【作者】 石磊；

【导师】 刘振东；

【作者基本信息】 中南大学 ， 临床医学， 2013， 硕士

【摘要】 目的：建立2型糖尿病大鼠动物骨折牵引模型,探讨骨痂中Wntβ-catenin、Wnt10b的表达变化与2型糖尿病骨折愈合障碍的关系。方法：将30只6周龄健康雄性SD大鼠,按随机数字表法分为对照组和实验组各15只,实验组喂以改良后的高糖高脂饲料,对照组喂以普通大鼠饲料。喂养8周后,实验组一次性腹腔注射小剂量STZ (30mg/kg),对照组腹腔注射等量枸橼酸盐缓冲液。以同上方法喂养2周后,选取空腹血糖高于16.7mmol/L者选入实验组继续实验,2组大鼠一同建立左侧胫骨骨折牵引成骨模型,术后第二天开始延长胫骨,持续至术后第十四天,以0.15mm/次,2次／天的方法持续延长胫骨,术后第15天停止延长收集血液标本后处死大鼠,无菌条件下收集左侧胫骨标本,迅速行左侧胫骨X线摄片检查。检测实验第8、10周及处死动物前血清标本中的三酰甘油(TG)、总胆固醇(TC)、胰岛素(FINs)、空腹血糖(FBG)。骨痂组织标本HE染色观察牵引间隙内骨痂生成情况,并进行组织学定量分析牵引间隙内骨痂新骨的形成面积以及骨髓腔内单位面积脂肪细胞的数量。将保存好的左胫骨骨痂标本分别做蛋白印迹法(Western-Blot)和实时荧光定量核酸扩增检测系统(Real-time Quantitative PCR Detecting System, QPCR)检测。结果：喂养8周后,实验组大鼠血清FINs、TG和TC较对照组升高(P<0.01), FBG则无明显差异(P>0.05)。实验10周及实验结束时,实验组大鼠TG、TC和FBG较对照组升高(P<0.01),而FINs则无明显差异(P>0.05)。X线摄片显示：两组大鼠胫骨牵引间隙内均有骨痂生成,但实验组骨折断端牵引间隙内骨痂生成较对照组明显减少。骨痂组织HE染色示：实验组较对照组的微骨柱(MCF)明显减少,且排列紊乱,微骨柱变短、变小；初始基质前沿(PMF)减少。而骨痂的组织学定量分析则显示：实验组骨痂内新骨的形成面积较对照组减少而髓腔内单位面积脂肪细胞数量增多(P<0.01)。W-B及QPCR检测则显示：实验组较对照组骨痂组织中Wntβ-catenin表达降低(P<0.05), Wnt10b表达降低(P<0.05)。结论：2型糖尿病大鼠骨折后愈合障碍,骨痂组织中Wntβ-catenin、Wnt10b表达降低,经典Wnt信号通路受到抑制,可能是造成2型糖尿病骨折愈合障碍的原因之一。更多还原

【Abstract】 Objective:To establish fracture traction model of type2diabet es rats and to discuss the relationship between expression changing of Wntβ-catenin, Wnt10b in the callus and diabetes fracture healing disorder.Methods:To divide30healthy SD rats with six weeks age into2groups with the same numbers by randomly,one group is name d experiment, the other is control group.The rats in experient group will be feed with high amount of sugar and fat,while the rats in c ontrol group will be raised with common feed. After eight weeks,ea ch rat will be evoked by abdominal cavity Injection with STZ(30m g/kg) in experiment group, while the rats will be injected by citrate with the same quantity and same way in control group. Two more weeks later, to select SD rats whose blood glucose is higher than16.7mmol/L in empty stomach state, then continue to carry on the following experiment and establish left side tibia fracture traction m odel in each group at the same time. Then to lengthen the left tib ia from the second day to the14th day after the operation with0.3mm/day and twice time per day. It stops to lengthen at the15th day and kill all the rats after collecting the serum sample, to colle ct left tibia under aseptic conditions and examine X-ray radiography immediatedly.To test TG, TC, FINs, FBG in serum samples at8th week,10th week and before killing. Observing callus generation in HE staining and to histologically quantitative analize forming area of new bone in callus and the number of fat cells per unit in the bone marrow cavity. To do Western blot and Real-time fluorescence Quantitative detection System for left tibia callus sample. Results.-After raising eight weeks,serum FINS, TG and TC in experirental g roup increased(P<0.01) comparising to the control group, FBG with no obvious difference(P>0.05);After raising10weeks and the over time of experient, TG, TC and FBG in experiental group increased (P<0.01) comparising to the control group, while FINs with no ob vious difference (P>0.05); X-ray radiography showed that the form ing callus in traction space of experiental groud obviously decreased comparising to the control group.The callus tissue HE staining sho wed that the MCF of experiental group obviously decreased compar ising to the control group with disordered arrangement, shorter and smaller.And the PMF decreased. While histological quantitative anal ysis to callus showed that forming size of new bone in callus in e xperiental group decreased comparising to the control group while t he number of fat cells per unit in the bone marrow cavity increase d (P<0.01). W-B and QPCR detection showed that Wntβ-catenin ex pression in callus decreased in experiental group(P<0.05) comparisin g to the control group and the same with Wnt10b(P<0.05).Conclusion:Type2diabetes rats healing disorder after fracture, the decrease of Wntβ-catenin, Wnt10b expression in callus and cla ssic Wnt signaling pathway has been restricted which may be one of the reasons causing type2diabetes fracture healing disorder.更多还原