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苏军,赵明兴,李芳,林乃锴,孙长英,鲁世金.多靶点沉默SOST基因修饰MC3T3-E1细胞注射对小鼠椎体微结构的影响[J].脊柱外科杂志,2022,20(5):327-333.
多靶点沉默SOST基因修饰MC3T3-E1细胞注射对小鼠椎体微结构的影响     点此下载全文 (Fulltext)
苏军1  赵明兴1  李芳1  林乃锴1  孙长英2  鲁世金3*
1. 杭州市余杭区第一人民医院骨科, 杭州 310000;
2. 长治医学院附属和平医院骨科, 长治 046000;
3. 广西中医药大学附属瑞康医院中西医结合转化医学中心, 南宁 530000
基金项目:山西省重点研发计划项目(201603D321075)
DOI:10.3969/j.issn.1672-2957.2022.05.008
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摘要:
      目的 利用多靶点SOST基因沉默技术修饰小鼠胚胎成骨细胞前体细胞MC3T3-E1,并将其注射到小鼠体内,观察其对小鼠椎体骨密度及微结构的影响。方法 取18只18月龄雌性C57BL/6小鼠在无菌条件下手术切除双侧卵巢制作骨质疏松模型,并分为3组(n=6):SOST基因沉默组注射采用多靶点SOST基因沉默技术修饰并同时转染CRISPR-sgRNA载体系统和SOST-RNAi修复模板载体假病毒颗粒的MC3T3-E1细胞;阴性组注射同时转染CRISPR-sgRNA载体系统和SOST-N修复模板载体假病毒颗粒的MC3T3-E1细胞;空白组注射不作任何处理的MC3T3-E1细胞。采用HE染色和Masson三色染色观察小鼠椎体骨小梁面积的变化,免疫组化染色观察骨组织中骨保护素(OPG)和RANKL表达的变化,实时荧光定量PCR检测观察各组细胞SOST及骨代谢相关细胞因子(RUNX2、β-catenin、RANKL、OPG)的表达变化。结果 L1 HE染色和L2 Masson三色染色结果显示,SOST基因沉默组骨小梁面积大于阴性组和空白组,差异均有统计学意义(P < 0.05)。L2免疫组织化学染色结果显示,SOST基因沉默组OPG表达量高于阴性组和空白组,RANKL表达量低于阴性组和空白组,差异均有统计学意义(P < 0.05)。L3实时荧光定量PCR检测结果显示,SOST基因沉默组SOST和RANKL表达量低于阴性组,RUNX2、β-catenin和OPG表达量高于阴性组,差异均有统计学意义(P < 0.05)。结论 多靶点沉默SOST基因修饰MC3T3-E1细胞尾静脉注射能够促进小鼠成骨细胞分化和分泌功能,抑制破骨细胞活性,有效改善小鼠椎体微结构。
关键词:脊柱  骨质疏松  成骨细胞  细胞分化  细胞增殖  小鼠
Effect of multi-target silencing of SOST gene modified MC3T3-E1 cell injection on bone microstructure of mouse vertebral bodies    Fulltext
Su Jun1  Zhao Mingxing1  Li Fang1  Lin Naikai1  Sun Changying2  Lu Shijin3*
1. Department of Orthopaedics, First People's Hospital of Yuhang District, Hangzhou 310000, Zhejiang, China;
2. Department of Orthopaedics, Heping Hospital, Changzhi Medical College, Changzhi 046000, Shanxi, China;
3. Department of Translational Medicine Center for Integrated Traditional Chinese and Western Medicine, Ruikang Hospital, Guangxi University of Traditional Chinese Medicine, Nanning 530000, Guangxi Zhuang Autonomous Region, China
Fund Project:
Abstract:
      Objective To observe the effect of injection of MC3T3-E1 cells modified by multi-target SOST gene silencing technique on bone mineral density and bone microstructure of mouse vertebral bodies.Methods Eighteen 18 month old female C57BL/6 mice received surgical removement of bilateral ovaries under sterile conditions to make an osteoporosis model,and were equally divided into 3 groups(n=6):the SOST gene silencing group was injected with MC3T3-E1 cells modified by multi-target SOST gene silencing technology and simultaneously transfected with CRISPR-sgRNA vector system and SOST RNAi repair template vector pseudovirus particles;the negative group was injected with MC3T3-E1 cells simultaneously transfected with CRISPR-sgRNA vector system and SOST-N repair template vector pseudovirus particles;the blank group was injected with MC3T3-E1 cells without any treatment.HE staining and Masson trichrome staining were used to observe the changes of trabecular bone area of mouse vertebrae;the immunohistochemical staining was used to observe the changes of osteoprotegerin(OPG) and RANKL expression in bone tissue;the real-time fluorescent quantitative PCR was used to detect and observe the expressions changes of the cell SOST and bone metabolism related cytokines(RUNX2,β-catenin,RANKL,OPG).Results The L1 HE staining and L2 Masson trichrome staining showed that the trabecular bone area of SOST gene silencing group was larger than that of negative and blank groups,and the differences were statistically significant(P < 0.05).The L2 immunohistochemical staining showed that the expression of OPG in SOST gene silencing group was higher than that in negative and blank groups,and the expression of RANKL was lower than that in negative and blank groups,and the differences were statistically significant(P < 0.05).The L3 real-time fluorescence quantitative PCR showed that the expressions of SOST and RANKL in SOST gene silencing group were lower than those in negative group,and the expressions of RUNX2,β-catenin and OPG were higher than those in negative group,and the differences were statistically significant(P < 0.05).Conclusion Tail vein injection of MC3T3-E1 cells modified by multi-target SOST gene silencing technique can promote the differentiation and secretion of mouse osteoblasts,inhibit the activity of osteoclasts,and effectively improve the bone microstructure of mouse vertebral bodies.
Keywords:Spine  Osteoporosis  Osteoblasts  Cell differentiation  Cell proliferation  Mice
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