| Objectives:1. To explore the best methods of extracting serum from kidney-tonifying traditional Chinese drug-fed rats;2. To investigate the biological effects of kidney-tonifying traditional Chinese drug-fed rats serum on the osteoclasts in vitro;3. To explore the biological effects of kidney-tonifying traditional Chinese drug-fed rats serum on the mRNA expression of RANK in osteoclasts;4. To investigate the biological effects of kidney-tonifying traditional Chinese drug-fed rats serum on mRNA and protein expression of osteoprotegerin(OPG),the ligand of osteoprotegerin(OPGL) in the osteoblast-osteoclast co-culture system, to observe the area of the resorption pits and the activity of TRAP on osteoclasts,to analyze the correlation between the rate of OPG/OPGL and the activity of osteoclasts.Methods:1. kidney-tonifying traditional Chinese drug was cooked traditionaly in water , obtained 252ml medicine liquid at the end , The SD rats were differed for four group at random[ control (naline2ml), high-dose( contained drug 7.08g/ml), middle-dose( contained drug 3.54g/ml), low-dose( contained drug 1.77g/ml)] and fed with different concentration medicine liquid above-mentioned for 2 times a day ,4 days in all, let blood at the end time ,obtained serum through centrifuge and dealed with 56 °C water for 30 min , and the second generation osteoblasts from the shull of newborn SD rats were mixed with the serum for the observation respectively on the proliferation and the differentiation;2. The serum of kidney-tonifying traditional Chinese drug-fed rats was administered to culture osteoclasts, the osteoclasts were divided into four groups, and three different concentrations of serum and the rat serum without drug were administered to culture them,the tartrate resistant acid phosphatase (TRAP) activities in the medium were measured by enzymetkinetics,the resorption pits were estained by toludineblue, the area and number of resorption pits were determined with the Leica Quantimet 500 system;3. The total RNA was obtained with the way of TRIzol from the osteoclastes,the mRNAexpression of RANK was examined by reverse transcriptase(RT) PCR;4. The osteoblasts and osteoclasts were isolated respectively from the skull and limb of SD rats and co-cultured in the same system ,The serum of kidney-tonifying traditional Chinese drug-fed rats was administered to the co-culture system, the mRNA expression of OPQOPGL was examined by reverse transcriptase(RT) PCR,The protein expression of OPG and OPGL was measured with Western blotting ,The tartrate resistant acid phosphatase (TRAP) activities in the medium were measured by enzyme kinetics,the resorption pits were stained by toludine blue, the area and number of resorption pits were determined with the Leica Quantimet 500 system, Comparisons of different trial group were made using analysis of variance (ANOVA) with multiple comparisons and a Student Newman Keuls test.Results:1. The osteoblast value of A570 without dealed with 56 °C water was 0.809 ± 0.261(HD),0.876 + 0.2749(MD),0.851 ± 0.299(LD),0.711 + 0.248(Control), and the value of A570 dealed with 56°Cwater was 0.669±0.179(HD),0.751±0.211(MD), 0.725±0.209(LD), 0.454±0.154(Control), comparion the results with SPSS10.0(pro vs post,p<0.01);2. TRAP activities of LD,MD.HD and control group in the medium were (1.36 + 0.14)U/L,(1.29±0.18)U/L,(1.31±0.24)U/L,(1.75±0.14)U/L(trial group vs control,P <0.01), the area of LD,MD.HD and control group were (293.23 +190.45) u m2, (335.40 + 202.33) w m2, (365.70 + 189.47) u m2, (495.25 + 210.37) u m2 (trial group vs control, P <0.01),and the number of resorption pits of LD,MD.HD and control group were 11.40 +1.50,12. 50+ 1.80,14.40± 2.42,, 18.40+ 2.32(trial group vs control, P3. RT-PCR displayed that the serum of kidney-tonifying traditional Chinese drug-fed ratscould regulated down the mRNAexpression of RANK;4. The serum of kidney-tonifying traditional Chinese drug-fed rats regulated up the expression of OPG but down OPGL on co-culture system;the area and number of resorption pits was restrained by the serum of kidney-tonifying traditional Chinese drug-fed rats , the output of correlation analyse indicated that the area of resorption pits was droped along with the rate of OPG/OPGL increasing.Conclusions:1. It had the tiptop element of medicine letting blood after the rats were fed with kidney-tonifying traditional Chinese drug at the last time 1 hour later, the serum that dealed with the 56 °C water for 30 min could fit for this study;2. The serum of kidney-tonifying traditional Chinese drug-fed rats could inhibitate the function of the resorption of osteoclast;3. The mRNA expression of RANK gene could be inhibitated by the serum of kidney-tonifying traditional Chinese drug-fed rats;4. The OPG/OPGL was the key facter Of regulating the activity of osteoclast ,the correlation between tge rate of OPG/OPGLwith the activity of osteoclast was negative .
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