【作者】 尚德阳；

【导师】 郑洪新；

【作者基本信息】 辽宁中医药大学 ， 中医基础理论， 2008， 博士

【摘要】 目的:通过研究摘除卵巢致肾虚骨质疏松症大鼠骨、肾、下丘脑组织中BMP6、BMP7、Smurf1、Smurf2的mRNA和蛋白的表达,揭示摘除卵巢致肾虚骨质疏松症的病理机制,并研究补肾壮骨益髓的补肾中药防治疗效及其作用机制。材料与方法:通过采用摘除大鼠双侧卵巢的方法,复制肾虚骨质疏松症模型。采用方证结合的办法,使用补肾中药对实验大鼠防治12周,同时用骨疏康颗粒剂、盖天力片(牡蛎钙)作为阳性对照组,正常大鼠、假手术组作为标准对照组,模型大鼠作为空白对照组,并与补脾中药(补中益气汤)作疗效的比较。12周后进行取材及实验指标的检测:使用双能X线骨密度仪检测离体大鼠左侧股骨的骨密度(BMD);用RT-PCR法及Western印迹法检测大鼠骨、肾、下丘脑组织中BMP6、BMP7、Smurf1、Smurf2的mRNA和蛋白表达。结果:(1)实验大鼠子宫指数比较:与正常组、假手术组比较,模型空白组明显降低,P<0.01。与模型空白组比较,补肾中药复方各组、盖天力组及骨疏康组有所升高,但均无显著性差异。(2)实验大鼠左侧股骨头骨密度BMD结果:与正常组、假手术组比较,模型空白组骨密度明显降低P<0.01。通过用药12周以后,与模型空白组比较,各用药组中补肾高剂量组、盖天力组可不同程度提高骨密度,其中补肾高剂量组效果最明显P<0.01。(3)RT-PCR法及Western法检测结果显示:正常大鼠骨、肾、下丘脑组织中存在BMP6的mRNA和蛋白表达;与正常组、假手术组比较,模空组BMP6 mRNA和蛋白的表达水平在骨、肾组织中明显降低P<0.01,而在下丘脑组织中表达明显升高P<0.01。用药12周后,与模空组比较,补肾高、低剂量组均可显著上调BMP6 mRNA和蛋白在骨、肾组织中的表达水平P<0.01;同时补肾高、低剂量组、补脾组、盖天力组、骨疏康组均可显著下调BMP6 mRNA和蛋白在下丘脑组织中的表达水平P<0.01。(4)RT-PCR法及Western法检测结果显示:正常大鼠骨、肾、下丘脑组织中存在BMP7的mRNA和蛋白表达;与正常组、假手术组比较,模空组BMP7 mRNA和蛋白的表达水平在骨、肾组织中明显降低P<0.01,而在下丘脑组织中表达明显升高P<0.01。用药12周后,与模空组比较,补肾高、低剂量组均可显著上调BMP7 mRNA和蛋白在骨、肾组织中的表达水平P<0.01;同时补肾高、低剂量组、补脾组、盖天力组、骨疏康组均可显著下调BMP7 mRNA和蛋白在下丘脑组织中的表达水平P<0.01。(5)RT-PCR法及Western法检测结果显示:大鼠骨、肾、下丘脑组织中存在Smurf1的mRNA和蛋白表达;与正常组、假手术组比较,模空组Smurf1 mRNA和蛋白的表达水平在骨、肾组织中明显降低P<0.01,而在下丘脑组织中表达明显升高P<0.01。用药12周后,与模空组比较,补肾高、低剂量组、补脾组均可明显上调Smurf1 mRNA和蛋白在骨、肾组织中的表达水平P<0.01;补肾高、低剂量组、补脾组、盖天力组、骨疏康组均可显著下调Smurf1 mRNA和蛋白在下丘脑组织中的表达水平P<0.01。(6)RT-PCR法及Western法检测结果显示:大鼠骨、肾、下丘脑组织中存在Smurf2的mRNA和蛋白表达,与正常组、假手术组比较,模空组Smurf2 mRNA和蛋白的表达水平在骨、肾组织中明显降低P<0.01,而在下丘脑组织中表达明显升高P<0.01。用药12周后,与模空组比较,补肾高、低剂量组可明显上调Smurf2 mRNA和蛋白在骨、肾组织中的表达水平P<0.01;补肾高、低剂量组、补脾组、盖天力组、骨疏康组均可明显下调Smurf2 mRNA和蛋白在下丘脑组织中的表达水平P<0.01。结论:(1)摘除大鼠双侧卵巢方法可以成功复制肾虚骨质疏松症模型。(2)正常大鼠骨、肾、下丘脑组织可以在基因、蛋白水平表达BMP6/BMP7、Smurf1/Smurf2,表明正常骨、肾、下丘脑组织中存在BMP6/BMP7、Smurf1/Smurf2,其在调节骨代谢过程中,起着重要的调控作用。(3)骨、肾组织中BMP6/BMP7、Smurf1/Smurf2的基因与蛋白表达下降和下丘脑组织中BMP6/BMP7、Smurf1/Smurf2的基因与蛋白表达升高,可能是肾虚骨质疏松症发生的重要机理之一。(4)补肾壮骨益髓方药可以提高骨、肾组织中BMP6/BMP7、Smurf1/Smurf2的基因与蛋白表达,同时可以降低下丘脑组织中BMP6/BMP7、Smurf1/Smurf2的基因与蛋白表达,起到防治肾虚骨质疏松症的作用。(5)BMP6/BMP7、Smurf1/Smurf2对骨代谢的调节作用,可能是中医学“肾主骨”调节机制之一。更多还原

【Abstract】 Purpose : In order to explore the etiological factors and pathologh of the osteoporosis due to kidney deficiency,We study the expression of BMP6/BMP7 and smurf1/smurf2 mRNA and proteins in the femur, kidney, hypothalamus of the rats of osteoporosis due to kidney deficiency by using ovariectomy.To explore the adjustable function of Chinese medicine and its regulating mechanism.Materials and Method:By using ovariectomy, we established the rat model of osteoporosis due to Kidney-deficiency. We treated the osteoporosis rat with tonifying kidney Chinese herbs (high dose, small dosage) for 12 weeks. Simultaneously,used GuShuKang granule and GaiTianLi tablets, tonifying spleen Chinese herbs, as positive control groups. And rats in the normal group and model group were not given any treatment. We used dual energy X-ray bone density instrument to detect the bone mineral density of the femur of the rats. We used the RT–PCR and Western Blotting method to investigate the expression of BMP6/BMP7 and smurf1/smurf2 mRNA and proteins in the femur, kidney,hypothalamus of the rats.Result:(1)The comparation of uterus exponent of the experimental rats: Comparing with the uterus exponent of the normal group and fake operational group , the uterus exponent of the model group is lower evidently. Comparing with the uterus exponent of the model group,the experimental drug groups(high, small dosage),the Gushukang granule group and the Gaitianli tablets group can increase the uterus exponent,and tonifying spleen Chinese herbs can decrease the uterus exponent,but all of them have no significance difference in statistics.(2)The comparison of the BMD of the femur of the experimental rats:After 12 weeks,comparing with the normal group and fake operational group, the BMD of the femur has evident tendency to reduce;comparing with the model group,the BMD of femur increased evidently in the experimental drug groups(high dosage) and the Gaitianli tablets group. (3)RT-PCR and Western blotting detection: the BMP6 existed in the normal femur, kidney, hypothalamus tissue. Comparing with the normal group and fake operational group,the level of the expression of the gene and protein of BMP6 in the femur and kidney tissue of model group decreased evidently,and the level of the expression of the gene and protein of BMP6 in the hypothalamus tissue of model group increased evidently.After the precaution treatment with the experimental drug groups(high,small dosage)、Gushukang granule、Gaitianli tablets group and tonifying spleen Chinese herbs,the experimental drug groups(high,small dosage) can increase evidently the mRNA and protein expression level of BMP6 in the femur and kidney tissue;and the experimental drug groups(high,small dosage)、Gushukang granule、Gaitianli tablets group and tonifying spleen Chinese herbs can decrease evidently the mRNA and protein expression level of BMP6 in the hypothalamus tissue.(4)RT-PCR and Western blotting detection: the BMP7 existed in the normal femur, kidney,hypothalamus tissue. Comparing with the normal group and fake operational group,the level of the expression of the gene and protein of BMP7 in the femur and kidney tissue of model group decreased evidently,and the level of the expression of the gene and protein of BMP7 in the hypothalamus tissue of model group increased evidently.After the precaution treatment with the experimental drug groups(high,small dosage),Gushukang granule , Gaitianli tablets group and tonifying spleen Chinese herbs,the experimental drug groups(high,small dosage) can increase evidently the mRNA and protein expression level of BMP7 in the femur and kidney tissue;and the experimental drug groups(high,small dosage),Gushukang granule , Gaitianli tablets group and tonifying spleen Chinese herbs can decrease evidently the mRNA and protein expression level of BMP7 in the hypothalamus tissue.(5)RT-PCR and Western blotting detection: the Smurf1 existed in the normal femur, kidney, hypothalamus tissue. Comparing with the normal group and fake operational group,the level of the expression of the gene and protein of Smurf1 in the femur and kidney tissue of model group decreased evidently,and the level of the expression of the gene and protein of Smurf1 in the hypothalamus tissue of model group increased evidently.After the precaution treatment with the experimental drug groups(high,small dosage),Gushukang granule , Gaitianli tablets group and tonifying spleen Chinese herbs can increase the mRNA and protein expression level of Smurf1 in the femur and kidney tissue in different degrees,and decreased the mRNA and protein expression level of Smurf1 in the hypothalamus tissue in different degrees.(6) RT-PCR and Western blotting detection: the Smurf2 existed in the normal femur, kidney, hypothalamus tissue. Comparing with the normal group and fake operational group,the level of the expression of the gene and protein of Smurf2 in the femur and kidney tissue of model group decreased evidently,and the level of the expression of the gene and protein of Smurf2 in the hypothalamus tissue of model group increased evidently.After the precaution treatment with the experimental drug groups(high,small dosage),Gushukang granule , Gaitianli tablets group and tonifying spleen Chinese herbs can increase the mRNA and protein expression level of Smurf2 in the femur and kidney tissue in different degrees,and decreased the mRNA and protein expression level of Smurf2 in the hypothalamus tissue in different degrees.Conclusion:(1).Using the method of ovariectomy, the rat model of osteoporosis due to Kidney-deficiency successfully can be established.(2). In normal rats,the tissue of the femur ,the kidney and the hypothalamus can express the BMP6/BMP7 and smurf1/smurf2 in the gene and protein level. That means normal the tissue of the femur ,the kidney and the hypothalamus exist BMP6/BMP7 and smurf1/smurf2,and it may have a very important contribution on adjusting osteal- metabolism.(3). The decreasing lever of BMP6/BMP7 and smurf1/smurf2 mRNA and protein expression in the femur and kidney tissue,and the increasing lever of BMP6/BMP7 and smurf1/smurf2 mRNA and protein expression in the hypothalamus tissue,may be one of the important occurring mechanism of the osteoporosis due to kidney deficiency.(4). The experimental Chinese herbs,which have the function of tonifying kidney Qi and strengthening essence ,can increase the level of the expression of the gene and protein of BMP6/BMP7 and smurf1/smurf2 in the femur and kidney tissue,and can decrease the level of the expression of the gene and protein of BMP6/BMP7 and smurf1/smurf2 in the hypothalamus tissue. It would be preventing and treating to the osteoporosis due to kidney deficiency.(5). The function of BMP6/BMP7 and smurf1/smurf2 to adjusting osteal metabolism would be one of the aspects of the kidney controlling bone in TCM.更多还原