【摘要】：目的： 在前期工作中,本研究組成員已初步獲得4個(gè)SCA3/MJD患者外周血清中異常表達(dá)的miRNAs,其中包括3個(gè)下調(diào)的miRNAs-miR-25, miR-29a及miR-125b,以及1個(gè)上調(diào)的miRNA-miR-34b。并且證實(shí),4個(gè)miRNAs對(duì)SCA3/MJD的致病基因ATXN3mRNA均無明顯影響,而miR-25可顯著降低野生型ataxin-3蛋白表達(dá)水平,其作用靶點(diǎn)為ATXN3mRNA的3'UTR的第259-266堿基區(qū)域。在前期工作基礎(chǔ)上,進(jìn)一步了解miR-25對(duì)擴(kuò)展突變型ATXN3mRNA及ataxin-3蛋白表達(dá)的影響；進(jìn)一步研究miR-25對(duì)SCA3/MJD轉(zhuǎn)基因細(xì)胞模型的活性的影響,為進(jìn)一步闡明SCA3/MJD等polyQ疾病的分子發(fā)病機(jī)制提供新思路,為深入探討SCA3/MJD等polyQ疾病的治療提供新線索。 方法： (1)利用實(shí)時(shí)熒光定量PCR (qRT-PCR)技術(shù)檢測(cè)miR-25對(duì)polyQ擴(kuò)展突變型及ataxin-3蛋白表達(dá)水平的影響； (2)利用免疫印跡(Western blot)技術(shù)檢測(cè)miR-25對(duì)擴(kuò)展突變型ATXN3mRNA表達(dá)水平的影響； (3)利用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴鹽比色法(MTT)檢測(cè)miR-25對(duì)SCA3/MJD轉(zhuǎn)基因細(xì)胞模型存活能力的影響； (4)利用流式細(xì)胞技術(shù)(FCM)檢測(cè)miR-25對(duì)SCA3/MJD轉(zhuǎn)基因細(xì)胞模型早期凋亡率的影響； (5)利用免疫熒光技術(shù)檢測(cè)miR-25對(duì)SCA3/MJD轉(zhuǎn)基因細(xì)胞模型核內(nèi)包涵體的影響。 結(jié)果： (1) qRT-PCR結(jié)果顯示：miR-25不影響擴(kuò)展突變型ATXN3mRNA的表達(dá)； (2) Western blot結(jié)果顯示：miR-25可顯著降低polyQ擴(kuò)展突變型ataxin-3蛋白的表達(dá)水平(P0.05)； (3)MTT結(jié)果顯示：轉(zhuǎn)染SCA3/MJD轉(zhuǎn)基因細(xì)胞模型48小時(shí)后,miR-25可顯著升高SCA3/MJD轉(zhuǎn)基因細(xì)胞模型存活能力(P0.05),且作用靶點(diǎn)作用靶點(diǎn)為3'UTR區(qū)； (4)FCM技術(shù)結(jié)果顯示：轉(zhuǎn)染SCA3/MJD轉(zhuǎn)基因細(xì)胞模型48小時(shí)后,miR-25可顯著降低SCA3/MJD轉(zhuǎn)基因細(xì)胞模型的早期凋亡率(P0.05),且作用靶點(diǎn)作用靶點(diǎn)為3'UTR區(qū)； (5)免疫熒光結(jié)果顯示：轉(zhuǎn)染SCA3/MJD轉(zhuǎn)基因細(xì)胞模型48小時(shí)后,miR-25可顯著降低SCA3/MJD轉(zhuǎn)基因細(xì)胞模型的核內(nèi)包涵體陽性細(xì)胞率(P0.05),且作用靶點(diǎn)作用靶點(diǎn)為3'UTR區(qū)；核內(nèi)包涵體多數(shù)位于細(xì)胞核周圍。 結(jié)論： (1)miR-25通過轉(zhuǎn)錄后水平影響擴(kuò)展突變型ATXN3的表達(dá)； (2)miR-25可顯著提高SCA3/MJD轉(zhuǎn)基因細(xì)胞模型的細(xì)胞活性； (3)miR-25可顯著降低SCA3/MJD轉(zhuǎn)基因細(xì)胞模型核內(nèi)包涵體的陽性細(xì)胞率。
[Abstract]:Objective: in the previous work, the members of the study group have obtained the abnormal expression of miRNAs, in the peripheral serum of 4 SCA3/MJD patients, including 3 down-regulated miRNAs-miR-25, miR-29a and miR-125b,. And an up-regulated miRNA-miR-34b.. Four miRNAs had no significant effect on ATXN3mRNA of SCA3/MJD, but miR-25 could significantly decrease the expression level of wild-type ataxin-3 protein. The target of miR-25 was 259-266bp region of 3'UTR of ATXN3mRNA. On the basis of previous work, the effect of miR-25 on the expression of extended mutant ATXN3mRNA and ataxin-3 protein was further studied. Further studies on the effect of miR-25 on the activity of SCA3/MJD transgenic cell model provide a new idea for further elucidating the molecular pathogenesis of polyQ diseases such as SCA3/MJD and providing new clues for further exploring the treatment of polyQ diseases such as SCA3/MJD. Methods: (1) Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the effect of miR-25 on polyQ extended mutation and ataxin-3 protein expression. (2) Western blot (Western blot) was used to detect the effect of miR-25 on the expression level of extended mutant ATXN3mRNA. (3) 3-(4,5-dimethylthiazole-2)-2,5-diphenyl tetrazolium bromide colorimetry (MTT) was used to detect the effect of miR-25 on the viability of SCA3/MJD transgenic cell model. (4) flow cytometry (FCM) was used to detect the effect of miR-25 on the early apoptosis rate of SCA3/MJD transgenic cell model. (5) the effect of miR-25 on the nuclear inclusion bodies of SCA3/MJD transgenic cell model was detected by immunofluorescence technique. Results: (1) the results of qRT-PCR showed that: (1) miR-25 did not affect the expression of extended mutant ATXN3mRNA (2) Western blot); (2) miR-25 significantly decreased the expression level of polyQ extended mutant ataxin-3 protein (P0.05); (3) the results of MTT showed that after 48 hours of transfection of SCA3/MJD transgenic cell model, miR-25 significantly increased the viability of SCA3/MJD transgenic cell model (P0.05), and the target was 3 'UTR region. (4) the results of FCM showed that after 48 hours of transfection of SCA3/MJD transgenic cell model, miR-25 could significantly reduce the early apoptosis rate of SCA3/MJD transgenic cell model (P0.05), and the target was 3 'UTR region. (5) the results of immunofluorescence showed that after 48 hours of transfection of SCA3/MJD transgenic cell model, miR-25 significantly decreased the rate of intracellular inclusion body positive cells in SCA3/MJD transgenic cell model (P0.05). The target of action was 3 'UTR region. Most of the inclusion bodies in the nucleus are located around the nucleus. Conclusion: (1) miR-25 affects the expression of extended mutant ATXN3 at post-transcriptional level, (2) miR-25 can significantly increase the cell activity of SCA3/MJD transgenic cell model. (3) miR-25 could significantly decrease the positive rate of inclusion bodies in the nucleus of SCA3/MJD transgenic cell model.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R744.7

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 王樹奇;徐斌;劉建;王吉鵬;胡薇;;日本血吸蟲醛糖還原酶基因RNA干擾效應(yīng)的初步研究[J];中國(guó)寄生蟲學(xué)與寄生蟲病雜志;2014年01期

，

本文編號(hào)：2471250