ADAM28 shRNA干扰载体对人牙周膜干细胞增殖、分化和凋亡的影响  

The effects of ADAM28 shRNA interference vector on the proliferation,differentiation and apoptosis of human periodontal ligament stem cells

作　　者：赵征[1] 李杰[1] 邱海燕[1] ZHAO Zheng;LI Jie;QIU Haiyan(Department of Geriatric Stomatology,Qingdao Stomatological Hospital Affiliated to Qingdao University,China,266001)

机构地区：[1]青岛大学附属青岛市口腔医院老年口腔科,266001

出　　处：《实用口腔医学杂志》2024年第5期619-624,共6页Journal of Practical Stomatology

基　　金：青岛市医疗卫生重点学科建设项目资助(编号:2022-2024);青岛市口腔疾病临床医学研究中心(编号:22-3-7-lczx-7-nsh)。

摘　　要：目的:探讨金属蛋白酶解离素28(ADAM28)shRNA干扰载体对人牙周膜干细胞(HPDLSCs)增殖、分化和凋亡的影响及作用机制。方法:用ADAM28 shRNA干扰载体转染HPDLSCs 48 h后检测转染效率,CCK-8法检测细胞增殖,流式细胞术(FCM)检测细胞周期。酶动力学法检测碱性磷酸酶(ALP)活性,Western blot检测分化相关基因CAP、Pax9蛋白的表达。FCM测定HPDLSCs凋亡率,Western blot检测Bax、Bcl-2蛋白的表达差异。用SPSS 21.0软件包对数据进行统计学分析。结果:重组干扰载体PGPU6/GFP/Neo-ADAM28-shRNA可高效转染HPDLSCs,干扰载体组的细胞增殖活性显著下降,S期、G 2+M期的细胞比例和细胞增殖指数(PI=S+G 2M)明显降低,ALP值明显降低,CAP、Pax9蛋白的表达水平下降,凋亡率显著提高,Bcl-2、Bax蛋白的表达水平上调。结论:ADAM28 shRNA干扰载体抑制HPDLSCs的增殖、分化,诱导HPDLSCs凋亡。其机制可能是干扰载体抑制金属蛋白酶域和类解离素域的催化裂解和类EGF功能域的促细胞增殖作用,阻碍Notch信号传递并参与细胞凋亡负反馈调节机制。Objective:To investigate the effects of disintegrin metalloproteinase 28(ADAM28)shRNA interference vector on the proliferation,differentiation and apoptosis of human periodontal ligament stem cells(HPDLSCs)and the related mechanism.Methods:Transfection efficiency of ADAM28 shRNA interference vector was detected 48 h after transfected into HPDLSCs.Cell proliferation was detected by CCK-8 and cell cycle was detected by flow cytometry(FCM).The activity of alkaline phosphatase(ALP)was tested by enzyme kinetics,and the expression of CAP and Pax9 were tested by Western blot.FCM was used to determine the apoptosis rate of HPDLSCs,and Western blot was used to detect the expression of Bax and Bcl-2 proteins.Statistical significance was assessed by SPSS-Windows version 21.0 program.Results:The recombinant interference vector PGPU6/GFP/Neo-ADAM28-shRNA was efficiently transfected into HPDLSCs.In the interference vector group,the cell proliferation decreased,and the cell percentage of S phase,G 2+M phase and cell proliferation index(PI=S+G 2M)in cell cycle decreased,the ALP value decreased,and the expression levels of CAP and Pax9 proteins decreased;the apoptosis rate of HPDLSCs increased,and the expression levels of Bcl-2 and Bax proteins increased.Conclusion:ADAM28 shRNA interference vector can restrain the proliferation and differentiation of HPDLSCs,and induce HPDLSCs apoptosis.ADAM28 shRNA interference vector may inhibit the catalytic cracking of metalloproteinase domain and disintegrin-like domain,and inhibit the promoting cell proliferation of epidermal growth factor(EGF)-like domain,block Notch signal transmission and participate in the negative feedback regulation mechanism of apoptosis.

关 键 词：金属蛋白酶解离素28 shRNA干扰载体 人牙周膜干细胞 增殖 分化 凋亡 

分 类 号：R781.4[医药卫生—口腔医学]