本文選題：內(nèi)耳　切入點(diǎn)：間充質(zhì)　出處：《皖南醫(yī)學(xué)院》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:通過建立胚胎期小鼠耳蝸感覺上皮的體外培養(yǎng)模型,研究耳周間充質(zhì)對耳蝸感覺前體細(xì)胞增殖和分化的影響,探討其可能機(jī)制。方法:本實(shí)驗(yàn)采用胚胎期12.5天(E12.5)和13.5天(E13.5)的孕鼠,取其腹中胚胎鼠,在顯微鏡下取出完整的耳蝸感覺上皮,進(jìn)行原代培養(yǎng)。對照組:帶有相鄰間充質(zhì)的耳蝸感覺上皮;實(shí)驗(yàn)照組:不帶間充質(zhì)的單純耳蝸感覺上皮。1).培養(yǎng)E12.5+6DIV或E13.5+5DIV,培養(yǎng)過程中采用倒置顯微鏡下觀察聽覺上皮的形態(tài)及生長狀態(tài),通過Myosin 7a標(biāo)記毛細(xì)胞并對毛細(xì)胞進(jìn)行計數(shù),比較在有、無間充質(zhì)的作用下,E12.5和E13.5的耳蝸感覺上皮的生長發(fā)育情況。2).采用RT-PCR技術(shù)測定Sox2和Math1基因的表達(dá)量。實(shí)驗(yàn)組和對照組的聽覺上皮分別體外培養(yǎng)24h和48h,測定在不同培養(yǎng)階段實(shí)驗(yàn)組和對照組耳蝸感覺上皮中Sox2及Math1基因的表達(dá)差異。3).在E12.5+6 DIV培養(yǎng)過程中加入6μg/ml BrdU,以BrdU標(biāo)記增殖細(xì)胞,Myosin 7a標(biāo)記毛細(xì)胞,定量研究間充質(zhì)對耳蝸感覺前體細(xì)胞增殖和分化的影響。結(jié)果:1.實(shí)驗(yàn)組(去間充質(zhì)組)中,E12.5和E13.5聽覺上皮分別培養(yǎng)6天和5天,其整體形態(tài)和細(xì)胞排列較對照組(保留間充質(zhì)組)均有一定差異。對照組E12.5天聽覺上皮在體外無血清培養(yǎng)過程中,形態(tài)發(fā)育良好,細(xì)胞排列呈一定規(guī)律性,細(xì)胞間的連接較為緊密,呈相對規(guī)則的弧形。內(nèi)毛細(xì)胞層和外毛細(xì)胞層之間的見有間隙,內(nèi)毛細(xì)胞為1-2層,外毛細(xì)胞為4-6層,內(nèi)毛細(xì)胞層相對于外毛細(xì)胞層較為紊亂。實(shí)驗(yàn)組E12.5聽覺上皮生長緩慢,細(xì)胞間排列明顯紊亂,無規(guī)則,細(xì)胞形態(tài)有一定的差異。毛細(xì)胞計數(shù)示:對照組723.75±79.54,實(shí)驗(yàn)組為515.35±45.63,兩組有顯著統(tǒng)計學(xué)(P0.05)。E13.5對照組聽覺上皮內(nèi)層毛細(xì)胞排列1-2層,排列整齊而緊密,外毛細(xì)胞也具有一定的規(guī)律性排列,尤其最外1-2層外毛細(xì)胞形態(tài)清晰可見,排列規(guī)整,實(shí)驗(yàn)組E13.5耳蝸感覺上皮毛細(xì)胞排列紊亂,連接較為疏松,內(nèi)層毛細(xì)胞和外層毛細(xì)胞之間的界限不清晰,毛細(xì)胞計數(shù)示:對照組為1216.67±183.47.實(shí)驗(yàn)組為1097.15±204.67,實(shí)驗(yàn)組和對照組毛細(xì)胞計數(shù)雖無明顯統(tǒng)計學(xué)差異,但實(shí)驗(yàn)組整體細(xì)胞數(shù)低于對照組。2.E12.5+24h聽覺上皮中,對照組Math1基因表達(dá)和實(shí)驗(yàn)組組之間無統(tǒng)計學(xué)差異(P0.05);E13.5+24h、E12.5+48h和E13.5+48h聽覺上皮中,對照組Math1基因表達(dá)高于實(shí)驗(yàn)組,實(shí)驗(yàn)組和對照組有統(tǒng)計學(xué)差異(P0.05)。E12.5+24h、E13.5+24h、E12.5+48h和E13.5+48h聽覺上皮中,對照組Sox2基因表達(dá)高于實(shí)驗(yàn)組,實(shí)驗(yàn)組和對照組有統(tǒng)計學(xué)差異(P0.05)。3.E12.5聽覺上皮體外培養(yǎng)6天后,BrdU+Myosin 7a雙標(biāo)陽性細(xì)胞:對照組18.9±2.77;實(shí)驗(yàn)組:13.7±2.91。實(shí)驗(yàn)組和對照組有統(tǒng)計學(xué)差異(P0.05)。結(jié)論:E12.5耳周間充質(zhì)能促進(jìn)耳蝸感覺前體細(xì)胞增殖,但更關(guān)鍵的是促進(jìn)耳蝸感覺前體細(xì)胞向毛細(xì)胞分化。胚胎鼠聽覺上皮形態(tài)發(fā)生過程中,耳周間充質(zhì)促進(jìn)Math1和Sox2基因表達(dá)上調(diào)。
[Abstract]:Objective: to study the effect of periauricular mesenchyma on the proliferation and differentiation of sensory precursor cells in mouse cochlea in vitro by establishing an in vitro culture model of mouse cochlear sensory epithelium. Methods: the pregnant mice of 12.5 and 13.5 days of embryonic stage were used in this study. The embryonic mice in their abdomen were taken out and the complete sensory epithelium of cochlea was removed under microscope. Primary culture. Control group: cochlear sensory epithelium with adjacent mesenchymal cells; In the experimental group, the sensory epithelium of cochlea was cultured without mesenchyma. The morphology and growth state of auditory epithelium were observed under inverted microscope. Hair cells were labeled with Myosin 7a and the hair cells were counted. There are more, The growth and development of cochlear sensory epithelium of E12.5 and E13.5 under the action of non-mesenchymal cells. The expression of Sox2 and Math1 genes was measured by RT-PCR technique. The auditory epithelium of the experimental group and the control group were cultured in vitro for 24 hours and 48 hours, respectively. The expression of Sox2 and Math1 genes in the sensory epithelium of the cochlea of the experimental group and the control group was different from that in the control group. 6 渭 g / ml BrdU was added into the culture process of E12.56 DIV, and the hair cells were labeled with BrdU labeled with Myosin 7a. The effects of mesenchymal cells on the proliferation and differentiation of cochlear sensory precursor cells were quantitatively studied. Results 1. In the experimental group (demesenchymal group), the auditory epithelium of E12.5 and E13.5 were cultured for 6 days and 5 days, respectively. The morphology and cell arrangement of the E12.5 day auditory epithelium in the control group were different from those in the control group (retention of mesenchymal cells), and the morphology of the auditory epithelium in the control group was well developed during the serum-free culture in vitro, and the cell arrangement was regular. There was a gap between the inner hair cell layer and the outer hair cell layer. The inner hair cell had 1-2 layers, and the outer hair cell had 4-6 layers. The inner hair cell layer was more disordered than the outer hair cell layer. In the experimental group, the auditory epithelium of E12.5 grew slowly, the intercellular arrangement was obviously disordered, and there was no rule. The number of hair cells was 723.75 鹵79.54 in the control group and 515.35 鹵45.63 in the experimental group. Especially, the outermost 1-2 layers of outer hair cells were clearly visible and arranged regularly. In the experimental group, the sensory hair cells of the E13.5 cochlea were arranged in disorder, the connection was looser, and the boundary between the inner hair cells and the outer hair cells was not clear. The number of hair cells in the control group was 1216.67 鹵183.47.The number of hair cells in the experimental group was 1097.15 鹵204.67. Although there was no significant difference between the experimental group and the control group, the total number of hair cells in the experimental group was lower than that in the control group. There was no significant difference between the expression of Math1 gene in the control group and that in the experimental group. The expression of Math1 gene in the auditory epithelium of the control group was higher than that in the experimental group. The expression of Math1 gene in the control group was higher than that in the experimental group. There was a significant difference between the experimental group and the control group. Sox2 gene expression in control group was higher than that in experimental group. There were significant differences between the experimental group and the control group (P 0.05N. 3.E12.5 auditory epithelium cultured in vitro for 6 days): BrdU Myosin 7a double labeled positive cells: control group 18.9 鹵2.77; experimental group 13.7 鹵2.91.There was significant difference between the experimental group and the control group (P0.050.Conclusion: 1. 05% E 12.5 ear filling can promote cochlea. Sensory precursor cell proliferation, However, it is more important to promote the differentiation of cochlear sensory precursor cells into hair cells. During the morphogenesis of auditory epithelium in embryonic mice, mesenchymal mesenchyma promotes the up-regulation of Math1 and Sox2 gene expression.
【學(xué)位授予單位】：皖南醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R764.43

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 蘇麗萍;王英;肖明振;余擎;阮禹松;;牙齒發(fā)育早期間充質(zhì)細(xì)胞的差異蛋白分析[J];臨床口腔醫(yī)學(xué)雜志;2008年05期

2 楊淑珍;;人的淋巴發(fā)育[J];四川解剖學(xué)雜志;1983年03期

3 蔡光浩,楊鎮(zhèn)洙,陳華勇;骨缺損修復(fù)過程中間充質(zhì)細(xì)胞的成骨作用[J];延邊大學(xué)醫(yī)學(xué)學(xué)報;2000年02期

4 黃彬;姜儻;;腎纖維化中上皮間充質(zhì)轉(zhuǎn)化的最新研究進(jìn)展[J];國際檢驗(yàn)醫(yī)學(xué)雜志;2006年03期

5 張鋒;鄧旭亮;梅芳;王新知;;空間微重力環(huán)境對人牙髓間充質(zhì)細(xì)胞的影響初探[J];科技導(dǎo)報;2007年02期

6 鄧蔓菁;陳小紅;孫雅娟;聶鑫;劉魯川;安建平;;牙源性上皮在外胚間充質(zhì)細(xì)胞向牙齒樣結(jié)構(gòu)形成中的作用[J];第三軍醫(yī)大學(xué)學(xué)報;2010年02期

7 胡逢春;陶謙;;上皮—間充質(zhì)轉(zhuǎn)化的生物學(xué)作用[J];國際口腔醫(yī)學(xué)雜志;2011年02期

8 鄧蔓菁,金巖,史俊南,劉源,趙宇;胚胎面突外胚間充質(zhì)細(xì)胞的培養(yǎng)鑒定及向平滑肌細(xì)胞的自分化研究[J];中國修復(fù)重建外科雜志;2003年05期

9 周澤淵,金巖,史俊南,張建平,軒昆,于淑湘;哺乳動物外胚間充質(zhì)細(xì)胞的表型和形態(tài)學(xué)研究[J];牙體牙髓牙周病學(xué)雜志;2004年05期

10 王立平,黨耕町;未分化間充質(zhì)細(xì)胞成骨作用的研究進(jìn)展[J];中華外科雜志;1998年02期

相關(guān)會議論文 前10條

1 閆征斌;劉磊;田衛(wèi)東;林云鋒;李志勇;;第一鰓弓外胚間充質(zhì)細(xì)胞牙向分化上調(diào)基因組抑制消減文庫的建立[A];2007年第七次全國牙體牙髓病學(xué)學(xué)術(shù)會議論文集[C];2007年

2 劉必成;;腎小管上皮間充質(zhì)轉(zhuǎn)變及其逆轉(zhuǎn)的機(jī)制研究進(jìn)展[A];中華醫(yī)學(xué)會腎臟病學(xué)分會2006年學(xué)術(shù)年會專題講座[C];2006年

3 李曉東;田衛(wèi)東;江宏兵;陳希哲;湯煒;鄭曉輝;劉磊;;外源性生長因子誘導(dǎo)胎鼠牙胚間充質(zhì)細(xì)胞牙向分化的實(shí)驗(yàn)研究[A];2004年中國口腔頜面修復(fù)重建外科學(xué)術(shù)會議論文匯編[C];2004年

4 劉必成;;上皮間充質(zhì)轉(zhuǎn)變(EMT)在腎臟纖維化中的作用[A];中華醫(yī)學(xué)會腎臟學(xué)分會2004年年會暨第二屆全國中青年腎臟病學(xué)術(shù)會議專題講座匯編[C];2004年

5 廖禮姝;鄭謙;盧勝軍;石冰;;不同濃度地塞米松對腭胚突間充質(zhì)細(xì)胞作用規(guī)律的研究[A];第七屆全國唇腭裂學(xué)術(shù)會議論文集[C];2009年

6 鄧蔓菁;劉魯川;陳小紅;安建平;匡桂英;AJ Smith;;多種生長因子對胚胎面突外胚間充質(zhì)細(xì)胞向成牙本質(zhì)樣細(xì)胞分化的誘導(dǎo)作用[A];2007年第七次全國牙體牙髓病學(xué)學(xué)術(shù)會議論文集[C];2007年

7 江宏兵;田衛(wèi)東;陳希哲;湯煒;劉磊;李曉東;;誘導(dǎo)顱神經(jīng)嵴向第一鰓弓外胚間充質(zhì)細(xì)胞分化的實(shí)驗(yàn)研究[A];2004年中國口腔頜面修復(fù)重建外科學(xué)術(shù)會議論文匯編[C];2004年

8 李曉東;田衛(wèi)東;江宏兵;陳希哲;劉磊;湯煒;鄭曉輝;;胎鼠牙胚間充質(zhì)細(xì)胞生物學(xué)特性的實(shí)驗(yàn)觀察[A];2004年中國口腔頜面修復(fù)重建外科學(xué)術(shù)會議論文匯編[C];2004年

9 鄭黎薇;葉玲;張艷;周學(xué)東;;人牙間充質(zhì)細(xì)胞的成牙誘導(dǎo)能力[A];全國第八次牙體牙髓病學(xué)學(xué)術(shù)會議論文匯編[C];2011年

10 張建平;金巖;鄧蔓菁;張光東;周澤淵;趙文明;;外胚間充質(zhì)干細(xì)胞的可塑性與應(yīng)用研究[A];全面建設(shè)小康社會：中國科技工作者的歷史責(zé)任——中國科協(xié)2003年學(xué)術(shù)年會論文集（上）[C];2003年

相關(guān)博士學(xué)位論文 前10條

1 閆飛;GLP-1抑制內(nèi)皮細(xì)胞間充質(zhì)化及Compound 21調(diào)控骨骼肌微循環(huán)灌注的機(jī)制研究[D];山東大學(xué);2015年

2 楊海平;TGF-β經(jīng)典信號通路及相關(guān)基因調(diào)控非小細(xì)胞肺癌細(xì)胞上皮—間充質(zhì)轉(zhuǎn)化和侵襲轉(zhuǎn)移機(jī)制研究[D];蘇州大學(xué);2015年

3 黃峰;小鼠磨牙間充質(zhì)成牙潛能的關(guān)鍵分子[D];福建師范大學(xué);2015年

4 閆征斌;第一鰓弓外胚間充質(zhì)細(xì)胞牙向分化差異基因的初步篩選[D];四川大學(xué);2005年

5 楊林;胞膜窖/窖蛋白在小鼠牙發(fā)育過程中作用的初步研究[D];四川大學(xué);2007年

6 連耀國;血管內(nèi)皮生長因子對TGF-β1誘導(dǎo)的腎小管上皮—間充質(zhì)細(xì)胞轉(zhuǎn)化的作用及機(jī)制探討[D];北京協(xié)和醫(yī)學(xué)院;2010年

7 高瞻;基于細(xì)胞膜片技術(shù)構(gòu)建組織工程化骨實(shí)驗(yàn)研究[D];第四軍醫(yī)大學(xué);2008年

8 周澤淵;頜突外胚間充質(zhì)干細(xì)胞的可塑性研究[D];第四軍醫(yī)大學(xué);2004年

9 周劍鋒;后腎間充質(zhì)細(xì)胞對大鼠缺血再灌注急性腎小管損傷修復(fù)作用的實(shí)驗(yàn)研究[D];第三軍醫(yī)大學(xué);2008年

10 畢凌云;胚胎后腎間充質(zhì)細(xì)胞轉(zhuǎn)染uPA基因移植治療單側(cè)輸尿管梗阻大鼠的實(shí)驗(yàn)研究[D];中南大學(xué);2008年

相關(guān)碩士學(xué)位論文 前10條

1 曾永芬;Wnt5a和Igf1對小鼠磨牙間充質(zhì)誘導(dǎo)成牙潛能的影響[D];福建師范大學(xué);2015年

2 王玉華;FUT6在TGF-β_1誘導(dǎo)的腎小管上皮—間充質(zhì)轉(zhuǎn)化中的作用研究[D];天津醫(yī)科大學(xué);2015年

3 李偉;間充質(zhì)對小鼠聽覺上皮發(fā)育的影響[D];皖南醫(yī)學(xué)院;2015年

4 孫吏聰;胰島素樣生長因子誘導(dǎo)外胚間充質(zhì)細(xì)胞向成骨細(xì)胞分化的實(shí)驗(yàn)研究[D];昆明醫(yī)學(xué)院;2008年

5 王曉英;維甲酸誘導(dǎo)腭裂小鼠出生后未分化腭間充質(zhì)細(xì)胞定位研究[D];大連醫(yī)科大學(xué);2011年

6 姜瑩;白蛋白超載對人腎小管上皮細(xì)胞—間充質(zhì)轉(zhuǎn)化及糖皮質(zhì)激素耐藥相關(guān)因素的影響[D];中南大學(xué);2009年

7 李旭艷;豬胚胎后腎帽狀間充質(zhì)細(xì)胞的發(fā)育及其對腎小管上皮細(xì)胞—間充質(zhì)轉(zhuǎn)分化的影響[D];中國人民解放軍醫(yī)學(xué)院;2013年

8 周強(qiáng)平;骨橋蛋白靶向干擾對環(huán)孢素A誘導(dǎo)的腎小管上皮細(xì)胞向間充質(zhì)轉(zhuǎn)變的影響[D];南京醫(yī)科大學(xué);2011年

9 彭U，

本文編號：1644648