本文關(guān)鍵詞： 股骨頭壞死 組蛋白去甲基化酶 JMJD 糖皮質(zhì)激素　出處：《廣州中醫(yī)藥大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：目的：研究組蛋白去甲基化酶JMJD家族在激素性股骨頭壞死的表達(dá)及其在成人骨髓間充質(zhì)干細(xì)胞成骨分化中的作用。方法：1.手術(shù)中從4例非激素性股骨頭壞死患者(對(duì)照組)和6例血瘀型激素性股骨頭壞死患者(壞死組)的股骨近端取出骨髓。采用密度梯度離心法提取成人骨髓間充質(zhì)干細(xì)胞(hBMSCs),經(jīng)過純化、培養(yǎng)、傳代后,取第四代(P4)的hBMSCs進(jìn)行實(shí)驗(yàn)。首先進(jìn)行hBMSCs的鑒定：采用流式細(xì)胞技術(shù)檢測(cè)hBMSCs的表面抗原(CD105、 CD29、CD73、CD44、CD34、CD45、CDllb/c),觀察hBMSCs的成骨分化能力(堿性磷酸酶染色和茜素紅染色)和成脂分化能力(油紅染色)。然后提取總RNA,分析組蛋白去甲基化酶JMJD家族的mRNA在血瘀型激素性股骨頭壞死中的表達(dá)。2.正常hBMSCs購(gòu)自Lonza,常規(guī)培養(yǎng)、傳代至P5進(jìn)行實(shí)驗(yàn)。收集正常hBMSCs成骨分化第0、3、7、14、21天的細(xì)胞,提取總RNA進(jìn)行RNA-seq測(cè)序分析,提取總蛋白進(jìn)行Western blot實(shí)驗(yàn)。構(gòu)建KDM4A基因沉默和基因超表達(dá)質(zhì)粒,轉(zhuǎn)染293T細(xì)胞并生產(chǎn)相應(yīng)病毒,觀察沉默或超表達(dá)KDM4A后對(duì)正常hBMSCs成骨分化的影響,包括堿性磷酸酶染色、堿性磷酸酶活性檢測(cè)、茜素紅染色、RNA-seq測(cè)序分析、Western blot實(shí)驗(yàn)等。利用JMJD家族基因廣譜抑制劑JIB-04,觀察不同濃度的JIB-04對(duì)正常hBMSCs成骨分化的影響,包括堿性磷酸酶染色、堿性磷酸酶活性檢測(cè)、茜素紅染色、RNA-seq測(cè)序分析、Western blot實(shí)驗(yàn)等。結(jié)果：1.從患者骨髓中提取的hBMSCs的表面抗原符合hBMSCs的特征,提取hBMSCs的形態(tài)呈長(zhǎng)梭形,具備成骨分化和成脂分化的能力。2.KDM6A和RUNX2基因mRNA的表達(dá)在血瘀型激素性股骨頭壞死組明顯低于對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。KDM4A、KDM4B、KDM4D的mRNA表達(dá)量隨著正常hBMSCs成骨分化的進(jìn)程呈現(xiàn)動(dòng)態(tài)的降低,KDM4C、KDM5B、KDM5C、KDM6A、KDM6B、UTY的mRNA表達(dá)量隨著成骨分化的進(jìn)程呈現(xiàn)動(dòng)態(tài)的升高。正常hBMSCs在成骨分化第0、3、7、14、21天KDM4A、KDM4B、KDM5A、KDM6B蛋白的表達(dá)隨著成骨分化時(shí)間的增加,其蛋白表達(dá)水平逐漸增加。3.沉默KDM4A以后,無論是在常規(guī)培養(yǎng)基中,還是在成骨分化的培養(yǎng)基中,shKDM4A#1、shKDM4A#2與shNeal(對(duì)照組)相比較,差異均無統(tǒng)計(jì)學(xué)意義(P0.05),說明沉默KDM4A對(duì)hBMSCs的增殖均無明顯的影響；hBMSCs在成骨分化第8天的堿性磷酸酶染色,shKDM4A#1和shKDM4A#2均比shNeal明顯減弱；hBMSCs在成骨分化第8天的堿性磷酸酶活性,shKDM4A#1、shKDM4A#2與shNeal相比較,差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。說明沉默KDM4A后,hBMSCs成骨分化第8天的堿性磷酸酶染色和活性均受到明顯的抑制；hBMSCs在成骨分化第14天的茜素紅染色,shKDM4A#1和shKDM4A#2均比shNeal明顯減弱,說明沉默KDM4A后,hBMSCs成骨分化的晚期礦化受到了明顯的抑制。RNA-seq結(jié)果顯示,沉默KDM4A以后,shKDM4A#1、shKDM4A#2與shNeal相比較,RUNX2、SPP1(OPN)、ALP等基因的mRNA表達(dá)明顯降低,這些基因相應(yīng)蛋白的表達(dá)水平也相應(yīng)的降低。超表達(dá)KDM4A以后,hBMSCs在成骨分化第8天的堿性磷酸酶染色,KDM4A組(野生型)和KDM4AH188A組(突變型)均比Vector組(對(duì)照組)明顯增強(qiáng)；hBMSCs在成骨分化第8天的堿性磷酸酶活性,KDM4A組、KDM4AH188A組與Vector組相比較,差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。說明超表達(dá)KDM4A后,hBMSCs成骨分化第8天的堿性磷酸酶染色和活性均明顯地升高；hBMSCs在成骨分化第14天的茜素紅染色,KDM4A組和KDM4AH188A組均比Vector組明顯增強(qiáng),說明超表達(dá)KDM4A可促進(jìn)hBMSCs成骨分化的晚期礦化。Western blot的結(jié)果顯示超表達(dá)KDM4A以后,KDM4A組、KDM4AH188A組與Vector組相比較,RUNX2和OPN蛋白的表達(dá)均升高了。4.在成骨分化培養(yǎng)基中,與對(duì)照組(DMSO)相比較,800nM的JIB-04抑制hBMSCs的增殖,差異有統(tǒng)計(jì)學(xué)意義(P0.05)；100nM、200nM、400nM的JIB-04對(duì)hBMSCs的增殖無影響,差異無統(tǒng)計(jì)學(xué)意義(P0.05)。hBMSCs在成骨分化第8天的堿性磷酸酶染色,50nM、100nM、200nM、400nM、800nM的JIB-04均比對(duì)照組明顯減弱,JIB-04濃度越高,堿性磷酸酶染色越弱；hBMSCs在成骨分化第8天的堿性磷酸酶活性,50nM、100nM、200nM、400nM、800nM的JIB-04均比對(duì)照組明顯降低,差異均有統(tǒng)計(jì)學(xué)意義(P0.05),JIB-04濃度越高,堿性磷酸酶活性越低。hBMSCs在成骨分化第16、19、21天的茜素紅染色,50nM、100nM、200nM、 400nM、800nM的JIB-04均比對(duì)照組明顯減弱,JIB-04濃度越高,茜素紅染色越弱。RNA-seq結(jié)果顯示,100nM、400nM的JIB-04與對(duì)照組相比較,RUNX2、SPP1(OPN)、ALP等基因的mRNA表達(dá)明顯降低,RUNX2和OPN蛋白的表達(dá)水平也相應(yīng)的降低。結(jié)論：KDM6A可能與血瘀型激素性股骨頭壞死發(fā)病機(jī)制有關(guān)。KMD4A是成人骨髓間充質(zhì)干細(xì)胞的成骨分化的重要調(diào)控基因,主要通過RUNX2-OPN/ALP信號(hào)通路調(diào)節(jié)成骨分化。JIB-04通過RUNX2-OPN信號(hào)通路抑制成人骨髓間充質(zhì)干細(xì)胞的成骨分化,JMJD家族基因的整體功能與成人骨髓間充質(zhì)干細(xì)胞的成骨分化密切相關(guān)。
[Abstract]:Objective: To study the histone demethylase JMJD family in steroid induced osteonecrosis of the femoral head and its expression in adult bone marrow mesenchymal stem cells into bone differentiation. Methods: 4 cases of non steroid induced necrosis of the femoral head from 1. patients with surgery (control group) and 6 cases of blood stasis type of steroid induced femoral head necrosis patients (necrosis group) of the proximal femur bone marrow removed. The extraction of adult bone marrow mesenchymal stem cells by density gradient centrifugation (hBMSCs), purified, cultured, passaged, take the fourth generation (P4) hBMSCs experiments. The first identification of hBMSCs: surface antigen flow cytometry using hBMSCs detection (CD105, CD29, CD73, CD44, CD34, CD45, CDllb/c), the capability of osteogenic differentiation was observed in hBMSCs (alkaline phosphatase staining and alizarin red staining) and the adipogenic differentiation ability (oil red staining). Then the total RNA was extracted, analysis of histone demethylase JMJD family in mRNA Blood stasis type of steroid induced necrosis of the femoral head in the normal expression of.2. hBMSCs was purchased from Lonza, conventional culture, the passage to the P5 experiment. HBMSCs cells collected from normal bone differentiation in 0,3,7,14,21 days, total RNA was extracted for RNA-seq sequencing, Western blot extracted total protein. Construction of KDM4A gene silencing and gene expression plasmid the production of the corresponding virus in 293T cells, transfection and observation, silence or overexpression of KDM4A after osteogenic differentiation of normal hBMSCs, including alkaline phosphatase staining, detection of alkaline phosphatase activity, alizarin red staining, RNA-seq sequencing, Western blot experiments. The use of broad-spectrum JMJD inhibitor JIB-04 gene family, the effects of different concentration of JIB-04 osteoblast on the differentiation of normal hBMSCs, including alkaline phosphatase staining, detection of alkaline phosphatase activity, alizarin red staining, RNA-seq sequencing, Western blot experiments. Results: from the 1. The extraction of surface antigen hBMSCs in the bone marrow of patients with the characteristics of hBMSCs and hBMSCs extraction were long fusiform, with the expression of osteogenic differentiation and the ability of.2.KDM6A and RUNX2 of mRNA gene and adipogenic differentiation in blood stasis type of steroid induced necrosis of the femoral head group was significantly lower than the control group, the difference was statistically significant (P0.05).KDM4A, KDM4B KDM4D, the expression of mRNA with normal hBMSCs osteogenic differentiation process presents a dynamic reduction, KDM4C, KDM5B, KDM5C, KDM6A, KDM6B, UTY and mRNA expression with the osteogenic differentiation process is a dynamic increase. Normal hBMSCs in osteogenic differentiation of the 0,3,7,14,21 KDM4B, KDM5A, KDM4A, KDM6B protein expression with the increase of time after osteogenic differentiation, the expression level of the protein gradually increased.3. silencing of KDM4A, both in the conventional culture medium, or shKDM4A#1 cultured in osteogenic differentiation, shKDM4A#2, and shNeal (control group) were compared , there were no significant differences (P0.05), that there was no obvious effect of KDM4A silencing on the proliferation of hBMSCs; hBMSCs in eighth days of differentiation of bone alkaline phosphatase staining, shKDM4A#1 and shKDM4A#2 were significantly decreased than shNeal; hBMSCs in eighth days of differentiation of bone alkaline phosphatase activity, shKDM4A#1, shKDM4A#2 compared with shNeal, the differences were statistically significant (P0.05). The results showed that silencing of KDM4A, hBMSCs and bone alkaline phosphatase activity eighth days of differentiation was inhibited; hBMSCs in osteogenic differentiation of fourteenth days of alizarin red staining, shKDM4A#1 and shKDM4A#2 were higher than shNeal significantly decreased, indicating silence after KDM4A hBMSCs late mineralized bone differentiation by the suppression of.RNA-seq obviously showed that silencing of KDM4A after shKDM4A#1, shKDM4A#2 and shNeal, RUNX2, SPP1 (OPN), the expression of ALP gene mRNA was significantly reduced, the corresponding gene The expression level of protein is low. After the overexpression of KDM4A and hBMSCs in eighth days of differentiation of bone alkaline phosphatase staining, KDM4A group (wild type) and group KDM4AH188A (mutant) were higher than Vector group (control group) increased significantly; hBMSCs in osteogenic differentiation of eighth days of alkaline phosphatase activity, KDM4A group, KDM4AH188A group compared with the Vector group, the differences were statistically significant (P0.05). The results indicated that the super expression of KDM4A, hBMSCs and alkaline phosphatase activity of bone differentiation for eighth days were significantly increased; hBMSCs in osteogenic differentiation of fourteenth days of alizarin red staining, KDM4A group and KDM4AH188A group were significantly increased than Vector group that, over expression of KDM4A can promote the osteogenic differentiation of hBMSCs.Western blot showed late mineralization after overexpression of KDM4A in KDM4A group, KDM4AH188A group compared with the Vector group, the expression of RUNX2 and OPN protein were increased.4. in osteogenic differentiation The culture medium, and the control group (DMSO) compared with 800nM, JIB-04 inhibited the proliferation of hBMSCs, the difference was statistically significant (P0.05); 100nM, 200nM, 400nM had no effect on JIB-04 proliferation and hBMSCs, there was no statistically significant difference (P0.05).HBMSCs in bone alkaline phosphatase staining eighth days of differentiation, 50nM 100nM, 200nM, 400nM, 800nM, JIB-04 were higher than that of the control group was significantly decreased, the higher the concentration of JIB-04, alkaline phosphatase staining is weak; hBMSCs in osteogenic differentiation of eighth days of alkaline phosphatase activity, 50nM, 100nM, 200nM, 400nM, 800nM, JIB-04 was significantly lower than control group, there were statistically significant differences (P0.05), the higher the concentration of JIB-04, alkaline phosphatase activity in the lower.HBMSCs in the first 16,19,21 days of osteogenic differentiation by alizarin red staining, 50nM, 100nM, 200nM, 400nM, 800nM and JIB-04 were higher than that of control group significantly decreased the concentration of JIB-04 is higher, the weaker the alizarin red staining showed.RNA-seq, 100N M, 400nM, JIB-04 compared with the control group, RUNX2, SPP1 (OPN), the expression of ALP gene of mRNA was significantly decreased, the expression level of RUNX2 and OPN protein also decreased. Conclusion: KDM6A may be related to blood stasis pathogenesis of steroid induced osteonecrosis of the femoral head is about.KMD4A of adult bone marrow mesenchymal stem regulation differentiation of gene into cells, mainly through the RUNX2-OPN/ALP signaling pathway to inhibit.JIB-04 differentiation of adult bone marrow mesenchymal stem cells to osteogenic differentiation by RUNX2-OPN pathway of JMJD gene family and the whole function of adult bone marrow mesenchymal stem cells osteoblast differentiation are closely related.

【學(xué)位授予單位】：廣州中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R274.9

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1 陳達(dá);基于組蛋白去甲基化酶JMJD家族探討血瘀型激素性股骨頭壞死的發(fā)病機(jī)制[D];廣州中醫(yī)藥大學(xué);2016年

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