力生长因子对牙周膜干细胞增殖分化的影响  

Effect of mechano⁃growth factor on proliferation and differentiation of periodontal ligament stem cells

作　　者：屠腾 刘艳丽 王惠 赵萤 张旻 TU Teng;LIU YanLi;WANG Hui;ZHAO Ying;ZHANG Min(State Key Laboratory of Military Stomatology,Nation-al Clinical Research Center for Oral Diseases,Shannxi International Joint Research Center for Oral Diseases,Depart-ment of General Dentistry and Emergency,School of Stomatology,the Fourth Military Medical University,Xi'an 710032,China;Department of Anesthesiology&Perioperative Medicine,Xi'an People's Hospital(Xi'an Fourth Hospital),People's Hospital Affiliated to Northwest University,Xi'an,Shaanxi 710100,China)

机构地区：[1]军事口腔医学国家重点实验室,国家口腔疾病临床医学研究中心陕西省口腔疾病国际联合研究中心,第四军医大学口腔医院急诊与综合临床科,陕西西安710032 [2]西北大学附属人民医院(西安市人民医院西安市第四医院),麻醉与围术期医学中心,陕西西安710100

出　　处：《口腔疾病防治》2023年第2期86-93,共8页Journal of Prevention and Treatment for Stomatological Diseases

基　　金：国家自然科学基金项目(31971248,81901052)。

摘　　要：目的探讨力生长因子(mechano⁃growth factor,MGF)对牙周膜干细胞增殖、分化的影响及其作用的分子机制。方法采用免疫磁珠法分选人牙周膜干细胞(periodontal ligament stem cells,PDLSCs),流式细胞仪检测表面标志物,并观察分选细胞的克隆形成能力及多向分化能力;CCK8法检测不同浓度MGF功能肽段(MGF⁃Ct24E肽)作用下PDLSCs的增殖活性;Western blot检测MGF⁃Ct24E肽作用下PDLSCs中的增殖细胞核抗原(pro⁃liferating cell nuclear antigen,PCNA)、碱性螺旋环螺旋转录因子Scleraxis、Ⅰ型胶原ɑ1(collagen type I alpha 1,COL1A1)、成骨细胞特异性转录因子Osterix、Yes相关蛋白(Yes⁃associated protein,YAP)、磷酸化YAP(phosphory⁃lation yes⁃associated protein,P⁃YAP)蛋白表达量;免疫荧光观察YAP的表达情况;siRNA干扰YAP表达后,观察PDLSCs中MGF⁃Ct24E作用下PCNA、Scleraxis和COL1A1的表达。结果免疫磁珠分选的PDLSCs高表达干细胞表面标志物CD29、CD90、CD105,低表达造血细胞表面标志物CD34、CD45;细胞具有较强的克隆形成能力,成骨诱导茜素红染色后可见红色钙结节,成脂诱导油红O染色后可见红色脂滴;50 ng/mL和100 ng/mL MGF⁃Ct24E肽作用24 h后,PDLSCs的增殖活性增强(P<0.05);细胞中PCNA、Scleraxis和COL1A1蛋白表达上调,而Osterix蛋白表达量下调(P<0.05);50 ng/mL MGF⁃Ct24E肽作用24 h后,YAP蛋白357位点磷酸化水平增强(P<0.05),免疫荧光染色观察发现,YAP蛋白在胞核聚集;MGF⁃Ct24E肽作用下,siRNA抑制YAP表达后,细胞中PCNA、Scler⁃axis蛋白表达量下调(P<0.05)。结论MGF通过激活YAP促进PDLSCs增殖及成纤维分化。Objective To investigate the effects and molecular mechanisms of mechano-growth factor(MGF) on the proliferation and differentiation of periodontal ligament stem cells(PDLSCs).Methods PDLSCs were obtained using magnetic bead sorting.Flow cytometry was performed to identify biomarkers.The clonogenicity and multidifferentiation potential of PDLSCs were identified by colony-forming unit,osteogenic and adipogenic differentiation assays.A CCK8 assay was used to detect the cell activity under different concentrations of mechano-growth factors(MGF-Ct24E).Western blot was used to detect the protein expression of proliferating cell nuclear antigen(PCNA),Scleraxis,collagen type Ⅰ alpha 1(COL1A1),Osterix,Yes-associated protein(YAP) and phosphorylation Yes-associated protein(P-YAP) in PDLSCs.YAP protein expression was observed by immunofluorescence.Knockdown of YAP expression by a siRNA,detected the expression of PCNA,Scleraxis and COL1A1 under MGF-Ct24E in PDLSCs.Results PDLSCs showed high expression of stem cell markers(CD29,CD90 and CD 105) and low expression of hematopoietic markers(CD34 and CD45).PDLSCs also have a strong ability to clone.Red calcium junctions were observed by Alizarin red staining,and red lipid droplets were observed by Oil red O staining.After treatment with 50 ng/mL and 100 ng/mL MGF-Ct24E for 24 h,the cell activity of periodontal ligament stem cells was significantly enhanced(P<0.05).The protein expression of PCNA,Scleraxis and COL1A1 was significantly upregulated,and the protein expression of Osterix was significantly downregulated(P<0.05).After treatment with 50 ng/mL mechano-growth factor for 24 h,the phosphorylation of YAP protein was significantly enhanced(P<0.05),and YAP protein was observed to accumulate in the nucleus by immunofluorescence.Following inhibition of YAP expression,PCNA and Scleraxis had significantly downregulated expression caused by MGFCt24E(P<0.05).Conclusion MGF promotes proliferation and fibrogenesis via upregulation of YAP activities.

关 键 词：力生长因子 牙周膜 牙周膜干细胞 牙周膜成纤维细胞 Yes相关蛋白 P⁃Yes相关蛋白 细胞增殖 成纤维分化 成骨分化 再生修复 

分 类 号：R78[医药卫生—口腔医学]