补肾健脾活血方含药血清干预过表达、沉默Beclin-1基因的MC-3T3-E1细胞增殖及碱性磷酸酶活性  

Effects of Bushen Jianpi Huoxue Recipe on cell proliferation and alkaline phosphatase activity in Beclin-1 overexpressing and silencing MC-3T3-E1 cells

作　　者：郑维蓬 胡伟坚 赵国源 魏合伟 刘治军 万雷 陈胜 廖志浩 Zheng Weipeng;Hu Weijian;Zhao Guoyuan;Wei Hewei;Liu Zhijun;Wan Lei;Chen Sheng;Liao Zhihao(The Third Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510375,Guangdong Province,China;Guangzhou University of Chinese Medicine,Guangzhou 510006,Guangdong Province,China)

机构地区：[1]广州中医药大学第三附属医院,广东省广州市510375 [2]广州中医药大学,广东省广州市510006

出　　处：《中国组织工程研究》2021年第29期4650-4655,共6页Chinese Journal of Tissue Engineering Research

基　　金：广东省中医药局科研项目(20191178),项目负责人:郑维蓬;广东省中医药局科研项目(20203011),项目负责人:魏合伟。

摘　　要：背景:成骨细胞是参与骨代谢动态平衡精细调节的重要细胞之一,其功能异常在骨质疏松症发病中极其重要。研究发现自噬基因Beclin-1敲除导致成骨细胞分化和矿化能力下降,提示Beclin-1在骨代谢中具有重要调控作用。目的:补肾健脾活血方含药血清对过表达、沉默Beclin-1基因的MC-3T3-E1细胞增殖及碱性磷酸酶活性的影响。方法:构建Beclin-1过表达及沉默重组慢病毒载体,制备补肾健脾活血方含药血清。将MC-3T3-E1成骨细胞分成6组,分别为空白血清+空病毒组、空白血清+Beclin-1过表达组、空白血清+Beclin-1沉默组、含药血清+空病毒组、含药血清+Beclin-1过表达组、含药血清+Beclin-1沉默组;根据组别不同进行干预,CCK-8法检测细胞增殖情况,流式细胞仪检测细胞周期,碱性磷酸酶试剂盒检测碱性磷酸酶活性。结果与结论:①与空白血清+空病毒组比较,含药血清+空病毒组、空白血清+Beclin-1过表达组及含药血清+Beclin-1过表达组的细胞增殖能力和碱性磷酸酶活性均显著增强,空白血清+Beclin-1沉默组、含药血清+Beclin-1沉默组的细胞增殖能力和碱性磷酸酶活性均降低,其中含药血清+Beclin-1过表达组、空白血清+Beclin-1沉默组的变化最明显;②空白血清+Beclin-1过表达组和含药血清+Beclin-1过表达组处于G2/M期的细胞比例较高;空白血清+空病毒组、空白血清+Beclin-1沉默组和含药血清+Beclin-1沉默组处于G0/G1期的细胞比例较高;空白血清+Beclin-1过表达组和含药血清+空病毒组处于S期的细胞比例较高;③结果表明,过表达Beclin-1、补肾健脾活血方含药血清可提高MC-3T3-E1细胞增殖能力及碱性磷酸酶活性,尤其二者同时干预时作用最强,而沉默Beclin-1则降低MC-3T3-E1细胞增殖能力及碱性磷酸酶活性,补肾健脾方含药血清在某种程度上可抑制Beclin-1沉默的不良影响。BACKGROUND:Osteoblasts are one of the important cells involved in the fine regulation of the dynamic balance of bone metabolism,and their dysfunction is extremely essential for the pathogenesis of osteoporosis.Studies have found that knockout of Beclin-1,an autophagy gene,leads to a decrease in osteoblast differentiation and mineralization,suggesting that Beclin-1 has an important regulatory role in bone metabolism.OBJECTIVE:To explore the effects of Bushen Jianpi Huoxue Recipe on cell proliferation and alkaline phosphatase activity in Beclin-1 overexpressing and silencing MC-3T3-E1 cells METHODS:Recombinant lentiviral vectors overexpression and silencing Beclin-1 were constructed,and drug-containing serum of Bushen Jianpi Huoxue Recipe was prepared.MC-3T3-E1 cells were divided into six groups:blank serum+blank virus group,blank serum+Beclin-1 overexpression group,blank serum+Beclin-1 silence group,drug serum+blank virus group,drug serum+Beclin-1 overexpression group,AND drug serum+Beclin-1 silence group.Interventions in each group were accordingly carried out.Cell proliferation was detected using cell counting kit-8,cell cycle was determined using flow cytometry,and alkaline phosphatase activity was measured using an alkaline phosphatase kit.RESULTS AND CONCLUSION:Compared with the blank serum+blank virus group,the cell proliferation and alkaline phosphatase activity were significantly increased in the drug serum+blank virus group,blank serum+Beclin-1 overexpression group,and drug serum+Beclin-1 overexpression group,and significantly decreased in the blank serum+Beclin-1 silence group and drug serum+Beclin-1 silence group.The most obvious changes were found in the drug serum+Beclin-1 overexpression group and blank serum+Beclin-1 silence group.From the perspective of cell cycle in each group,the proportion of cells in G2/M phase was relatively higher in the blank serum+Beclin-1 overexpression group and drug serum+Beclin-1 overexpression group;the proportion of cells in G0/G1 phase was relatively higher in the bla

关 键 词：MC-3T3-E1细胞 慢病毒 转染 BECLIN-1 自噬 中药 碱性磷酸酶 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学]