稳定性骨折愈合对软骨细胞增生和凋亡的影响

Influence of stable fracture healing on proliferation and apoptosis of chondrocytes

目的:骨折愈合过程中骨折断端间稳定性对软骨细胞增生、分化与凋亡的影响。方法:实验于2004-04/11在第三军医大学大坪医院野战外科研究所创伤实验室完成。选择8～10周的野生型小鼠48只,分成两组,每组24只。制作小鼠胫骨不稳定模型(不稳定骨折组)和稳定模型(稳定骨折组)。于3,5,7,10,14,21d取材,每组各时间点4只。用X射线片、苏木素-伊红染色、5-溴-2脱氧尿苷染色、原位杂交等手段检测软骨细胞在整体、组织、细胞水平中软骨细胞增生、分化与凋亡的差异。5-溴-2脱氧尿苷阳性细胞为棕色的细胞核,代表正在增生期的细胞;原位杂交阳性细胞为胞浆内蓝色的颗粒,Ⅱ型胶原探针为阳性表示增殖和分化的软骨细胞,Ⅹ型胶原探针为阳性表示肥大的软骨细胞。结果:48只小鼠均进入结果分析。①两组小鼠胫骨愈合X射线检测结果:骨折后第21天不稳定骨折组骨折断端间仍有较多骨痂,稳定骨折组已基本完成骨痂的重新塑性。②软骨细胞组织学观察:第21天苏木精-伊红染色可见不稳定骨折组骨折断端间软骨细胞正被成骨细胞替代,而稳定骨折组基本完成骨痂的重新塑性,与X射线结果相符合。③软骨细胞增生的检测结果:小鼠骨折后的5-溴-2脱氧尿苷染色第3,10,14天可见不稳定骨折组有大量软骨细胞增生,增生的成骨细胞并不多,而稳定骨折组增生的软骨细胞较少,被大量增生活跃的成骨细胞替代。④原位杂交检测软骨细胞标记物结果:两组骨痂中的软骨细胞在出现和消失的时间上没有明显的差别。结论:骨折断端间的稳定固定可以减慢软骨细胞的增生,加速其凋亡,但对软骨细胞的分化没有明显影响。

AIM:To investigate the influence of stability of fractured ends in the fractu re healing on the proliferation,differentiation and apoptosis of chondrocytes. METHODS:The experiment was conducted in the Laboratory for Traumatology,Resea rch Institute of Surgery,Daping Hospital,Third Military Medical University from April to November 2004.Forty- eight wild- type mice at the age from 8 to 10 we eks were equally divided into 2 groups.The mice were modeled on the tibia into u nstable fracture group and stable fracture group.On the 3rd,5th,7th,10th,14th an d 21st days,4 mice were chosen at each timepoint to dectect the differences of p roliferation,differentiation and apoptosis of chondrocytes in whole,tissue and c ellular level by using the methods such as X- ray,hematoxylin and eosin(HE) sta ining,5- bromodeoxyuridine(BrdU) and in situ hybridization.The positive cells d etected by BrdU manifesting brown nuclei were proliferating cells.When the posit ive cells detected by in situ hybridization manifested blue granules in cytoplas m,the positive type Ⅱ collagen probe represented proliferative and differentia ting chondrocytes,and the positive type X collagen probe represented hypertrophi c chondrocytes. RESULTS:All the 48 mice were included in the result analysis.① As shown in t he X- ray examination of tibial fracture healing in the two groups on the 21st day,there were still many calluses between the fractured ends in the unstable fr acture group,but nearly reconstructured calluses in the stable fracture group.② It was shown from the histological observation of chondrocytes by HE staining o n the 21st day that chondrocytes were substituted by osteoblasts between the fra ctured ends in the unstable fracture group,but nearly reconstructured calluses i n the stable fracture group,which were similar to those shown by X- ray examina tion.③ Shown by the detection of proliferative chondrocytes,chondrocytes could be found on the 3rd,10th and 14th days by BrdU in the fractured mice.In the unst able fracture group,there were a lot of proliferative chondrocytes,but a few ost eoblasts;However in the stable fracture group,there were a few proliferative cho ndrocytes,but a lot of active osteoblasts.④ The difference in the presence and disappearance of chondrocytes were unobvious between the two groups in the detec tion of chondrocyte markers by in situ hybridization. CONCLUSION:The stability of fracture healing can slow the proliferation of ch ondrocytes and accelerate the apoptosis of chondrocytes,but has no influence on the their differentiation.