[1]杨黎丽sup>,黄胜杰,李媚,等.温阳补肾药对抗骨髓间充质干细胞凋亡的实验研究[J].中医正骨,2013,25(02):3-7.
　HUANG Sheng-jie*,YANG Li-li,LI Mei,et al.Mechanism of action of warming recuperating kidney medicine against the apoptosis of bone marrow stem cells[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2013,25(02):3-7.
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温阳补肾药对抗骨髓间充质干细胞凋亡的实验研究()
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《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第25卷
期数:
2013年02期
页码:
3-7
栏目:
基础研究
出版日期:
2013-02-20

文章信息/Info

Title:
Mechanism of action of warming recuperating kidney medicine against the apoptosis of bone marrow stem cells
作者:
杨黎丽<sup>1; 黄胜杰2; 李媚1; 王和鸣1
1.福建中医药大学,福建 福州 350003;
2.马来西亚南方大学,柔佛 新山 81300
Author(s):
HUANG Sheng-jie*; YANG Li-li; LI Mei; WANG He-ming.
*Fujian University of Traditional Chinese Medicine,Fuzhou 350003,Fujian,China
关键词:
细胞凋亡 温补肾阳 骨髓间充质干细胞
Keywords:
Apoptosis;  WARMING RECUPERATING KIDNEY;  Bone marrow stem cell
摘要:
目的:探讨温阳补肾药对骨髓间充质干细胞凋亡的影响及作用机制。方法:建立白细胞介素1β诱导体外培养的SD大鼠骨髓间充质干细胞凋亡体系,分别向其中加入巴戟天含药血清(A组)、鹿角胶含药血清(B组)、淫羊藿含药血清(C组)、骨碎补含药血清(D组)和空白血清(F组),E组不加入血清。各组细胞培养48 h后,采用RT-PCR法检测bcl-2和Bax的mRNA表达量,采用电泳法检测骨髓间充质干细胞凋亡体系的建立,采用Western Blot法检测bcl-2和Bax的蛋白表达量。结果:①骨髓间充质干细胞表面标记结果。流式细胞仪检测结果显示CD34、CD45表达阴性,CD90表达阳性。②bcl-2和Bax的mRNA表达量。各组bcl-2mRNA表达量比较,差异有统计学意义(22.25±1.15,15.18±1.51,15.36±0.72,16.12±0.95,16.79±1.39,16.02±1.14; F=182.000,P=0.000)。A组bcl-2mRNA表达量高于B组、C组、D组、E组和F组(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000); E组高于B组(P=0.030)。各组BaxmRNA表达量比较,差异有统计学意义(19.17±0.67,15.37±1.02,18.18±0.23,17.37±0.72,28.36±0.93,23.52±1.26; F=28.422,P=0.000)。A组BaxmRNA表达量高于B组和D组(P=0.001,P=0.018),低于E组(P=0.001); B组低于C组、E组和F组(P=0.013,P=0.000,P=0.000),B组与D组比较,差异无统计学意义(P=0.052); D组低于F组(P=0.000); E组高于C组和F组(P=0.000,P=0.000)。③bcl-2DNA和BaxDNA电泳结果。电泳图谱显示,E组条带与A组、B组、C组、D组有明显差异。④bcl-2和Bax的蛋白表达量。各组bcl-2蛋白表达量比较,差异有统计学意义(11 526.18±697.38,20 096.88±953.73,16 784.88±504.36,14 856.65±546.90,8 549.29±313.63,9 004.79±399.12; F=341.740,P=0.000)。A组bcl-2蛋白表达量低于B组、C组和D组(P=0.000,P=0.000,P=0.000),高于E组和F组(P=0.000,P=0.000); B组高于C组、D组、E组和F组(P=0.000,P=0.000,P=0.000,P=0.000); C组高于D组、E组和F组(P=0.000,P=0.000,P=0.000); D组高于E组和F组(P=0.000,P=0.000)。各组Bax蛋白表达量比较,差异有统计学意义(12 435.09±1002.35,4 590.96±71.45,11 721.90±855.08,10 393.54±662.79,13 874.83±541.16,12 774.15±674.40; F=136.305,P=0.000)。A组Bax蛋白表达量高于B组和D组(P=0.000,P=0.039); B组低于C组、D组、E组和F组(P=0.000,P=0.000,P=0.000,P=0.000); C组低于E组(P=0.010); D组低于E组和F组(P=0.000,P=0.002)。结论:温阳补肾药含药血清有对抗白细胞介素1β诱导的骨髓间充质干细胞凋亡的作用,其机制主要是上调抑制凋亡基因Bcl-2的表达,下调促进凋亡基因Bax的表达,抑制其蛋白酶活性从而降低细胞凋亡率。
Abstract:
Objective:To explore the effect and mechanism of warming recuperating kidney medicine against the apoptosis of bone marrow stem cell(BMSC).Methods:The apoptosis system of SD rats BMSC cultured in vitro was established,and the apoptosis was induced by interleukin-1β(IL-1β),then the cells were respectively added with morinda root medicated serum(group A),deer-horn glue medicated serum(group B),epimedium herb medicated serum(group C),drynaria fortunei medicated serum(group D)and blank serum(group F),while group E without serum.After culturing 48 h for cells in each group,RT-PCR assay was used to detect mRNA expression levels of bcl-2 and Bax; electrophoresis method used to test the establishment of BMSC apoptosis system; and Western Blot method used to determine the protein expression levels of bcl-2 and Bax.Results:①The results of BMSC surface marker:the test results from flow cytometer were shown that CD34 and CD45 with negative expression,while CD90 with positive expression.②mRNA expression levels of bcl-2 and Bax:there was statistical difference in bcl-2mRNA expression level among all the groups(22.25±1.15,15.18±1.51,15.36±0.72,16.12±0.95,16.79±1.39,16.02±1.14; F=182.000,P=0.000).bcl-2mRNA expression level of group A was higher than that of group B,C,D,E and group F respectivrly(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000); group E was higher than group B(P=0.030).There was statistical difference in Bax mRNA expression level among all the groups(19.17±0.67,15.37±1.02,18.18±0.23,17.37±0.72,28.36±0.93,23.52±1.26; F=28.422,P=0.000).Bax mRNA expression level of group A was higher than that of group B and group D respectively(P=0.001,P=0.018),while lower than group E(P=0.001); group B was lower than group C,E and group F(P=0.013,P=0.000,P=0.000),while there was no statistical difference between group B and group D(P=0.052); group D was lower than group F(P=0.000); group E was higher than group C and group F respectively(P=0.000,P=0.000).③Electrophoresis results of bcl-2DNA and BaxDNA:there was significant difference in band between group E and group A,B,C and group D respectively through electrophoresis patterns.④Protein expression levels of bcl-2 and Bax:there was statistical difference in protein expression level of bcl-2 among all the groups(11 526.18±697.38,20 096.88±953.73,16 784.88±504.36,14 856.65±546.90,8 549.29±313.63,9 004.79±399.12; F=341.740,P=0.000).Protein expression level of bcl-2 of group A was lower than that of group B,C and group D respectively(P=0.000,P=0.000,P=0.000),while higher than that of group E and group F respectively(P=0.000,P=0.000); group B was higher than group C,D,E and group F respectively(P=0.000,P=0.000,P=0.000,P=0.000); group C was higher than group D,E and group F respectively(P=0.000,P=0.000,P=0.000); group D was higher than group E and group F respectively(P=0.000,P=0.000).There was statistical difference in protein expression level of Bax among all the groups(12 435.09±1 002.35,4 590.96±71.45,11 721.90±855.08,10 393.54±662.79,13 874.83±541.16,12 774.15±674.40; F=136.305,P=0.000).Protein expression level of Bax of group A was higher than that of group B and group D respectively(P=0.000,P=0.039); group B was lower than group C,D,E and group F respectively(P=0.000,P=0.000,P=0.000,P=0.000); group C was lower than group E(P=0.010); group D was lower than group E and group F respectively(P=0.000,P=0.002).Conclusion:The medicated serum of warming recuperating kidney medicine has the effect of inhibiting BMSC apoptosis induced by IL-1β,and its main mechanism is to inhibiting proteinase activity through up-regulation the expression of Bcl-2 while down-regulation the expression of Bax,then reduce the apoptosis rate.

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备注/Memo

备注/Memo:
基金项目:国家自然科学基金项目(30973763,81173283),教育部博士点基金-博导类项目(20093519110001)
通讯作者:王和鸣 E-mail:whm27@163.com
更新日期/Last Update: 2013-02-20