本文選題：絕經(jīng)后骨質(zhì)疏松　切入點(diǎn)：雌激素受體α　出處：《廣州中醫(yī)藥大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:探究骨組織中雌激素受體α(ERα)基因啟動子甲基化與腎虛型絕經(jīng)后骨質(zhì)疏松癥(PMOP)的關(guān)系以及補(bǔ)腎中藥干預(yù)對成骨細(xì)胞內(nèi)ERα基因啟動子甲基化、細(xì)胞增殖及分化的影響。方法:(1)收集行髖、膝手術(shù)的PMOP女性患者20例作為觀察組,行髓、膝手術(shù)的非PMOP女性患者20例作為對照組。通過問卷調(diào)查和體檢的方法收集患者的一般資料和體格資料,應(yīng)用雙能X線骨密度分析儀檢測患者腰椎2-4骨密度,應(yīng)用甲基化PCR(MSP)檢測患者骨組織中ERα基因啟動子甲基化情況。統(tǒng)計(jì)學(xué)分析觀察組與對照組患者骨密度、骨組織中ERα基因啟動子甲基化差異及骨密度與ERα基因啟動子甲基化程度的相關(guān)性。(2)二次酶消化法分離培養(yǎng)大鼠原代成骨細(xì)胞,用六味地黃丸和金匱腎氣丸分別以10、50、100ng/μL的濃度干預(yù)處理。MSP法檢測各組細(xì)胞內(nèi)ERα基因啟動子甲基化狀況,RT-PCR與WB法檢測補(bǔ)腎中藥干預(yù)后ERαmRNA和蛋白表達(dá)變化,CCK8試劑盒檢測細(xì)胞增殖活性,ALP試劑盒檢測堿性磷酸酶(ALP)表達(dá),細(xì)胞免疫組織化學(xué)染色法檢測成骨細(xì)胞標(biāo)志物膠原I和骨保護(hù)素的表達(dá)變化,WB法檢測影響成骨細(xì)胞增殖分化的信號通路關(guān)鍵分子AMPK和β-catenin的表達(dá)變化。結(jié)果:(1)與正常對照組患者相比,觀察組患者骨密度值降低、骨組織內(nèi)ERα基因啟動子甲基化增加,統(tǒng)計(jì)學(xué)分析表明,患者骨密度與ERα基因啟動子甲基化程度呈現(xiàn)負(fù)相關(guān)性。(2)補(bǔ)腎中藥干預(yù)能夠抑制成骨細(xì)胞內(nèi)ERα基因啟動子甲基化并增加ERαmRNA和蛋白表達(dá),其中,金匱腎氣丸的作用更為明顯。應(yīng)用CCK8試劑盒檢測細(xì)胞增殖結(jié)果顯示:六味地黃丸干預(yù)后,成骨細(xì)胞的增殖活性沒有明顯變化;金匱腎氣丸以50、100 ng/μL的濃度干預(yù)處理48 h時,能夠顯著地增加細(xì)胞增殖活性。同時,細(xì)胞組化檢測結(jié)果顯示:對于成骨細(xì)胞早期分化標(biāo)志物ALP與膠原I的表達(dá),六味地黃丸以100 ng/μL濃度干預(yù)時能夠增加其表達(dá),金匱腎氣丸以50 ng/μL和100 ng/μL濃度干預(yù)處理時能夠顯著增加其表達(dá),且金匱腎氣丸促成骨細(xì)胞早期分化效果更為明顯。此外,WB檢測補(bǔ)腎中藥干預(yù)對影響成骨細(xì)胞增殖分化的信號通路關(guān)鍵分子表達(dá)變化的結(jié)果顯示金匱腎氣丸干預(yù)處理能夠降低成骨細(xì)胞內(nèi)p-AMPK蛋白的表達(dá),增加Wnt通路中β-catenin蛋白的表達(dá)。結(jié)論:患者骨密度值與骨組織內(nèi)ERα基因啟動子甲基化程度呈負(fù)相關(guān);補(bǔ)腎中藥金匱腎氣丸能夠顯著抑制ERα基因啟動子甲基化及增加ERαmRNA和蛋白表達(dá),并通過此途徑所介導(dǎo)的AMPK和β-catenin的作用促進(jìn)成骨細(xì)胞的增殖分化。
[Abstract]:Objective: to investigate the relationship between the methylation of estrogen receptor 偽 er 偽) gene promoter and PMOP in postmenopausal osteoporosis of kidney deficiency type and the methylation of ER 偽 promoter in osteoblasts after intervention of Chinese herbs for tonifying the kidney. Methods 20 female PMOP patients undergoing hip and knee surgery were collected as the observation group. Twenty non-#en0# female patients undergoing knee surgery were used as the control group. The general and physical data of the patients were collected by questionnaire and physical examination. The 2-4 bone mineral density of lumbar vertebrae was measured by dual energy X-ray absorptiometry. The methylation of ER 偽 gene promoter in bone tissue was detected by methylated PCRG MSP.The bone mineral density (BMD) of patients in observation group and control group was analyzed statistically. The methylation difference of ER 偽 gene promoter and the correlation between bone mineral density and methylation degree of ER 偽 gene promoter in bone tissue. Using Liuwei Dihuang Pill and Jinkui Shenqi Pill to detect the methylation status of ER 偽 Gene Promoter in the cells of each Group by 100 ng / 渭 L concentration of 100.50g / 渭 L; RT-PCR and WB methods to detect the changes of ER 偽 mRNA and protein expression after intervention of Kidney tonifying herbs. The expression of alkaline phosphatase (ALP) was detected by ALP kit. Expression of osteoblast marker collagen I and osteoprotegerin were detected by immunohistochemical staining and the expression of AMPK and 尾 -catenin, the key molecules of signal pathway affecting osteoblast proliferation and differentiation, were detected by WB method. Compared with the patients in the normal control group, In the observation group, the bone mineral density decreased and the promoter methylation of ER 偽 gene increased in bone tissue. There was a negative correlation between bone mineral density (BMD) and methylation degree of promoter of ER 偽 gene. (2) intervention of traditional Chinese medicine for tonifying kidney could inhibit methylation of promoter of ER 偽 gene and increase expression of ER 偽 mRNA and protein in osteoblasts. The effect of Jingui Shenqi Pill was more obvious. The results of CCK8 assay showed that the proliferation activity of osteoblasts did not change after intervention of Liuwei Dihuang pills, and the proliferation activity of Jingui Shenqi Pill was treated with 50,100 ng/ 渭 L for 48 h. At the same time, the expression of ALP and collagen I in the early differentiation of osteoblasts could be increased by the intervention of Liuwei Dihuang pills with 100 ng/ 渭 L concentration, and the results of cell histochemical analysis showed that the expression of ALP and collagen I in the early differentiation of osteoblasts could be increased when the concentration of Liuwei Dihuang pills was 100 渭 L. Jingui Shenqi Pill could significantly increase its expression when it was treated with 50 ng/ 渭 L and 100 ng/ 渭 L concentration. Moreover, the effect of Jinkui Shenqi pills on the early differentiation of osteoblasts was more obvious. In addition, the effect of intervention of Chinese medicine for tonifying the kidney on the expression of key molecules of signal pathway affecting the proliferation and differentiation of osteoblasts was more obvious. Li can reduce the expression of p-AMPK protein in osteoblasts. Conclusion: bone mineral density is negatively correlated with the degree of methylation of ER 偽 gene promoter in bone tissue, Jinkui Shenqi Pill can significantly inhibit the methylation of ER 偽 gene promoter and increase the expression of ER 偽 mRNA and protein in bone tissue, and Jingui Shenqi Pill can significantly inhibit the methylation of the promoter of ER 偽 gene and increase the expression of ER 偽 mRNA and protein. The effects of AMPK and 尾-catenin mediated by this pathway promote the proliferation and differentiation of osteoblasts.
【學(xué)位授予單位】：廣州中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R259

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