犬上颌窦黏膜干细胞的培养和成骨性能的鉴定  

Culture and identification of the osteogenic properties of beagle maxillary sinus membrane stem cells

作　　者：张静 朱双喜[2] 彭伟[2] 李祥[2] 荣琼 陈松龄[2] ZHANG Jing;ZHU Shuangxi;PENG Wei;LI Xiang;RONG Qiong;CHEN Songling(Department of Stomatology,Cliford Hospital,Guangzhou University of Chinese Medicine,Guangzhou 511495,China;Department of Stomatology,First Affiliated Hospital,Sun Yat-sen University,Guangzhou 510080,China;Department of Stomatology,First People＇ s Hospital of Yunnan Province,Affiliated Hospital of Kunming University of Science and Technology,Kunming 650032,China)

机构地区：[1]广州中医药大学祈福医院口腔科,广东广州511495 [2]中山大学附属第一医院口腔科,广东广州510080 [3]云南省第一人民医院口腔科昆明理工大学附属医院口腔科,云南昆明650032

出　　处：《口腔疾病防治》2018年第7期422-427,共6页Journal of Prevention and Treatment for Stomatological Diseases

基　　金：国家自然科学基金(81371111);云南省科技厅-昆明医科大学应用基础研究联合专项(2017FE468-168)

摘　　要：目的探讨犬上颌窦黏膜干细胞的成骨性能。方法取健康Beagle犬上颌窦黏膜,免疫磁珠法分选CD146阳性细胞,培养犬上颌窦黏膜干细胞。流式细胞术检测一代细胞的表面抗原CD146和CD44、CD34。犬上颌窦黏膜干细胞经基础培养(基础培养组)和成骨诱导培养(成骨诱导组)后,应用Real-time PCR、免疫组化法检测2组成骨相关基因mRNA和蛋白的表达,应用ALP测试盒检测碱性磷酸酶(alkaline phosphatase,ALP)活性,应用茜素红染色、Von KOSSA染色观察成骨诱导后矿化结节的形成。结果成功培养犬上颌窦黏膜干细胞,流式细胞术检测显示CD146和CD44阳性、CD34阴性。成骨诱导组犬上颌窦黏膜干细胞Runt相关转录因子2(runt-related transcription factor 2,RUNX2)(t=14.44,P<0.001)、骨桥蛋白(osteopontin,OPN)(t=7.85,P=0.001)和ALP mRNA(t=14.27,P<0.001)表达量明显高于基础培养组,差异均有统计学意义;成骨诱导组RUNX2和OPN蛋白表达水平增强。犬上颌窦黏膜干细胞经成骨诱导后,与基础培养组相比,ALP活性增高,3 d(t=8.79,P<0.001)、7 d(t=9.75,P<0.001)、14 d(t=12.14,P<0.001)、21 d(t=19.62,P<0.001)、28 d(t=17.53,P<0.001)时,成骨诱导组明显高于基础培养组;犬上颌窦黏膜干细胞经成骨诱导后茜素红和Von KOSSA染色可见到明显的矿化结节。结论犬上颌窦黏膜干细胞具有成骨能力。Objective To investigate the osteogenic properties of maxillary sinus membrane stem cells（MSMSCs）.Methods Beagle maxillary sinus mucosa was collected, immunomagnetic bead method was applied for isolation of CD146~＋cells, and MSMSCs were harvested and cultured from the canine maxillary sinus floor mucosa. The levels of the cell surface antigens CD44, CD146, and CD34 were determined at passage one by flow cytometry. Cells at passage one were cultured in basal medium and osteogenic inductive medium. Real-time PCR, immunohistochemical staining, alkaline phosphatase activity, alizarin red staining and Von Kossa staining were used to investigate the osteogenic properties in vitro. Results The canine MSMSCs were cultured successfully. The results of flow cytometry were positive for CD146 and CD44 expression but negative for CD34 expression. The relative mRNA expression of runt-related transcription factor 2（RUNX2）（t = 14.44,P〈0.001）, osteopontin（OPN）（t = 7.85,P = 0.001） and alkaline phosphatase alkaline phosphatase（t = 14.27,P〈0.001） was apparently higher in the osteoinductive medium group than in the basal medium group, the differences in relative mRNA expression between the groups were significant. The protein levels of RUNX2 and OPN increased in the osteoinductive medium group. The alkaline phosphatase activity of the MSMSCs increased when the cells were cultured in osteoinductive medium; the activity increased to a level that was significantly higher than that in basal medium, particularly at days 3（t = 8.79, P〈0.001）, 7（t = 9.75,P〈0.001）, 14（t = 12.14,P〈0.001）, 21（t = 19.62,P〈0.001） and 28（t = 17.53,P〈0.001）. Obvious mineralized nodules were observed by alizarin red staining or Von Kossa staining. Conclusion Maxillary sinus membrane stem cells exhibit osteogenic ability.

关 键 词：上颌窦黏膜 干细胞 成骨 碱性磷酸酶 Runt相关转录因子2 骨桥蛋白 矿化结节 

分 类 号：R783.4[医药卫生—口腔医学]