Wnt经典通路β-catenin过表达对人牙周膜干细胞增殖能力及成骨能力的影响

Effects of canonical Wnt/β-catenin over expression on proliferation and osteogenic differentiation of human periodontal ligament stem cells

目的:探讨Wnt经典通路β-连环蛋白(β-catenin)过表达对人牙周膜干细胞(periodontal ligament stem cells,PDLSCs)增殖能力及成骨能力的影响。方法:收集因治疗需要拔除的健康前磨牙和第三磨牙8颗,分离、培养PDLSCs。通过构建慢病毒过表达载体,转染β-catenin基因使实验组过表达β-catenin。转染后显微镜下观察常规培养及成骨诱导7d后细胞形态;MTT测定转染前后PDLSCs的增殖情况;实时定量PCR技术检测淋巴增强因子-1(LEF1)、T淋巴细胞因子-4(TCF4)、Runt相关转移因子-2(Runt-related transcription factor 2,Runx2)、碱性磷酸酶(alkaline phosphatase,ALP)、人Ⅰ型胶原蛋白-1(Colossians 1,COL-1)及P38丝裂原活化蛋白激酶(P38mitogen activated protein kinases,P38 MAPK)mRNA表达。成骨诱导7d后,提取RNA,检测成骨相关基因Runx2、ALP、COL-1、P38 MAPK mRNA表达。结果:转染后细胞表达绿色荧光(green fluorescent protein,GFP)。MTT结果示:β-catenin过表达组PDLSCs增殖能力较空转染组增强。β-catenin过表达组LEF1表达水平显著上升(P<0.05),TCF4表达水平无显著性差异(P>0.05);与空转染组相比β-catenin过表达组成骨诱导7d后Runx2、ALP、COL-1及P38表达水平显著降低(P<0.05)。结论:经典Wnt/β-catenin通路在PDLSCs增殖及成骨分化过程中发挥重要作用,核心蛋白β-catenin过表达可上调LEF1的表达促进PDLSCs的增殖能力,并可通过下调RUNX2、ALP、COL-1、P38的表达抑制其成骨能力。

Objective： To construct Wnt/ β-catenin gene overexpressed vector and to investigate the effects of its over expression on proliferation and osteogenic ability of human periodontal ligament stem cells. Methods： 8 healthy premolars and third molars extracted for treatment were isolated and cultured for human healthy individuals （PDLSCs）. Amplify the Wnt/β-catenin gene from PDLSCs, and cloned it into pLV-EGFP-Nvector, to construct Wnt/β-catenin overexpressed vector, then to transfect it into PDLSCs by lipofection method. Cell morphology was observed under fluorescent microscope after transfection of PDLSCs in conventional and osteogenic culture condition on the seventh day. We tested MTT results of PDLSCs. lymphoid enhancer factor-1 （LEF1）, T cell factor-4（LEF4）, Runt-related transfer factor-2（Runx2）, alkaline phosphatase （ALP）, human type I collagen （COL-l） and P38 mitogen activated protein kinase （P38 MAPK） were detected by real-time quantitative PCR. Results： GFP was expressed efficiently in PDLSCs after transfection. MTT results showed proliferation of PDLSCs after transfection was boosted specifically and efficiently than before.Real-time PCR results showed the expression of LEF1 mRNA was significantly increased than before, while there is no difference in TCF4. Simultaneously, Runx2, ALP, COL-1 and p38 were significantly decreased of PDLSCs transfected with Wnt/β-catenin overexpressed vector cultured in osteogenic differentiation condition. Conclusion： Classic Wnt/β-catenin pathway plays an important role in PDLSCs proliferation and osteogenic differentiation. Overexpression of the core protein Wnt/β-catenin can upregulate the expression of LEF1, promote the proliferation of PDLSCs, and inhibit the osteogenic ability of PDLSCs by down regulating the expression of RUNX2, ALP, COL-1 and P38.