摘要
目的探讨超表达Notch胞内结构域(Notch intracellular domain,NICD)对人牙周膜干细胞(human periodontal ligament stem cells,hPDLSC)增殖和成骨分化的影响,为牙周病骨缺损的治疗提供依据。方法以第3代稳定超表达NICD的hPDLSC为实验组,以正常hPDLSC为阴性对照组,转染空载体的hPDLSC为空白对照组,通过细胞计数试剂盒(cell counting kit-8,CCK-8)检测超表达NICD对hPDLSC增殖能力的影响;通过茜素红染色和实时荧光定量PCR(real-time quantitative PCR,qPCR)检测超表达NICD对hPDLSC成骨相关基因:牙骨质附着蛋白(cementum attachment proteins,CAP)、骨钙蛋白、Runt相关转录因子2(Runt-related transcription factor2,RUNX2),Notch信号通路受体Notch1的影响;通过蛋白质免疫印迹法检测超表达NICD对hPDLSC成骨蛋白RUNX2和flag标记蛋白(用以标记NICD)的影响。结果CCK-8结果显示,3组1~2d的A值相比差异均无统计学意义(P>0.05);实验组3~7d的细胞数量(A值分别为0.203±0.016、0.364±0.014、0.449±0.020、0.549±0.020及0.570±0.020)与其他两组相比显著增加(P<0.05)。茜素红染色结果示,与空白对照组及阴性对照组相比,实验组染色的矿化结节形成范围小颜色浅;qPCR结果显示,实验组14及21dCAP基因表达量(0.751±0.058、0.887±0.025)、骨钙蛋白基因表达量(0.592±0.051、0.670±0.045)及RUNX2基因表达量(0.319±0.038、0.684±0.055),均较阴性对照组及空白对照组显著降低(P<0.05),但14及21d的Notch1表达水平(2.507±0.047、4.041±0.219)显著高于阴性对照组及空白对照组(P<0.05)。蛋白质印迹法结果显示,实验组14及21dflag标记蛋白表达量(0.167±0.007、0.204±0.010)均显著高于阴性对照组及空白对照组(P<0.05),但RUNX2的蛋白表达量(0.075±0.006、0.074±0.013)均显著低于阴性对照组(0.092±0.003、0.118±0.008)及空白对照组(0.174±0.006、0.212±0.008)(P<0.05)。结论超表达NICD可以促进hPDLSC的增殖能力,同时抑制其成骨分化。
Objective To investigate the effect of overexpression of Notch intracellular domain (NICD) on proliferation and osteogenic differentiation of human periodontal ligament stem cells (hPDLSC).Methods The third generation hPDLSC with stable overexpressing of NICD were assigned as experimental group,normal hPDLSC were as negative control group and hPDLSC transfected with empty vector were as blank control group.The effect of overexpressing NICD on proliferation ability of hPDLSC was detected by using cell counting kit-8 (CCK-8).Alizarin Red staining and real-time quantitative PCR (qPCR) were used to detect the effects of NICD on cementum attachment proteins (CAP),osteocalcin (OCN),Runt-related transcription factor 2 (RUNX2) and Notch signal pathway receptor Notchl.The effect of overexpressing NICD on hPDLSC osteogenic protein RUNX2 and flag marker protein (used to label NICD) were detected by using Western blotting.Results CCK-8 results showed that there were no significant differences in A values amongst the three groups for 1-2 days (P>0.05).The number of cells in the experimental group was significantly increase than that of the two control groups from the third to seventh days (A values were 0.203±0.016,0.364 ± 0.014,0.449 ± 0.020,0.549 ± 0.020 and 0.570±0.020,respectively) (P<0.05).Alizarin red staining showed that compared with the blank control group and negative control group,the mineralized nodules in the experimental group had smaller formation range and lighter color,and the differences were statistically significant (P<0.05).The expressions of CAP gene (0.751 ± 0.058,0.887 ± 0.025),osteocalcin gene (0.592±0.051,0.670±0.045) and RUNX2 gene (0.319±0.038,0.684±0.055) at 14 and 21 days in the experimental group were significantly lower than those in the negative control group respectively (P<0.05).However,the expression levels of Notchl gene at 14 and 21 days (2.507 ±0.047,4.041 ± 0.219) were significantly higher than those of negative and blank control groups (P<0.05).The results of Western blotting showed that the expressions of flag marker protein (0.167±0.007,0.204±0.010) at 14 and 21 days in the experimental group were significantly higher than those in the negative and blank control groups (P<0.05).However,the expressions of RUNX2 protein (0.075 ± 0.006,0.074 ± 0.013) at 14 and 21 days were significantly lower than that in the negative control group (0.092±0.003,0.118±0.008) and blank control group (0.174±0.006,0.212±0.008) (P<0.05).Conclusions Overexpression of NICD can promote the proliferation capacity of hPDLSC and inhibit its osteogenic differentiation.
作者
邱申彩
龙晏
陈晓燕
舍玉秀
吴佩玲
Qiu Shencai;Long Yan;Chen Xiaoyan;She Yuxiu;Wu Peiling(Department of Stomatology, The Second Affiliated Hospital of Xinjiang Medical Liniversity, Urumqi 830063, China)
出处
《中华口腔医学杂志》
CAS
CSCD
北大核心
2019年第5期315-321,共7页
Chinese Journal of Stomatology
基金
国家自然科学基金(81460103)。
关键词
细胞增殖
超表达
Notch胞内结构域
人牙周膜干细胞
成骨分化
Cell proliferation
Overexpression
Notch intracellular domain
Human periodontal ligament stem cells
Osteogenic differentiation
