摘要
目的:探讨补肾中药吸收后血清对离体大鼠成骨细胞骨形成蛋白(BMP 6、BMP 7)、Smad 1、Smad 7、Smurf1、Smurf2等基因与蛋白表达,探讨骨形成蛋白的泛素-蛋白水解酶复合体通路(UPP)—Smad信号转导通路相关基因和蛋白表达的影响,研究肾虚骨质疏松症证候病机,观察补肾中药对其防治疗效及其作用机理。
材料与方法:应用多次胶原酶消化法获得新生大鼠颅盖骨的成骨细胞进行培养,采用Gomori改良钙钴法测定其碱性磷酸酶表达;随机将实验大鼠分为正常组、补肾中药组、补脾中药组、骨疏康组,通过血清药理学方法,采用灌胃8天后各组大鼠血清,培养成骨细胞48小时;并且,另行加入UPP通路抑制剂MG132以及正常组、补肾中药组、补脾中药组、骨疏康组大鼠血清,培养成骨细胞48小时;用MTT法检测成骨细胞增殖,同时用RT-PCR法、Western印迹法检测成骨细胞BMP6、7、Smad1、7、Smurf1、
2 mRNA与蛋白表达。
结果:
1.用酶消化法进行大鼠成骨细胞培养,方便易行,可培养大量高纯度成骨细胞供研究使用。
2.补肾中药血清作用48小时后可明显促进成骨细胞的增殖(P＜0.01),同时加MG132血清可诱导抑制成骨细胞的增殖率(P＜0.05)。
3.成骨细胞BMP6 mRNA与蛋白表达情况:与正常组比较,补肾中药组基因表达水平无显著差异(P＞0.05)而蛋白表达增高(P＜0.05),其它各组基因与蛋白表达水平明显降低(P＜0.05,P＜0.01)。使用药物血清各组比较,补肾中药组表达水平高于补脾中药组与骨疏康组(P＜0.01)。加入MG132药物血清组间,无明显差异(P＞0.05)。
4.成骨细胞BMP7 mRNA与蛋白表达情况:与正常组比较,补肾中药组基因表达水平增高(P＜0.01)而蛋白表达无显著差异(P＞0.05),其它各组基因与蛋白表达水平明显降低(P＜0.01),使用药物血清各组比较,补肾中药组表达水平高于补脾中药组与骨疏康组(P＜0.01)。加入MG132药物血清各组间,MG132+正常组表达水平最高(P＜0.05)。
5.成骨细胞Smad1 mRNA与蛋白表达情况:与正常组比较,补肾中药组基因表达水平无显著差异(P＞0.05)而蛋白表达降低(P＜0.01),其它各组基因与蛋白表达水平明显降低(P＜0.01)。使用药物血清各组比较,补肾中药组表达水平高于补脾中药组与骨疏康组(P＜0.01)。加入MG132药物血清各组比较,MG132+补肾中药组基因与蛋白表达水平明显高于MG132+补脾中药组(P＜0.01,P＜0.05)。
6.成骨细胞Smad7 mRNA与蛋白表达情况:与正常组比较,各组均可明显上调表达水平(P＜0.01,P＜0.05)。使用药物血清各组比较,补脾中药组基因与蛋白表达水平最高(P＜0.05)。加入MG132药物血清各组比较,MG132+补脾中药组基因表达水平明显低于其它加MG132药物血清各组(P＜0.01),而MG132+补肾中药组蛋白表达水平明显上调其他加MG132药物血清各组(P＜0.01)。
7.成骨细胞Smurf1 mRNA与蛋白表达情况:与正常组比较,其它各组表达水平明显下降(P＜0.01),使用药物血清各组比较,补肾中药组表达水平明显高于补脾中药组与骨疏康组(P＜0.01)。加入MG132药物血清组间,MG132+正常组基因表达水平最高(P＜0.05),MG132+骨疏康组蛋白表达水平最高(P＜0.05)。
8.成骨细胞Smurf2 mRNA与蛋白表达情况:与正常组比较,补肾中药组基因表达水平上调(P＜0.01)而蛋白表达水平无明显差异(P＞0.05),其它各组明显低于正常组(P＜0.01)。加入MG132药物血清组间,基因表达无明显差异(P＞0.05),MG132+骨疏康组蛋白表达水平最高(P＜0.05)。
结论:
1.采用药物血清作用于体外培养新生大鼠颅盖骨成骨细胞的方法是较为稳定可靠的血清药理学方法。
2.体外培养新生大鼠颅盖骨成骨细胞中存在泛素-蛋白水解酶复合体通路(UPP)-Smad蛋白其信号转导通路。
3.应用补肾中药血清可明显促进成骨细胞增殖,明显上调成骨细胞BMP6、BMP7、Smad1、Smurf1、Smurf2的基因与蛋白表达,下调抑制性信号转导因子Smad7的基因与蛋白表达。
4.应用UPP通路抑制剂MG132可明显抑制成骨细胞增殖,并明显抑制成骨细胞中BMP6、BMP7、Smad1、Smad7、Smurf1、Smurf2的基因与蛋白表达。
5.应用补肾中药血清具有干预MG132抑制对成骨细胞增殖的作用,对成骨细胞中BMP6、BMP7、Smad1、Smad7、Smurf1、Smurf2的基因与蛋白表达水平具有明显上调作用,明显优于MG132+补脾中药组。
Purpose:To inquire into the gene and protein expressions of bone morphogenetic protein (BMP6,BMP7),Smad1,Smad7,Smurf1,Smurf2 ofex vivo osteoblasts from rots, using the serum containing nourishing-kidney herbs after being absorbed.To research the effect of related gene and protein expressions about bone morphogenetic protein ubiquitinproteinase complex pathway(UPP) on Smad pathway.To observe the syndrome and pathogennesis of kidney deficiency type osteoporosis,as well as the curative effect and mechanism of action of nourishing-kidney herbs.
Material and method:To procure the osteoblast from newborn rats cranium by using the method of collagenase digestion for many times.Using modified Gomori calcium-cobalt method to detect the expressions of alkaline phosphatase.Experimental rats were randomly divided into normal group,nourishing-kidney herbs group,reinforcing-spleen herbs group, GUSHUKANG group.Through serum pharmacology method,after intragastric administration for 8 days,using the serum of each group rats to carry out osteoblast culture for 48 hours. In addition,by adding a separate UPP pathway inhibitor MG132 and normal group, nourishing-kidney herbs group,reinforcing-spleen herbs group,GUSHUKANG group, cultured for 48 hours of osteoblasts.To detect the proliferation of osteoblasts by using MTT method,to detect the mRNA and protein expression of BMP6,BMP7,Smad1,Smad7, Smurf1,Smurf2 in the osteoblast by using RT-PCR and Western blot method.
Results:
1.It is convenient to carry out the osteoblast culture of rats by enzyme digestion,and a large number of high purity osteoblast can be obtained for research.
2.The proliferation of osteoblast is obvious after adding the serum containing nourishingkidney herbs being absorbed for 48 hours(P＜0.01),at the same time adding MG 132 into the culture can inhibit the growth rate of osteoblast(P＜0.05).
3.Osteoblasts BMP6 mRNA and protein expression:Compared with normal group,the gene expressions of nourishing-kidney herbs group have no significant deviation(P＞0.05),and protein expression increased(P＜0.05),the gene and protein expressions of other groups decreased obviously(P＜0.05,P＜0.05).Compared between each group serum containing herbs,the expression level of nourishing-kidney herbs group are higher than reinforcingspleen herbs group and GUSHUKANG group(P＜0.01).It has no obvious difference between each group serum containing herbs after added MG132 into the culture(P＞0.05).
4.Osteoblasts BMP7 mRNA and protein expression:Compared with normal group,the nourishing-kidney herbs group increased the level of gene expressions(P＜0.01),protein expression and have no significant deviation(P＞0.05),the gene expression and protein expression level of other groups decreased obviously(P＜0.01).Compared between each group serum containing herbs,the expression level of nourishing-kidney herbs group are higher than reinforcing-spleen herbs group and GUSHUKANG group(P＜0.01).The expression level of normal group is the highest among each group serum containing berbs after added MG132 into the culture(P＜0.05).
5.Osteoblasts Smad1 mRNA and protein expression:Compared with normal group,the gene expressions of nourishing-kidney herbs group have no significant deviation(P＞0.05),and protein expression lower(P＜0.01),the gene and protein expressions level of the other groups decreased obviously(P＜0.01).Compared between each group serum containing herbs,the expression level of nourishing-kidney herbs group are higher than reinforcing-spleen herbs group and GUSHUKANG group(P＜0.01).Compared between each group serum containing herbs after added MG132,nourishing-kidney herbs group after added MG132 the level of gene and protein expressions was significantly higher than the reinforcing-spleen herbs group after added MG132(P＜0.01,P＜0.05).
6.Osteoblasts Smad7 mRNA and protein expression:Compared with normal group,the expression lever of the other group can be up-regulated(P＜0.01,P＜0.05).Compared between each group serum containing herbs,gene and protein expressions level of reinforcingspleen herbs group is the highest(P＜0.05).Compared between each group serum containing herbs after added MG132,the gene express(?)on level of reinforcing-spleen herbs group is lower obviously than the other group after added MG132 into the culture(P＜0.01),and the protein expression level of nourishing-kidney herbs group is higher obviously than the other group after added MG132 into the culture(P＜0.01).
7.Osteoblasts Smurfl mRNA and protein expression:Compared with normal group,the expression lever of the other groups decreased obviously(P＜0.01).Compared between each group serum containing herbs,the expression level of nourishing-kidney herbs group are higher obviously than reinforcing-spleen herbs group and GUSHUKANG group(P＜0.01). Compared between each group serum containing herbs after added MG132,the gene expression normal group after added MG132 the highest,and the protein expression level of GUSHUKANG group after added MG132 the highest(P＜0.05).
8.Osteoblasts Smurf2 mRNA and protein expression:Compared with normal group,the nourishing-kidney herbs group increased the level of gene expressions(P＜0.01),protein expression and have no significant deviation(P＞0.05),the expression lever of the other group are lower obviously than normal group(P＜0.01).Compared between each group serum containing herbs after added MG132,gene expression and have no significant deviation(P＞0.05),and the protein expression level of GUSHUKANG group after added MG132 the highest(P＜0.05).
Conclusion:
1.It is reliable serum pharmacology method that adding the serum containing herbs into osteoblast culture in vitro of neonate rat cranium.
2.The UPP- Smad protein signal transduction pathway can be finded in the osteoblast culture in vitro of neonate rat' cranium.
3.By using the serum containing tonifying-kidney,replenishing-essence and strengthening- bone herbs,the proliferation of osteoblast can be promoted obviously,and the gene and protein expression of BMP6.BMP7.Smad1.Smurf1.Smurf2 in osteoblast can be upregulated obviously,at the same time the gene and protein expression of Smad7 which is rejection capability transduced element of osteoblast can be down-regulated.
4.The using of MG132 which is suppressive agent of UPP pathway,the proliferation of osteoblast can be inhibited obviously,and the gene and protein expression of BMP6,BMP7, Smad1,Smurf1,Smurf2 can be inhibited obviously.
5.The using of the serum containing nourishing-kidney herbs,replenishing-essence and strengthening-bone herbs can interfere in the inhibition of MG132 to the proliferation of osteoblast,and can up-regulated the gene and protein expression of BMP6.BMP7.Smad1. Smad7.Smurf1.Smurf2 in osteoblast obviously which are obviously surpass the reinforcingspleen herbs group after adding MG132.
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