本文選題：縫隙連接蛋白 + 轉(zhuǎn)基因小鼠��； 參考：《華中科技大學(xué)》2014年博士論文

【摘要】：第一部分 出生后不同時(shí)間點(diǎn)Connexin26基因敲除小鼠模型的建立和鑒定 目的：建立及鑒定出生后不同時(shí)間點(diǎn)Connexin26基因敲除小鼠模型。 方法：通過遺傳學(xué)手段,將Cx26loxp/loxp轉(zhuǎn)基因小鼠與工具鼠Rosa26CreER雜交獲得Cx26lop/WT;Rosa26CreER子一代小鼠。子一代小鼠相互交配,獲得Cx26loxp/loxp; Rosa26CreER子二代小鼠。分別在上述子二代小鼠出生后第1天(P1),第6天(P6)和第12天(P12)給予頸背部單次皮下他莫昔芬(tamoxifen, TMX)注射,獲得出生后不同時(shí)間點(diǎn)Connexin26基因敲除小鼠模型。使用基因分型(genotyping)、蛋白印記(Western Blot)和免疫熒光技術(shù)檢測(cè)小鼠的基因背景和蛋白敲除情況。使用聽性腦干反應(yīng)(auditory brainstem responses, ABR)檢測(cè)小鼠聽力。 結(jié)果：Cx26loxp/loxp;Rosa26CreER小鼠可以穩(wěn)定繁殖,體重和發(fā)育正常,毛色光亮,無明顯退化現(xiàn)象。蛋白印記和免疫熒光技術(shù)確認(rèn)3種不同時(shí)間點(diǎn)打藥的小鼠耳蝸內(nèi)Connexin26被廣泛敲除。ABR結(jié)果顯示P1和P6敲除組小鼠早期出現(xiàn)中重度耳聾,耳聾隨時(shí)間推移稍有加重。P12敲除組小鼠早期聽力損傷較輕,后期呈進(jìn)行性加重的聽力損傷。 結(jié)論：我們成功的建立了出生后不同時(shí)間點(diǎn)Connexin26基因敲除小鼠的動(dòng)物模型。 第二部分 出生后不同時(shí)間點(diǎn)Connexin26基因敲除小鼠耳蝸細(xì)胞長(zhǎng)時(shí)程損傷模式的研究 目的：研究和比較出生后不同時(shí)間點(diǎn)Connexin26小鼠耳蝸毛細(xì)胞損傷、Corti器(organ of Corti, OC)形態(tài)結(jié)構(gòu)變化和繼發(fā)性螺旋神經(jīng)節(jié)細(xì)胞(spiral ganglion cells, SGCs)死亡的模式。 方法：使用基底膜鋪片(n=3)和耳蝸軸位切片蘇木素-伊紅染色(n=3)來觀察P1、P6和P12敲除組小鼠出生后1、3和5個(gè)月時(shí)耳蝸毛細(xì)胞凋亡模式和Corti器形態(tài)變化。采用螺旋神經(jīng)節(jié)計(jì)數(shù)(n=3)的方法,比較上述不同實(shí)驗(yàn)組和對(duì)照組耳蝸螺旋神經(jīng)節(jié)細(xì)胞不同時(shí)間的損傷程度。 結(jié)果：基底膜鋪片顯示,P1和P6敲除組在出生后1個(gè)月耳蝸中回出現(xiàn)毛細(xì)胞大量死亡,隨時(shí)間推移毛細(xì)胞死亡范圍逐漸擴(kuò)大到底回,中回?fù)p傷進(jìn)一步加重。P12敲除組在出生后3個(gè)月出現(xiàn)耳蝸底回的少量毛細(xì)胞損傷。切片HE染色提示,P1組中回Corti隧道;tunnel of Corti, TC)不能開放并隨時(shí)間推移出現(xiàn)支持細(xì)胞(supporting cell,SC)大量死亡,而P6和P12組可以正常打開。上述3組光鏡下未見血管紋(striae vascularis, SV)、外側(cè)壁和其他部位細(xì)胞明顯異常。耳蝸螺旋神經(jīng)節(jié)細(xì)胞計(jì)數(shù)提示,在毛細(xì)胞損傷對(duì)應(yīng)部位,P1和P6敲除組出現(xiàn)繼發(fā)性螺旋神經(jīng)節(jié)細(xì)胞死亡,P12組未見明顯螺旋神經(jīng)節(jié)細(xì)胞死亡。 結(jié)論：出生后Connexin26早期敲除組小鼠毛細(xì)胞、支持細(xì)胞和螺旋神經(jīng)節(jié)細(xì)胞的損傷程度和范圍明顯大于出生后晚期敲除組,提示Connexin26在出生后耳蝸的早期發(fā)育中起到重要作用,而在出生后耳蝸晚期發(fā)育中的作用是可替代的。 第三部分 出生后早期Connexin26敲除小鼠耳蝸細(xì)胞損傷與葡萄糖轉(zhuǎn)運(yùn)障礙的研究 目的：探究出生后早期Connexin26敲除小鼠耳蝸細(xì)胞損傷與葡萄糖轉(zhuǎn)運(yùn)子(glucose transporter, GLUT)變化和細(xì)胞能量代謝的關(guān)系。 方法：使用蛋白印記技術(shù)檢測(cè)1月齡出生后第一天(P1)降低Connexin26表達(dá)組小鼠(n=5)與對(duì)照組(n=5)耳蝸磷酸化AMPK蛋白表達(dá)變化情況。采用實(shí)時(shí)定量PCR技術(shù)檢測(cè)上述小鼠耳蝸不同葡萄糖轉(zhuǎn)運(yùn)子的表達(dá)變化。 結(jié)果：1月齡P1敲除組小鼠和對(duì)照組小鼠耳蝸磷酸化MPK蛋白表達(dá)差異無統(tǒng)計(jì)學(xué)意義。P1敲除組小鼠和對(duì)照組小鼠耳蝸GLUT1、GLUT8和GLUT10在mRNA水平上表達(dá)無統(tǒng)計(jì)學(xué)意義。 結(jié)論：出生后第一天敲除組小鼠耳蝸細(xì)胞損傷可能與細(xì)胞葡萄糖能量代謝障礙無明顯相關(guān)性。出生后降低Connexin26表達(dá)對(duì)耳蝸葡萄糖轉(zhuǎn)運(yùn)子GLUT1、GLUT8和GLUT10表達(dá)無明顯調(diào)節(jié)作用。
[Abstract]:Part one
Establishment and identification of Connexin26 knockout mice at different time points after birth
Objective: to establish and identify a Connexin26 knockout mouse model at different time points after birth.
Methods: through genetic method, the Cx26loxp/loxp transgenic mice were hybridized with the tool mouse Rosa26CreER to obtain Cx26lop/WT; Rosa26CreER offspring mice. The offspring of the offspring were mating each other to obtain Cx26loxp/loxp; the Rosa26CreER son two generation mice. First days after the birth of the above two generation mice (P1), sixth days (P6) and twelfth days (P12) were given to the neck respectively. A single subcutaneous tamoxifen (TMX) injection was used to obtain a Connexin26 knockout mouse model at different time points after birth. The gene typing (genotyping), protein imprint (Western Blot) and immunofluorescence technique were used to detect the gene background and protein knockout of mice. The auditory brainstem response (auditory brainstem responses, auditory) was used. ABR) test the hearing of mice.
Results: Cx26loxp/loxp, Rosa26CreER mice can reproduce steadily, body weight and development are normal, hair color is bright, and there is no obvious degeneration. Protein imprint and immunofluorescence technique confirm that the Connexin26 in the cochlea of mice with 3 different time points is widely knocked out of.ABR, and the results of the early severe deafness in the P1 and P6 knockout mice are in the early stage of the deafness and deafness. Time lapse slightly aggravated the early hearing impairment of.P12 knockout mice.
Conclusion: we successfully established the animal model of Connexin26 knockout mice at different time points after birth.
The second part
Long term damage patterns of cochlear cells in Connexin26 knockout mice at different time points after birth
Objective: To study and compare the damage of the cochlear hair cells in Connexin26 mice, the morphological changes of the Corti (organ of Corti, OC) and the mode of secondary spiral ganglion cells (spiral ganglion cells, SGCs) in different time points at different postnatal points.
Methods: the mode of apoptosis and the morphological changes of the cochlear hair cells in P1, P6 and P12 knockout mice were observed by using basal membrane (n=3) and cochlear axis sectioning with hematoxylin eosin staining (n=3). The spiral ganglion count (n=3) method was used to compare the spiral ganglion of the cochlear cochlear group and the control group with the method of spiral ganglion count (n=3). The degree of damage at different times of the cell.
Results: the basal membrane lay showed that the hair cells in the P1 and P6 knockout groups died in the middle of the cochlea 1 months after birth. The death range of the hair cells gradually expanded to the end as time went on. The middle gyrus damage further aggravated the few hair cell damage in the.P12 knockout group at the 3 month after birth. The slice HE staining suggested that the P1 group returned to C. The Orti tunnel; tunnel of Corti, TC) can not be open and appear to be in large number of death cells (supporting cell, SC) with time, and P6 and P12 groups can be opened normally. The 3 groups of light mirrors have no vascular lines (striae vascularis,), the lateral wall and other parts of the cells are obviously abnormal. Cochlear spiral ganglion cell count suggests, in hair cells In the P1 and P6 knockout group, secondary spiral ganglion cells died, but no obvious spiral ganglion cells were found in P12 group.
Conclusion: the damage degree and scope of mouse hair cells, supporting cells and spiral ganglion cells in the early Connexin26 knockout group were significantly greater than those in the late postnatal knockout group, suggesting that Connexin26 plays an important role in the early development of the cochlea after birth, and the role in the late development of the cochlea after birth is alternative.
The third part
Damage of cochlear cells and impaired glucose transport in Connexin26 knockout mice at early postnatal stage
Objective: To explore the relationship between the damage of the cochlear cells and the changes of the glucose transporter (glucose transporter, GLUT) and the energy metabolism in the cochlear cells of Connexin26 knockout mice.
Methods: the protein imprinting technique was used to detect the changes in the expression of phosphorylated AMPK protein in the cochlea of the Connexin26 expression group (n=5) and the control group (n=5) after 1 month old days of birth (P1). The changes in the expression of different glucose transporters in the cochlea of the mice were detected by real-time quantitative PCR.
Results: there was no significant difference in the expression of Phosphorated MPK protein in the cochlea between the 1 month old P1 knockout mice and the control group. There was no significant difference in the expression of the cochlea GLUT1 in the.P1 knockout mice and the control mice, and the expression of GLUT8 and GLUT10 at the mRNA level was not statistically significant.
Conclusion: the damage of the cochlear cells in the first day knockout group of mice may not be significantly correlated with the glucose energy metabolism disorder. The expression of Connexin26 after birth has no obvious regulating effect on the expression of GLUT1, GLUT8 and GLUT10 in the cochlear glucose transporter.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R764.43

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;Conditional gene manipulation:Cre-ating a new biological era[J];Journal of Zhejiang University-Science B(Biomedicine & Biotechnology);2012年07期

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本文編號(hào)：1950415