Chinese Journal of Tissue Engineering Research ›› 2016, Vol. 20 ›› Issue (29): 4269-4276.doi: 10.3969/j.issn.2095-4344.2016.29.002
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Zhou Qiang1, Lu Hua1, Wang Zhan-chao2, Zhang Hao-jie2, Jiang Lei-sheng1
Received:
2016-04-24
Online:
2016-07-08
Published:
2016-07-08
Contact:
iang Lei-sheng, M.D., Chief physician, Doctoral supervisor, Department of Orthopedics, Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China
About author:
Zhou Qiang, Master, Attending physician, Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China
CLC Number:
Zhou Qiang, Lu Hua, Wang Zhan-chao, Zhang Hao-jie, Jiang Lei-sheng. Insulin effects on fracture healing and cytokines in the osteotylus in experimental diabetic rats[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(29): 4269-4276.
静脉注射5%四氧嘧啶,选取连续3 d血糖> 16.7 mmol/L的糖尿病造模成功大鼠进行实验。成功造模74只,建模成功率82.2%。将建模成功的大鼠分为2组,糖尿病组37只,胰岛素治疗组37只。 2.2 不同时期的血糖水平变化 分别于建模前和建模后的48 h以及第1,4,6,8周测量空腹血糖值,见表1。造模前,对照组、糖尿病组和胰岛素治疗组之间的基础血糖没有差异。大鼠注射四氧嘧啶48 h后,血糖浓度与对照组比较显著升高(P < 0.01),达到(28.71±2.20) mmol/L。骨折造模1周后,胰岛素治疗组血糖值明显低于糖尿病组,且糖尿病组相较于胰岛素治疗组和对照组,血糖值差异有显著性意义(P < 0.01)。结果表明,实验成功建立了糖尿病和胰岛素治疗组的大鼠骨折模型。 2.3 各组大鼠X射线片观察骨折愈合情况 见图1。骨折后第1周(图1A)骨折端骨折线清楚,骨膜增厚,对照组和胰岛素治疗组可见形成少量骨痂,然而糖尿病组中并未观察到明显的骨痂形成现象。骨折后第4周(图1B)对照组和胰岛素治疗组可以看到,骨折区域的X射线片透亮度显著下降,骨量较多,并能观察到骨小梁发生连接,而在糖尿病组观察到骨折区域的X射线片透亮度较高,骨量较少,仍能观察到清晰的骨折线。骨折后第8周(图1C)对照组和胰岛素治疗组可以看到,骨折区域的骨量进一步增加,观察到的骨折线模糊不清,而在糖尿病组的骨折区域可见大量的骨痂包裹,但相比对照组和胰岛素治疗组骨折线仍较清晰。 2.4 生物力学检测结果 将对照组、糖尿病组、胰岛素治疗组造模后的第4,6,8周大鼠取出完整胫骨干置于力学测试机的夹具上固定测定。破坏性三点弯曲力学测试结果见图2A,B,扭转强度测试结果见图2C,D。由计算机软件自动计算弯曲强度、扭转强度和每对标本的参数比率。结果显示,糖尿病组生物力学特征的数值相比对照组差异有显著性意义(P < 0.01),而胰岛素治疗后检测数值明显上升,差异有显著性意义(P < 0.01)。 2.5 免疫荧光检测相关细胞因子的表达 为了检测糖尿病和胰岛素干预治疗对糖尿病大鼠骨折愈合的影响,实验通过免疫荧光技术检测骨痂内多种常见细胞因子的表达情况。在第4周,分别选取对照组、糖尿病组、胰岛素治疗组中骨形态发生蛋白2、碱性成纤维细胞生长因子、转化生长因子、血管内皮生长因子的免疫荧光结果(图3)。骨形态发生蛋白2、碱性成纤维细胞生长因子、血管内皮生长因子呈绿色荧光的阳性表达,可以明显观察到对照组和胰岛素治疗组的荧光强度较高,糖尿病组中则较弱;转化生长因子呈红色荧光的阳性表达,同样可以观察到对照组和胰岛素治疗组的荧光强度较高,糖尿病组中则较弱。结果表明,糖尿病组中各细胞因子骨形态发生蛋白2、碱性成纤维细胞生长因子、转化生长因子、血管内皮生长因子相比于对照组免疫染色结果均下降,而糖尿病组进行胰岛素治疗后,骨形态发生蛋白2、碱性成纤维细胞生长因子、转化生长因子、血管内皮生长因子的免疫染色显著增强,且与对照组染色结果区别不大。 2.6 原位杂交检测相关细胞因子的表达 为了进一步观察3组大鼠骨折愈合过程中细胞因子的动态变化,实验运用原位杂交技术检测骨形态发生蛋白2、碱性成纤维细胞生长因子、转化生长因子、血管内皮生长因子在造模后1,2,3,4,6,8周的表达情况(图4A),并计算平均灰度值,代表各细胞因子的含量。对照组和胰岛素治疗组的骨形态发生蛋白2在第1周和第2周的阳性表达逐步升高,第2周达顶峰,第3,4,6,8周逐渐呈下降趋势,而糖尿病组骨形态发生蛋白2阳性表达第3周达顶峰,且阳性表达明显低于对照组(P < 0.01)(图4B)。对照组和胰岛素治疗组的碱性成纤维细胞生长因子表达自第1周至第3周逐步提高,呈现高阳性的持续表达,第3周达表达顶峰,在第4,6,8周表达逐渐降低,而糖尿病组碱性成纤维细胞生长因子阳性表达在第4周达顶峰,且阳性表达明显低于对照组(P < 0.01)(图4C)。对照组和胰岛素治疗组的转化生长因子阳性表达自第1周至第3周逐步升高,第3周达顶峰,第4,6,8周逐渐下降,而糖尿病组转化生长因子阳性表达在第4周达顶峰,且阳性表达明显低于对照组(P < 0.01)(图4D)。对照组和胰岛素治疗组的血管内皮生长因子表达第1至3周逐步升高,呈持续性的高阳性表达,在第3周时达顶峰,第4,6,8周逐渐下降,而糖尿病组血管内皮生长因子阳性表达在第4周达顶峰,且阳性表达明显低于对照组(P < 0.01)(图4E)。"
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