龟鹿二仙胶治疗骨质疏松症的机制研究

Study on the mechanism of guiluerxianjiao treatment of osteoporosis

目的:研究龟鹿二仙胶对成骨细胞I型胶原及TGF-β表达的影响,探索龟鹿二仙胶治疗骨质疏松症的机制。方法:SD大鼠随机分为对照组、龟鹿二仙胶低剂量组、中剂量组、高剂量组、骨化三醇组,每天灌胃1次,持续7 d,末次给药2 h后腹主动脉采血,制成含药血清作用于大鼠成骨样细胞ROS17_2.8株。用MTT法及活细胞形态观察含药血清对成骨细胞增殖及凋亡的影响。用各组含药血清条件培养基培养成骨细胞,应用Western Blot法检测各组成骨细胞I型胶原及TGF-β蛋白表达水平,采用RT-PCR法检测各组成骨细胞I型胶原及TGF-βmRNA的表达。结果:(1)MTT结果显示:龟鹿二仙胶各组的成骨细胞无明显增殖。骨化三醇组的成骨细胞有明显增值(P<0.05)。细胞染色提示:骨化三醇组及龟鹿二仙胶组的成骨细胞无显著的凋亡。(2)RTPCR法结果显示:骨化三醇组与龟鹿二仙胶各组COL1A1及COL1A2mRNA的水平均有增加(P<0.01);龟鹿二仙胶中、高剂量组COL1A1及COL1A2mRNA水平增加较明显(P<0.05),龟鹿二仙胶高剂量组较中剂量组COL1A1及COL1A2mRNA水平增加不明显(P>0.05);骨化三醇组TGF-β1及TGF-β2mRNA水平无明显变化(P>0.05);龟鹿二仙胶各组TGF-β1及TGF-β2mRNA水平均有增加(P<0.01);且龟鹿二仙胶中、高剂量组TGF-β1、TGF-β2mRNA水平增加差异无统计学意义(P>0.05)。(3)Western blot结果提示:第24 h,龟鹿二仙胶高剂量组能使成骨细胞COL1A1及COL1A2蛋白合成增加(P<0.05)。第48 h,骨化三醇组、龟鹿二仙胶各组均能使成骨细COL1A1及COL1A2蛋白合成增加(P<0.01);龟鹿二仙胶高、中剂量组之间比较差异没有统计学意义(P>0.05);第24、48 h,骨化三醇组TGF-β1及TGF-β2蛋白合成无明显变化(P>0.05);在24 h,龟鹿二仙胶高剂量组TGF-β1及TGF-β2蛋白合成有显著增强(P<0.01),龟鹿二仙胶低剂量和中剂量组TGF-β1及TGF-β2蛋白合成无明显变化(P>0.05);48 h,龟鹿二仙胶各组TGF-β1及TGF-β2蛋白合成增加(P<0.01)。龟鹿二仙胶高中剂量组之间无显著差异(P>0.05)。结论:龟鹿二仙胶治疗骨质疏松症机制可能通过调节细胞因子TGF-β,促进I型胶原的表达,为探讨中药促胶原形成治疗骨质疏松提供细胞及分子生物学依据。

Objective： To study the effect of guiluerxianjiao collagen I and TGF-expression,explore the mechanism of guiluerxianjiao treatment of osteoporosis. Methods： SD rats were randomly divided into control group,guiluerxianjiao low dose group,medium dose group,high dose group,ossification in three alcohol group,gavage 1 times every day,for7 days,2h after the last administration of abdominal aortic blood serum made in rat osteoblast like cells ROS17_2. 8strain. To observe the effect of serum on proliferation and apoptosis of osteoblasts by cell morphology and MTT method.Cultured osteoblasts with serum containing culture conditions in each group,the expression level of Western Blot was detected by cell type I collagen and TGF-protein expression was detected by RT-PCR,the composition of bone cells of type I collagen and TGF-beta mRNA. Results： the results showed that：（ 1） MTT guiluerxianjiao group osteoblasts without obvious proliferation. Osteogenic cells of the three alcohol group had significant value added（ P〈0. 05）. Cell staining showed： ossification in three alcohol group and guiluerxianjiao group no significant apoptosis of osteoblasts.（ 2）the results of RT-PCR showed that the level of ossification in three alcohol group and guiluerxianjiao groups COL1A1 and COL1A2 mRNA increased（ P〈0. 01）; guiluerxianjiao in the high dose group of COL1A1 and COL1A2 mRNA lev-els increased obviously（ P〈0. 05）,guiluerxianjiao high dose group compared with middle dose group and COL1A1COL1A2 mRNA level was not significantly increased（ P〉0. 05）; no significant change of ossification in three alcohol group TGF-beta 1 and beta 2 level TGF-mRNA（ P〉0. 05）; guiluerxianjiao group TGF-beta 1 and beta 2 TGF-mRNA level increased significantly（ P〈0. 01）; and guiluerxianjiao in middle and high dose group TGF-beta 1,TGF-beta 2 increased mRNA levels showed no significant difference（ P〉0. 05）.（ 3） Western blot results suggest： twenty-fourth hours,guiluerxianjiao can make the high dose group of osteoblast COL1A1 and increased COL1A2 protein synthesis（ P〈0. 05）. Forty-eighth hours of ossification in three alcohol group,guiluerxianjiao groups can make bone fine COL1A1 and COL1A2 increased protein synthesis（ P〈0. 01）; guiluerxianjiao,between the high dose group compared the difference was not statistically significant（ P〉0. 05）; twenty-fourth,48 hours,ossification in three alcohol group TGF-beta 1 and beta 2 TGF-no significant changes in protein synthesis（ P〉0. 05）; in 24 hours,guiluerxianjiao high dose group TGF-beta 1 and beta 2 TGF-protein synthesis was significantly increased（ P〈0. 01）,guiluerxianjiao low and middle dose group TGF-beta 1 and beta 2 TGF-protein synthesis had no significant change（ P〈0. 05）;48 hours,guiluerxianjiao group TGF-beta 1 and beta 2 TGF-increased protein synthesis（ P〈0. 01）. There was no significant difference between guiluerxianjiao high dose group（ P〉0. 05）. Conclusion： guiluerxianjiao treatment of osteoporosis mechanism may be by regulating cytokine TGF-beta,promote the expression of type I collagen,promote collagen formation for TCM treatment of osteoporosis to provide cellular and molecular biology basis.