激素性股骨头坏死囊性变组织骨修复机制分析与活血通络胶囊含药血清对肥大软骨细胞成骨分化影响的实验研究  

Mechanism of bone repair in the cystic lesion tissues of steroid-induced osteonecrosis of the femoral head and the effects of Huoxue Tongluo Jiaonang(活血通络胶囊)medicated serum on the osteogenic differentiation of hypertrophic chondrocytes:an experimental study

作　　者：彭鹏 肖欢 方伟华 田佳庆 林锟 姚放鸣 肖方骏 何敏聪 何伟 魏秋实 PENG Peng;XIAO Huan;FANG Weihua;TIAN Jiaqing;LIN Kun;YAO Fangming;XIAO Fangjun;HE Mincong;HE Wei;WEI Qiushi(The Third Clinical Medical College of Guangzhou University of Chinese Medicine,Guangzhou 510006,Guangdong,China;Bijie Traditional Chinese Medicine Hospital,Bijie 551700,Guizhou,China;The Third Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510378,Guangdong,China)

机构地区：[1]广州中医药大学第三临床医学院,广东广州510006 [2]毕节市中医医院,贵州毕节551700 [3]广州中医药大学第三附属医院,广东广州510378

出　　处：《中医正骨》2024年第9期19-28,共10页The Journal of Traditional Chinese Orthopedics and Traumatology

基　　金：国家自然科学基金项目(82274544,82004392);广东省基础与应用基础研究基金项目(2023A1515010551);毕节市科学技术局2022年度“揭榜挂帅”项目(毕科合重大专项[2022]1号);广东省中医骨伤研究院开放课题重点项目(GYH202101-01,GYH202101-04)。

摘　　要：目的:分析激素性股骨头坏死(steroid-induced osteonecrosis of the femoral head,SONFH)囊性变组织的骨修复机制,并观察活血通络胶囊含药血清对肥大软骨细胞成骨分化的影响。方法:(1)SONFH囊性变组织的骨修复机制分析。纳入6例采用全髋关节置换术治疗的SONFH患者,术后收集坏死股骨头6个,其中3个股骨头用于囊性变组织的单细胞转录组测序,采用生物信息学方法分析囊性变组织中的细胞分类、软骨细胞的细胞亚群、肥大软骨细胞参与的生物过程;另外3个股骨头用于囊性变组织的病理学检查,分别采用苏木素-伊红染色、阿利新蓝染色、番红O-固绿染色观察囊性变组织中软骨细胞、肥大软骨细胞的分布与特征,采用免疫组织化学染色观察囊性变组织中肥大软骨细胞标志蛋白Ⅹ型胶原α1(collagen typeⅩα1,Col10α1)、基质金属蛋白酶(matrix metalloproteinase,MMP)13和成骨分化相关蛋白Runt相关转录因子2(Runt-related transcription factor 2,RUNX2)、骨桥蛋白(osteopontin,OPN)的表达情况。(2)活血通络胶囊含药血清对肥大软骨细胞成骨分化影响的分析。采用活血通络胶囊(活血通络胶囊粉末溶于蒸馏水)给大鼠灌胃,制备活血通络胶囊含药血清。取小鼠膝关节软骨,消化分离后进行培养。将小鼠软骨细胞接种于含有不同浓度的活血通络胶囊含药血清的完全培养基中,筛选活血通络胶囊含药血清的最佳干预浓度。取处于对数生长期的第1代小鼠软骨细胞,行肥大软骨细胞诱导成功后,分为成骨诱导组、5%含药血清干预组和对照组,成骨诱导组更换成骨诱导培养基培养,5%含药血清干预组更换含5%活血通络胶囊含药血清的成骨诱导培养基培养,对照组继续采用肥大诱导培养基培养;培养7 d后,分别采用碱性磷酸酶(alkaline phosphatase,ALP)染色和茜素红染色,观察细胞形态特征,分别采用实时定量PCR和蛋白质印迹法检测Ⅰ型Objective:To analyze the mechanism of bone repair in cystic lesion tissues of steroid-induced osteonecrosis of the femoral head(SONFH),and to observe the effects of Huoxue Tongluo Jiaonang(活血通络胶囊,HXTLJN)medicated serum on the osteogenic differentiation of hypertrophic chondrocytes(HCs).Methods:①Analyzing the mechanism of bone repair in SONFH-associated cystic lesion tissues.Six patients who underwent total hip arthroplasty for SONFH were enrolled in the study,and their necrotic femur heads were collected after the surgery.Among the 6 femur heads,3 ones were used for single-cell RNA sequencing(scRNA-seq)on cystic lesion tissues to analyze the cell categories,chondrocyte subsets,and biological processes involving in HCs in cystic lesion tissues by bioinformatics methods;while,the other 3 ones were used for pathological examination on the cystic lesion tissues to observe the distributions and characteristics of chondrocytes and HCs in cystic lesion tissues by using hematoxylin-eosin(HE)staining,Alcian blue staining,and safranin O-fast green(SO-FG)staining,respectively,and detect the expression of hypertrophic chondrocyte marker protein including collagen typeⅩα1(Col10α1)and matrix metalloproteinase(MMP)13,as well as the osteogenic differentiation-related protein including Runt-related transcription factor 2(RUNX2)and osteopontin(OPN)in the cystic lesion tissues by using immunohistochemical staining.②Analyzing the effects of HXTLJN medicated serum on the osteogenic differentiation of HCs.The rats were intervened by intragastric administration with HXTLJN(HXTLJN powders were dissolved into the distilled water)for making HXTLJN medicated serum.The knee articular cartilages were harvested from the sacrificed mice,and were digested and separated for chondrocytes,which were then cultured in DMEM/F-12.Furthermore,the chondrocytes were inoculated into complete medium containing different concentrations of HXTLJN medicated serum for determining the optimal intervention concentration of HXTLJN medicated se

关 键 词：股骨头坏死 糖皮质激素 囊性变 RNA测序 活血通络胶囊 软骨细胞 细胞分化 骨生成 

分 类 号：R274.9[医药卫生—中西医结合]