| Background and Objective:The global drug abuse and dependence is the most significant problem for our society. The number of drug addicts raised continently, especially the number of female drug abuser has increased substantially in absolute and relative to the society, families and individuals has brought about tremendous hazards. Many experiments have shown that not only the role of endogenous opioid peptides in the central nervous system (hypothalamus, pituitary, etc.) caused by changes in the target organ, but also a direct effect on the peripheral system (uterus, ovary, bone, etc.), but the latter is less research. The subject will culture and identify the functions of female osteoblasts in vitro, the cells were harvested from the trabecular bone of normal subjects. To evaluate the effect of morphine on expression of MOR mRNA and ER mRNA of human Osteoblasts and investigate the direct effect of morphine on osteoblas.Materials and Methods:Test materials from 15 cases of bone fracture surgery in female patients (20-45 years old). These cases were from No.1 Hospital of Shanxi Medical University from March,2009 to June. All patients were excluded from endocrine diseases and systemic diseases, as well as the first 3 months of treatment does not take over hormone drugs. The materials were collected under sterile conditions and cultured within 30 minutes. The human osteoblasts cell (HOBs) were isolated from the cancellous bone by trypsin and collagenase digestion and identified by alizarin red staining and modified Gomonri method. The cells were divided into two parts. Cell proliferation assay:cells were divided into control group,17-βestradiol group,17-βestradiol+morphine group,17-βestradiol+morphine+naloxone groups, each with 30 holes. We carried on the cell counting by MTT method according the time(1,2,3,5,7 days), then drawed the growth cures. RT-PCR experiments:(1) the effects of morphine on the expression of MORmRNA:cells were divided into control group,morphine group(10-4 10-5,10-6,10-7mol/L),morphine+naloxone group.(2) the effects of morphine on ERmRNA expression:cells were divided into control group,morphine group(10-4,10-5,10-6,10-7mol/L),morphine+naloxone group,17-βestradiol+morphine group,17-βestradiol +morphine+naloxone groups,17-βestradiol group. Control group is blank control, morphine group added morphine. In other groups, the final concentration of morphine and naloxone were 10-5mol/L, the 17-βestradiol is 10-8mol/L. After cultured 24 hours, we collected all cells and analyzed the expression of MOR,ERmRNA by RT-PCR method. Statistical calculations were done by SPSS13.0 and the standard isa=0.05.Results:Newly inoculated cells were spherical, three days later, almost all of osteoblast adhesion,8-15 days is almost covered with cells, some cells interconnected by processes. Cell synthesis of modified calcium-cobalt method of alkaline phosphatase (Gomori method) stained brownish-black fine particles; cells in the conditioned medium cultured for 10 days, the cells merge into overlapping cobblestone growth, it formulates non-transparent light mineralized nodules and Alizarin red staining showed mineralized nodules showed red. We can see from the growth curve, E2 group compared with the control group increased cell number, E2+M group of cells were decreased compared with E2, the difference was significant (P<0.05), E2+M+ N group of cells with the E2 group, There was no significant difference (P> 0.05). RT-PCR shows characteristic strip could be found at 342 bp and 249 bp for MOR and ER respectively.It proves the existence of MOR with the ER. RT-PCR showed that comparing with the control group, expression of MORmRNA and ERmRNA in different concentration morphine groups decreased significantly (P<0.05),and they had negative correlation with the concentration of morphine (r=-0.946, P<0.05; r=-0.962, P＜0.05). There was significant difference in morphine group(P<0.05). E2 group compared with control group differences in ER mRNA expression was significantly (P<0.05), morphine+naloxone group and control group MOR mRNA, ER mRNA expression was no significant difference (P> 0.05).Conclusion:1. Human osteoblast cells on the presence of MOR, ER.2. Morphine can directly affect the effect of estrogen on human osteoblast proliferation-promoting role, while this role may be blocked by naloxone, which is the specific opioid receptor antagonist.3. Morphine can cause MOR, ER decreased the number of down-regulation and gene expression, and negatively correlated with the concentration of morphine, this effect can also be blocked by naloxone...
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