本文關(guān)鍵詞：丹參酮ⅡA對人食管癌細(xì)胞系Eca-109增殖抑制作用及機(jī)制研究　出處：《河南中醫(yī)學(xué)院》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 食管癌 TanⅡA 細(xì)胞周期 細(xì)胞凋亡

【摘要】：研究背景據(jù)報道,在我國每年死于食管癌的人數(shù)約為21萬人,男性人數(shù)多于女性,40歲以上的發(fā)病率高,它嚴(yán)重威脅著我國人民的健康和生命[1]。其治療方法主要有手術(shù)切除、腫瘤的放療和化療等,單一的任何一種方法都不是理想的,因此多采用綜合治療,在手術(shù)切除后配合藥物化療�，F(xiàn)在的化療新藥層出不窮,但因化療產(chǎn)生的副反應(yīng)仍然較大,并且耐藥性不斷增加,病人的的5年存活率依然很低。因此,利用我國的優(yōu)勢,研究中藥及其提取的有效成分在腫瘤治療方面的作用是以后發(fā)展的方向之一。本研究探討了食管癌可能的發(fā)生機(jī)制及中藥丹參中的揮發(fā)油-丹參酮ⅡA(TanⅡA)對人食管癌細(xì)胞株Eca-109的作用。TanⅡA是中藥丹參的主要成分。丹參酮在臨床上已被廣泛應(yīng)用于非腫瘤領(lǐng)域,主要用來治療心血管疾病,在抗菌消炎等[1]方面也有顯著療效。近年來,學(xué)者對丹參酮IIA的抗腫瘤活性產(chǎn)生了極大興趣,并進(jìn)行了多種腫瘤細(xì)胞的體內(nèi)外試驗,但對食管癌的抗癌作用卻鮮見報道。目的通過對人食管癌Eca-109細(xì)胞的培養(yǎng),用不同濃度的TanⅡA干預(yù)其生長,運(yùn)用現(xiàn)代分子生物學(xué)技術(shù),探討TanⅡA促進(jìn)Eca-109細(xì)胞凋亡的機(jī)制,為TanⅡA更深入的研究做準(zhǔn)備。方法1.MTT法:通過培養(yǎng)人食管癌Eca-109細(xì)胞,用不同劑量的TanⅡA作用于Eca-109細(xì)胞,通過四甲基偶氮唑鹽法(MTT法)測定各組的吸光值(OD值),并計算出各組細(xì)胞的增殖抑制率,繪制增殖抑制率曲線。2.顯微形態(tài)觀察:藥物組細(xì)胞和正常組細(xì)胞培養(yǎng)24小時后,在顯微鏡下觀察正常細(xì)胞和藥物干預(yù)細(xì)胞的生長狀態(tài)和形態(tài)變化。3.細(xì)胞熒光觀察:藥物組細(xì)胞和正常組細(xì)胞培養(yǎng)24小時后,應(yīng)用AO/EB染色,在熒光顯微鏡下觀察兩組細(xì)胞的形態(tài)變化。4.流式細(xì)胞術(shù):采用140μg/ml、160μg/ml的TanⅡA干預(yù)細(xì)胞生長24和48小時,設(shè)置陰性對照,用流式檢測細(xì)胞周期和細(xì)胞凋亡。5.免疫蛋白印跡法:采用140μg/ml、160μg/ml的TanⅡA干預(yù)細(xì)胞生長24小時,提取細(xì)胞的總蛋白,用BCA法進(jìn)行蛋白定量,采用蛋白免疫印跡技術(shù)(western blot)檢測Caspase-3、Caspase-9、Cyclin A和CDK2的表達(dá),以β-actin作為內(nèi)參,用BIO-RAD軟件分析條帶的灰度值。結(jié)果1.MTT檢測結(jié)果顯示:正常組細(xì)胞活力明顯高于藥物組,并且在一定的范圍內(nèi),隨藥物的濃度增加,細(xì)胞的活力下降,最高的抑制率可達(dá)66.164%,與陰性對照組相比較,差異有統(tǒng)計學(xué)意義(P㩳0.05)。2.顯微鏡觀察顯示:經(jīng)過TanⅡA處理后的細(xì)胞與正常組細(xì)胞相比較,藥物組細(xì)胞有明顯的凋亡形態(tài)的改變,出現(xiàn)細(xì)胞漂浮,細(xì)胞核固縮,細(xì)胞膜結(jié)構(gòu)破壞出現(xiàn)“小泡”現(xiàn)象。3.細(xì)胞熒光觀察結(jié)果顯示:與正常組細(xì)胞相比較,藥物組細(xì)胞出現(xiàn)核染色質(zhì)著綠色或紅色并呈固縮狀、圓珠狀。4.流式結(jié)果表明:1)140μg/ml TanⅡA干預(yù)Eca-109細(xì)胞24小時后,結(jié)果顯示S期的細(xì)胞數(shù)升高,達(dá)到54.47%;同時G0/G1期的細(xì)胞數(shù)下降,由51.317%降至41.787%,與陰性對照相比,差異具有統(tǒng)計學(xué)意義(P㩳0.05)。2)采用140μg/ml和160μg/ml的TanⅡA培養(yǎng)細(xì)胞24小時,結(jié)果顯示藥物組凋亡指數(shù)隨著藥物濃度的增加,凋亡指數(shù)增大,由陰性的8.319增加到TanⅡA140、160μg/ml組的31.982和52.937。用藥組和陰性對照組相比較,差異具有統(tǒng)計學(xué)意義(P㩳0.01)。5.Western blot結(jié)果顯示:藥物培養(yǎng)24小時后,細(xì)胞內(nèi)Cyclin A、CDK2表達(dá)量降低;Caspase-3、Caspase-9的表達(dá)量上升,和正常組相比較,差異具有統(tǒng)計學(xué)意義(P㩳0.05)。結(jié)論1.TanⅡA可以抑制人食管癌細(xì)胞的增殖活力,并且這種抑制作用隨藥物濃度的增加會更明顯。2.在TanⅡA作用于食管癌細(xì)胞系Eca-109的過程中,隨著藥物濃度的升高,細(xì)胞在S期的比例逐漸升高,S期檢查點的調(diào)控因子Cyclin A和CDK2的相對表達(dá)量降低。3.TanⅡA抑制人食管癌Eca-109細(xì)胞的增殖并促進(jìn)其凋亡的機(jī)制可能與通過下調(diào)Cyclin A和CDK2,將細(xì)胞阻滯于S期和上調(diào)Caspase-9和Caspase-3促進(jìn)細(xì)胞凋亡增加有關(guān)。
[Abstract]:According to reports in the background, the number of China's annual died of esophageal cancer is about 210 thousand people, the number of men more than women over the age of 40, the incidence rate is high, it is a serious threat to our health and life of the people [1]. the main treatments include surgical resection, radiotherapy and chemotherapy of tumor, any single method are not ideal, so we adopt comprehensive treatment, after surgical removal combined with chemotherapy. Chemotherapy drugs now because chemotherapy side effects emerge in an endless stream, the resistance is still large, and increasing the patient's 5 year survival rate is still very low. Therefore, the use of our advantages, effect of effective components Chinese herbal medicine and its extract in the treatment of cancer is one of the future direction of development. This study explores the possible occurrence of esophageal cancer and mechanism of volatile oil of Salvia miltiorrhiza in tanshinone A (Tan A) on human esophageal carcinoma fine The role of Eca-109 in cell line.Tan II A is the main component of Salvia miltiorrhiza. Tanshinone in clinical practice has been widely used in the field of non tumor, mainly used in the treatment of cardiovascular diseases, anti inflammatory [1] also had significant effect. In recent years, the antitumor activity of tanshinone IIA have generated great interest, and in vivo test of tumor cells to anticancer effect on esophageal cancer, but is rarely reported. The culture of human esophageal carcinoma Eca-109 cells, the growth of the intervention with different concentrations of Tan II A, using modern molecular biology technology, discuss the mechanism of Eca-109 Tan II A promote cell apoptosis, prepare for study of Tan A deeply. 1.MTT method by cultured human esophageal cancer Eca-109 cells, with different doses of Tan II A in Eca-109 cells by four methyl thiazolyl tetrazolium method (MTT method) were measured by light absorption value (OD), And calculate the inhibition rate of cell proliferation, rendering the proliferation rate of observation of.2. morphology curve: drug group and normal group cells cultured for 24 hours, to observe the growth state of normal cells and drug intervention cells and morphological changes of.3. cell fluorescence microscope observation: drug group and normal group cells cultured for 24 hours the application, AO/EB staining to observe the morphological changes in two groups of.4. cells under the fluorescence microscope and flow cytometry using 140 g/ml Tan II A 160 g/ml intervention on cell growth of 24 and 48 hours, set the negative control, using flow cytometry to detect the cell cycle and apoptosis of.5. cells by Western blot: 140 g/ml, Tan II A 160 g/ml intervention on cell growth in 24 hours, the total protein extracted from cells, proteins were quantified by BCA assay, Western blot technique (Western blot) Caspase-9, Cyclin detection of Caspase-3, A and CDK2 The expression of beta -actin as reference, using BIO-RAD software to analyze the gray value of bands. 1.MTT results showed that: in normal group, the cell viability was significantly higher than that of the drug group, and in a certain range, with the drug concentration increased, cell viability decreased, the highest inhibition rate reached 66.164%, compared with the negative control group, the difference was statistically significant (P? 0.05).2. microscope observation showed that: after Tan II A treated cells compared with normal cells, the drug group cells have obvious apoptotic morphology changes, cell floating, karyopyknosis, "vesicles" phenomenon of.3. cell fluorescence observation results show that the damage of the cell membrane structure: compared with normal group cells, drug group cells showed nuclear chromatin with green or red and showed pyknosis, bead like.4. flow cytometry results showed that: 1) 140 g/ml Tan II A Eca-109 cells 24 hours after intervention, the results showed that S The number of cells increased, reaching 54.47%; at the same time, the number of cells in G0/G1 phase decreased from 51.317% to 41.787%, compared with the negative control, the difference was statistically significant (P.2)? 0.05) by 140 g/ml and 160 g/ml Tan II A cells cultured for 24 hours. The results showed that the drug group with apoptosis index the increase of drug concentration, the apoptosis index increased, increased from 8.319 to Tan II A140160 negative g/ml group 31.982 and 52.937. group and negative control group compared the difference was statistically significant (P? 0.01).5.Western blot results showed that the drug culture after 24 hours, Cyclin A cells, CDK2 expression was decreased Caspase-3; the expression of Caspase-9 was increased, compared with normal group, the difference was statistically significant (P? 0.05). Conclusion 1.Tan II A can inhibit human esophageal cancer cell proliferation, and this inhibition effect will be more obvious in.2. Tan II with the increase of drug concentration In the process of esophageal cancer cell line Eca-109 A, with the increase of drug concentration, the cells gradually increased in the proportion of S phase, S phase checkpoint regulation factor Cyclin A and CDK2 decreased the relative expression mechanism and promote the apoptosis of.3.Tan II A inhibited the proliferation of Eca-109 cells may be related to downregulation by Cyclin A and CDK2, arrest the cells in S and upregulation of Caspase-9 and Caspase-3 to promote the increase of cell apoptosis.

【學(xué)位授予單位】：河南中醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R735.1

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