两种斯赤列提取物促进成骨细胞的增殖与分化  

Proliferation and differentiation of osteoblasts promoted by the two extracts of Sambucus adnata Wall.

作　　者：郑梅梅 郑俭彬 江自鲜 贾朝玉 王涛[2] 王文静 Zheng Meimei;Zheng Jianbin;Jiang Zixian;Jia Zhaoyu;Wang Tao;Wang Wenjing(Yunnan University of Chinese Medicine,Kunming 650500,Yunnan Province,China;Department of Orthopedics,Kunming Municipal Hospital of Traditional Chinese Medicine,Kunming 650051,Yunnan Province,China)

机构地区：[1]云南中医药大学,云南省昆明市650500 [2]昆明市中医医院骨伤科,云南省昆明市650051

出　　处：《中国组织工程研究》2023年第11期1677-1682,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金资助项目(81660716),项目负责人:王文静;云南省科技计划项目[2017FF117(-021)],项目参与人:王文静。

摘　　要：背景:在骨稳态中,成骨细胞和破骨细胞通过释放多种调节因子在骨重塑中发挥重要作用,一旦两种细胞功能和活性的动态平衡被打破,则易引起骨质疏松等骨骼疾病。彝族人民常用斯赤列治疗跌打损伤、骨折创伤等,作者所在课题组前期研究发现斯赤列的水提取物(SAW-A)和二氯甲烷提取物(SAW-B)为其治疗骨折的活性部位,具有促进骨折愈合、改善骨质疏松的作用,但其活性部位对成骨细胞增殖、分化作用的影响尚不清楚。目的:探讨斯赤列改善骨质疏松的活性部位——水提取物(SAW-A)和二氯甲烷提取物(SAW-B)对成骨细胞增殖与分化的影响。方法:胶原酶消化法提取新生SD大鼠颅骨成骨细胞,CCK-8试剂盒检测SAW-A和SAW-B的细胞毒性;分别给予SAW-A(312.5 mg/L)和SAW-B(156.15 mg/L)干预大鼠第3代成骨细胞,于24,48,72 h用CCK-8试剂盒检测成骨细胞增殖活性;流式细胞仪检测SAW-A、SAW-B干预6 d后对细胞活性的影响,通过碱性磷酸酶染色、茜素红染色检测SAW-A、SAW-B干预后成骨细胞的增殖分化情况,实时荧光定量PCR检测成骨相关基因RUNX2、骨涎蛋白、骨钙蛋白和Ⅰ型胶原蛋白A1的相对表达量。结果与结论:①实验结果表明,SAW-A和SAW-B干预后48 h及72 h,均可明显提高成骨细胞的增殖能力,且SAW-B的作用要强于SAW-A;②流式细胞仪结果表明,SAW-A和SAW-B分别干预成骨细胞6 d后,二者均有抑制细胞凋亡的作用;③碱性磷酸酶和茜素红的染色结果显示,SAW-A和SAW-B均可促进成骨细胞成骨矿化;④SAW-A、SAW-B处理后,成骨细胞分化标记基因RUNX2、骨涎蛋白、骨钙蛋白和Ⅰ型胶原蛋白A1相对表达量均有上升,而且SAW-B促进骨涎蛋白和Ⅰ型胶原蛋白A1基因表达的作用比SAW-A强;⑤表明斯赤列提取物SAW-A和SAW-B均具有促进成骨细胞分化的作用。BACKGROUND:Osteoblasts and osteoclasts play an important role in bone remodeling through the release of regulatory factors to remain the bone homeostasis.When the balance between functions and activities of the two types of cells is disturbed,bone diseases such as osteoporosis and osteoarthritis will occur.Sombucus odnota Wall.is commonly used by the ethnic Yi people to treat injuries caused by falls and fractures.Our previous studies have found that the water extract(SAW-A) and dichlo romethane extract(SAW-B) of Sombucus odnata Wall.can promote fracture healing and improve osteoporosis in the active parts of fractures.However,their effects on osteoblast prolife ration and differentiation in the active parts are still unclear.OBJECTIVE:To investigate the biological effects of SAW-A and SAW-B extracts from Sambucus adnata Wall.on the proliferation and differentiation of osteoblasts.METHODS:Osteoblasts were extracted from the skull of newborn Sprague-Dawley rats by collagenase digestion,and cell counting kit-8 was used to detect the cytotoxicity of SAW-A and SAW-B.The proliferation activity of osteoblasts was detected by cell counting kit-8 at 24,48,and 72 hours after extra ct intervention,to investigate the effects of 312.5 mg/L SAW-A and 156.15 mg/L SAW-B on osteoblast proliferation.Flow cytometry was used to detect the effect of SAW-A and SAW-B on cell activity at 6 days of intervention.The proliferation and differentiation of osteoblasts after SAW-A and SAW-B intervention were detected by alkaline phosphatase staining and alizarin red staining.Real-time fluorescence quantitative PCR assay was performed to detect the relative expression of osteogenesis-related genes RUNX2,bone sialoprotein,osteocalcin and type Ⅰ collagen A1.RESULTS AND CONCLUSION:SAW-A and SAW-B could significantly improve the proliferation of osteoblasts at 48 and 72 hours after intervention,and SAW-B was stronger than SAW-A.Flow cytometry results showed that SAW-A and SAW-B inhibited apoptosis in osteoblasts after 6 days of intervention.The

关 键 词：斯赤列 水提取物 二氯甲烷提取物 成骨细胞 细胞增殖 细胞分化 骨稳态 骨质疏松 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学] R285