Bcl2促进UMR-106细胞BMP-2、OPG表达及补肾健脾活血方对其影响  

Bcl2 promotes expression of BMP-2 and OPG and the effect of nourishing kidney,invigorating spleen,and promoting blood flow recipe on the protein expression in UMR-106 cells

作　　者：王吉利 张志海[3,2] 黄宏兴[3,2] 万雷[3,2] 刘海全[3,2] 柴爽[1,2] 汪悦东 WANG Jili1,3 , ZHANG Zhihai2,3 , HUANG Hongxing2,3, WAN Lei2,3 , LIU Haiquan2,3 , CHAI Shuang1,3, WANG Yuedong1,3(1. The Third Clinical Medical School, Guangzhou University of Chinese Medicine, Guangzhou 510405,China 2. The Third Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine, Guangzhou 510200,China 3. The Lab of Orthopedics and Traumatology, Lingnan Chinese Medicine Research Center, Guangzhou University of Chinese Medicine, Guangzhou 510405 ,Chin)

机构地区：[1]广州中医药大学第三临床医学院,广东广州510405 [2]广州中医药大学岭南医学研究中心中医骨伤科学实验室,广东广州510405 [3]广州中医药大学附属骨伤科医院,广东广州510200

出　　处：《中国骨质疏松杂志》2018年第7期841-846,873,共7页Chinese Journal of Osteoporosis

基　　金：国家自然科学基金项目(81373653,81674004,81673786);广东省科技计划项目(2016ZC0096,2016A020216024);广东省自然科学基金项目(2015A030313367);广州市荔湾区科技计划项目(201704041)

摘　　要：目的基于构建Bcl2沉默和过表达腺病毒观察Bcl2对成骨细胞骨形态发生蛋白2(bone morphogenetic protein-2,BMP-2)、骨保护素(osteoprotegerin,OPG)蛋白的调节作用及补肾健脾活血方对其影响。方法将培养的UMR-106细胞分为空载腺病毒组(沉默Bcl2空载腺病毒NC-bcl2,过表达Bcl2空载腺病毒WT-bcl2)、Bcl2腺病毒组(沉默Bcl2腺病毒sh-bcl2,过表达Bcl2腺病毒Ad-bcl2),空载腺病毒+空白血清组(NC-bcl2,WT-bcl2)、空载腺病毒+补肾健脾活血方含药血清组(NC-bcl2+ME,WT-bcl2+ME)、腺病毒+空白血清组(sh-bcl2,Ad-bcl2)、腺病毒+补肾健脾活血方血清组(sh-bcl2+ME,Ad-bcl2+ME),用RT-q PCR检测Bcl2、OPG mRNA的变化、应用免疫印迹(Western Blot)检测Bcl2、OPG、BMP-2的变化。结果 (1)荧光倒置显微镜观察包装腺病毒转染UMR-106细胞;(2)在正常培养基中,与NC-bcl2比较,sh-bcl2可以降低Bcl2、OPG、BMP-2蛋白的表达(P均<0.05),与WT-bcl2比较,Ad-bcl2可以提高Bcl2、OPG、BMP-2蛋白的表达(P均<0.05);(3)与NC-bcl2比较,RTq PCR显示sh-bcl2可以降低Bcl2 mRNA的表达,但OPG mRNA无统计学意义;(4)在大鼠血清干预中,与空载腺病毒比较,补肾健脾活血方含药血清均可以增加BMP-2、OPG蛋白表达(P均<0.05);在sh-bcl2干预的细胞中,补肾健脾活血方含药血清提高BMP-2、OPG蛋白的表达(P均<0.05);在Ad-bcl2干预的细胞中,中药干预后,BMP-2、OPG未见明显变化。结论 Bcl2可以影响BMP-2、OPG蛋白的表达,补肾健脾活血方可能通过作用于Bcl2影响成骨细胞成骨相关蛋白的表达。Objective By the construction of the silencing and overexpression adenovirus of Bcl2,to obverse the effect Bcl2 on bone morphogenetic protein-2（BMP-2） and osteoprotegerin（OPG）,and the influence of nourishing kidney,invigorating spleen,and promoting blood flow recipe（Recipe）. Methods UMR-106 cells were divided into vector group（silencing vector Bcl2,NCbcl2,and overexpression vector Bcl2,WT-bcl2）,Bcl2 adenovirus group（silencing Bcl2,sh-bcl2,and overexpression Bcl2,Adbcl2）,vector adenovirus ＋ blank serum group（NC-bcl2 and WT-bcl2）,vector adenovirus ＋ Recipe serum group（NC-bcl2 ＋ ME and WT-bcl2 ＋ ME）,adenovirus ＋ blank serum group（sh-bcl2 and Ad-bcl2）,adenovirus ＋ Recipe serum group（sh-bcl2 ＋ ME and Ad-bcl2 ＋ ME）. Bcl2 and OPG mRNA were detected with RT-qPCR. The portion expressions of Bcl2,OPG,and BMP-2 weredetected with Western blotting. Results 1） Adenovirus fluorescent in UMR-106 cells was observed with fluorescent inverted microscope. 2） In normal medium,compared with NC-bcl2,sh-bcl2 reduced the protein expressions of Bcl2,OPG,and BMP-2（P 〈 0. 05）. Compared with WT-bcl2,the protein expressions of Bcl2,OPG,and BMP-2 were significantly improved in Ad-bcl2（P 〈 0. 05）. 3） Compared with NC-bcl2,the mRNA expression of bcl2 decreased in sh-bcl2. However,mRNA expression of OPG had no significance. 4） Compared with vector adenovirus in rat serum intervention,the protein expressions of BMP-2 and OPG increased in Recipe serum（P 〈 0. 05）. The protein expressions of BMP-2 and OPG increased in the Recipe serum and sh-bcl2 intervention（P 〈 0. 05）. The protein expressions of BMP-2 and OPG had no significance in Ad-bcl2 intervention. Conclusion Bcl2 could affect the protein expressions of BMP-2 and OPG,and Recipe may affect the osteoclast-related protein expressions through Bcl2.

关 键 词：补肾健脾活血方 UMR-106细胞 B淋巴细胞瘤-2 过表达/沉默 骨形态发生蛋白2 骨保护素 

分 类 号：R274[医药卫生—中医骨伤科学]