二膦酸盐对糖尿病大鼠下颌骨骨折愈合影响的实验研究

发布时间：2018-03-12 12:02

本文选题：二膦酸盐　切入点：糖尿病　出处：《河北联合大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的本实验通过建立大鼠Ⅰ型糖尿病下颌骨骨折模型，观察阿仑膦酸钠与胰岛素联合应用于Ⅰ型糖尿病大鼠下颌骨骨折愈合的过程，探讨阿仑膦酸钠在糖尿病干扰下对大鼠下颌骨骨折愈合的影响，为糖尿病颌骨骨折筛选临床用药提供依据及动物实验数据。方法选用6～8周龄，体重300±20gWistar大鼠96只，随机分为4组，空白对照组（A组），糖尿病骨折组（B组），糖尿病骨折+胰岛素组（C组），糖尿病骨折+胰岛素+阿仑膦酸钠组（D组），每组24只。适应性喂养两周后，应用链脲佐菌素（STZ）大剂量一次性腹腔注射，建立I型糖尿病动物模型。三天后，，测定随机血糖浓度，根据相关文献，血糖浓度16.7mmol/L大鼠，视为造模成功。恢复饲养3d后，10%水合氯醛全身麻醉下行大鼠左侧下颌骨骨折模型造模术。术后，除空白对照组及糖尿病骨折组外，其余各组给予胰岛素控糖。术后1d起糖尿病骨折+胰岛素+阿仑膦酸钠组给予35mg/kg/w阿仑膦酸钠灌胃，其余各组给予等量的生理盐水灌胃。术后在2w，4w，6w，8w时每组各处死大鼠6只，取材，应用ELISA方法检测血清中OPG及TRACP-5b含量，取出下颌骨行骨密度检测和X线片检查后，放入4％多聚甲醛固定48h，再用10％乙二胺四乙酸（EDTA）将骨组织脱钙60d。脱钙完成后，组织块石蜡包埋行HE染色组织形态学观察，OPG免疫组织化学检测及TRACP-5b特异性染色。结果1糖尿病造模成功后，大鼠出现典型的糖尿病“三多一少”症状；骨折模型建立后，伤口周围软组织红肿，无感染症状，实验过程中糖尿病大鼠的血糖水平较稳定，建立糖尿病大鼠骨折模型成功。2造模成功后，2w，4w，6w，8w各组间进行X线比较：2w时各组间X线片显示大鼠骨折线密度均较低；4w，6w，8w时空白对照组及糖尿病骨折+胰岛素+阿仑膦酸钠组骨折区域密度相对较高，骨折线模糊。骨密度比较：术后2w，4w，糖尿病骨折+胰岛素+阿仑膦酸钠组骨密度明显高于糖尿病骨折组及糖尿病骨折+胰岛素组，有统计学意义。组织形态学观察：糖尿病骨折+胰岛素+阿仑膦酸钠组在骨折2w，4w，6w时破骨细胞数目较少、骨小梁数量、骨小梁宽度、骨小梁长度等优于糖尿病骨折组。免疫组化检测：抗酒石酸酸性磷酸酶-5b在骨痂区2w，4w，6w时糖尿病骨折+胰岛素+阿仑膦酸钠组表达低于糖尿病骨折+胰岛素组及糖尿病骨折组有统计学意义（P0.05）。骨保护素：在骨痂区各组间均有表达，2w，4w，6w时糖尿病骨折+胰岛素+阿仑膦酸钠组表达均高于糖尿病骨折+胰岛素组及糖尿病骨折组，有统计学意义（P0.05），8w虽有表达，但其数值无统计学意义（P0.05）。结论1一次性大剂量腹腔注射STZ以及外科手术方法的应用可成功建立大鼠I型糖尿病下颌骨骨折模型。2糖尿病大鼠骨折愈合质量差，比较容易出现愈合延迟甚至不愈合。3阿仑膦酸钠联合胰岛素在糖尿病大鼠下颌骨骨折模型中可起到促进骨折愈合的作用，其可能机制为阿仑膦酸钠促进OPG的表达及抑制TRACP-5b的释放，抑制破骨细胞在糖尿病骨折愈合区域的过度增殖从而促进骨愈合。
[Abstract]:Objective to establish a type I diabetic rat mandibular fracture model through the experiment, the process of observation of the combined application of alendronate and insulin on mandibular fracture healing in diabetic rats, to investigate the alendronate effects on fracture healing in diabetic rat mandibular fracture disturbance, provide the basis for screening clinical and animal experiments the data for diabetic jaws.
Methods 6~8 week old, weight 300 + 96 20gWistar rats were randomly divided into 4 groups, control group (group A), diabetic group (group B), fracture fracture and diabetes + insulin group (C group), diabetic fracture + insulin + alendronate group (D group), each group 24. To two weeks after feeding, streptozotocin (STZ) intraperitoneal injection of high dose, the establishment of animal models of type I diabetes. Three days later, the determination of random blood glucose levels, according to the related literature, the blood glucose concentration in 16.7mmol/L rats, as a successful model. The recovery process after 3D, 10% chloral hydrate aldehyde under general anesthesia in rats model of left mandible fracture model. After the operation, in addition to the control group and diabetic fracture group, the rest groups were given insulin glucose control. Postoperative 1D diabetes fracture + insulin + alendronate group were given alendronate 35mg/kg/w intragastric administration of saline, other groups were given the amount of gastric lavage After operation in 2W, 4W, 6W, 8W in each group and 6 rats were detected by ELISA OPG in serum and the content of TRACP-5b, remove the mandible bone density testing and X-ray examination, fixed in 4% paraformaldehyde and 10% 48h EDTA (EDTA) will decalcified bone tissue 60d. after the completion of decalcified tissue, paraffin embedded for HE staining and histological observation, OPG immunohistochemistry and TRACP-5b staining.
Results 1 rats, rats showed typical diabetic symptoms; fracture model, soft tissue swelling around the wound, no symptoms of infection, diabetic rats during the experiment the blood glucose level is relatively stable, the establishment of diabetic rat fracture model of.2 after successful modeling, 2W, 4W. 6W, X-ray comparison: 8W group 2W group rats X-ray showed the fracture line density were low; 4W, 6W, 8W of blank control group and DM + fracture insulin + alendronate group fracture area density is relatively high, the fracture lines blurred. Bone mineral density: 2W 4W, diabetes, fracture + insulin + alendronate group bone density was significantly higher than that of diabetic group and diabetic fracture fracture + insulin group, was statistically significant. Histological observation: diabetes fracture + insulin + alendronate group in 2W 4W, 6W fracture, osteoclast The cell number and the number of trabecular bone, trabecular thickness, trabecular bone fracture length is better than that of diabetic group. Immunohistochemical detection: tartrate resistant acid phosphatase -5b in callus area 2W, 4W, 6W + + insulin diabetes fracture in the alendronate group was lower than that of diabetes fracture + insulin group diabetes and fracture group was statistically significant (P0.05). Osteoprotegerin expression in each group, the callus area between 2W, 4W, 6W + + insulin diabetes fracture in the alendronate group expression were higher in diabetic group and diabetic insulin + fracture fracture group, there was statistical significance (P0.05), 8W is the expression, but no statistically significant values (P0.05).
Conclusion the application of 1 single dose intraperitoneal injection of STZ and surgical methods can successfully establish rat mandibular fracture type I diabetes model of.2 diabetic rats poor quality of healing fractures, more prone to delayed healing or even nonunion.3 alendronate combined with insulin in the rat mandibular fracture diabetes can promote fracture healing in the model, the possible mechanism for the expression of alendronate promote OPG and inhibiting the release of TRACP-5b, and inhibit the proliferation of osteoclasts in bone fracture healing area of diabetes can promote bone healing.

【学位授予单位】：河北联合大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R782.4;R587.1

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