本文選題：順鉑 + 耳蝸　； 參考：《遼寧醫(yī)學(xué)院》2011年碩士論文

【摘要】：目的 建立新生昆明小鼠離體順鉑耳毒性模型,研究順鉑對(duì)離體培養(yǎng)小鼠耳蝸毛細(xì)胞凋亡和鈣蛋白酶(calpain)表達(dá)的影響,為進(jìn)一步探討順鉑的耳毒性機(jī)制及其防護(hù)辦法提供實(shí)驗(yàn)依據(jù)。 方法 分離生后3～5 d的昆明小鼠耳蝸基底膜,在含有1%牛血清白蛋白的Dulbecco改良Eagle培養(yǎng)基/ F12(Dulbecco's Modified Eagle Media: Nutrient Mixture F-12,DMEM / F12)中培養(yǎng)24h,然后棄去原培養(yǎng)基。對(duì)照組加入2ml新鮮培養(yǎng)基,順鉑組分別加入含有不同濃度順鉑(4μg/ml、8μg/ml、16μg/ml和64μg/ml )的新鮮培養(yǎng)基,繼續(xù)培養(yǎng)24h。采用異硫氰酸四甲基羅丹明(tetramethylrhodamine isothiocyanate,TRITC)標(biāo)記的鬼筆環(huán)肽熒光染色方法觀察小鼠耳蝸毛細(xì)胞的形態(tài)改變,耳蝸毛細(xì)胞計(jì)數(shù)檢測(cè)毛細(xì)胞缺失情況;同時(shí)應(yīng)用Hoechst 33258熒光染色方法觀察耳蝸毛細(xì)胞的凋亡,耳蝸凋亡毛細(xì)胞計(jì)數(shù)檢測(cè)毛細(xì)胞凋亡率;并采用免疫熒光染色和免疫印跡技術(shù)檢測(cè)calpain 1(μ-calpain)和calpain 2(m-calpain)在小鼠耳蝸毛細(xì)胞的表達(dá)。 結(jié)果 1. TRITC-鬼筆環(huán)肽熒光染色結(jié)果顯示,對(duì)照組耳蝸內(nèi)毛細(xì)胞(inner hair cell,IHC)和外毛細(xì)胞(outer hair cell,OHC)均結(jié)構(gòu)完好,纖毛排列有序。順鉑組小鼠耳蝸毛細(xì)胞出現(xiàn)纖毛倒伏、排列紊亂等形態(tài)改變。耳蝸毛細(xì)胞計(jì)數(shù)結(jié)果顯示,當(dāng)順鉑濃度為4μg/ml時(shí),IHC和OHC缺失率均明顯增大,與對(duì)照組比較有顯著性差異(P0.01);并且隨著順鉑濃度的增高,IHC和OHC缺失率亦顯著增大,呈現(xiàn)明顯的量效關(guān)系(P0.01)。 2. Hoechst 33258熒光染色結(jié)果顯示,對(duì)照組小鼠耳蝸毛細(xì)胞的細(xì)胞核呈圓形,淡藍(lán)色,核內(nèi)染色質(zhì)分布均勻。順鉑組小鼠耳蝸毛細(xì)胞的細(xì)胞核則出現(xiàn)固縮,核內(nèi)染色質(zhì)由于濃集而呈亮藍(lán)色,核呈分葉、碎片狀;并且隨著順鉑濃度的增高,致密濃染和破碎的細(xì)胞核亦顯著增多。耳蝸凋亡毛細(xì)胞計(jì)數(shù)結(jié)果顯示,當(dāng)順鉑濃度為4μg/ml時(shí),毛細(xì)胞凋亡率明顯增大,與對(duì)照組比較有顯著性差異(P0.01);并且隨著順鉑濃度的增高,毛細(xì)胞凋亡率亦顯著增大,呈現(xiàn)明顯的量效關(guān)系(P0.01)。 3. Calpain免疫熒光染色結(jié)果顯示,μ-calpain和m-calpain在對(duì)照組耳蝸毛細(xì)胞均呈微弱紅色熒光;不同濃度順鉑組耳蝸毛細(xì)胞μ-calpain和m-calpain紅色熒光均較對(duì)照組有所增強(qiáng),但順鉑組間μ-calpain熒光強(qiáng)度基本相同,而m-calpain熒光則隨順鉑濃度的增高而明顯增強(qiáng)。 4. Calpain免疫印跡檢測(cè)結(jié)果顯示,對(duì)照組有μ-calpain和m-calpain的微弱表達(dá);不同濃度順鉑組μ-calpain和m-calpain的表達(dá)均較對(duì)照組明顯增強(qiáng)(P0.01),但順鉑組間μ-calpain的表達(dá)無顯著性差異,而m-calpain的表達(dá)則隨順鉑濃度的增高而明顯增強(qiáng)(P0.01)。 結(jié)論 應(yīng)用耳蝸螺旋器離體培養(yǎng)技術(shù)可以建立新生昆明小鼠離體順鉑耳毒性模型;順鉑可通過凋亡機(jī)制對(duì)小鼠耳蝸毛細(xì)胞造成損傷,并且順鉑可影響calpain在離體培養(yǎng)小鼠耳蝸毛細(xì)胞的表達(dá),隨著順鉑濃度的增高,m-calpain的表達(dá)亦逐漸增強(qiáng),提示順鉑可通過calpain通路誘導(dǎo)小鼠耳蝸毛細(xì)胞的凋亡,進(jìn)一步證實(shí)了凋亡可能是順鉑的耳毒性機(jī)制之一。
[Abstract]:Purpose

To study the effect of cisplatin on apoptosis and calpain expression of cochlear hair cells in vitro and to provide experimental basis for further exploring the mechanism of ototoxicity of cisplatin .

method

The cochlear basement membrane was cultured in Dulbecco ' s Modified Eagle Medium / F12 ( Dulbecco ' s Modified Eagle Media , Dulbecco ' s Modified Eagle Media , Dulbecco ' s Modified Eagle Media , Dulbecco ' s Modified Eagle Media , Dulbecco ' s Modified Eagle Media , Dulbecco ' s Modified Eagle Media : Dulbecco ' s Modified Eagle Media : Fluorescence Mixture F - 12 , DMEM / F12 ) for 24 h .

Results

1 . The results showed that both the inner hair cell ( IHC ) and outer hair cell ( OHC ) of the control group were intact and the ciliary arrangement was orderly . When the concentration of cisplatin was 4 渭g / ml , the level of IHC and OHC deletion increased significantly ( P0.01 ) , and the IHC and OHC deletion rate increased significantly with the increase of cisplatin concentration ( P0.01 ) .

2 . The results showed that the nuclei of the cochlear hair cells in the control group were round , pale blue and homogeneous in the nucleus . The nuclei of the cochlear hair cells in the cisplatin group were significantly increased . The apoptosis rate of the hair cells increased significantly with the increase of cisplatin concentration ( P0.01 ) .

3 . Calpain immunofluorescence staining showed that 渭 - calpain and m - calpain were in weak red fluorescence in the control group . The fluorescence intensity of 渭 - calpain and m - calpain in the cochlear hair cells in the cisplatin group was significantly higher than that in the control group , but the fluorescence intensity of 渭 - calpain in the cisplatin group was almost the same , while the m - calpain fluorescence increased obviously with the increase of the platinum concentration .

4 . Calpain Western blot showed that the expression of 渭 - calpain and m - calpain in the control group was significantly higher than that in the control group ( P0.01 ) , but the expression of 渭 - calpain and m - calpain in the cisplatin group was significantly higher than that in the control group ( P0.01 ) .

Conclusion

It is suggested that cisplatin can induce apoptosis of cochlear hair cells in mice by calpain pathway , and it is suggested that cisplatin can induce apoptosis of cochlear hair cells in mice by calpain pathway , and it is further confirmed that apoptosis may be one of the ototoxicity mechanisms of cisplatin .

【學(xué)位授予單位】：遼寧醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R764.43

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 王蘋;杜波;杜寶東;;鈣蛋白酶活性及其誘導(dǎo)內(nèi)耳細(xì)胞死亡[J];國際耳鼻咽喉頭頸外科雜志;2006年06期

2 路虹,劉硯星,薛麗華,楊逸,劉輝,張喜琴;順鉑對(duì)豚鼠耳蝸毛細(xì)胞損害的形態(tài)及機(jī)理研究[J];河北醫(yī)科大學(xué)學(xué)報(bào);2003年02期

3 許濤;王愛梅;劉雙月;牟華;;卡那霉素對(duì)體外培養(yǎng)小鼠耳蝸毛細(xì)胞的損傷作用[J];江西醫(yī)學(xué)院學(xué)報(bào);2009年11期

4 郭曄;郭慕依;;順鉑的細(xì)胞和分子藥理學(xué)研究進(jìn)展[J];復(fù)旦學(xué)報(bào)(醫(yī)學(xué)版);2006年01期

5 費(fèi)文彬;江洋;吳展元;;順鉑耳毒性的研究[J];聽力學(xué)及言語疾病雜志;2007年06期

6 孫偉霞;王愛梅;包翠芬;惠麗杰;常志杰;;順鉑對(duì)小鼠耳蝸毒性及caspase-3表達(dá)的影響[J];西安交通大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2009年05期

7 鄧姍;鉑類的耳毒性[J];腫瘤學(xué)雜志;2003年01期

8 孫偉霞;王愛梅;;順鉑耳毒性機(jī)制及其防護(hù)的研究進(jìn)展[J];醫(yī)學(xué)綜述;2008年22期

9 孫偉霞;王愛梅;包翠芬;惠麗杰;常志杰;;順鉑對(duì)小鼠耳蝸螺旋神經(jīng)節(jié)細(xì)胞凋亡及caspase-3表達(dá)的影響[J];中國應(yīng)用生理學(xué)雜志;2010年03期

10 丁大連;亓衛(wèi)東;張梅;王蘋;蔣海燕;Richard Salvi;;順鉑及其耳毒性[J];中華耳科學(xué)雜志;2008年02期

，

本文編號(hào)：1780892