本文關(guān)鍵詞：毛葉香茶菜素F對(duì)人食管癌EC9706細(xì)胞生長(zhǎng)的抑制作用及其機(jī)制初步研究　出處：《鄭州大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 毛葉香茶菜素F 食管癌EC9706細(xì)胞 生長(zhǎng)抑制 細(xì)胞周期、凋亡 Western blot

【摘要】：背景和目的食管癌是最常見的消化道疾病之一,其死亡率幾乎與胃癌相當(dāng),治療效果較差,5年存活率還不足10%,嚴(yán)重威脅著人類健康。食管癌分為食管鱗癌和食管腺癌,我國(guó)以食管鱗癌居多,食管腺癌較少,尤其是河南安陽(yáng)、林州等地是食管癌的高發(fā)地區(qū)。目前食管癌還沒有特效治療藥物,而且其治療方案的選擇也與發(fā)病時(shí)期有關(guān),處于發(fā)病早期和部分中期的患者可以采用手術(shù)治療,而晚期患者只能采用放化療方案,并且效果都不是特別理想。毛葉香茶菜是唇形科植物,主要含有二萜、三萜、木質(zhì)素和黃酮類等化合物,已有體內(nèi)外研究表明,毛葉香茶菜素A、G和E在體外對(duì)艾氏腹水癌(ECA)的增殖具有明顯的抑制作用,在體內(nèi)同樣表現(xiàn)出一定的抗腫瘤效應(yīng),毛葉香茶菜素B對(duì)L1210白血病小鼠模型的生命延長(zhǎng)表現(xiàn)出可觀的效果,毛葉香茶菜素C和D則沒有抗腫瘤作用。目前對(duì)毛葉香茶菜素F的研究較少,關(guān)于其對(duì)食管癌的作用文獻(xiàn)中尚未見報(bào)道,本研的目的在于初步探討毛葉香茶菜素F對(duì)人食管癌EC9706細(xì)胞增殖的抑制作用及機(jī)制。材料和方法1.MTT法粗測(cè)樣品活性以食管癌EC9706細(xì)胞為模型,試驗(yàn)組分別加100、80、50、40、30μg/ml的毛葉香茶菜素F樣品,陰性對(duì)照組加相同體積的完全培養(yǎng)基,在37℃5%的CO2培養(yǎng)箱中分別培養(yǎng)24h、48h、72h后每孔加20μl MTT溶液,繼續(xù)培養(yǎng)4h后吸掉上清并每孔加入160μl DMSO,用酶標(biāo)儀檢測(cè)吸光度。2.觀察樣品對(duì)細(xì)胞生長(zhǎng)狀態(tài)的影響試驗(yàn)組分別加入80、50、40、30μg/ml的毛葉香茶菜素F樣品,陰性對(duì)照組加相同體積的完全培養(yǎng)基,在37℃5%的CO2培養(yǎng)箱中培養(yǎng)72h后在顯微鏡下觀察細(xì)胞生長(zhǎng)狀態(tài)并拍照。3.細(xì)胞劃痕試驗(yàn)陰性對(duì)照組不加藥,試驗(yàn)組分別加入50、40、30μg/ml的毛葉香茶菜素F樣品,陽(yáng)性對(duì)照組加入50μg/ml順鉑,作用24h后,在顯微鏡下觀察、取樣、拍照并測(cè)量各劃痕區(qū)域的面積。4.Hoechst/PI細(xì)胞凋亡檢測(cè)試驗(yàn)組分別加入80、50、40、30μg/ml的毛葉香茶菜素F樣品,陰性對(duì)照組加相同體積的完全培養(yǎng)基,在37℃5%的CO2培養(yǎng)箱中分別培養(yǎng)24h、48h、72h后加入熒光染料,然后用流式細(xì)胞儀檢測(cè)細(xì)胞凋亡率。5.細(xì)胞周期檢測(cè)試驗(yàn)組分別加入50、40、30μg/ml的毛葉香茶菜素F樣品,陰性對(duì)照組加相同體積的完全培養(yǎng)基,在37℃5%的CO2培養(yǎng)箱中分別培養(yǎng)24h、48h、72h后加入熒光染料,然后用流式細(xì)胞儀檢測(cè)細(xì)胞周期分布情況。6.Western blot檢測(cè)樣品對(duì)EC9706細(xì)胞中5種蛋白表達(dá)的影響試驗(yàn)組按照50、40、30μg/ml的濃度加入毛葉香茶菜素F樣品,陰性對(duì)照組加相同體積的完全培養(yǎng)基,在37℃5%的CO2培養(yǎng)箱中分別培養(yǎng)72h后,提取相關(guān)蛋白,用Western blot方法檢測(cè)毛葉香茶菜素F作用后Bax、Bcl-2、Caspase-3、m TOR及P53五種蛋白表達(dá)水平的變化。7.SPSS統(tǒng)計(jì)學(xué)處理采用統(tǒng)計(jì)學(xué)處理軟件SPSS17.0,數(shù)據(jù)資料采用x±s的表示形式,每組試驗(yàn)均重復(fù)三次以上。結(jié)果1.MTT法檢測(cè)結(jié)果顯示,毛葉香茶菜素F作用24h、48h和72h后對(duì)EC9706細(xì)胞的生長(zhǎng)均表現(xiàn)出明顯的抑制作用,其IC50分別為46.7μg/ml、25.4μg/ml和20.6μg/ml,且隨著濃度的增大和時(shí)間的延長(zhǎng)抑制作用也呈增強(qiáng)的趨勢(shì)。2.試驗(yàn)組在不同濃度的毛葉香茶菜素F樣品作用72h后與對(duì)照組相比,細(xì)胞體積變小,細(xì)胞質(zhì)皺縮,呈較小的顆粒狀。3.在細(xì)胞劃痕試驗(yàn)中,毛葉香茶菜素F終濃度為50μg/ml組細(xì)胞培養(yǎng)24h后劃痕面積約為3.10×105,與對(duì)照組2.52×105相比明顯增大,并且差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。4.當(dāng)濃度為50μg/ml時(shí),毛葉香茶菜素F作用24h、48h和72h后細(xì)胞凋亡率約分別為20.20%、21.65%和53.21%,當(dāng)作用時(shí)間為72h,隨著濃度的增大細(xì)胞凋亡率分別為10.37%、17.84%、53.21%和86.63%,;當(dāng)濃度為50μg/ml時(shí),毛葉香茶菜素F作用24h、48h和72h后細(xì)胞周期G1期細(xì)胞比例分別為58.53%、57.37%和52.37%,S期細(xì)胞比例分別為40.06%、41.16%和45.62%,當(dāng)作用時(shí)間為72h,隨著濃度的增大G1期細(xì)胞比例分別為59.65%、56.43%和52.37%,S期細(xì)胞比例分別為39.71%、42.35%和45.62%,均呈時(shí)間和濃度依賴性,且組間差異具有統(tǒng)計(jì)學(xué)意義(P0.05),G2期細(xì)胞無(wú)明顯變化。5.毛葉香茶菜素F對(duì)EC9706細(xì)胞5種蛋白表達(dá)水平的影響毛葉香茶菜素F作用72h后,隨著濃度的增大,Bax蛋白灰度值分別為0.91、1.20和1.26、Caspase-3蛋白灰度值分別為0.77、0.83和1.11,而Bcl-2蛋白灰度值分別為1.01、0.95和0.92,m TOR蛋白灰度值分別為1.08、0.84和0.71,P53(突變型)蛋白的灰度值分別為0.94、0.72和0.58,與對(duì)照組相比發(fā)現(xiàn)其可以上調(diào)Bax和Caspase-3蛋白的表達(dá)、下調(diào)Bcl-2、m TOR及P53(突變型)蛋白的表達(dá)。結(jié)論毛葉香茶菜素F可以上調(diào)Bax、Caspase-3蛋白的表達(dá)水平,下調(diào)Bcl-2、m TOR及P53(突變型)蛋白的表達(dá)水平,能夠抑制食管癌EC9706細(xì)胞的增殖、遷移,將細(xì)胞生長(zhǎng)阻滯于S期,促進(jìn)細(xì)胞的凋亡,這些可能是毛葉香茶菜素F產(chǎn)生抗腫瘤作用的機(jī)制之一。
[Abstract]:Background and objective: esophageal cancer is one of the most common gastrointestinal disease, the mortality rate of gastric cancer and almost equivalent to that of poor treatment, 5 year survival rate is less than 10%, a serious threat to human health. Esophageal cancer is divided into squamous cell carcinoma of the esophagus and esophageal adenocarcinoma, squamous cell carcinoma of the esophagus in our country with the majority, food tube adenocarcinoma is less, especially Henan Anyang, Linzhou and other places is the high incidence of esophageal cancer. At present there is no effective drugs for the treatment of esophageal cancer, and the treatment options are also associated with the incidence of early onset period, in the middle part and can be used in patients with surgical treatment, and patients can only use chemotherapy, and the effect is not ideal. Their hair tea dish is Labiatae, mainly containing two terpenes, three terpenes, lignin and flavonoid compounds, existing research shows that in vivo, Mao Yexiang Nervosin A, G and E in vitro on Ehrlich ascites carcinoma (ECA). Cloning has obvious inhibitory effect, in vivo showed the same anti-tumor effect, life Ye Xiang Mao Nervosin B on L1210 leukemia mouse model extension showed considerable effect, Mao Ye Xiang Nervosin C and D have no antitumor activity. The Ye Xiang Mao tea dish less research in F. On the role of esophageal cancer has not been reported in the literature, this study aims to investigate the hair Ye Xiang Nervosin inhibitory effect of F on proliferation of human esophageal carcinoma EC9706 cells and its mechanism. Materials and methods 1.MTT crude sample activity in esophageal carcinoma EC9706 cells as the model, the experimental groups were g/ml and 100,80,50,40,30 the hair Ye Xiang Nervosin F samples, negative control group and the same volume of complete medium, incubator were cultured in 24h, 48h at 37 DEG C, 5% CO2, 72h 20 l per hole adding MTT solution to 4h after culture and absorb the supernatant per hole adding 160 L DM SO, 80,50,40,30 were added to g/ml by ELISA test group were detected.2. observation sample absorbance effect on cell growth of hair Ye Xiang Nervosin F samples, negative control group and the same volume of culture medium, 72h culture box under a microscope to observe cell growth state and cell scratch test camera.3. the negative control group without dosing at 37 C for 5% CO2, the test group were added with 50,40,30 g/ml hair Ye Xiang Nervosin F samples, 50 g/ml of cisplatin with the positive control group, 24h, under the microscope observation, sampling, photo and test group of apoptosis.4.Hoechst/PI cells to detect the scratch area area were added 80,50,40,30 g/ml Ye Xiang Nervosin F samples, the negative control group and the same volume of complete medium, incubator were cultured in 24h, 48h at 37 DEG C, 5% CO2, 72h after adding fluorescent dye, followed by flow cytometry The apoptosis rate of.5. cell cycle detector test group were added to 50,40,30 g/ml their hair Nervosin F samples, negative control group and the same volume of complete medium, incubator were cultured in 24h, 48h at 37 DEG C, 5% CO2, 72h after adding fluorescent dye, and then detected by flow cytometry samples cell cycle distribution was detected by.6.Western blot in 5 EC9706 cell protein expression in the experimental group were in accordance with the concentration of 50,40,30 g/ml with their hair Nervosin F samples, negative control group and the same volume of culture medium, CO2 culture in 37 C 5% a 72h box were cultured after extraction related protein using Western, blot method to detect their hair Nervosin after F Bax, Bcl-2, Caspase-3,.7.SPSS were analyzed by statistical software SPSS17.0 m and P53 TOR expression of five proteins, said the data was analyzed using X + S Form, each test was repeated three times or more. The results of 1.MTT assay showed that their hair Nervosin F role of 24h, 48h and 72h on the growth of EC9706 cells significantly inhibited, the IC50 were 46.7 g/ml, 25.4 g/ml and 20.6 g/ml, and with the increase of concentration and prolonged inhibition also showed the trend of the.2. test group increased in different concentrations of their hair Nervosin F samples after 72h compared with the control group, the cell size, cytoplasmic shrinkage, a small granular.3. in cell scratch test, their hair Nervosin F final concentration of 50 mu g/ml group cell culture after 24h scratch area of about 3.10 x 105, 2.52 x 105 compared with the control group increased significantly, and the difference was statistically significant (P0.05) when the.4. concentration is 50 g/ml, their gross Nervosin F 24h, 48h 72h and apoptosis rate were 20.20%, 21.65% and 5 3.21%, when the time is 72h, with the increase of the concentration of cell apoptosis rate were 10.37%, 17.84%, 53.21% and 86.63%; when the concentration is 50 g/ml, their hair Nervosin F role of 24h, 48h and 72h after the proportion of cells in the G1 phase of the cell cycle were 58.53%, 57.37% and 52.37%, the proportion of S cells respectively 40.06%, 41.16% and 45.62%, when the time is 72h, with the increase of the concentration, the proportion of cells in G1 phase were 59.65%, 56.43% and 52.37%, the proportion of cells in S phase were 39.71%, 42.35% and 45.62%, were in a time and concentration dependent, and the difference between groups was statistically significant (P0.05). The cells in G2 phase of.5. had no obvious change in their gross Nervosin F of 5 EC9706 cell protein expression level influence their hair Nervosin F 72h, with the increase of the concentration of Bax protein, the gray value of 0.91,1.20 and 1.26 respectively, the gray value of Caspase-3 protein were 0.77,0.83 and 1.11, Bcl-2 protein The gray value of 1.01,0.95 and 0.92 respectively, m TOR protein gray values were 1.08,0.84 and 0.71, P53 (mutant) protein gray values were 0.94,0.72 and 0.58 respectively, compared with the control group found its expression, up regulation of Bax and down-regulation of Caspase-3 protein Bcl-2, m TOR and P53 (mutant) protein expression. Conclusion their hair Nervosin F can upregulate Bax expression, down-regulation of Caspase-3 protein Bcl-2, m TOR and P53 (mutant) protein expression, can inhibit EC9706 esophageal cancer cell proliferation, migration, cell growth arrest in S phase, promote cell apoptosis, which may be one of the mechanisms of their hair Nervosin F has antitumor effect.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R285

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