微小RNA-206及其靶基因GJA1（间隙连接蛋白43基因）在激素性股骨头坏死中的作用研究
发布时间：2019-03-21 17:03
【摘要】：miRNA是生物体内源长度约为21个核苷酸左右的非编码小RNA,通过与靶mRNA分子3’末端非翻译区域(3'-untranslated region,3' UTR)互补配对而在转录水平上对基因的表达进行负调控,导致mRNA的翻译抑制或者降解从而发挥生物调控功能。现有研究表明骨组织的发生、生长、代谢同样与miRNA有着密切的关联。连接子蛋白43(connexin 43, Cx43)是骨组织中主要的间隙连接(gap junction)蛋白和半通道(hemichannel)蛋白,由Cx43形成的间隙连接及半通道实现了骨组织细胞间的直接通讯,对骨组织的正常发育、骨重建过程的建立与平衡起重要作用。已有研究表明miRNA-206以间隙连接蛋白43为靶基因对成骨细胞的生物学行为产生影响[4]。而成骨细胞是骨发育重建过程中最重要细胞之一,在骨骼发育成熟和修复重建中具有重要作用,其数量、功能的改变与股骨头坏死密切相关。我们假设激素性股骨头坏死的病理发生发展与Cx43/miRNA-206调控的成骨细胞分化通路相关。本实验以miRNA-206为中心,从分子生物学水平、细胞水平对Cx43/miRNA-206调控的成骨细胞分化通路在激素性股骨头坏死中的作用深入研究,为研究激素性股骨头缺血性坏死的发病机制提供新的思路。实验方法与主要结果一、miRNA-206及其靶基因GJAl(间隙连接蛋白43基因)在激素性股骨头坏死骨组织中的表达变化方法：(1)收集激素性股骨头坏死与股骨颈骨折行全髋置换手术患者的股骨头标本46例,其中激素性股骨头缺血性坏死(实验组)26例；新鲜股骨颈骨折(对照组)20例(2)HE染色观察两组股骨头组织形态变化(3)免疫组织化学检测CX43蛋白在股骨头的表达(4)提取患者股骨头总RNA实时荧光定量PCR检测两组股骨头miRNA-206及CX43mRNA的表达情况(5)提取患者股骨头总蛋白质,western blot定量检测股骨头CX43蛋白的表达研究结果：(1)HE染色：实验组：骨小梁坏死,细胞核消失,骨髓造血及微循环障碍；对照组：骨小梁内有少量坏死骨细胞,骨髓腔造血细胞增生活跃。(2)免疫组化：Cx43蛋白阳性表达于骨细胞、成骨细胞及细胞与细胞之间的胞膜和胞浆上。(3)荧光定量PCR:miRNA-206在实验组的表达量是对照组的4.3倍,两组具有统计学差异(P0.05), CX43mRNA在对照组的表达是实验组的2.4倍,两者间具有统计学差异(P0.05)。(4) Western blot:CX43蛋白的表达在实验组中低于对照组,两者具有统计学差异(P0.05)。二、miRNA-206通过抑制Cx43的表达而参与成骨细胞的调控方法：(1)成骨细胞株人SV40转染成骨细胞株购于(中国科学院细胞库)；(2)向原代成骨细胞内转染miRNA-206的mimic,(模拟剂)和inhibitor(抑制剂)及阴性对照N.C;(3)采用qRT-PCR检测miRNA-206、Cx43的mRNA表达及Western-blot方法检测Cx43蛋白表达水平,CCK-8法检测细胞增殖能力,ALP底物显色反应来评价转染后成骨细胞功能变化。研究结果：(1)转染miRNA-206 mimic后,细胞中miRNA-206表达明显增加,Cx43蛋白表达下降,与阴性对照比较具有统计学差异(P0.05), Cx43mRNA表达不变,与阴性对照比较无统计学差异(P0.05)；成骨细胞数量与N.C.对照组相比均显著下降,差异具有统计学意义(P0.05)；成骨细胞特异性分化标志物ALP活性下降,差异具有统计学意义(P0.05)。(2)转染miRNA-206inhibitor后,细胞中miRNA-206表达明显下降,Cx43蛋白表达增加,与阴性对照比较具有统计学差异(P0.05)Cx43mRNA表达不变,与阴性对照比较无统计学差异(P0.05)；成骨细胞数量与正常对照组和N.C.对照组相比均显著升高,差异具有统计学意义(P0.05)；成骨细胞增殖能力增强,成骨细胞特异性分化标志物ALP活性增加,差异具有统计学意义(P0.05)。实验结论：(1)在激素性股骨头坏死骨组织中,miRNA-206表达上调,CX43表达下调；(2) miRNA-206通过抑制Cx43的表达,从而抑制成骨细胞增殖、分化；(3) miRNA-206可能通过调控其靶基因CAJ1目的蛋白Cx43表达调控成骨细胞增殖、分化进而参与的激素性骨头坏死的发生与发展。
[Abstract]:The miRNA is a non-coding small RNA of the endogenous length of the organism about 21 nucleotides, and the expression of the gene is negatively regulated at the transcription level by complementary pairing with the non-translation region (3 '-untranslated region,3' UTR) of the 3 '-end non-translation region (3' UTR) of the target mRNA molecule, Resulting in a translation inhibition or degradation of the mrna to exert a biological regulatory function. The existing studies have shown that the occurrence, growth and metabolism of bone tissue are closely associated with miRNAs. The connexin 43 (Cx43) is a main gap junction protein and a half-channel protein in the bone tissue, and the gap connection and the half-channel formed by the Cx43 realize the direct communication between the bone tissue cells and the normal development of the bone tissue. The establishment and balance of bone reconstruction plays an important role. It has been shown that miRNA-206 has an effect on the biological behavior of osteoblasts in the presence of a gap-linked protein 43 as a target gene[4]. Osteoblasts are one of the most important cells in the process of bone development and reconstruction. We assume that the pathology of steroid-induced femoral head necrosis is associated with the osteoblast differentiation pathway that is regulated by Cx43/ miRNA-206. In this experiment, by using miRNA-206 as the center, the role of the osteoblast differentiation pathway regulated by Cx43/ miRNA-206 from the level of molecular biology and the cell level in the necrosis of the steroid-induced femoral head was studied, and a new idea was provided to study the pathogenesis of the steroid-induced avascular necrosis of the femoral head. The experimental method and the main results 1, miRNA-206 and its target gene GJAl (gap junction protein 43 gene) expression in the bone tissue of the steroid-induced femoral head necrosis: (1) collecting 46 cases of the head of the femoral head of the femoral head necrosis and the total hip replacement operation of the femoral neck fracture, There were 26 cases of ischemic necrosis (experimental group) of steroid-induced avascular necrosis of the femoral head. The expression (5) of the two groups of femoral head miRNA-206 and CX43mRNA was detected by real-time fluorescence quantitative PCR (RT-PCR) in 20 (2) patients with fresh femoral neck fracture (control group) in 20 (2) HE staining. The results of the study on the expression of the CX43 protein of the femoral head were measured by western blot: (1) HE staining: the experimental group: the bone trabecula necrosis, the disappearance of the nucleus, the bone marrow hemopoietic and the microcirculation disturbance; the control group: there was a small amount of necrotic bone cells in the bone trabecula, The bone marrow cavity hematopoietic cell proliferation is active. (2) Immunohistochemistry: The positive expression of Cx43 protein in the cell membrane and the cytoplasm between the bone cells, the osteoblasts and the cells and the cells. (3) The fluorescence quantitative PCR: the expression of miRNA-206 in the experimental group was 4.3 times that of the control group, the two groups had statistical difference (P0.05), and the expression of the CX43mRNA in the control group was 2.4 times that of the experimental group, and there was a statistical difference between the two groups (P0.05). (4) Western blot: The expression of CX43 protein in the experimental group was lower than that in the control group (P0.05). 2. The miRNA-206 is involved in the control of the osteoblast by inhibiting the expression of Cx43: (1) the osteoblast strain is transfected by the osteoblast strain SV40 (the cell bank of Chinese Academy of Sciences); (2) the mimetic of the miRNA-206, (the mimetic) and the inhibitor (inhibitor) and the negative control N.C. are transfected into the primary osteoblast; (3) The expression of Cx43 and the expression of Cx43 were detected by using qRT-PCR and the expression level of Cx43 was detected by Western-blot. Results: (1) The expression of the miRNA-206 in the cells increased significantly after transfection of the miRNA-206mmic, and the expression of Cx43 in the cells decreased, and the expression of Cx43 mRNA was not the same as that of the negative control (P0.05). The number of osteoblasts decreased significantly compared with that of the control group (P <0.05). The difference of ALP activity of the specific differentiation markers of the osteoblast was statistically significant (P0.05). (2) The expression of the miRNA-206 in the cells decreased significantly after transfection of the miRNA-206 inhitor, and the expression of the Cx43 protein increased, and the expression of the Cx43 mRNA in the cell was not the same as that of the negative control (P0.05); the number of the osteoblast was significantly higher than that of the normal control group and the control group of the N. C. The difference was statistically significant (P0.05); the proliferation ability of the osteoblast was enhanced, and the ALP activity of the osteoblast-specific differentiation marker was increased, and the difference was statistically significant (P0.05). Conclusion: (1) The expression of miRNA-206 is up-regulated and the expression of CX43 is down-regulated in the bone tissue of steroid-induced femoral head necrosis. (2) miRNA-206 inhibits the expression of Cx43, thereby inhibiting the proliferation and differentiation of osteoblast. (3) miRNA-206 may be used to regulate the occurrence and development of the hormone-induced bone necrosis that regulates the proliferation and differentiation of the osteoblast by regulating the expression of the protein Cx43 of the target gene CAJ1.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R681.8
本文编号：2445134
[Abstract]:The miRNA is a non-coding small RNA of the endogenous length of the organism about 21 nucleotides, and the expression of the gene is negatively regulated at the transcription level by complementary pairing with the non-translation region (3 '-untranslated region,3' UTR) of the 3 '-end non-translation region (3' UTR) of the target mRNA molecule, Resulting in a translation inhibition or degradation of the mrna to exert a biological regulatory function. The existing studies have shown that the occurrence, growth and metabolism of bone tissue are closely associated with miRNAs. The connexin 43 (Cx43) is a main gap junction protein and a half-channel protein in the bone tissue, and the gap connection and the half-channel formed by the Cx43 realize the direct communication between the bone tissue cells and the normal development of the bone tissue. The establishment and balance of bone reconstruction plays an important role. It has been shown that miRNA-206 has an effect on the biological behavior of osteoblasts in the presence of a gap-linked protein 43 as a target gene[4]. Osteoblasts are one of the most important cells in the process of bone development and reconstruction. We assume that the pathology of steroid-induced femoral head necrosis is associated with the osteoblast differentiation pathway that is regulated by Cx43/ miRNA-206. In this experiment, by using miRNA-206 as the center, the role of the osteoblast differentiation pathway regulated by Cx43/ miRNA-206 from the level of molecular biology and the cell level in the necrosis of the steroid-induced femoral head was studied, and a new idea was provided to study the pathogenesis of the steroid-induced avascular necrosis of the femoral head. The experimental method and the main results 1, miRNA-206 and its target gene GJAl (gap junction protein 43 gene) expression in the bone tissue of the steroid-induced femoral head necrosis: (1) collecting 46 cases of the head of the femoral head of the femoral head necrosis and the total hip replacement operation of the femoral neck fracture, There were 26 cases of ischemic necrosis (experimental group) of steroid-induced avascular necrosis of the femoral head. The expression (5) of the two groups of femoral head miRNA-206 and CX43mRNA was detected by real-time fluorescence quantitative PCR (RT-PCR) in 20 (2) patients with fresh femoral neck fracture (control group) in 20 (2) HE staining. The results of the study on the expression of the CX43 protein of the femoral head were measured by western blot: (1) HE staining: the experimental group: the bone trabecula necrosis, the disappearance of the nucleus, the bone marrow hemopoietic and the microcirculation disturbance; the control group: there was a small amount of necrotic bone cells in the bone trabecula, The bone marrow cavity hematopoietic cell proliferation is active. (2) Immunohistochemistry: The positive expression of Cx43 protein in the cell membrane and the cytoplasm between the bone cells, the osteoblasts and the cells and the cells. (3) The fluorescence quantitative PCR: the expression of miRNA-206 in the experimental group was 4.3 times that of the control group, the two groups had statistical difference (P0.05), and the expression of the CX43mRNA in the control group was 2.4 times that of the experimental group, and there was a statistical difference between the two groups (P0.05). (4) Western blot: The expression of CX43 protein in the experimental group was lower than that in the control group (P0.05). 2. The miRNA-206 is involved in the control of the osteoblast by inhibiting the expression of Cx43: (1) the osteoblast strain is transfected by the osteoblast strain SV40 (the cell bank of Chinese Academy of Sciences); (2) the mimetic of the miRNA-206, (the mimetic) and the inhibitor (inhibitor) and the negative control N.C. are transfected into the primary osteoblast; (3) The expression of Cx43 and the expression of Cx43 were detected by using qRT-PCR and the expression level of Cx43 was detected by Western-blot. Results: (1) The expression of the miRNA-206 in the cells increased significantly after transfection of the miRNA-206mmic, and the expression of Cx43 in the cells decreased, and the expression of Cx43 mRNA was not the same as that of the negative control (P0.05). The number of osteoblasts decreased significantly compared with that of the control group (P <0.05). The difference of ALP activity of the specific differentiation markers of the osteoblast was statistically significant (P0.05). (2) The expression of the miRNA-206 in the cells decreased significantly after transfection of the miRNA-206 inhitor, and the expression of the Cx43 protein increased, and the expression of the Cx43 mRNA in the cell was not the same as that of the negative control (P0.05); the number of the osteoblast was significantly higher than that of the normal control group and the control group of the N. C. The difference was statistically significant (P0.05); the proliferation ability of the osteoblast was enhanced, and the ALP activity of the osteoblast-specific differentiation marker was increased, and the difference was statistically significant (P0.05). Conclusion: (1) The expression of miRNA-206 is up-regulated and the expression of CX43 is down-regulated in the bone tissue of steroid-induced femoral head necrosis. (2) miRNA-206 inhibits the expression of Cx43, thereby inhibiting the proliferation and differentiation of osteoblast. (3) miRNA-206 may be used to regulate the occurrence and development of the hormone-induced bone necrosis that regulates the proliferation and differentiation of the osteoblast by regulating the expression of the protein Cx43 of the target gene CAJ1.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R681.8
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2 贾岩波;刘万林;任逸众;;激素性股骨头坏死发病机制中细胞凋亡的研究进展[J];中国组织工程研究;2012年13期
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