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。
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-1 β（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 CDgo 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）. ③Eleetrophoresis results of bel-2DNA and BaxDNA：there was significant difference in band between group E and group A, B, C and group D respectively through eleetrophoresis patterns. ④Protein expression levels of bcl-2 and Bax： there was statistical difference in protein expression level of bel-2 among all the groups（ll 526.18 ±697.38,20096.88 ±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 respeetively（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.
出处
《中医正骨》
2013年第2期3-7,共5页
The Journal of Traditional Chinese Orthopedics and Traumatology
基金
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)
关键词
细胞凋亡
温补肾阳
骨髓间充质干细胞
Apoptosis
WARMING RECUPERATING KIDNEY
Bone marrow stem cell
