本文選題：喉腫瘤　切入點(diǎn)：鱗狀細(xì)胞癌　出處：《山西醫(yī)科大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：【目的】從前期應(yīng)用microRNA基因芯片技術(shù)檢測(cè)喉鱗癌癌組織及癌旁組織差異性表達(dá)microRNA譜中，通過qRT-PCR技術(shù)發(fā)現(xiàn)喉鱗狀細(xì)胞癌石蠟包埋組織中has-mir-93-5p的表達(dá)較對(duì)應(yīng)癌旁切緣組織明顯上調(diào)。擬利用體外轉(zhuǎn)染hsa-miR-93-5p inbibitor，探討其對(duì)人喉鱗癌細(xì)胞株Hep-2細(xì)胞細(xì)胞功能的影響。 【方法】 1、取對(duì)數(shù)生長(zhǎng)期的體外培養(yǎng)人喉鱗狀細(xì)胞癌Hep-2細(xì)胞株進(jìn)行試驗(yàn)。 2、將hsa-miR-93-5p Inhibitor序列（即hsa-miR-93-5p mimisc反向互補(bǔ)序列）轉(zhuǎn)染至Hep-2細(xì)胞中作為Inhibitor轉(zhuǎn)染組，以未經(jīng)任何轉(zhuǎn)染處理的Hep-2細(xì)胞作為空白對(duì)照組。 3、檢測(cè)hsa-miR-93-5p Inhibitor轉(zhuǎn)染組和空白對(duì)照組Hep-2細(xì)胞增殖能力，采用MTS法、平板克隆形成實(shí)驗(yàn)。 4、采用Edu法檢測(cè)細(xì)胞分裂增殖能力。 5、采用細(xì)胞遷移實(shí)驗(yàn)，Transwell侵襲實(shí)驗(yàn)檢測(cè)Inhibitor轉(zhuǎn)染組和空白對(duì)照組細(xì)胞遷移侵襲能力。 6、采用PI染料法檢測(cè)細(xì)胞周期。 7、采用Annexin V-FITC/PI雙染法檢測(cè)細(xì)胞凋亡。 8、采用SPSS13.0軟件對(duì)所得數(shù)據(jù)進(jìn)行統(tǒng)計(jì)學(xué)分析，，計(jì)量資料采用均值±標(biāo)準(zhǔn)差表示，對(duì)結(jié)果進(jìn)行t檢驗(yàn)，P0.05認(rèn)為差異有統(tǒng)計(jì)學(xué)意義。 【結(jié)果】 1、細(xì)胞轉(zhuǎn)染48h后，熒光顯微鏡檢測(cè)Hep-2細(xì)胞中miR-93轉(zhuǎn)染效率。隨機(jī)計(jì)數(shù)100個(gè)細(xì)胞，其中有熒光的細(xì)胞約占90%，符合繼續(xù)實(shí)驗(yàn)的標(biāo)準(zhǔn)。 2、MTS法檢測(cè)顯示hsa-miR-93-5p Inhibitor轉(zhuǎn)染的Hep-2細(xì)胞抑制率較空白對(duì)照組抑制率明顯升高，并且隨著轉(zhuǎn)染后培養(yǎng)時(shí)間的延長(zhǎng)，細(xì)胞抑制率更加明顯(P0.05)，但增值率相比變化不明顯。 3、不同處理的Hep-2細(xì)胞Edu法檢測(cè)細(xì)胞增殖，顯示Inhibitor轉(zhuǎn)染組較空白對(duì)照組明顯降低，細(xì)胞生長(zhǎng)活力得到明顯抑制。 4、克隆形成實(shí)驗(yàn)結(jié)果示Inhibitor轉(zhuǎn)染組的存活分?jǐn)?shù)明顯低于空白對(duì)照組(P0.05)。 5、流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期結(jié)果，hsa-miR-93-5p抑制物轉(zhuǎn)染的Hep-2細(xì)胞處于G2期的細(xì)胞數(shù)量（9.62±0.71）明顯高于空白對(duì)照組（0.04±0.06），差異具有統(tǒng)計(jì)學(xué)意義（P0.05）。 6、流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡結(jié)果，Hep-2細(xì)胞Inhibitor轉(zhuǎn)染組總凋亡率（1.97±0.11）明顯高于空白對(duì)照組（1.49±0.11），差異具有統(tǒng)計(jì)學(xué)意義（P0.05）。 7、細(xì)胞遷移實(shí)驗(yàn)結(jié)果，Hep-2細(xì)胞Inhibitor轉(zhuǎn)染組細(xì)胞遷移數(shù)均明顯低于Hep-2細(xì)胞空白對(duì)照組，差異具有統(tǒng)計(jì)學(xué)意義（P0.05）。 8、Transwell侵襲實(shí)驗(yàn)檢測(cè)結(jié)果，Hep-2細(xì)胞Inhibitor轉(zhuǎn)染組細(xì)胞侵襲能力（101.5±11.84）明顯低于空白對(duì)照組（158.67±20.82），差異具有統(tǒng)計(jì)學(xué)意義（P0.05）。 【結(jié)論】 1、轉(zhuǎn)染hsa-miR-93-5p Inhibitor序列的喉鱗癌Hep-2細(xì)胞的增殖與遷移侵襲能力明顯低于未經(jīng)任何處理的喉鱗癌Hep-2細(xì)胞，提示hsa-miR-93-5p可能是喉鱗癌的發(fā)病機(jī)制中一個(gè)重要分子 2、轉(zhuǎn)染hsa-miR-93-5p Inhibitor序列可以抑制喉鱗癌Hep-2細(xì)胞的生長(zhǎng)，使細(xì)胞阻滯于G2期（P0.05）。提示hsa-miR-93-5p可能通過調(diào)控喉鱗癌細(xì)胞的細(xì)胞周期進(jìn)而發(fā)揮其生物學(xué)功能。
[Abstract]:[objective] to detect the differential expression of microRNA in laryngeal squamous cell carcinoma (LSCC) and adjacent tissues by using microRNA gene chip technique. The expression of has-mir-93-5p in paraffin embedded laryngeal squamous cell carcinoma (LSCC) was found to be significantly up-regulated than that in adjacent incisor tissue by qRT-PCR technique. The effect of hsa-miR-93-5p inbibitor transfection on the function of Hep-2 cell line was studied by in vitro transfection of hsa-miR-93-5p into human laryngeal squamous cell carcinoma (LSCC) cell line. [methods]. 1. Human laryngeal squamous cell carcinoma (Hep-2) cell line was cultured in logarithmic growth period. 2. Hsa-miR-93-5p Inhibitor sequence (hsa-miR-93-5p mimisc reverse complementary sequence) was transfected into Hep-2 cells as Inhibitor transfection group, and Hep-2 cells without any transfection were used as blank control group. 3. The proliferative ability of Hep-2 cells in hsa-miR-93-5p Inhibitor transfection group and blank control group was detected by MTS assay. 4. The ability of cell division and proliferation was detected by Edu assay. 5. Transwell invasion assay was used to detect the ability of cell migration and invasion in Inhibitor transfection group and blank control group. 6. Pi dye method was used to detect cell cycle. 7. Apoptosis was detected by Annexin V-FITC / Pi double staining. 8. SPSS13.0 software was used to analyze the data. The measurement data was expressed by mean 鹵standard deviation, and the difference was statistically significant by t test (P0.05). [results]. 1. After 48 hours of transfection, the transfection efficiency of miR-93 in Hep-2 cells was detected by fluorescence microscope. 2the inhibition rate of Hep-2 cells transfected with hsa-miR-93-5p Inhibitor was significantly higher than that of the control group, and with the extension of culture time after transfection, the inhibition rate of Hep-2 cells was more obvious than that of the control group, but there was no significant change in the proliferation rate. 3. The proliferation of Hep-2 cells was detected by Edu assay with different treatments. The results showed that the Inhibitor transfection group was significantly lower than the blank control group, and the cell growth activity was significantly inhibited. 4. The results of clone formation test showed that the survival fraction of Inhibitor transfected group was significantly lower than that of control group (P 0.05). 5. The cell cycle of Hep-2 cells transfected with hsa-miR-93-5p inhibitor was significantly higher than that of control group (9.62 鹵0.71), which was significantly higher than that of control group (0.04 鹵0.06), and the difference was statistically significant (P 0.05). 6. The apoptotic rate of Hep-2 cells in Inhibitor transfection group was 1.97 鹵0.11, which was significantly higher than that in control group (1.49 鹵0.11), and the difference was statistically significant (P 0.05). 7. The results of cell migration assay showed that the number of cell migration in Inhibitor transfected group was significantly lower than that in Hep-2 cell blank control group, and the difference was statistically significant (P 0.05). The invasive ability of Hep-2 cells transfected with Inhibitor was 101.5 鹵11.84, which was significantly lower than that of control group (158.67 鹵20.82), and the difference was statistically significant (P 0.05). [conclusion]. 1. The proliferation and migration ability of Hep-2 cells transfected with hsa-miR-93-5p Inhibitor sequence was significantly lower than that of untreated Hep-2 cells, suggesting that hsa-miR-93-5p might be an important molecule in the pathogenesis of laryngeal squamous cell carcinoma. 2. Transfection of hsa-miR-93-5p Inhibitor sequence can inhibit the growth of laryngeal squamous cell carcinoma Hep-2 cells and block the cells at G2 phase P0.05, suggesting that hsa-miR-93-5p may play its biological function by regulating the cell cycle of laryngeal squamous carcinoma cells.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R739.65

【參考文獻(xiàn)】

相關(guān)期刊論文 前6條

1 王蘋;付濤;王緒銳;祝威;;應(yīng)用微陣列芯片分析喉鱗狀細(xì)胞癌miRNA與正常黏膜表達(dá)差異的初步研究[J];臨床耳鼻咽喉頭頸外科雜志;2010年12期

2 胡安;金曉杰;;mir-21與mir-375在喉鱗狀細(xì)胞癌中的表達(dá)[J];臨床耳鼻咽喉頭頸外科雜志;2012年18期

3 鐘琦;房居高;黃志剛;陳曉紅;周維國(guó);陳學(xué)軍;許洪波;馬泓智;;喉鱗狀細(xì)胞癌組織miRNA表達(dá)的初步研究[J];中華腫瘤防治雜志;2010年14期

4 揭偉;王曉燕;羅泊濤;黃培春;敖啟林;申志華;;應(yīng)用微陣列芯片分析人不同分化鼻咽癌細(xì)胞株miRNA的表達(dá)[J];華中科技大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2010年03期

5 李文晰;梁琳慧;何祥火;萬大方;顧健人;;Hsa-miR-93在肝癌中的作用機(jī)制研究[J];胃腸病學(xué)和肝病學(xué)雜志;2008年06期

6 徐興偉;嵇武;;microRNA-155的研究進(jìn)展[J];中國(guó)免疫學(xué)雜志;2012年05期

相關(guān)博士學(xué)位論文 前2條

1 劉英;鼻咽癌易感基因miRNA結(jié)合位點(diǎn)多態(tài)性研究[D];南方醫(yī)科大學(xué);2009年

2 魏明輝;喉鱗癌及癌旁正常組織中microRNA的差異表達(dá)分析[D];中國(guó)協(xié)和醫(yī)科大學(xué);2009年

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