基于组蛋白去甲基化酶JMJD家族探讨血瘀型激素性股骨头坏死的发病机制

发布时间：2018-03-01 19:20

  本文关键词： 股骨头坏死 组蛋白去甲基化酶 JMJD 糖皮质激素　出处：《广州中医药大学》2016年博士论文　论文类型：学位论文

【摘要】：目的：研究组蛋白去甲基化酶JMJD家族在激素性股骨头坏死的表达及其在成人骨髓间充质干细胞成骨分化中的作用。方法：1.手术中从4例非激素性股骨头坏死患者(对照组)和6例血瘀型激素性股骨头坏死患者(坏死组)的股骨近端取出骨髓。采用密度梯度离心法提取成人骨髓间充质干细胞(hBMSCs),经过纯化、培养、传代后,取第四代(P4)的hBMSCs进行实验。首先进行hBMSCs的鉴定：采用流式细胞技术检测hBMSCs的表面抗原(CD105、 CD29、CD73、CD44、CD34、CD45、CDllb/c),观察hBMSCs的成骨分化能力(碱性磷酸酶染色和茜素红染色)和成脂分化能力(油红染色)。然后提取总RNA,分析组蛋白去甲基化酶JMJD家族的mRNA在血瘀型激素性股骨头坏死中的表达。2.正常hBMSCs购自Lonza,常规培养、传代至P5进行实验。收集正常hBMSCs成骨分化第0、3、7、14、21天的细胞,提取总RNA进行RNA-seq测序分析,提取总蛋白进行Western blot实验。构建KDM4A基因沉默和基因超表达质粒,转染293T细胞并生产相应病毒,观察沉默或超表达KDM4A后对正常hBMSCs成骨分化的影响,包括碱性磷酸酶染色、碱性磷酸酶活性检测、茜素红染色、RNA-seq测序分析、Western blot实验等。利用JMJD家族基因广谱抑制剂JIB-04,观察不同浓度的JIB-04对正常hBMSCs成骨分化的影响,包括碱性磷酸酶染色、碱性磷酸酶活性检测、茜素红染色、RNA-seq测序分析、Western blot实验等。结果：1.从患者骨髓中提取的hBMSCs的表面抗原符合hBMSCs的特征,提取hBMSCs的形态呈长梭形,具备成骨分化和成脂分化的能力。2.KDM6A和RUNX2基因mRNA的表达在血瘀型激素性股骨头坏死组明显低于对照组,差异有统计学意义(P0.05)。KDM4A、KDM4B、KDM4D的mRNA表达量随着正常hBMSCs成骨分化的进程呈现动态的降低,KDM4C、KDM5B、KDM5C、KDM6A、KDM6B、UTY的mRNA表达量随着成骨分化的进程呈现动态的升高。正常hBMSCs在成骨分化第0、3、7、14、21天KDM4A、KDM4B、KDM5A、KDM6B蛋白的表达随着成骨分化时间的增加,其蛋白表达水平逐渐增加。3.沉默KDM4A以后,无论是在常规培养基中,还是在成骨分化的培养基中,shKDM4A#1、shKDM4A#2与shNeal(对照组)相比较,差异均无统计学意义(P0.05),说明沉默KDM4A对hBMSCs的增殖均无明显的影响；hBMSCs在成骨分化第8天的碱性磷酸酶染色,shKDM4A#1和shKDM4A#2均比shNeal明显减弱；hBMSCs在成骨分化第8天的碱性磷酸酶活性,shKDM4A#1、shKDM4A#2与shNeal相比较,差异均有统计学意义(P0.05)。说明沉默KDM4A后,hBMSCs成骨分化第8天的碱性磷酸酶染色和活性均受到明显的抑制；hBMSCs在成骨分化第14天的茜素红染色,shKDM4A#1和shKDM4A#2均比shNeal明显减弱,说明沉默KDM4A后,hBMSCs成骨分化的晚期矿化受到了明显的抑制。RNA-seq结果显示,沉默KDM4A以后,shKDM4A#1、shKDM4A#2与shNeal相比较,RUNX2、SPP1(OPN)、ALP等基因的mRNA表达明显降低,这些基因相应蛋白的表达水平也相应的降低。超表达KDM4A以后,hBMSCs在成骨分化第8天的碱性磷酸酶染色,KDM4A组(野生型)和KDM4AH188A组(突变型)均比Vector组(对照组)明显增强；hBMSCs在成骨分化第8天的碱性磷酸酶活性,KDM4A组、KDM4AH188A组与Vector组相比较,差异均有统计学意义(P0.05)。说明超表达KDM4A后,hBMSCs成骨分化第8天的碱性磷酸酶染色和活性均明显地升高；hBMSCs在成骨分化第14天的茜素红染色,KDM4A组和KDM4AH188A组均比Vector组明显增强,说明超表达KDM4A可促进hBMSCs成骨分化的晚期矿化。Western blot的结果显示超表达KDM4A以后,KDM4A组、KDM4AH188A组与Vector组相比较,RUNX2和OPN蛋白的表达均升高了。4.在成骨分化培养基中,与对照组(DMSO)相比较,800nM的JIB-04抑制hBMSCs的增殖,差异有统计学意义(P0.05)；100nM、200nM、400nM的JIB-04对hBMSCs的增殖无影响,差异无统计学意义(P0.05)。hBMSCs在成骨分化第8天的碱性磷酸酶染色,50nM、100nM、200nM、400nM、800nM的JIB-04均比对照组明显减弱,JIB-04浓度越高,碱性磷酸酶染色越弱；hBMSCs在成骨分化第8天的碱性磷酸酶活性,50nM、100nM、200nM、400nM、800nM的JIB-04均比对照组明显降低,差异均有统计学意义(P0.05),JIB-04浓度越高,碱性磷酸酶活性越低。hBMSCs在成骨分化第16、19、21天的茜素红染色,50nM、100nM、200nM、 400nM、800nM的JIB-04均比对照组明显减弱,JIB-04浓度越高,茜素红染色越弱。RNA-seq结果显示,100nM、400nM的JIB-04与对照组相比较,RUNX2、SPP1(OPN)、ALP等基因的mRNA表达明显降低,RUNX2和OPN蛋白的表达水平也相应的降低。结论：KDM6A可能与血瘀型激素性股骨头坏死发病机制有关。KMD4A是成人骨髓间充质干细胞的成骨分化的重要调控基因,主要通过RUNX2-OPN/ALP信号通路调节成骨分化。JIB-04通过RUNX2-OPN信号通路抑制成人骨髓间充质干细胞的成骨分化,JMJD家族基因的整体功能与成人骨髓间充质干细胞的成骨分化密切相关。
[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.

【学位授予单位】：广州中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R274.9

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1 陈达;基于组蛋白去甲基化酶JMJD家族探讨血瘀型激素性股骨头坏死的发病机制[D];广州中医药大学;2016年

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