消瘀止痛合剂联合骨髓间充质干细胞对股骨颈骨折兔骨愈合的影响及机制  

Effect of mixture for resolving blood stasis and relieving pain combined with bone marrow mesenchymal stem cells promotes bone healing in rabbits with femoral neck fracture

作　　者：韦志豪 袁振中 胡耶芳[2] 陈炜坚 钟健[1,2] 郭诗雯 唐刚健 WEI Zhihao;YUAN Zhenzhong;HU Yefang;CHEN Weijian;ZHONG Jian;GUO Shiwen;TANG Gangjian(Guangxi University of Chinese Medicine,Nanning 530200,Guangxi,China;Guilin Hospital of Traditional Chinese Medicine,Guilin 541002,Guangxi,China)

机构地区：[1]广西中医药大学,广西南宁530200 [2]桂林市中医医院,广西桂林541002

出　　处：《现代中西医结合杂志》2023年第20期2781-2787,2797,共8页Modern Journal of Integrated Traditional Chinese and Western Medicine

基　　金：吴阶平医学基金临床科研专项资助项目(320.6750.2022-20-32);桂林市科学研究与技术开发计划项目(2020011208-1);广西中医药大学研究生教育创新计划项目(YCSY2022052)。

摘　　要：目的研究消瘀止痛合剂联合骨髓间充质干细胞(BMSCs)对股骨颈骨折兔骨愈合的影响,并基于转录组测序技术探讨其可能作用机制。方法取36只新西兰白兔建立股骨颈骨折模型,将造模成功白兔随机分为空白组、BMSCs组、消瘀止痛合剂组和消瘀止痛合剂+BMSCs组,每组9只。BMSCs组、消瘀止痛合剂+BMSCs组于造模后第1天在骨折端周围注射1 mL大白兔BMSCs悬液(BMSCs 5×10^(6)个/mL),其他组局部注射等剂量DMEM。于术后第2天开始,消瘀止痛合剂组及消瘀止痛合剂+BMSCs组给予消瘀止痛合剂4.2 mL/kg灌胃,空白组、BMSCs组给予等量生理盐水灌胃,均1次/d。连续干预4周后,ELISA法检测血清中碱性磷酸酶(ALP)及骨钙素(OCN)水平;处死白兔后取造模侧股骨,观察股骨组织变化,HE染色观察骨组织病理学形态,实时荧光定量PCR法检测股骨组织中ALP及OCN mRNA表达情况;转录组测序及生物信息学分析空白组与消瘀止痛合剂+BMSCs组差异表达基因情况。结果消瘀止痛合剂+BMSCs组血清ALP、OCN水平及骨组织中ALP、OCN mRNA相对表达量均明显高于空白组及BMSCs组(P均<0.05),消瘀止痛合剂组血清ALP、OCN水平及骨组织中ALP、OCN mRNA相对表达量均明显高于空白组(P均<0.05)。消瘀止痛合剂组及消瘀止痛合剂+BMSCs组骨折端及周围有大量骨痂生成,可见明显凸起,伴肥大增生;HE染色显示消瘀止痛合剂+BMSCs组骨小梁宽度、骨小梁密集程度及钙化程度均优于其余3组。转录组测序结果分析显示,消瘀止痛合剂联合BMSCs治疗股骨颈骨折主要涉及钙离子信号通路、Ras信号通路、破骨细胞分化、趋化因子信号通路、Toll样受体信号通路等,可能与调控Toll样受体4(TLR4)、TYRO蛋白酪氨酸激酶结合蛋白(TYROBP)、集落刺激因子-1受体(CSF1R)、基质金属蛋白酶-9(MMP-9)等基因有关。结论消瘀止痛合剂联合BMSCs可促进新西兰白兔股骨颈骨折愈合,其机制可能与�Objective It is to study the effect of mixture for resolving blood stasis and relieving pain(MRBSRP)combined with bone marrow mesenchymal stem cells(BMSCs)on bone healing in rabbits with femoral neck fracture,and to explore the possible mechanism of action based on transcriptome sequencing technique.Methods Thirty-six New Zealand white rabbits were taken to establish models of femoral neck fracture,and the successfully modeled white rabbits were randomly divided into blank group,BMSCs group,MRBSRP group,and MRBSRP+BMSCs group,with 9 rabbits in each group.The BMSCs group and MRBSRP+BMSCs group were injected with 1 mL of large rabbit BMSCs suspension(BMSCs 5×10^(6)/mL)around the fracture site on the first day after modeling,and the other groups were injected with an equal dose of DMEM locally.Starting from the second postoperative day,the MRBSRP group,and MRBSRP+BMSCs group were given MRBSRP 4.2 mL/kg by gavage,the blank group and BMSCs group were given an equal amount of normal saline by gavage,all once daily.After 4 weeks of continuous intervention,the serum levels of alkaline phosphatase(ALP)and osteocalcin(OCN)were detected by ELISA;the femur on the modeling side was taken from the white rabbits after execution to observe the changes in femoral tissues,and observe the pathologic morphology of bone histology by HE staining,the expressions of ALP and OCN mRNA in the femoral tissues were detected by real-time fluorescence PCR;the differentially expressed genes in the blank group and MRBSRP+BMSCs group were analyzed by transcriptome sequencing and bioinformatics.Results The serum levels of ALP and OCN and the relative expressions of ALP and OCN mRNA in bone tissues in the MRBSRP+BMSCs group were significantly higher than those in the blank group and BMSCs group(all P<0.05),the serum levels of ALP and OCN and the relative expressions of ALP and OCN mRNA in bone tissues in the MRBSRP group were significantly higher than those in the blank group(all P<0.05).There were a large number of callus formation at and around

关 键 词：股骨颈骨折 骨愈合 消瘀止痛合剂 骨髓间充质干细胞 转录组测序 

分 类 号：R-332[医药卫生]