【摘要】：睪丸支持細胞位于睪丸曲細精管基底部,與各種不同發(fā)育狀態(tài)的生精細胞共同構(gòu)成曲細精管壁。支持細胞具有為生精細胞提供營養(yǎng),啟動精原干細胞分化,維持精子發(fā)生,吞噬精子發(fā)生過程中的殘余胞質(zhì)等重要功能。睪丸支持細胞的增殖決定了動物個體睪丸大小和成熟后精子質(zhì)量和數(shù)量。并且成熟的支持細胞數(shù)量又取決于未成熟的支持細胞數(shù)量。因此,未成熟的支持細胞對動物睪丸發(fā)育和生精能力也有著至關(guān)重要的作用。QKI是一種RNA結(jié)合蛋白,屬于高度保守的STAR家族成員,QKI主要表達QKI-5,QKI-6和QKI-7三種異構(gòu)體。QKI能通過結(jié)合靶m RNA的QRE序列,影響RNA的代謝,從而參與細胞增殖、凋亡、分化等過程。本研究的目的是探討QKI-5對ST細胞凋亡的影響以及QKI-5對WT1和Caspase8的轉(zhuǎn)錄后調(diào)控作用。首先,確認(rèn)豬的QKI基因具有QKI-5、QKI-6和QKI-7這三種剪接模式。根據(jù)人的QKI-5,QKI-6和QKI-7序列分別設(shè)計三對特異性引物,以豬的c DNA為模版,成功克隆出三條目的片段。它們分別具有QKI-5、QKI-6和QKI-7特異的C末端,并且,通過分析發(fā)現(xiàn)QKI-5具有典型的核定位信號(NLS)。隨后,研究了QKI-5對ST細胞凋亡的影響。首先成功構(gòu)建QKI-5的過表達載體并合成了4條干擾載體(sh RNAs)。在ST細胞中進行過表達和沉默QKI-5,MTS結(jié)果表明,過表達QKI-5后,細胞數(shù)顯著高于對照組,而干擾QKI-5后,細胞數(shù)出現(xiàn)顯著降低。流式細胞術(shù)結(jié)果表明,過表達QKI-5時,細胞凋亡率為2.32±0.11%,而沉默QKI-5時,細胞凋亡率為25.64±0.68%,差異顯著(p0.01),表明QKI-5具有抑制ST細胞凋亡的功能。然后,驗證了QKI-5抑制細胞凋亡的分子機制。生物信息學(xué)分析發(fā)現(xiàn)Caspase8基因的3’UTR上有1個可被QKI結(jié)合的QRE元件(1009bp-1014bp,ACTAAC),而WT1有2個,分別是QRE-1(2046bp-2052bp,ACTAAC)和QRE-2(2211bp-2217bp,ACTAAC)。為了驗證QKI-5是否與Caspase8和WT1的3’UTR結(jié)合,成功構(gòu)建了Caspase8的野生型雙熒光素酶報告基因載體Caspase8-3’UTR-WT和突變型載體Caspase8-3’UTR-MUT;以及WT1的野生型載體WT1-3’UTR-WT和WT1-3’UTR-MUT1,WT1-3’UTR-MUT2和WT1-3’UTR-MUT3三種突變型載體。雙熒光素活性檢測發(fā)現(xiàn),QKI-5能與Caspase8的3’UTR結(jié)合,但只與WT1基因的QRE-1結(jié)合。最后,驗證了QKI-5對Caspase8和WT1的轉(zhuǎn)錄后調(diào)控作用。實驗結(jié)果表明,在加入放線菌素D 0-2h到6-8h期間,過表達QKI-5有助于WT1 m RNA的穩(wěn)定,過表達QKI-5還能顯著促進WT1及其下游基因Bcl-2的表達量;而且,當(dāng)sh QKI3與pBI-WT1共轉(zhuǎn)染ST細胞時,WT1基因可以彌補因沉默QKI-5引起的Bcl-2基因表達水平降低的現(xiàn)象。沉默QKI-5時,結(jié)果相反。在過表達QKI-5細胞中,加入放線菌素D 0-2h到6-8h期間,QKI-5能加快Caspase8的m RNA降解。能顯著抑制Caspase8和Caspase3的表達水平,沉默QKI-5時,結(jié)果相反。結(jié)論:QKI-5具有抑制ST細胞的凋亡的功能。其分子機制是通過轉(zhuǎn)錄后調(diào)控維持WT1 m RNA穩(wěn)定,促進WT1及其下游基因Bcl-2的表達量;QKI-5可加快Caspase8 m RNA的降解,并顯著抑制Caspase8和Caspase3的表達水平,從而起到抑制ST細胞的凋亡功能。
[Abstract]:Testis supporting cells are located in the basement of the seminiferous tubule of the testis, and together with a variety of spermatogenic cells with different developmental states together to form the wall of the fine seminiferous tubule. Support cells have the important function of providing nutrition for spermatogenic cells, starting spermatogonial stem cell differentiation, maintaining spermatogenesis, phagocytosis and the important function of residual cytoplasm in the process of spermatogenesis. Colonization determines the size of the testis and the quantity and quantity of sperm after maturity. And the number of mature supporting cells depends on the number of immature support cells. Therefore, immature support cells also play a vital role in the development and spermatogenesis of animal testis..QKI is a kind of RNA binding protein, which is highly conserved STAR. Family members, QKI, mainly express QKI-5, QKI-6 and QKI-7 three isomers.QKI can affect the metabolism of RNA by combining the QRE sequence of the target m RNA, thus participating in the process of cell proliferation, apoptosis and differentiation. The purpose of this study is to explore the effect of QKI-5 on the apoptosis of ST cells and the post transcriptional regulation of QKI-5. The QKI gene has three splicing patterns of QKI-5, QKI-6 and QKI-7. According to human QKI-5, QKI-6 and QKI-7 sequences, three pairs of specific primers were designed respectively. The three target fragments were cloned successfully with C DNA as a template. They had QKI-5, QKI-6, and QKI-7 specific ends respectively. (NLS). Then, the effect of QKI-5 on the apoptosis of ST cells was studied. First, the overexpression vector of QKI-5 was constructed and 4 interference carriers (SH RNAs) were synthesized. The expression and silence of QKI-5 in ST cells showed that the number of cells was significantly higher than that of the control group after the overexpression of QKI-5, and the number of cells decreased significantly after the interference QKI-5. The cell apoptosis rate was 2.32 + 0.11%, and the cell apoptosis rate was 2.32 + 0.11%, while the cell apoptosis rate was 25.64 + 0.68%, the difference was significant (P0.01), indicating that QKI-5 had the function of inhibiting the apoptosis of ST cells. Then, the molecular mechanism of QKI-5 inhibition of apoptosis was verified. Bioinformatics analysis found that there were 1 on the 3 'UTR of the Caspase8 gene. QRE components (1009bp-1014bp, ACTAAC) that can be combined by QKI, and WT1 have 2, QRE-1 (2046bp-2052bp, ACTAAC) and QRE-2 (2211bp-2217bp, ACTAAC). E8-3 'UTR-MUT; and the three mutant vectors of WT1-3' UTR-WT and WT1-3 'UTR-MUT1, WT1-3' UTR-MUT2 and WT1-3 'UTR-MUT3. Double fluorescein activity detection found that QKI-5 can be combined with 3' but only combined with the transcriptional regulation of the genes. The results showed that overexpression of QKI-5 could contribute to the stability of WT1 m RNA during the addition of actinomycin D 0-2h to 6-8h, and the overexpression of QKI-5 also significantly promoted the expression of WT1 and its downstream gene Bcl-2. When silent QKI-5, the result is opposite. In over expression QKI-5 cells, QKI-5 can accelerate m RNA degradation of Caspase8 by adding actinomycin D 0-2h to 6-8h. It can significantly inhibit the expression level of Caspase8 and Caspase3, when silent QKI-5, the result is opposite. Controlling the stability of WT1 m RNA and promoting the expression of WT1 and its downstream gene Bcl-2; QKI-5 can accelerate the degradation of Caspase8 m RNA, and significantly inhibit the expression level of Caspase8 and Caspase3, thus inhibiting the apoptosis function of ST cells.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S828

【參考文獻】

相關(guān)期刊論文 前6條

1 許兵;劉慧;許應(yīng)星;吳巖;肖魯偉;童培建;;成骨細胞中經(jīng)典Wnt/β-catenin通路研究進展[J];生命科學(xué);2011年05期

2 韓妲麗;邢新;王曉云;;睪丸支持細胞介導(dǎo)局部免疫耐受的進展[J];中國美容醫(yī)學(xué);2008年07期

3 廖尚英,朱寶長;雄性學(xué)研究(1)[J];生物學(xué)通報;2004年06期

4 曹博,鄭俊波,郭筠秋;大鼠睪丸支持細胞的分離純化與鑒定[J];解剖科學(xué)進展;2004年01期

5 上官芳芳,史小林;睪丸支持細胞的功能及其應(yīng)用的探討[J];解剖學(xué)報;2002年04期

6 高伯生;睪丸支持細胞功能及調(diào)節(jié)[J];國外醫(yī)學(xué)(計劃生育分冊);1994年04期

相關(guān)碩士學(xué)位論文 前1條

1 劉薇;RNA結(jié)合蛋白QKI在血管緊張素Ⅱ誘導(dǎo)的心肌細胞肥大中的作用[D];第四軍醫(yī)大學(xué);2012年

，

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