大麻二酚对人牙周膜干细胞增殖和成骨分化的影响

Effect of cannabidiol on proliferation and osteogenic differentiation of human periodontal ligament stem cells

目的探究大麻二酚(CBD)对人牙周膜干细胞(hPDLSCs)增殖和成骨分化的影响。方法从健康牙周膜组织中分离培养hPDLSCs,通过流式细胞仪检测hPDLSCs表面标志物(STRO-1、CD-146、CD34和CD45)的表达。通过MTT试剂盒检测不同浓度的大麻二酚(0.1,0.5,1,2,5,10,20,40μmol/L CBD)培养24 h后hPDLSCs的活性。将hPDLSCs分为4组:对照组、0.5μmol/L大麻二酚组(0.5μmol/L组)、1μmol/L大麻二酚组(1μmol/L组)、10μmol/L大麻二酚组(10μmol/L组)。将hPDLSCs用成骨分化诱导培养基和各组对应浓度的大麻二酚联合培养21 d,然后检测各组hPDLSCs的碱性磷酸酶(ALP)活性,通过茜素红染色观察钙化结节,通过qRT-PCR和Western blot检测Collagen Ⅰ、Runt相关转录因子2(RUNX2)和骨钙素(OCN)的mRNA和蛋白表达。结果PDLSCs主要呈长梭形,hPDLSCs中STRO-1(98.58%)和CD146(99.20%)主要为阳性表达,CD34(0.36%)和CD45(0.31%)主要为阴性表达。与0μmol/L比,0.5,1,2μmol/L CBD处理hPDLSCs后相对细胞活力均升高(P<0.05),10,20,40μmol/L CBD处理hPDLSCs后相对细胞活力均降低(P<0.05)。与对照组相比,0.5μmol/L组和1μmol/L组hPDLSCs的ALP活性、钙化结节形成量、Collagen Ⅰ、RUNX2和OCN的mRNA和蛋白水平均显著升高(P<0.05)。与对照组相比,10μmol/L组hPDLSCs中ALP活性、钙化结节形成量、CollagenⅠ和RUNX2的mRNA和蛋白水平均显著降低(P<0.05)。结论适当浓度的大麻二酚可促进hPDLSCs的增殖和成骨分化。

Objective To reveal the effect of cannabidiol(CBD)on the proliferation and the osteogenic differentiation of human periodontal ligament stem cells(hPDLSCs).Methods The hPDLSCs were isolated and cultured from healthy periodontal ligament tissue,and the expression of hPDLSCs surface markers(STRO-1,CD-146,CD34 and CD45)was detected by flow cytometry.The activity of hPDLSCs was detected after cultured with different concentrations of cannabidiol(0.1,0.5,1,2,5,10,20,40μmol/L)for 24 h by MTT kit.The hPDLSCs were divided into four groups:control group,0.5μmol/L cannabidiol group(0.5μmol/L group),1μmol/L cannabidiol group(1μmol/L group),and 10μmol/L cannabidiol group(10μmol/L group).The hPDLSCs were co-cultured with osteogenic differentiation induction medium and corresponding concentration of cannabidiol for 21 d.Then the alkaline phosphatase(ALP)activity in hPDLSCs was detected.The calcified nodules were observed by Alizarin red staining.The mRNA and protein expression levels of Collagen Ⅰ,Runt-related transcription factor 2(RUNX2)and osteocalcin(OCN)were detected by qRT-PCR and Western blot.Results The hPDLSCs were mainly long fusiform.In hPDLSCs,STRO-1(98.58%)and CD146(99.20%)were mainly positive,and CD34(0.36%)and CD45(0.31%)were mainly negative.Compared with untreated hPDLSCs,the relative cell viability of hPDLSCs was increased in 0.5,1,2μmol/L groups(P<0.05),but the relative cell viability of hPDLSCs was decreased in 10,20,40μmol/L groups(P<0.05).The relative cell viability of hPDLSCs was not significantly different between 5μmol/L group and 0μmol/L group(P>0.05).Compared with control group,ALP activity,calcified nodule formation,mRNA and protein levels of Collagen Ⅰ,RUNX2 and OCN in hPDLSCs were significantly increased in 0.5μmol/L group and 1μmol/L group(P<0.05).Compared with control group,ALP activity,calcified nodule formation,mRNA and protein levels of Collagen Ⅰ and RUNX2 were significantly decreased in 10μmol/L group(P<0.05).Conclusion Appropriate concentration of cannabidiol can promote the proliferation and the osteogenic differentiation of hPDLSCs.