| 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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