| 通过体外细胞培养观察补体C3对骨形成影响的实验研究 |
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投稿时间：2020-06-20
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| 期刊信息：《中国骨伤》2022年，第35卷，第6期，第548-554页 |
| DOI：10.12200/j.issn.1003-0034.2022.06.009 |
| 基金项目:国家自然基金面上项目(编号:81973884);浙江省自然科学基金(编号:LY19H290004);浙江省中医药重点研究项目(编号:2022zz020);浙江省自然科学基金(编号:91873884) |
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中文摘要:
目的:探讨慢病毒介导补体C3沉默载体转染人B淋巴细胞Raji-成骨细胞系MG63共培养体系对共培养体系成骨能力的影响及其作用机制。
方法:构建慢病毒补体C3沉默载体,转染人B淋巴细胞Raji后建立体外Raji-成骨细胞系MG63共培养体系,分为空白对照组(不做特殊处理)、补体C3沉默组(慢病毒补体C3沉默载体转染共培养体系)与模型组(慢病毒空载载体转染共培养体系)。培养24 h后,采用反转录酶-聚合酶链锁反应(reverse transcription-polymerase chain reaction,RT-PCR)法及Western-Blot法检测各组补体C3表达水平;各组分别培养0、3、6、12、24、48、72 h后采用CCK-8检测各组成骨细胞系MG63增殖能力;培养24 h后分别采用流式细胞术检测各组成骨细胞系MG63的细胞凋亡情况,采用碱性磷酸酶(alkaline phosphatase,AKP)检测盒检测各组MG63细胞AKP活力,采用Western-Blot法检测各组成骨细胞系MG63的骨保护素(osteoprotegerin,OPG)蛋白表达水平。
结果:RT-PCR法检测结果显示补体C3沉默组C3表达水平低于空白对照组和模型组;Western-Blot法检测结果显示补体C3沉默组C3表达水平低于空白对照组和模型组;CCK-8检测结果提示培养3、6 h后补体C3沉默组MG63增殖能力与空白对照组、模型组比较差异无统计学意义;培养12 h后补体C3沉默组成骨细胞系MG63增殖能力高于空白对照组和模型组;培养24、48、72 h后补体C3沉默组成骨细胞系MG63增殖能力高于空白对照组和模型组;流式细胞术检测结果提示补体C3沉默组成骨细胞系MG63的细胞凋亡水平低于空白对照组和模型组;AKP检测盒检测结果提示补体C3沉默组AKP活力高于空白对照组和模型组;Western-Blot法检测结果提示补体C3沉默组OPG蛋白表达水平高于空白对照组和模型组。
结论:补体C3的沉默能够增强成骨细胞系MG63的骨形成能力,并且发挥这种能力需要时间的累积。补体C3可能通过改变OPG/RANKL/RANK轴来影响成骨能力。 |
| 【关键词】补体C3|骨生成|细胞培养技术|碱性磷酸酶 |
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| Experimental study on effect of complement C3 on bone formation observed by cell culture in vitro |
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ABSTRACT
Objective: To explore effect of lentivirus-mediated complement C3 silencing vector on the osteogenic ability of human B lymphocyte Raji-osteoblast cell line MG63 co-culture system and its mechanism.
Methods: The lentiviral complement C3 silencing vector was constructed and transfected into human B lymphocyte Raji to establish in vitro Raji-osteoblast cell line MG63 co-culture system. The cells were divided into blank control group (without special treatment),complement C3 silencing group (lentiviral complement C3 silencing vector transfection co-culture system) and model group(lentiviral vector transfection co-culture system). The expression of complement C3 in each group was detected by reverse transcription-polymerase chain reaction(RT-PCR) and Western-Blot at 24 h after culture,proliferation of MG63 cells was detected by CCK-8 at 0,3,6,12,24,48 and 72 h,apoptosis of MG63 cells in each group was detected by flow cytometry at 24 h after culture,and alkaline phosphatase(AKP) activity of MG63 cells in each group was detected by AKP detection kit,and osteoprotegerin (OPG) protein expression of MG63 cells in each group was detected by Western- Blot method.
Results: RT-PCR results showed that the expression level of C3 in complement C3 silencing group was lower than that in blank control group and model group,Western-Blot results showed that expression of C3 in complement C3 silencing group was lower than that in blank control group and model group,CCK-8 results showed that there was no difference in proliferation ability of MG63 among complement C3 silencing group and blank control group and model group at 3 and 6 h after culture;at 12 h after culture,proliferation ability of MG63 cells with C3 silencing was higher than that of blank control group and model group;at 24,48 and 72 h after culture,proliferation ability of MG63 cell line with complement C3 silencing group were higher than that of blank control group and model group. Flow cytometry resluts showed that apoptosis of proliferation ability of MG63 cell line with complement C3 silencing group was lower than that of blank control group and model group;AKP detection kit suggested that AKP activity in complement C3 silencing group was higher than that in blank control group and model group,Western-Blot results showed that expression level of OPG protein in complement C3 silencing groupwas higher than that in blank control groupand model group.
Conclusion: Silencing of complement C3 could enhance bone formation ability of osteoblast MG63,and it takes time to accumulate this ability. Complement C3 may affect osteogenesis by altering OPG/RANKL/RANK axis. |
| KEY WORDS Complement C3|Osteogenesis|Cell culture techniques|Alkaline phosphatase |
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| 引用本文，请按以下格式著录参考文献： |
| 中文格式: | 刘康,童铭豪,唐彬彬,曹延广,史晓林,余阳.通过体外细胞培养观察补体C3对骨形成影响的实验研究[J].中国骨伤,2022,35(6):548~554 |
| 英文格式: | LIU Kang,TONG Ming-hao,TANG Bin-bin,CAO Yan-guang,SHI Xiao-lin,YU Yang.Experimental study on effect of complement C3 on bone formation observed by cell culture in vitro[J].zhongguo gu shang / China J Orthop Trauma ,2022,35(6):548~554 |
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