發(fā)布時間：2018-07-01 18:49

  本文選題：Prestin + 基因敲入��； 參考：《山東大學(xué)》2011年碩士論文

【摘要】：哺乳動物的聽力極為敏感,這是由于耳蝸外毛細(xì)胞(Outer Hair Cells, OHC)中有一種生物放大器。這種放大器通過外毛細(xì)胞胞體長度的改變將基底膜的振動放大100倍,從而使人聽到聲音。外毛細(xì)胞的胞體長度的改變是由某種電動蛋白驅(qū)動的,多年來人們一直在尋找能夠引起外毛細(xì)胞胞體振蕩的電動蛋白。在2000年P(guān)restin基因被克隆后,研究者利用基因敲除技術(shù)證明了Prestin就是內(nèi)耳外毛細(xì)胞中的電動蛋白。Prestin是聽力放大的核心基因,它與人類耳聾疾病密切相關(guān),至今已發(fā)現(xiàn)兩種引起入耳聾的Prestin點突變,但目前對Prestin突變的分子病理機(jī)制知之甚少。 關(guān)于Prestin在人類中的突變致聾已有文獻(xiàn)報道：2003年在第2內(nèi)含子和第3外顯子(ATG)交匯處一種點突變(IVS2-2A/G)被發(fā)現(xiàn),它處于Prestin mRNA剪切相關(guān)的區(qū)域；2007年在第6外顯子中,發(fā)現(xiàn)了另一個點突變(R150Q)。這兩種點突變均能引起人聽力的損失。 鑒于人類耳聾是發(fā)病率相對較高的一種疾病,利用制作相應(yīng)疾病動物模型的方法研究治病機(jī)理具有極高的價值。我們利用基因敲入的方法,制作Prestin基因敲入小鼠模型,以期精確模擬導(dǎo)致人類耳聾疾病的Prestin基因第6號外顯子上的點突變(R150Q)。我們可以通過對這種小鼠模型的表型分析和研究,避免了在人體實驗材料以及實驗技術(shù)不可操作性的限制。本研究選擇對第6外顯子進(jìn)行特定的點突變(R150Q),原因是利用它不僅可以發(fā)現(xiàn)Prestin突變致聾的分子病理機(jī)制,還可以在Prestin功能的基礎(chǔ)研究上有新的突破,因為Prestin蛋白在耳蝸放大功能中的基礎(chǔ)研究雖然取得了很大進(jìn)展,但也存在許多尚未解決的重要問題,我們準(zhǔn)備制作的小鼠模型將在解決這些問題中起到關(guān)鍵作用。 實驗方法：第一,我們設(shè)計并構(gòu)建了基因敲入的打靶載體；第二,通過電轉(zhuǎn)化的方式,把線性化好的載體電轉(zhuǎn)化到胚胎干細(xì)胞(Embryonic Stem cells, ES)中,用含有G418的培養(yǎng)基篩選培養(yǎng)ES細(xì)胞,細(xì)胞長到一定的數(shù)量時,收集細(xì)胞；第三,提取細(xì)胞的基因組DNA,用先前設(shè)計好的篩選引物進(jìn)行PCR擴(kuò)增篩選,如果同源重組成功,PCR就可以擴(kuò)增出特異的條帶；第四,我們把經(jīng)過篩選得到的ES細(xì)胞通過顯微注射的方式,注射到小鼠囊胚期的胚胎中,最終獲得嵌合體小鼠；第五,通過雜交來確定是否具有生殖系轉(zhuǎn)移；最后,如果得到了具有生殖系轉(zhuǎn)移的小鼠,通過雜交和自交得到純和體后代,進(jìn)行生理、生化、蛋白等方面的分析。 結(jié)果：成功的制作了Prestin Konck-in載體,轉(zhuǎn)染到ES中。通過篩選得到了發(fā)生正確同源重組的克隆,并通過顯微操作技術(shù)將得到的ES細(xì)胞注射到囊胚,最終獲得高嵌合率的小鼠。本研究的工作為進(jìn)一步闡明外毛細(xì)胞中聽覺發(fā)生的分子機(jī)制奠定了基礎(chǔ)。
[Abstract]:The hearing of mammals is very sensitive. This is due to a biological amplifier in the Outer Hair Cells (OHC). This amplifier amplifies the vibration of the basement membrane by 100 times that of the cell body length of the outer hair cell, thus making people hear the sound. The changes in the length of the cell body of the outer hair cells are driven by a certain electroprotein. After the Prestin gene was cloned in 2000, the researchers used gene knockout technique to prove that Prestin is the core gene of the hearing amplification, which is closely related to the deafness disease of human beings. It has been developed so far. There are two Prestin point mutations that cause deafness, but little is known about the molecular pathogenesis of Prestin mutations.
The mutation induced deafness of Prestin in humans has been reported in the literature: a point mutation (IVS2-2A/G) was found at the intersection of second introns and third exons (ATG) in 2003. It was found in the region of Prestin mRNA shear related; in 2007, another point mutation (R150Q) was found in exon Sixth. These two point mutations all cause human hearing. Loss.
In view of human deafness is a relatively high incidence of disease, it is of great value to use the method of making the animal model of the corresponding disease to study the mechanism of the disease. We use the method of gene knock to make the Prestin gene into the mouse model in order to accurately simulate the point on the exon sixth of the Prestin gene that leads to the deafness of human beings. Mutation (R150Q). We can avoid the limitation on the non operability of experimental materials and experimental techniques by analyzing and studying the phenotype of this mouse model. This study chooses a specific point mutation (R150Q) for sixth exons, because it can be used not only to find the molecular pathological mechanism of the deafness caused by Prestin mutation but also to find the molecular mechanism of the deafness. There are new breakthroughs in the basic research on Prestin function, because the basic research of the Prestin protein in the cochlear enlargement has made great progress, but there are many important problems that have not been solved. The mouse model we are preparing to produce will play a key role in solving these problems.
Experimental methods: first, we designed and constructed the targeting vector of gene knocking; second, through the electrical transformation, the linearized carrier was converted into Embryonic Stem cells (ES), and ES cells were screened and cultured with G418 medium, and the cells were collected when the cell was long to a certain number; and third, extract fine. The genomic DNA of the cell is screened by PCR amplification with previously designed primers. If the homologous recombination is successful, the specific bands can be amplified by PCR; fourth, we inject the selected ES cells into the mouse blastocyst embryos by microinjection, and finally get the chimerism mice; fifth, through hybridization. To determine whether or not a reproductive system is transferred; finally, if a mouse with a reproductive system has been transferred, the analysis of physiological, biochemical, protein, and other aspects is carried out by cross and self crossbreeding of pure and body offspring.
Results: the Prestin Konck-in vector was successfully produced and transfected into ES. The correct homologous recombination was obtained by screening, and the ES cells were injected into the blastocyst by micromanipulation, and the mice with high chimerism were finally obtained. The work of this study is to further clarify the molecular mechanism of auditory occurrence in the outer hair cells. Set the foundation.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R764;R-332

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