根尖牙乳头干细胞外泌体联合成纤维细胞生长因子-2对人牙髓干细胞生长活性及牙源性分化的作用

Role of SCAP-derived exosomes combined with FGF-2 in growth activity and odontogenic differentiation of dental pulp stem cells

目的 分离根尖牙乳头干细胞(stem cells from apical papilla, SCAP)来源的外泌体(exosomes, EXO),探讨SCAP来源的EXO联合成纤维细胞生长因子-2(fibroblast growth factor-2, FGF-2)对人牙髓干细胞生长活性与牙源性分化的影响。方法 取正畸患者拔除的根尖孔未闭合的第三磨牙,收集根尖牙乳头,培养SCAP;采用流式细胞术检测SCAP表面标志物CD29、CD31、CD34、CD44、CD45、CD90抗体表达情况;采用茜素红染色观察SCAP成骨能力,油红O染色观察SCAP成脂能力。采用超速离心法提取SCAP来源EXO,应用透射电镜观察EXO形态,采用Western blot法检测EXO特异性标志蛋白CD9、CD63、CD81、TSG101阳性表达情况。取人牙髓干细胞分为SCAP-EXO组、FGF-2组、SCAP-EXO+FGF-2组、对照组,SCAP-EXO组加入10 mg/L SCAP来源EXO,FGF-2组加入50μg/L FGF-2,SCAP-EXO+FGF-2组加入10 mg/L SCAP来源EXO和50μg/L FGF-2,对照组正常培养不进行任何处理,培养14 d后,采用CCK-8法检测各组细胞增殖率,采用比色法测定各组细胞碱性磷酸酶活性,采用茜素红染色观察各组细胞成骨能力,采用Western blot法检测各组细胞成骨分化相关标志物DSPP、OPN、BSP、OCN蛋白相对表达量。结果 SCAP表面标志物CD29、CD44、CD90抗体均呈阳性表达,CD31、CD34、CD45抗体均呈阴性表达,茜素红染色的SCAP内可见明显的红色至暗红色沉淀,有钙结节形成;油红O染色的SCAP内可见明显红色脂滴染色团块,有成脂分化,SCAP细胞分离成功;透射电镜可见SCAP外泌物呈杯状或球形囊泡,直径为40~100 nm,粒径峰值为50 nm;EXO标志蛋白CD9、CD63、CD81和TSG101均表达阳性,SCAP-EXO分离成功。SCAP-EXO+FGF-2组[(138.14±12.46)%、13.44±1.23]、FGF-2组[(120.47±11.79)%、10.69±1.03]、SCAP-EXO组[(119.68±10.95)%、11.54±1.10]细胞增殖率、碱性磷酸酶活性均高于对照组[(100.00±9.64)%、7.36±0.58](P<0.05),SCAP-EXO+FGF-2组均高于FGF-2组、SCAP-EXO组(P<0.05)。茜素红染色的SCAP-EXO组、FGF-2组、SCAP-EXO+FGF-2组细胞内布满红色至暗红色结节块,颜色较深,数量较对照组增多;SCAP-EXO+FGF-2组内红色至暗红色结节块数量多于SCAP-EXO组、FGF-2组。SCAP-EXO组(0.45±0.04、0.74±0.06、0.28±0.02、0.41±0.03)、FGF-2组(0.47±0.04、1.17±0.10、0.30±0.03、0.45±0.04)、SCAP-EXO+FGF-2组(1.24±0.11、1.27±0.12、1.18±0.11、1.22±0.10)DSPP、OCN、OPN、BSP蛋白相对表达量均高于对照组(0.08±0.01、0.29±0.02、0.04±0.00、0.27±0.02)(P<0.05),SCAP-EXO+FGF-2组高于SCAP-EXO组、FGF-2组(P<0.05)。结论 SCAP-EXO联合FGF-2可增加人牙髓干细胞的生长活性,促进牙源性分化。

Objective To isolate stem cells from apical papilla(SCAP)-derived exosomes(EXO), and investigate the effect of SCAP-EXO combined with fibroblast growth factor-2(FGF-2) on the growth activity and odontogenic differentiation of human dental pulp stem cells. Methods The third molars with open apical foramen were extracted from orthodontic patients, the apical papillae were collected, and SCAP were cultured. The expressions of SCAP surface markers(CD29, CD31, CD34, CD44, CD45 and CD90 antibodies) were detected by flow cytometry. Alizarin red staining was used to observe the osteogenic ability of SCAP,and Oil Red O staining was used to observe the adipogenic potentials of SCAP.The SCAP-EXO were extracted by ultracentrifugation,the morphology of EXO was observed by transmission electron microscopy,and Western blot was used to detect the positive expressions of EXO specific marker proteins(CD9,CD63,CD81and TSG101).SCAP were divided into SCAP-EXO group(cultured with 10mg/L SCAP-EXO),FGF-2group(cultured with 50μg/L FGF-2),SCAP-EXO+FGF-2group(cultured with 10 mg/L SCAP-EXO and 50μg/L FGF-2)and control group(normally cultured without any treatment).After culture for 14d,the cell proliferation rate of each group was detected by CCK-8method,the alkaline phosphatase activity of the cells in each group was determined by colorimetric method,Alizarin red staining was used to detect the osteogenic ability,and Western blot was used to detect the relative expressions of DSPP,OPN,BSP and OCN proteins.Results CD29,CD44and CD90antibodies were all positively expressed,while CD31,CD34and CD45antibodies were all negatively expressed.The obvious red to dark red precipitates were seen in SCAP,and calcium nodules were formed.There were obvious red lipid droplet staining clumps in SCAP stained with Oil Red O,with adipogenic differentiation,and the SCAP cells were successfully separated.Transmission electron microscopy showed that the SCAP exudates were cup-shaped or spherical vesicles with a diameter of 40to 100nm and a peak size of 50nm.CD9,CD63,CD81and TSG101proteins were all positive,and SCAP-EXO were successfully isolated.The cell proliferation rate and alkaline phosphatase activity were higher in SCAP-EXO+FGF-2group[(138.14±12.46)%,13.44±1.23],FGF-2group[(120.47±11.79)%,10.69±1.03]and SCAP-EXO group[(119.68±10.95)%,11.54±1.10]than those in control group[(100.00±9.64)%,7.36±0.58](P<0.05),and higher in SCAP-EXO+FGF-2group than those in FGF-2group and SCAP-EXO group(P<0.05).The cells in SCAP-EXO group,FGF-2group,and CAP-EXO+FGF-2group were covered with red to dark red nodules,which were darker in color and increased in number compared with control group;the cells in SCAP-EXO+FGF-2group were red,and the number of dark red nodules was more than that in SCAP-EXO group and FGF-2group.The relative expressions of DSPP,OCN,OPN and BSP proteins were higher in SCAP-EXO group(0.45±0.04,0.74±0.06,0.28±0.02,0.41±0.03),FGF-2group(0.47±0.04,1.17±0.10,0.30±0.03,0.45±0.04)and SCAP-EXO+FGF-2group(1.24±0.11,1.27±0.12,1.18±0.11,1.22±0.10)than those in control group(0.08±0.01,0.29±0.02,0.04±0.00,0.27±0.02)(P<0.05),and higher in SCAP-EXO+FGF-2group than those in SCAP-EXO group and FGF-2group(P<0.05).Conclusion SCAP-EXO combined with FGF-2can significantly promote the growth and odontogenic differentiation of dental pulp stem cells.