雌激素受體β介導植物雌激素骨代謝效應的流行病學與細胞學研究
發(fā)布時間：2019-06-11 04:36
【摘要】：背景:絕經(jīng)后骨質(zhì)疏松癥(PMOP)是由更年期后女性機體雌激素水平下降引起,現(xiàn)階段臨床防治手段多使用外源性補充雌激素,但是該替代療法可能產(chǎn)生諸如乳腺癌,子宮內(nèi)膜癌的等惡性腫瘤毒副作用。而植物雌激素作為安全的雌激素替代物防治PMOP正逐漸受到關注。我國膳食攝取植物雌激素量雖然顯著高于西方歐美人口,但是PMOP發(fā)病率并未明顯降低。我們認為這可能涉及遺傳因素。本課題組前期研究已經(jīng)發(fā)現(xiàn)絕境后婦女ERα基因多態(tài)性與膳食中植物雌激素攝入的骨密度效應有關,且細胞研究發(fā)現(xiàn)植物雌激素對ERβ親和力顯著高于ERα,但是ERβ基因多態(tài)性是否與膳食植物雌激素攝入的骨密度效應有關并未見關報道。目的:本研究擬從流行病學水平探討ERβ基因多態(tài)性與膳食植物雌激素骨密度調(diào)節(jié)效應的關系。方法:對選取符合調(diào)查要求的300名南昌市絕經(jīng)后婦女,用PCR-RFLP技術(shù)檢測ERβ基因Rsa I和Alu I限制性酶切位點基因多態(tài)性;根據(jù)調(diào)查數(shù)據(jù)比較ERβ不同基因型時骨密度差異,并分析不同基因型時植物雌激素攝入量與不同部位BMD骨密度關系。結(jié)果:在比較各基因型樣本間一般情況與各部位骨密度發(fā)現(xiàn),在校正體重指數(shù)、年齡、絕經(jīng)時間后,RR基因型L4的BMD高于Rr、rr組(P0.05),L2,L3髖骨總體及股骨頸、大轉(zhuǎn)子、ward三角在各攝入量組內(nèi)無統(tǒng)計學差異。將各基因型樣本依據(jù)膳食植物雌激素攝入量三分位數(shù)分組后進一步分析發(fā)現(xiàn),在校正體重指數(shù)、年齡、絕經(jīng)時間后,Alu I,Rsa I基因型樣本植物雌激素攝入量與低中高三組不同骨組織的骨密度比較,差別均無統(tǒng)計學意義。結(jié)論:1.ERβ基因Rsa I位點的RR基因型樣本L4 BMD高于Rr,rr組,其余部位BMD在個基因型組間無顯著性差異。2.ERβ基因Alu I位點多態(tài)性與BMD無關。3.在ERβ基因Rsa I,Alu I位點的各基因型樣本中,各部位與膳食植物雌激素攝入量無關。目的:本課題組流行病學研究顯示ERα而非ERβ基因多態(tài)性影響膳食植物雌激素與BMD關聯(lián)性,現(xiàn)有報道認為植物雌激素的生物學效應主要通過作用于ERβ,這與我們的結(jié)果相矛盾。本研究擬從細胞水平觀察植物雌激素代表藥金雀異黃酮(genestein,GEN)對成骨細胞和破骨細胞效應是否由ERα、ERβ所介導,從而為流行病學研究結(jié)果提供依據(jù)。。方法:體外誘導培養(yǎng)小鼠前成骨細胞MC3T3-E1分別給予各組細胞10-9-10-7mol/l的GEN,以及ERα阻斷劑MPP,ERβ阻斷劑PHTPP處理。成骨細胞骨形成效應,通過堿性磷酸酶(ALP)染色法進行觀察;并通過礦化結(jié)節(jié)茜素紅染色再次驗證同時體外誘導培養(yǎng)小鼠單核/巨噬細胞系RAW264.7細胞分化為破骨細胞,分別給予10-9-10-7mol/l GEN,以及ERα阻斷劑MPP,ERβ阻斷劑PHTPP處理。通過抗酒石酸酸性磷酸酶(TRAP)染色實驗與骨吸收陷窩實驗觀察破骨細胞的骨吸收效應。結(jié)果:ALP染色結(jié)果顯示,與對照組相比,不同濃度的GEN可增加成功細胞的生成,差異有統(tǒng)計學意義(p0.01)。在加入MPP,PHTPP后,可取消GEN的這種效應(p0.05)。礦化結(jié)節(jié)染色實驗結(jié)果與ALP染色實驗類似。破骨細胞實驗方面,TRAP染色試驗中,加入不同濃度的GEN均能減少成熟破骨細胞的生成(p0.01),且呈濃度依賴性,加入MPP后可逆轉(zhuǎn)GEN的破骨細胞抑制效應。骨吸收陷窩實驗結(jié)果顯示與對照組相比,加入不同濃度的GEN均可減少骨吸收陷窩面積,且呈濃度依賴性。而這一效應可以被MPP而非PHTPP所逆轉(zhuǎn)。結(jié)論:1.金雀異黃酮(GEN)的促MC3T3-E1骨形成效應是由ERα、ERβ所共同介導。2.金雀異黃酮(GEN)的抗RAW264.7分化為破骨細胞是主要由ERα介導,其對RAW264.7分化破骨細胞的骨吸收陷窩抑制效應由ERα而非ERβ所介導。
[Abstract]:BACKGROUND: The post-menopausal osteoporosis (PMOP) is caused by the decrease of the estrogen level of the female body after the menopause. At present, the external complementary estrogen is used in the present clinical prevention means, but the replacement therapy can produce the malignant and toxic side effects such as breast cancer and endometrial cancer. The treatment of PMOP with phytoestrogen as a safe estrogen replacement is becoming more and more concerned. Although the amount of estrogen in the dietary intake of China was significantly higher than that of the Western European and American population, the incidence of PMOP was not significantly reduced. We think this may involve genetic factors. in that early study of the research group, the polymorphism of ER gene in the postmenopausal women has been found to be related to the bone mineral density effect of the estrogen intake of the plant in the diet, and the cell study found that the affinity of the phytoestrogen to ER is significantly higher than that of ER, However, whether the ER gene polymorphism is related to the bone mineral density effect of the dietary phytoestrogen intake is not reported. Objective: To study the relationship between the polymorphism of ER gene and the regulation of the bone mineral density in dietary plant from the level of epidemiology. Methods: The polymorphisms of the ER gene Rsa I and Alu I restriction enzyme sites were detected by PCR-RFLP in 300 women with post-menopausal women in Nanchang, which were in accordance with the survey requirements. The relationship between the intake of phytoestrogen and the BMD of different parts in different genotypes was also analyzed. Results: The BMD of RR genotype L4 was higher than that of Rr, rr group (P0.05), L2, L3 hip bone and femoral neck and large rotor after comparing the general conditions between the genotype samples and the bone mineral density of each site. There was no statistical difference in the intake group. according to the three-digit number of the estrogen intake of the dietary plant, each genotype sample is further analyzed and found, after the body weight index, the age and the menopause time are corrected, the estrogen intake of the Alu I and the Rsa I genotype sample is compared with the bone density of the three different bone tissues in the low middle and high three groups, The difference was not statistically significant. Conclusion:1. The BMD of the Rsa I site in the ER gene is higher than that of the Rr, rr group, and the BMD of the rest is no significant difference between the genotypes. In each genotype sample of the ER gene Rsa I, the Alu I site, each site was independent of the dietary phytoestrogen intake. Objective: To study the relationship between ER and non-ER gene polymorphism on the relationship between the estrogen and BMD of dietary plant, and the present report is that the biological effect of phytoestrogen is mainly applied to ER, which is in contradiction with our results. In this study, it is proposed to observe whether the effect of phytoestrogen on osteoblast and osteoclast effect is mediated by ER antigen and ER antigen from the cell level, so as to provide the basis for epidemiological study. Methods: The pre-cultured mouse osteoblast MC3T3-E1 in vitro was treated with 10-9-10-7 mol/ l of GEN and ER-blocking agent MPP, ER-blocking agent, PHTPP, respectively. Osteoblastic bone formation effect was observed by alkaline phosphatase (ALP) staining method, and the mouse mononuclear/ macrophage-based RAW264.7 cells were re-validated by the mineralization of nodular and red staining. The cells were differentiated into osteoclasts,10-9-10-7 mol/ l GEN and ER-type blocking agent MPP, respectively. The ER antagonist PHTPP was treated. The bone resorption of osteoclasts was observed by anti-tartrate-acid phosphatase (TRAP) staining and bone resorption. Results: The results showed that, compared with the control group, the different concentrations of GEN could increase the production of successful cells and the difference was of statistical significance (p0.01). This effect of GEN can be eliminated after the addition of MPP, PHTPP (p0.05). The results of the staining of the mineralized nodules were similar to those of the ALP staining. In the experiment of osteoclast, in the TRAP staining test, the generation of mature osteoclasts (p0.01) can be reduced by the addition of a different concentration of GEN, and the inhibitory effect of the osteoclast can be reversed after the addition of MPP. The results of bone resorption and depression showed that, compared with the control group, the addition of the different concentrations of GEN could decrease the area of the bone resorption pit and be in a concentration-dependent manner. And this effect can be reversed by the mpp rather than the pHTPP. Conclusion:1. The MC3T3-E1 bone formation effect of the genistein (GEN) is mediated by ER antigen and ER antigen. The anti-RAW264.7 differentiation of the genistein (GEN) is mainly mediated by ER, and its inhibitory effect on the bone resorption pit of the differentiation osteoclasts of RAW264.7 is mediated by ER antigen rather than ER antigen.
【學位授予單位】：南昌大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R580
本文編號：2497019
[Abstract]:BACKGROUND: The post-menopausal osteoporosis (PMOP) is caused by the decrease of the estrogen level of the female body after the menopause. At present, the external complementary estrogen is used in the present clinical prevention means, but the replacement therapy can produce the malignant and toxic side effects such as breast cancer and endometrial cancer. The treatment of PMOP with phytoestrogen as a safe estrogen replacement is becoming more and more concerned. Although the amount of estrogen in the dietary intake of China was significantly higher than that of the Western European and American population, the incidence of PMOP was not significantly reduced. We think this may involve genetic factors. in that early study of the research group, the polymorphism of ER gene in the postmenopausal women has been found to be related to the bone mineral density effect of the estrogen intake of the plant in the diet, and the cell study found that the affinity of the phytoestrogen to ER is significantly higher than that of ER, However, whether the ER gene polymorphism is related to the bone mineral density effect of the dietary phytoestrogen intake is not reported. Objective: To study the relationship between the polymorphism of ER gene and the regulation of the bone mineral density in dietary plant from the level of epidemiology. Methods: The polymorphisms of the ER gene Rsa I and Alu I restriction enzyme sites were detected by PCR-RFLP in 300 women with post-menopausal women in Nanchang, which were in accordance with the survey requirements. The relationship between the intake of phytoestrogen and the BMD of different parts in different genotypes was also analyzed. Results: The BMD of RR genotype L4 was higher than that of Rr, rr group (P0.05), L2, L3 hip bone and femoral neck and large rotor after comparing the general conditions between the genotype samples and the bone mineral density of each site. There was no statistical difference in the intake group. according to the three-digit number of the estrogen intake of the dietary plant, each genotype sample is further analyzed and found, after the body weight index, the age and the menopause time are corrected, the estrogen intake of the Alu I and the Rsa I genotype sample is compared with the bone density of the three different bone tissues in the low middle and high three groups, The difference was not statistically significant. Conclusion:1. The BMD of the Rsa I site in the ER gene is higher than that of the Rr, rr group, and the BMD of the rest is no significant difference between the genotypes. In each genotype sample of the ER gene Rsa I, the Alu I site, each site was independent of the dietary phytoestrogen intake. Objective: To study the relationship between ER and non-ER gene polymorphism on the relationship between the estrogen and BMD of dietary plant, and the present report is that the biological effect of phytoestrogen is mainly applied to ER, which is in contradiction with our results. In this study, it is proposed to observe whether the effect of phytoestrogen on osteoblast and osteoclast effect is mediated by ER antigen and ER antigen from the cell level, so as to provide the basis for epidemiological study. Methods: The pre-cultured mouse osteoblast MC3T3-E1 in vitro was treated with 10-9-10-7 mol/ l of GEN and ER-blocking agent MPP, ER-blocking agent, PHTPP, respectively. Osteoblastic bone formation effect was observed by alkaline phosphatase (ALP) staining method, and the mouse mononuclear/ macrophage-based RAW264.7 cells were re-validated by the mineralization of nodular and red staining. The cells were differentiated into osteoclasts,10-9-10-7 mol/ l GEN and ER-type blocking agent MPP, respectively. The ER antagonist PHTPP was treated. The bone resorption of osteoclasts was observed by anti-tartrate-acid phosphatase (TRAP) staining and bone resorption. Results: The results showed that, compared with the control group, the different concentrations of GEN could increase the production of successful cells and the difference was of statistical significance (p0.01). This effect of GEN can be eliminated after the addition of MPP, PHTPP (p0.05). The results of the staining of the mineralized nodules were similar to those of the ALP staining. In the experiment of osteoclast, in the TRAP staining test, the generation of mature osteoclasts (p0.01) can be reduced by the addition of a different concentration of GEN, and the inhibitory effect of the osteoclast can be reversed after the addition of MPP. The results of bone resorption and depression showed that, compared with the control group, the addition of the different concentrations of GEN could decrease the area of the bone resorption pit and be in a concentration-dependent manner. And this effect can be reversed by the mpp rather than the pHTPP. Conclusion:1. The MC3T3-E1 bone formation effect of the genistein (GEN) is mediated by ER antigen and ER antigen. The anti-RAW264.7 differentiation of the genistein (GEN) is mainly mediated by ER, and its inhibitory effect on the bone resorption pit of the differentiation osteoclasts of RAW264.7 is mediated by ER antigen rather than ER antigen.
【學位授予單位】：南昌大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R580
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