独正岗接骨膏对体外培养兔骨髓间充质干细胞成骨活性的影响
|
摘要
目的观察独正岗接骨膏对骨髓间充质干细胞(BMSCs)成骨活性的影响。方法分离、培养兔骨髓间充质干细胞,选取生长良好第3代BMSCs分组培养。依据培养基中加入影响因子的不同分为4组:对照组,不加任何影响因子;独正岗接骨膏组,分别加入三种不同浓度独正岗接骨膏水提液(5%、10%、20%);Chordin干预组,分别于三种不同浓度独正岗接骨膏水提液(5%、10%、20%)中均加入Chordin (100μg/L)进行干预;Noggin干预组,分别于三种不同浓度独正岗接骨膏水提液(5%、10%、20%)中加入Noggin(100μg/L)进行干预。各组分别于培养第4、7、10天进行指标检测。采用MTT法测定各组BMSCs细胞相对数量;采用磷酸苯二钠法测定各组细胞ALP活性;采用Real-time PCR法检测各组细胞中BMP-2,7,9基因的表达情况。结果加药干预4、7 d后,与对照组相比,各组细胞均有明显增殖趋势(P <0.05),其中各组内10%浓度细胞增殖趋势显著高于同期5%、20%浓度(P <0.05),20%浓度细胞增殖趋势高于同组5%浓度(P <0.05);加药干预10 d后,各组细胞增殖趋势低于对照组(P <0.05);加药干预4、7、10 d后,除对照组外,各组细胞ALP活性、BMP-2,7,9基因表达量均在7 d时达到最高,随后开始下降(P <0.05);各组间比较,在各时间段(4、7、10 d),独正岗接骨膏组ALP活性、BMP-2,7,9基因表达量均高于同期Chordin干预组和Noggin干预组(P <0.05);结论独正岗接骨膏可能通过促进BMP的早期表达,促进BMSCs向OB分化,达到促进骨折愈合的作用。
Objective To observe the eff ect of Duzhenggang Jiegu ointment on the osteogenic activity of bone marrow mesenchymal stem cells(BMSCs). Methods Bone marrow mesenchymal stem cells were isolated and cultured, and BMSCs of the 3 rd generation were selected for subculture. According to the difference of adding influencing factors to the medium, they were divided into four groups: 1) control group: no influencing factors were added. 2) In the Duzhenggang Jiegu ointment group, three diff erent concentrations of Duzhenggang Jiegu ointment solution(5%, 10%, 20%) were added respectively. 3) Chordin intervention group: Chordin(100 μg/L) was added to three diff erent concentrations of water extract of Duzhenggang Jiegu ointment(5%, 10%, 20%) for intervention. 4) In the noggin intervention group, noggin(100 nm g/L) was added to the aqueous extract of Duzhenggang Jiegu ointment at three diff erent concentrations(5%, 10%, 20%). The indexes were tested on the fourth, seventh and tenth day of culture. The relative number of BMSCs cells in each group was determined by MTT method. The activity of ALP in cells of each group was determined by phenyldisodium phosphate. The expression of BMP-2, 7, 9 in cells of each group was detected by real-time PCR. Statistical analysis of the differences in various indicators between groups. Results After dosing intervention for 4 and 7 days, cells in each group showed significant proliferation trend compared with the control group(P < 0.05). The proliferation trend of cells with 10% concentration in each group was significantly higher than that of 5% and 20% during the same period(P <0.05). The tendency of cell proliferation at 20% concentration was higher than that in the same group of 5%(P < 0.05). After 10 d of dosing intervention, the proliferation trend of cells in each group was lower than that of the control group(P < 0.05). After dosing intervention 4, 7 and 10 days, ALP activity, BMP 2, 7 and 9 gene expression in cells of each group reached the highest at 7 days, and then began to decline(P < 0.05). Comparison between the groups: at each time period(4, 7 and 10 d), the ALP activity, BMP 2, 7 and 9 gene expression level in the Duzhenggang Jiegu ointment group was higher than that in the Chordin intervention group and Noggin intervention group, and the diff erence was statistically significant(P < 0.05). Conclusion By promoting early expression of BMP, Duzhenggang Jiegu ointment may promote BMSCs' diff erentiation to OB and promote fracture healing.
Objective To observe the eff ect of Duzhenggang Jiegu ointment on the osteogenic activity of bone marrow mesenchymal stem cells(BMSCs). Methods Bone marrow mesenchymal stem cells were isolated and cultured, and BMSCs of the 3 rd generation were selected for subculture. According to the difference of adding influencing factors to the medium, they were divided into four groups: 1) control group: no influencing factors were added. 2) In the Duzhenggang Jiegu ointment group, three diff erent concentrations of Duzhenggang Jiegu ointment solution(5%, 10%, 20%) were added respectively. 3) Chordin intervention group: Chordin(100 μg/L) was added to three diff erent concentrations of water extract of Duzhenggang Jiegu ointment(5%, 10%, 20%) for intervention. 4) In the noggin intervention group, noggin(100 nm g/L) was added to the aqueous extract of Duzhenggang Jiegu ointment at three diff erent concentrations(5%, 10%, 20%). The indexes were tested on the fourth, seventh and tenth day of culture. The relative number of BMSCs cells in each group was determined by MTT method. The activity of ALP in cells of each group was determined by phenyldisodium phosphate. The expression of BMP-2, 7, 9 in cells of each group was detected by real-time PCR. Statistical analysis of the differences in various indicators between groups. Results After dosing intervention for 4 and 7 days, cells in each group showed significant proliferation trend compared with the control group(P < 0.05). The proliferation trend of cells with 10% concentration in each group was significantly higher than that of 5% and 20% during the same period(P <0.05). The tendency of cell proliferation at 20% concentration was higher than that in the same group of 5%(P < 0.05). After 10 d of dosing intervention, the proliferation trend of cells in each group was lower than that of the control group(P < 0.05). After dosing intervention 4, 7 and 10 days, ALP activity, BMP 2, 7 and 9 gene expression in cells of each group reached the highest at 7 days, and then began to decline(P < 0.05). Comparison between the groups: at each time period(4, 7 and 10 d), the ALP activity, BMP 2, 7 and 9 gene expression level in the Duzhenggang Jiegu ointment group was higher than that in the Chordin intervention group and Noggin intervention group, and the diff erence was statistically significant(P < 0.05). Conclusion By promoting early expression of BMP, Duzhenggang Jiegu ointment may promote BMSCs' diff erentiation to OB and promote fracture healing.
引文
[1]袁昌锦,付天明,刘敏,等.中草药接骨膏对新西兰大白兔创伤性骨折愈合作用的实验研究[J].四川中医, 2005,23(10):34-35.
[2]刘小琴.活血通络法在外伤性骨折与筋伤中的运用—土家族验方接骨膏接骨疗伤作用机理研究[D].恩施:湖北民族学院, 2010.
[3]方秀统. BMP-2和VEGF-165双基因共转染大鼠骨髓基质干细胞体内成骨的实验研究[D].长春:吉林大学, 2006.
[4]李安娜. BMP-Smad信号通路在成骨与破骨细胞生成中的作用[D].济南:山东大学, 2014.
[5]李丽莉. BMP-Smad信号通路的调节及其在成骨分化中的作用[D].济南:山东大学, 2013.
[6]高毅.淫羊藿苷对成骨细胞增殖分化的影响及机制探讨[D].石家庄:河北医科大学, 2013.
[7]吴荣华.碱性磷酸酶、C反应蛋白、降钙素原联合检测对于判断骨折术后感染的临床价值研究[J].中国医药指南,2013, 11(36):433-434.
[8]乌日汗,敖强.骨形态发生蛋白研究进展[J].内蒙古民族大学学报(自然科学版), 2011, 26(2):203-206.
[9]马雨楠,游颖,沈欢欢,等. Noggin基因沉默对BMP和Wnt信号通路表达的影响[J].中国实验动物学报, 2016,24(5):475-480.
[10]马雨楠.干预Noggin对BMP和Wnt信号通路作用的初步研究[D].北京:中国人民解放军军事医学科学院,2016.
[11]朱海燕,林正梅.骨形态发生蛋白信号通路的负向调节[J].国际口腔医学杂志, 2008(6):647-649, 653.
[12]梁源,常新.骨形态发生蛋白的信号传导通路及其拮抗因子[J].大连医科大学学报, 2011, 33(2):187-191.
[13]KANG Q, SUN MH, CHENG H, et al. Characterization of the distinct or thotopic Bone-forming activity of 14BMPs using recombinant adenovirus-mediated gene delivery[J].Gene Ther, 2004, 11(17):1312-1320.
[14]张思波,甚晖,杨久云,等. HPLC法测定土家药独正岗中齐墩果酸的含量[J].中国民族医药杂志, 2010,16(4):42-43.
[15]郑玲玲.土家药刺老苞根皮干预骨折愈合机制研究[D].北京:中央民族大学, 2012.
[16]刘忠英.中药化学成分在炮制、配伍和提取过程中的化学变化及其转运机制的研究[D].长春:吉林大学, 2005.
[2]刘小琴.活血通络法在外伤性骨折与筋伤中的运用—土家族验方接骨膏接骨疗伤作用机理研究[D].恩施:湖北民族学院, 2010.
[3]方秀统. BMP-2和VEGF-165双基因共转染大鼠骨髓基质干细胞体内成骨的实验研究[D].长春:吉林大学, 2006.
[4]李安娜. BMP-Smad信号通路在成骨与破骨细胞生成中的作用[D].济南:山东大学, 2014.
[5]李丽莉. BMP-Smad信号通路的调节及其在成骨分化中的作用[D].济南:山东大学, 2013.
[6]高毅.淫羊藿苷对成骨细胞增殖分化的影响及机制探讨[D].石家庄:河北医科大学, 2013.
[7]吴荣华.碱性磷酸酶、C反应蛋白、降钙素原联合检测对于判断骨折术后感染的临床价值研究[J].中国医药指南,2013, 11(36):433-434.
[8]乌日汗,敖强.骨形态发生蛋白研究进展[J].内蒙古民族大学学报(自然科学版), 2011, 26(2):203-206.
[9]马雨楠,游颖,沈欢欢,等. Noggin基因沉默对BMP和Wnt信号通路表达的影响[J].中国实验动物学报, 2016,24(5):475-480.
[10]马雨楠.干预Noggin对BMP和Wnt信号通路作用的初步研究[D].北京:中国人民解放军军事医学科学院,2016.
[11]朱海燕,林正梅.骨形态发生蛋白信号通路的负向调节[J].国际口腔医学杂志, 2008(6):647-649, 653.
[12]梁源,常新.骨形态发生蛋白的信号传导通路及其拮抗因子[J].大连医科大学学报, 2011, 33(2):187-191.
[13]KANG Q, SUN MH, CHENG H, et al. Characterization of the distinct or thotopic Bone-forming activity of 14BMPs using recombinant adenovirus-mediated gene delivery[J].Gene Ther, 2004, 11(17):1312-1320.
[14]张思波,甚晖,杨久云,等. HPLC法测定土家药独正岗中齐墩果酸的含量[J].中国民族医药杂志, 2010,16(4):42-43.
[15]郑玲玲.土家药刺老苞根皮干预骨折愈合机制研究[D].北京:中央民族大学, 2012.
[16]刘忠英.中药化学成分在炮制、配伍和提取过程中的化学变化及其转运机制的研究[D].长春:吉林大学, 2005.