股密葆含药血清对成骨细胞分化成熟的影响及其作用机制

Effect of GUMIBAO medicated serum on osteoblastic differentiation and maturation and its mechanism of action

目的:探讨股密葆含药血清对成骨细胞分化成熟的影响及其作用机制。方法:将20只2月龄雌性SD大鼠随机分成4组,对照组8只,高、中、低浓度股密葆组各4只。对照组按10 mL·kg-1体质量以生理盐水灌胃,高浓度组按10 mL·kg-1体质量以2 430 mg·mL-1股密葆混悬液灌胃,中浓度组按10 mL·kg-1体质量以1 215 mg·mL-1股密葆混悬液灌胃,低浓度组按10mL·kg-1体质量以607.5 mg·mL-1股密葆混悬液灌胃。每天灌胃1次,连续3 d。末次灌胃后1 h提取各组实验大鼠含药血清,低温保存备用。将10只新生SD大鼠处死,取其颅骨,采用多次酶消化法分离出成骨细胞。将第1代成骨细胞分别在含有对照组大鼠血清及高、中、低浓度股密葆含药血清的培养基中培养1周后,采用染色法检测碱性磷酸酶表达情况,采用Western Blotting法检测Ⅰ型胶原、Runt相关转录因子2、β连环素及转录因子4的蛋白表达情况。结果:①成骨细胞中碱性磷酸酶的表达。与对照组比较,各浓度股密葆含药血清组碱性磷酸酶染色较深,其中中浓度组染色最深。②成骨细胞中Ⅰ型胶原蛋白的表达。对照组、高浓度组、中浓度组、低浓度组Ⅰ型胶原蛋白的表达量比较,差异有统计学意义[(1.172±0.047),(2.023±0.131),(2.792±0.737),(2.235±0.525),F=6.466,P=0.016]。组间两两比较,中浓度组、低浓度组高于对照组(P=0.003,P=0.022);高浓度组与对照组比较,差异无统计学意义(P=0.052);其余各组间比较,差异均无统计学意义(P>0.05)。③成骨细胞中Runt相关转录因子2蛋白的表达。各组Runt相关转录因子2蛋白的表达量比较,差异有统计学意义[(1.968±0.263),(2.255±0.286),(2.675±0.181),(2.652±0.211),F=6.066,P=0.019]。组间两两比较,中浓度组、低浓度组高于对照组(P=0.007,P=0.008);高浓度组与对照组比较,差异无统计学意义(P=0.180);其余各组间比较,差异均无统计学意义(P>0.05)。④成骨细胞中β连环蛋白的表达。各组β连环蛋白表达量比较,差异有统计学意义[(1.571±0.140),(2.264±0.265),(2.783±0.432),(2.860±0.576),F=6.980,P=0.013]。组间两两比较,中、低浓度组均高于对照组(P=0.005,P=0.004);高浓度组与对照组比较,差异无统计学意义(P=0.061);其余各组间比较,差异均无统计学意义(P>0.05)。⑤成骨细胞中转录因子4蛋白的表达。各组转录因子4蛋白表达量比较,差异有统计学意义[(3.268±0.298),(3.941±0.360),(4.446±0.443),(4.186±0.249),F=6.431,P=0.016]。组间两两比较,高、中、低浓度组均高于对照组(P=0.044,P=0.003,P=0.012);其余各组间比较,差异均无统计学意义(P>0.05)。结论:适当浓度股密葆含药血清可促进成骨细胞的分化,其作用机制或许与Wnt/β连环素信号通路的激活有关。

Objective ： To explore the effect of GUMIBAO medicated serum on osteoblastic differentiation and maturation and its mecha- nism of action. Methods：Twenty two-month-old female SD rats were randomly divided into 4 groups ,8 cases in control group and 4 cases in each of other groups. Rats in control group were intragastric administrated with normal saline（ l0 mL/kg） , while other rats were intragastric administrated with GUMIBAO suspension（ l0 mL/kg） with dose of 2 430 mg/mL（ high-concentration group）, 1 215 mg/mL（ middle-con- centration group） and 607.5 mg/mL（ low-concentration group） , once a day for consecutive 3 days. The medicated serum were extracted from rats one hour after the last intragastric administration and were reserved at low temperature. Then ten newborn SD rats were executed and the skulls were fetched out for separating osteoblasts by muhiple enzyme digestion. The expression of alkaline phosphatase（ALP） were detected by staining, and the protein expression of collagen type I , Runt-related transcription factor 2, beta catenin and transcription factor 4 were detected by Western Blotting after the first-generation osteoblasts were cultured in medicated serum for 1 week. Results：The ALP staining in the GUMIBAO medicated serum groups with different concentration were darker than that of control group,and the ALP staining was the darkest in middle concentration group compared to other groups. There were statistical differences in the expression of collagen type I be- tween the groups （ （ 1.172 ＋/- 0.047）, （2. 023 4/- O. 131 ）, （2. 792 -t/- O. 737 ）, （ 2. 235 4/- 0. 525 ）, F = 6. 466, P = 0. 016 ）. Further pairwise comparison showed that the expression of collagen type ~ in middle and low concentration group were higher than that of high con- centration group（P = 0. 003,P = 0. 022）. There were no statistical differences in the expression of collagen type I between high concentra- tion group and control group（ P = 0. 052） ,while there were no statistical differenees between rest groups （P 〉 0.05 ）. There were statistical differences in the protein expression of Runt-related transcription factor 2 between the groups （ （ 1. 968 4,/- 0. 263 ）, （2. 255 4/- 0. 286 ） , （2. 675 ＋/- 0. 181 ） , （2. 652 ＋/- 0.211 ） , F = 6. 066, P = 0. 019）. Further pairwise comparison showed that middle and low concentration group surpassed the control group（ P =0. 007 ,P = 0. 008 ） , and there were no statistical differences between high concentration group and control group （P = 0.180 ） , while there were no statistical difference between rest groups （P 〉 0.05 ）. There were statistical differences in the expression of beta catenin between the groups （ （ 1. 571 4/- 0. 140 ）, （ 2. 264 4/- 0. 265 ）, （ 2. 783 4/- 0. 432 ）, （ 2. 860 4/- 0. 576 ）, F = 6. 980,P = 0. 013 ）. Further pairwise comparison showed that the expression of beta catenin in middle and low concentration group were higher than that of control group （ P = 0. 005, P = 0. 004 ） , and there were no statistical differences between high concentration group and control group（ P = 0. 061 ）,while there were no statistieal differences between rest groups （P 〉 0.05 ）. There were statistical differences in the expression of transcription factor 4 between the groups（ （3.268 -4/-0.298）, （3.941 4/-0. 360）, （4.446 ＋/-0.443 ）, （4. 186 4/-0. 249） ,F = 6. 431 ,P =0.016）. Further pairwise comparison showed that the expression of transcription factor 4 in high, middle and low concentration group were higher than that of control group （ P = 0. 044, P = 0. 003, P -- 0. 012）, while there were no statistical differences between rest groups（P 〉0.05）. Conclusion： GUMIBAO medicated serum with suitable concentration can significantly promote osteoblastic differentia- tion and it may concerned the activation of Wnt/beta catenin signaling pathway.