微小RNA-206及其靶基因GJA1（間隙連接蛋白43基因）在激素性股骨頭壞死中的作用研究
發(fā)布時(shí)間：2019-03-21 17:03
【摘要】：miRNA是生物體內(nèi)源長(zhǎng)度約為21個(gè)核苷酸左右的非編碼小RNA,通過與靶mRNA分子3’末端非翻譯區(qū)域(3'-untranslated region,3' UTR)互補(bǔ)配對(duì)而在轉(zhuǎn)錄水平上對(duì)基因的表達(dá)進(jìn)行負(fù)調(diào)控,導(dǎo)致mRNA的翻譯抑制或者降解從而發(fā)揮生物調(diào)控功能。現(xiàn)有研究表明骨組織的發(fā)生、生長(zhǎng)、代謝同樣與miRNA有著密切的關(guān)聯(lián)。連接子蛋白43(connexin 43, Cx43)是骨組織中主要的間隙連接(gap junction)蛋白和半通道(hemichannel)蛋白,由Cx43形成的間隙連接及半通道實(shí)現(xiàn)了骨組織細(xì)胞間的直接通訊,對(duì)骨組織的正常發(fā)育、骨重建過程的建立與平衡起重要作用。已有研究表明miRNA-206以間隙連接蛋白43為靶基因?qū)Τ晒羌?xì)胞的生物學(xué)行為產(chǎn)生影響[4]。而成骨細(xì)胞是骨發(fā)育重建過程中最重要細(xì)胞之一,在骨骼發(fā)育成熟和修復(fù)重建中具有重要作用,其數(shù)量、功能的改變與股骨頭壞死密切相關(guān)。我們假設(shè)激素性股骨頭壞死的病理發(fā)生發(fā)展與Cx43/miRNA-206調(diào)控的成骨細(xì)胞分化通路相關(guān)。本實(shí)驗(yàn)以miRNA-206為中心,從分子生物學(xué)水平、細(xì)胞水平對(duì)Cx43/miRNA-206調(diào)控的成骨細(xì)胞分化通路在激素性股骨頭壞死中的作用深入研究,為研究激素性股骨頭缺血性壞死的發(fā)病機(jī)制提供新的思路。實(shí)驗(yàn)方法與主要結(jié)果一、miRNA-206及其靶基因GJAl(間隙連接蛋白43基因)在激素性股骨頭壞死骨組織中的表達(dá)變化方法：(1)收集激素性股骨頭壞死與股骨頸骨折行全髖置換手術(shù)患者的股骨頭標(biāo)本46例,其中激素性股骨頭缺血性壞死(實(shí)驗(yàn)組)26例；新鮮股骨頸骨折(對(duì)照組)20例(2)HE染色觀察兩組股骨頭組織形態(tài)變化(3)免疫組織化學(xué)檢測(cè)CX43蛋白在股骨頭的表達(dá)(4)提取患者股骨頭總RNA實(shí)時(shí)熒光定量PCR檢測(cè)兩組股骨頭miRNA-206及CX43mRNA的表達(dá)情況(5)提取患者股骨頭總蛋白質(zhì),western blot定量檢測(cè)股骨頭CX43蛋白的表達(dá)研究結(jié)果：(1)HE染色：實(shí)驗(yàn)組：骨小梁壞死,細(xì)胞核消失,骨髓造血及微循環(huán)障礙；對(duì)照組：骨小梁內(nèi)有少量壞死骨細(xì)胞,骨髓腔造血細(xì)胞增生活躍。(2)免疫組化：Cx43蛋白陽性表達(dá)于骨細(xì)胞、成骨細(xì)胞及細(xì)胞與細(xì)胞之間的胞膜和胞漿上。(3)熒光定量PCR:miRNA-206在實(shí)驗(yàn)組的表達(dá)量是對(duì)照組的4.3倍,兩組具有統(tǒng)計(jì)學(xué)差異(P0.05), CX43mRNA在對(duì)照組的表達(dá)是實(shí)驗(yàn)組的2.4倍,兩者間具有統(tǒng)計(jì)學(xué)差異(P0.05)。(4) Western blot:CX43蛋白的表達(dá)在實(shí)驗(yàn)組中低于對(duì)照組,兩者具有統(tǒng)計(jì)學(xué)差異(P0.05)。二、miRNA-206通過抑制Cx43的表達(dá)而參與成骨細(xì)胞的調(diào)控方法：(1)成骨細(xì)胞株人SV40轉(zhuǎn)染成骨細(xì)胞株購(gòu)于(中國(guó)科學(xué)院細(xì)胞庫)；(2)向原代成骨細(xì)胞內(nèi)轉(zhuǎn)染miRNA-206的mimic,(模擬劑)和inhibitor(抑制劑)及陰性對(duì)照N.C;(3)采用qRT-PCR檢測(cè)miRNA-206、Cx43的mRNA表達(dá)及Western-blot方法檢測(cè)Cx43蛋白表達(dá)水平,CCK-8法檢測(cè)細(xì)胞增殖能力,ALP底物顯色反應(yīng)來評(píng)價(jià)轉(zhuǎn)染后成骨細(xì)胞功能變化。研究結(jié)果：(1)轉(zhuǎn)染miRNA-206 mimic后,細(xì)胞中miRNA-206表達(dá)明顯增加,Cx43蛋白表達(dá)下降,與陰性對(duì)照比較具有統(tǒng)計(jì)學(xué)差異(P0.05), Cx43mRNA表達(dá)不變,與陰性對(duì)照比較無統(tǒng)計(jì)學(xué)差異(P0.05)；成骨細(xì)胞數(shù)量與N.C.對(duì)照組相比均顯著下降,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)；成骨細(xì)胞特異性分化標(biāo)志物ALP活性下降,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。(2)轉(zhuǎn)染miRNA-206inhibitor后,細(xì)胞中miRNA-206表達(dá)明顯下降,Cx43蛋白表達(dá)增加,與陰性對(duì)照比較具有統(tǒng)計(jì)學(xué)差異(P0.05)Cx43mRNA表達(dá)不變,與陰性對(duì)照比較無統(tǒng)計(jì)學(xué)差異(P0.05)；成骨細(xì)胞數(shù)量與正常對(duì)照組和N.C.對(duì)照組相比均顯著升高,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)；成骨細(xì)胞增殖能力增強(qiáng),成骨細(xì)胞特異性分化標(biāo)志物ALP活性增加,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。實(shí)驗(yàn)結(jié)論：(1)在激素性股骨頭壞死骨組織中,miRNA-206表達(dá)上調(diào),CX43表達(dá)下調(diào)；(2) miRNA-206通過抑制Cx43的表達(dá),從而抑制成骨細(xì)胞增殖、分化；(3) miRNA-206可能通過調(diào)控其靶基因CAJ1目的蛋白Cx43表達(dá)調(diào)控成骨細(xì)胞增殖、分化進(jìn)而參與的激素性骨頭壞死的發(fā)生與發(fā)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.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R681.8
本文編號(hào)：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.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R681.8
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相關(guān)期刊論文 前2條
1 暴淑英;趙慶國(guó);畢黎琦;;激素性股骨頭壞死早期細(xì)胞凋亡相關(guān)基因表達(dá)及阿侖磷酸鈉的干預(yù)[J];中國(guó)組織工程研究與臨床康復(fù);2008年46期
2 賈巖波;劉萬林;任逸眾;;激素性股骨頭壞死發(fā)病機(jī)制中細(xì)胞凋亡的研究進(jìn)展[J];中國(guó)組織工程研究;2012年13期
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