良好控制的大鼠表现出更高的整合素α5β1及纤维粘连蛋白的表达。
[Abstract]:Background and purpose: oral implant is the most effective way to treat the patients with tooth deficiency. The "bone binding Osseointegration" concept proposed by B ranemark in 1971 has become the most important criterion for the success of the implant. A stable bone binding depends on the adhesion and proliferation of osteoblasts on the surface of the implant. This process is affected by a variety of local and systemic factors. Diabetes as a relative contraindication of dental implant therapy has attracted the attention of scholars. It is generally recognized as one of the most important factors affecting implant bone union. This study intends to observe in vitro high glucose on MC3T3-E1 cell proliferation, osteogenic differentiation, and analysis of PI3K/Akt signal pathway in the regulation mechanism; from in vivo experiments, through the model of diabetic rats, detect the blood glucose levels of rats bone around the implant and changes of integrin alpha 5 beta 1 and fibronectin (the expression level of FN), comprehensive evaluation of the impact of the mechanisms of diabetes mellitus combined with bone implant. Materials and methods: in vitro: the PI3K/AKT pathway in osteogenic differentiation of MC3T3-E1 cells under high glucose condition according to the different literature selected four kinds of radical concentrations of glucose: 5.5mmol/L, 15.5mmol/L, 25.5mmol/L, 35.5mmol/L, to simulate the normal physiological concentration of glucose in the body and in different degrees of high blood glucose concentration, and according to whether adding PI3K/AKT to LY294002 pathway inhibitors into eight groups: 1) 5.5mM glucose concentration group (5.5mM-); 2) 15.5mM sugar concentration group (15.5mM-); 3) 25.5mM sugar concentration group (25.5mM-); 4) 35.5mM sugar concentration group (35.5mM-); 5) 5.5mM sugar concentration +LY294002 group (5.5mM+); 6) +LY294002 group (1 5.5mM glucose concentration 15.5mM+); 7) 25.5mM sugar concentration +LY294002 group (25.5mM+); 8) 35.5mM sugar concentration +LY294002 group (35.5mM+); the 5.5mM sugar concentration group (5.5mM) as the control group. Methyl thiazolyl four triazole (MTT) method for the determination of 1D, 3D cell proliferation; alkaline phosphatase (ALP) detection of 3D activity, cell differentiation of 7D; alizarin red staining 7d, 14d cells secrete calcium nodules; Determination of fluorescence real-time quantitative PCR gene Run mRNA and OCN mRNA were expressed in 3D and 7d Western blot (Western; blot) for detection of 3D and phosphorylation of 7D protein kinase (P Akt) expression. In vivo experiment: the mechanism of osseointegration around implants in diabetic rats. A total of 33 healthy male ZDF (Zucker diabetic fatty) rats were randomly divided into 3 groups, 11 rats in each group, and 2 implants in each of the 3 month old rats. A group: control group, ZDF rats were injected with normal saline, direct implants (11 rats, 22 implants); group B: ZDF rats received exenatide release nanospheres treatment (subcutaneous injection, 0.74%, 0.1m]/100g), and at the same time implants (11 rats rats, 22 implants); group C: ZDF rats received exenatide release nanospheres treatment (subcutaneous injection, 0.74%, 0.1ml/100g), and serum glucose maintained at a constant level (less than 16mmol/L), and then implants (11 rats, 22 implants). Implant after surgery, in 7d, 14d, 30d, 60d rats were killed in batches, surgical removal of bilateral femur to net muscle, mucosa and soft tissue, then use bone implant drill cutting range cut near 3mm, resulting in a rectangular bone implant containing specimens (section 7d, the number of specimens of n = 4 / n, 14d, 30d, 60d, the number of specimens were n=6 / N). The bone metabolism status around the implants was observed by hard tissue slices and HE staining. The expression of integrin alpha 5 beta 1 and fibronectin around the implants were detected by immunohistochemistry. Results: MTT results showed that in the 24h, compared with the control group, 15.5mM- group and 25.5mM- absorbance values showed no significant change (PO.05); however, the absorbance of 35.5mM- group was significantly lower than the control group, the difference was statistically significant (P0.05); in the 72h, compared with the control group, the absorbance of 15.5mM- the value of the group increased significantly, the difference was statistically significant (P0.05); however, the absorbance of 25.5mM- and 35.5mM- group was significantly lower than the control group, and the difference was statistically significant (P0.05). In addition, at each time point, the absorbance using four groups of cells treated with LY294002 values were significantly decreased compared with the control group, the difference was statistically significant (P0.05) ALP experimental results show that, in the 3D, compared with the control group, 15.5mM- group, 25.5mM- group and 35.5mM- group absorbance values were not significant change, no statistically significant difference between the absorbance (P0.05), however, adding four groups of cells treated with LY294002 were significantly lower than the control group, the difference was statistically significant (P0.05); in the 7d, the 15.5mM- group showed the highest absorbance value, and compared with the control group with significant difference (P0.05), however. 25.5mM- group and mM- group of 35.5 absorbance values significantly decreased, the difference was statistically significant (P0.05). In addition, the absorbance values of the four groups treated by LY294002 were significantly lower than those of the control group (P0.05), and the absorbance values of the 15.5 mM+ group were higher than those of the 5.5 mM+ group, the 25.5 mM+ group and the 35.5 mM+ group, and the difference was statistically significant (P0.05). Alizarin red staining showed that in the 7d group, 15.5mM- integral absorbance value of calcium nodules was significantly higher than the control group and the experimental group, the difference was statistically significant (P0.05), at the same time, the integral absorbance of 25.5mM- group and 35.5mM- group of calcium nodules value is higher than that of the control group, and the difference had statistical differences (P0.05) in addition. The integral absorbance of calcium nodules, four groups of cells treated with LY294002 were significantly lower than the control group, the difference was statistically significant (P0.05); in the 14d, compared with the control group, 15.5mM- group of integral absorbance value to calcium nodules increased significantly, the difference was statistically significant (P0.05), however, the integral absorbance of 25.5mM- group 35.5mM- group and calcium nodules were compared with the control group had no significant difference (P0.05), also, the calcium nodules integral absorbance of four groups of cells treated with LY294002 were significantly lower than the control group, the difference has statistical significance Yi (P
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R587.2;R783

【共引文献】