| 应用影像导航精准注射小干扰RNA-Piezo1修饰骨髓间充质干细胞来源外泌体在骨关节炎动物模型中的修复研究 |
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Received:September 06, 2021
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| 作者 | Author | 单位 | Unit | E-Mail |
| 厉玲玲 |
LI Ling-ling |
浙江大学附属金华医院 金华市中心医院影像科, 浙江 金华 321000 |
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| 李晓飞 |
LI Xiao-fei |
浙江大学附属金华医院 金华市中心医院骨科, 浙江 金华 321000 |
Department of Orthopaedics, the Jinhua Hospital Affiliated to Zhejiang University, Jinhua 321000, Zhejiang, China |
professorlxf@163.com |
| 张建一 |
ZHANG Jian-yi |
青岛大学附属医院科教部中心实验室, 山东 青岛 266001 |
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| 周勇伟 |
ZHOU Yong-wei |
浙江大学附属金华医院 金华市中心医院骨科, 浙江 金华 321000 |
Department of Orthopaedics, the Jinhua Hospital Affiliated to Zhejiang University, Jinhua 321000, Zhejiang, China |
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| 杨骐宁 |
YANG Qi-ning |
浙江大学附属金华医院 金华市中心医院骨科, 浙江 金华 321000 |
Department of Orthopaedics, the Jinhua Hospital Affiliated to Zhejiang University, Jinhua 321000, Zhejiang, China |
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| 期刊信息：《中国骨伤》2021年34卷，第12期，第1171-1178页 |
| DOI：10.12200/j.issn.1003-0034.2021.12.015 |
| 基金项目:浙江省自然科学基金项目（编号：Q20H060058）；浙江省医药卫生科技项目（编号：2020KY343）；金华市公益类项目（编号：2019-4-002） |
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目的：探讨小干扰RNA （small interference RNA，siRNA） Piezo1沉默质粒转染骨髓间充质干细胞（bone marrow mesenchymal stem cells，BMSCs）来源外泌体对骨关节炎（osteoarthritis，OA）动物模型的作用。
方法：选取雄性SD大鼠SPF级20只，月龄5.46~6.96（6.21±0.75）个月，体重385.76~428.66（407.21±21.45） g，提取大鼠的BMSCs，利用siRNA技术构建Piezo1的siRNA沉默质粒，慢病毒转染BMSCs后，提取外泌体。在细胞层面，根据是否转染siRNA-Piezo1，将BMSCs分成空白质粒组和siRNA沉默质粒组，利用RT-PCR和Western blot法检测siRNA-Piezo1对BMSCs的成骨诱导能力。在动物模型层面，利用手术切除膝关节交叉韧带的方案制备OA动物模型，根据处理方案不同，将SD大鼠分为4组，空白对照组，模型组，BMSCs组和外泌体组。空白对照组大鼠不做处理；模型组大鼠切除交叉韧带后，构建OA动物模型；BMSCs组大鼠OA模型建成后，在CT引导下膝关节腔内注射BMSCs，细胞量为5×105个/ml，加载量2 ml，每周2次，持续4周；外泌体组大鼠OA模型建成后，加入siRNA-BMSCs来源的100 μg外泌体，每周2次，持续4周。采用苏木精-伊红（HE）染色和番红-固绿染色检测动物模型的软骨情况，并用改良Minkin评分和国际骨关节炎研究学会（Osteoarthritis Research Society International，OARSI）评分量化处理。反转录-聚合酶链反应（reverse transcription-polymerase chain reaction，RT-PCR）检测软骨组织中多聚蛋白多糖（Aggrecan）Ⅱ型胶原蛋白（CollagenⅡ）的mRNA相对表达水平。
结果：慢病毒转染效率为（92.11±4.22）%。RT-PCR结果显示，空白质粒组Piezo1 mRNA的相对表达量为1.07±0.06，与siRNA沉默质粒组0.31±0.01比较，差异有统计学意义（t=2.907，P<0.05）。HE染色和番红-固绿染色结果显示，空白对照组大鼠膝关节软骨结构存在，软骨面光滑。模型组大鼠膝关节结构已经完全破坏，BMSCs组大鼠膝关节软骨结构欠清晰，外泌体组大鼠膝关节软骨下骨成分存在。膝关节HE染色的改良Minkin评分结果和番红固绿染色的OARSI评分结果比较差异有统计学意义（F=15.903，19.005；P<0.05），外泌体组修复效果优于BMSCs组和模型组（P<0.05）。RT-PCR结果显示，BMSCs组中Aggrecan的mRNA相对表达量高于模型组（P<0.05），外泌体组中Aggrecan的mRNA相对表达量高于BMSCs组和模型组（P<0.05）。BMSCs组中CollagenⅡ的mRNA相对表达量高于模型组（P<0.05），外泌体组中CollagenⅡ的mRNA相对表达量高于BMSCs组和模型组（P<0.05）。
结论：Piezo1 siRNA沉默载体可以促进BMSCs向软骨细胞分化，并且可以有效抑制OA的进展，延缓OA的病情。 |
| [关键词]:骨关节炎,膝 慢病毒感染 外泌体 动物实验 |
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| Repair of osteoarthritis in animal model with exosomes derived from BMSCs transfected by the siRNA-Piezo1 through CT navigation |
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Abstract:
Objective: To investigate the effect of the exosomes from bone marrow mesenchymal stem cells(BMSCs) transfected with silence plasmid of Piezol small interference RNA(siRNA)on osteoarthritis(OA) animal model.
Methods: Twenty male SD rats with specific pathogen free (SPF) were selected,ranging in age from 5.46 to 6.96 months,with a mean of(6.21±0.75) months;and ranging in weight from 385.76 g to 428.66 g,with a mean of (407.21±21.45) g. BMSCs were extracted. The siRNA silencing plasmid of piezo1 was constructed by siRNA technology. After lentivirus was transfected into BMSCs,the exosomes were extracted. At the cellular level,BMSCs were divided into blank plasmid group and siRNA silencing plasmid group according to whether siRNA-Piezo1 was transfected or not. The osteogenic induction ability of siRNA-Piezo1 on BMSCs was detected by RT-PCR and Western blot. At the animal model level,the OA model was established by surgical resection of cruciate ligament of knee joint.According to different treatment schemes,SD rats were divided into 4 groups:blank control group,model group,BMSCs group and exosome group. SD rats in the blank control group were not treated. In the model group,the cruciate ligaments of rats were excised and OA animal model was established. In BMSCs group,BMSCs were injected into knee joint under CT guidance after OA model establishment,and the cell volume was 5×105/ml,loading amount of 2 ml,twice a week for 4 weeks. In the exosome group,100 μg exosomes from siRNA BMSCs were added twice a week for 4 weeks after OA model establishment. The cartilage of the animal model was detected by hematoxylin eosin (HE) staining and safranin solid green staining,and quantified by the modified Minkin score and the score of the international society for osteoarthritis research(OARSI). Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the relative mRNA expression level of aggrecan type II collagen in cartilage.
Results: The lentivirus transfection efficiency was(92.11±4.22)%. RT-PCR showed that the relative expression of Piezo1 mRNA in blank plasmid group was 1.07±0.06,which was significantly different from that of 0.31±0.01 in siRNA silencing plasmid group (t=2.907,P<0.05). The results of HE staining and safranine solid green staining showed that there was cartilage structure and smooth cartilage surface in the knee joint of SD rats in the blank control group. The knee joint structure in the model group had been completely destroyed,the knee joint cartilage structure in the BMSCs group was not clear,and there were subchondral bone components in the OA rats in the exosomes group. There was significant difference between the modified Minkin score of HE staining and the OARSI score of safranin solid green staining (F=15.903,19.005;P<0.05). Among them,the repair effect of exosome group was significantly better than that of BMSCs group and model group (P<0.05). RT-PCR results showed that the relative expression of aggrecan mRNA in BMSCs group was significantly higher than that in model group (P<0.05),and the relative expression of aggrecan mRNA in exosome group was higher than that in BMSCs group and model group (P<0.05). The relative expression of CollagenⅡmRNA in BMSCs group was higher than that in model group(P<0.05),and the relative expression of CollagenⅡmRNA in exosomes group was higher than that in BMSCs group and model group(P<0.05).
Conclusion: Piezo1 siRNA silencing vector can promote the differentiation of BMSCs into chondrocytes and effectively inhibit the progression of OA,so as to delay the disease of OA. |
| KEYWORDS:Osteoarthritis,knee Lentivirus infections Exosomes Animal experimentation |
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| 引用本文，请按以下格式著录参考文献： |
| 中文格式: | 厉玲玲,李晓飞,张建一,周勇伟,杨骐宁.应用影像导航精准注射小干扰RNA-Piezo1修饰骨髓间充质干细胞来源外泌体在骨关节炎动物模型中的修复研究[J].中国骨伤,2021,34(12):1171~1178 |
| 英文格式: | LI Ling-ling,LI Xiao-fei,ZHANG Jian-yi,ZHOU Yong-wei,YANG Qi-ning.Repair of osteoarthritis in animal model with exosomes derived from BMSCs transfected by the siRNA-Piezo1 through CT navigation[J].zhongguo gu shang / China J Orthop Trauma ,2021,34(12):1171~1178 |
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