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

【摘要】：【目的】從前期應用microRNA基因芯片技術檢測喉鱗癌癌組織及癌旁組織差異性表達microRNA譜中，通過qRT-PCR技術發(fā)現(xiàn)喉鱗狀細胞癌石蠟包埋組織中has-mir-93-5p的表達較對應癌旁切緣組織明顯上調。擬利用體外轉染hsa-miR-93-5p inbibitor，探討其對人喉鱗癌細胞株Hep-2細胞細胞功能的影響。 【方法】 1、取對數(shù)生長期的體外培養(yǎng)人喉鱗狀細胞癌Hep-2細胞株進行試驗。 2、將hsa-miR-93-5p Inhibitor序列（即hsa-miR-93-5p mimisc反向互補序列）轉染至Hep-2細胞中作為Inhibitor轉染組，以未經(jīng)任何轉染處理的Hep-2細胞作為空白對照組。 3、檢測hsa-miR-93-5p Inhibitor轉染組和空白對照組Hep-2細胞增殖能力，采用MTS法、平板克隆形成實驗。 4、采用Edu法檢測細胞分裂增殖能力。 5、采用細胞遷移實驗，Transwell侵襲實驗檢測Inhibitor轉染組和空白對照組細胞遷移侵襲能力。 6、采用PI染料法檢測細胞周期。 7、采用Annexin V-FITC/PI雙染法檢測細胞凋亡。 8、采用SPSS13.0軟件對所得數(shù)據(jù)進行統(tǒng)計學分析，，計量資料采用均值±標準差表示，對結果進行t檢驗，P0.05認為差異有統(tǒng)計學意義。 【結果】 1、細胞轉染48h后，熒光顯微鏡檢測Hep-2細胞中miR-93轉染效率。隨機計數(shù)100個細胞，其中有熒光的細胞約占90%，符合繼續(xù)實驗的標準。 2、MTS法檢測顯示hsa-miR-93-5p Inhibitor轉染的Hep-2細胞抑制率較空白對照組抑制率明顯升高，并且隨著轉染后培養(yǎng)時間的延長，細胞抑制率更加明顯(P0.05)，但增值率相比變化不明顯。 3、不同處理的Hep-2細胞Edu法檢測細胞增殖，顯示Inhibitor轉染組較空白對照組明顯降低，細胞生長活力得到明顯抑制。 4、克隆形成實驗結果示Inhibitor轉染組的存活分數(shù)明顯低于空白對照組(P0.05)。 5、流式細胞術檢測細胞周期結果，hsa-miR-93-5p抑制物轉染的Hep-2細胞處于G2期的細胞數(shù)量（9.62±0.71）明顯高于空白對照組（0.04±0.06），差異具有統(tǒng)計學意義（P0.05）。 6、流式細胞術檢測細胞凋亡結果，Hep-2細胞Inhibitor轉染組總凋亡率（1.97±0.11）明顯高于空白對照組（1.49±0.11），差異具有統(tǒng)計學意義（P0.05）。 7、細胞遷移實驗結果，Hep-2細胞Inhibitor轉染組細胞遷移數(shù)均明顯低于Hep-2細胞空白對照組，差異具有統(tǒng)計學意義（P0.05）。 8、Transwell侵襲實驗檢測結果，Hep-2細胞Inhibitor轉染組細胞侵襲能力（101.5±11.84）明顯低于空白對照組（158.67±20.82），差異具有統(tǒng)計學意義（P0.05）。 【結論】 1、轉染hsa-miR-93-5p Inhibitor序列的喉鱗癌Hep-2細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細胞，提示hsa-miR-93-5p可能是喉鱗癌的發(fā)病機制中一個重要分子 2、轉染hsa-miR-93-5p Inhibitor序列可以抑制喉鱗癌Hep-2細胞的生長，使細胞阻滯于G2期（P0.05）。提示hsa-miR-93-5p可能通過調控喉鱗癌細胞的細胞周期進而發(fā)揮其生物學功能。
[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.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R739.65

【參考文獻】

相關期刊論文 前6條

1 王蘋;付濤;王緒銳;祝威;;應用微陣列芯片分析喉鱗狀細胞癌miRNA與正常黏膜表達差異的初步研究[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在喉鱗狀細胞癌中的表達[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;陳學軍;許洪波;馬泓智;;喉鱗狀細胞癌組織miRNA表達的初步研究[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;;應用微陣列芯片分析人不同分化鼻咽癌細胞株miRNA的表達[J];華中科技大學學報(醫(yī)學版);2010年03期

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

6 徐興偉;嵇武;;microRNA-155的研究進展[J];中國免疫學雜志;2012年05期

相關博士學位論文 前2條

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

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

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