本文選題：蒿甲醚(ARE) + 人結(jié)直腸癌裸鼠移植瘤�。� 參考：《昆明醫(yī)科大學(xué)》2017年碩士論文

【摘要】：[目的]:大量研究發(fā)現(xiàn)青蒿素及其衍生物除具有抗腫瘤活性外,還能增加腫瘤的放療敏感性,但其具體機制尚有很多未知的方面。本課題通過研究蒿甲醚(artemether,ARE)對放療敏感人結(jié)直腸癌HCT116細(xì)胞及放化療抵抗人結(jié)直腸癌HCT116-CRR (chemoradioresistant,CRR)細(xì)胞裸鼠移植瘤放療效果的影響,并探討其可能的作用機制是否與調(diào)控上皮-間質(zhì)轉(zhuǎn)化(epithelial mesenchymal transition,EMT)相關(guān),為蒿甲醚作為結(jié)直腸癌放療增敏藥物提供實驗依據(jù)。[方法] :1、建立人結(jié)直腸癌裸鼠移植瘤模型:培養(yǎng)放療敏感人結(jié)直腸癌HCT116細(xì)胞及放化療抵抗人結(jié)直腸癌HCT116-CRR細(xì)胞,按二代移植瘤接種的方法,分別建立BALB/c-nu裸小鼠人結(jié)直腸癌皮下移植瘤模型,待腫瘤長至約5 × 5 ×5mm3時,將各部分裸鼠隨機分為四組,分別為對照組(Control)、蒿甲醚組(ARE)、放療組(RT)、聯(lián)合組(RT+ARE),按不同分組進(jìn)行治療;2、觀察各組裸鼠的成瘤率、生存狀態(tài)、體重變化等;3、隔日監(jiān)測腫瘤長短徑,計算腫瘤體積抑制率及增敏系數(shù)(efficiency factor,EF),繪制瘤體積變化曲線;4、治療結(jié)束處死各組裸鼠取瘤塊,稱重并計算抑瘤率;5、通過流式細(xì)胞儀檢測各組瘤組織的凋亡率及壞死率;6、取各組瘤組織10%甲醛固定,常規(guī)石蠟包埋、切片,HE染色,光鏡下觀察;7、免疫組化檢測各組瘤組織中EMT相關(guān)因子E-cadherin、N-cadherin、Vimentin、Snail、Slug、Twist、β -Catenin 的表達(dá)情況;8、RT-PCR 檢測各組瘤組織中 E-cadherin、N-cadherin、Vimentin、Snail、Slug、Twist、β-Catenin基因的表達(dá)水平;9、Western blot 檢測各組瘤組織中 E-cadherin、N-cadherin、Vimentin、Snail、Slug、Twist、β -Catenin蛋白的表達(dá)水平;10、采用SPSS22.0軟件進(jìn)行數(shù)據(jù)統(tǒng)計分析,數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差(x±s)表示,滿足方差齊性檢驗要求的數(shù)據(jù)采用方差分析,事后兩兩比較采用LSD-t檢驗;不滿足方差齊性檢驗的數(shù)據(jù)采用Brown-Forsythe分析,事后兩兩比較采用Dunnett's T3 檢驗。[結(jié)果]:1. HCT116及HCT116-CRR細(xì)胞裸鼠成瘤率為100%;各組間裸鼠體重?zé)o明顯差異,治療期間放療組及聯(lián)合組裸鼠活動明顯減少,精神稍差。2. HCT116細(xì)胞各組裸鼠瘤體積治療前無明顯差異,治療后聯(lián)合組裸鼠瘤體積小于放療組(P0.05),聯(lián)合組腫瘤體積抑制率91.66%明顯高于放療組88.13%,蒿甲醚對HCT116細(xì)胞裸鼠移植瘤的EF為3.20; HCT116-CRR細(xì)胞各組裸鼠瘤體積治療前無明顯差異,治療后聯(lián)合組裸鼠瘤體積小于放療組(P0.05),聯(lián)合組腫瘤體積抑制率80.84%明顯高于放療組65.04%,蒿甲醚對HCT116-CRR細(xì)胞裸鼠移植瘤的EF為2.43。3. HCT116細(xì)胞聯(lián)合組裸鼠平均瘤重小于放療組(P0.05),聯(lián)合組抑瘤率91.20%明顯高于放療組88.83%; HCT116-CRR細(xì)胞聯(lián)合組裸鼠平均瘤重小于放療組(P0.05),聯(lián)合組抑瘤率83.84%明顯高于放療組67.24%。4. HCT116細(xì)胞聯(lián)合組裸鼠瘤組織凋亡率(41.353±2.628) %大于放療組(30.47±0.738)%(P0.05),各組裸鼠瘤組織壞死率無明顯差異;HCT116-CRR細(xì)胞聯(lián)合組裸鼠瘤組織凋亡率(24.280±1.883) %大于放療組(12.803±0.788) %(P0.05),各組裸鼠瘤組織壞死率無明顯差異。5.各組瘤組織HE染色后光鏡下觀察,HCT116細(xì)胞對照組裸鼠瘤組織中心可見明顯的大片出血壞死液化區(qū),壞死區(qū)域內(nèi)有血細(xì)胞浸潤,各治療組裸鼠瘤組織壞死區(qū)域明顯擴大,以聯(lián)合組最為顯著;HCT116-CRR細(xì)胞對照組裸鼠瘤組織可見部分腫瘤性壞死,腫瘤間質(zhì)增生明顯,各治療組裸鼠瘤組織壞死區(qū)域有不同程度的擴大,以聯(lián)合組顯著。6.免疫組化檢測各組裸鼠瘤組織中EMT相關(guān)因子的表達(dá)情況。HCT116細(xì)胞各組裸鼠瘤組織中,上皮標(biāo)志物E-cadherin、β-Catenin呈強陽性表達(dá),與放療組比較,聯(lián)合組表達(dá)上調(diào);間質(zhì)標(biāo)志物N-cadherin在各組中呈陰性表達(dá),Vimentin、Snail、Slug、Twist呈弱陽性表達(dá),與放療組比較,聯(lián)合組表達(dá)下調(diào)。HCT116-CRR細(xì)胞各組裸鼠瘤組織中,上皮標(biāo)志物E-cadherin在放療組呈陰性表達(dá),在聯(lián)合組呈弱陽性表達(dá),β-Catenin在各組中呈弱陽性表達(dá),與放療組比較,聯(lián)合組表達(dá)上調(diào);間質(zhì)標(biāo)志物N-cadherin、Slug在各組中呈弱陽性表達(dá),Vimentin、Snail、Twist呈強陽性表達(dá),與放療組比較,聯(lián)合組表達(dá)下調(diào)。7. RT-PCR檢測各組裸鼠瘤組織中EMT相關(guān)因子的基因相對表達(dá)量。HCT116細(xì)胞聯(lián)合組裸鼠瘤組織與放療組比較,β-Catenin的基因表達(dá)量較放療組升高(P0.05),Vimentin的基因表達(dá)量較放療組下降(P0.05)。HCT116-CRR細(xì)胞聯(lián)合組裸鼠瘤組織與放療組比較,E-cadherin、β-Catenin的基因表達(dá)量較放療組升高(P0.05),N-cadherin、Vimentin、Snail、Slug、Twist 的基因表達(dá)量較放療組下降(P0.05)。8. Western blot檢測各組裸鼠瘤組織中EMT相關(guān)因子的蛋白表達(dá)水平。HCT116細(xì)胞聯(lián)合組裸鼠瘤組織與放療組比較,β-Catenin的蛋白表達(dá)量較放療組升高(P0.05),Vimentin的蛋白表達(dá)量較放療組下降(P0.05)。HCT116-CRR細(xì)胞聯(lián)合組裸鼠瘤組織與放療組比較,E-cadherin、β-Catenin的蛋白表達(dá)量較放療組升高(P0.05),N-cadherin、Vimentin、Snail、Slug、Twist 的蛋白表達(dá)量較放療組下降(P0.05)。[結(jié)論]:1. ARE聯(lián)合放療能顯著抑制人結(jié)直腸癌HCT116細(xì)胞裸鼠移植瘤的生長,從而增強其放療效果;ARE聯(lián)合放療能顯著抑制人結(jié)直腸癌HCT116-CRR細(xì)胞裸鼠移植瘤的生長,從而增強其放療效果,逆轉(zhuǎn)其放化療抵抗性;2. ARE聯(lián)合放療能促進(jìn)人結(jié)直腸癌HCT116細(xì)胞裸鼠移植瘤的凋亡及壞死,從而增強其放療效果;ARE聯(lián)合放療能促進(jìn)人結(jié)直腸癌HCT116-CRR細(xì)胞裸鼠移植瘤的凋亡及壞死,從而增強其放療效果,逆轉(zhuǎn)其放化療抵抗性;3. ARE聯(lián)合放療能上調(diào)人結(jié)直腸癌HCT116細(xì)胞裸鼠移植瘤上皮標(biāo)志物β-Catenin的表達(dá),下調(diào)間質(zhì)標(biāo)志物Vimentin的表達(dá),提示ARE可能通過阻滯EMT增強人結(jié)直腸癌HCT116細(xì)胞裸鼠移植瘤放療效果;4. ARE聯(lián)合放療能上調(diào)人結(jié)直腸癌HCT116-CRR細(xì)胞裸鼠移植瘤上皮標(biāo)志物 E-cadherin、β-Catenin 的表達(dá),下調(diào)間質(zhì)標(biāo)志物 N-cadherin、Vimentin、Snail、Slug、Twist的表達(dá),提示ARE可能通過逆轉(zhuǎn)EMT增強人結(jié)直腸癌HCT116-CRR細(xì)胞裸鼠移植瘤放療效果,逆轉(zhuǎn)其放化療抵抗性;5. ARE能增強人結(jié)直腸癌裸鼠移植瘤放療效果,可能成為一種低毒高效的放射增敏劑,應(yīng)用于結(jié)直腸癌放射治療當(dāng)中。
[Abstract]:[Objective]: a large number of studies have found that artemisinin and its derivatives can increase the radiosensitivity of tumor in addition to anti-tumor activity, but there are still many unknown mechanisms in its specific mechanism. The study was conducted by the study of artemether (ARE) for HCT116 cells in colorectal cancer sensitive human colorectal cancer and HCT116-CRR (CHEM) for colorectal cancer. The effect of oradioresistant, CRR) on the radiotherapy effect of transplanted tumor in nude mice and whether its possible mechanism is related to the regulation of epithelial mesenchymal transition (epithelial mesenchymal transition, EMT), and provide experimental evidence for artemether as a radiotherapy sensitizing drug for colorectal cancer. [method] 1, a model of human colorectal carcinoma in nude mice was established. The HCT116 cells of colorectal cancer sensitive human colorectal cancer and HCT116-CRR cells of radiochemotherapy resistant human colorectal cancer were cultured in accordance with the method of inoculation of two generations of transplanted tumor. The tumor model of human colorectal carcinoma in BALB/c-nu mice was established respectively. When the tumor grew to about 5 x 5 x 5mm3, the nude mice were randomly divided into four groups, the control group (Control), Artemisia Artemisia, respectively. Methyl ether group (ARE), radiotherapy group (RT), combined group (RT+ARE), treated with different groups, 2, observe the tumor formation rate, survival state, body weight change of nude mice, 3, monitor the long diameter of tumor on the other day, calculate the tumor volume inhibition rate and increase the sensitivity coefficient (efficiency factor, EF), draw the volume change curve of the tumor; 4, take the nude mice at the end of the treatment and take the tumor. Block, weighing and calculating the tumor suppressor rate; 5, the rate of apoptosis and necrosis of the tumor tissues were detected by flow cytometry; 6, the tumor tissues were fixed with 10% formaldehyde, conventional paraffin embedding, slicing, HE staining, observed under light microscope; 7, immunohistochemistry was used to detect E-cadherin, N-cadherin, Vimentin, Snail, Slug, Twist, and beta -Catenin in each group of tumor tissues. 8, RT-PCR detected the expression level of E-cadherin, N-cadherin, Vimentin, Snail, Slug, Twist, and beta -Catenin in the tumor tissues, and 9, Western blot to detect the expression level of the tumor tissue, and 10. The data were expressed with mean standard deviation (x + s). The data that met the requirements of variance homogeneity test were analyzed by variance, and LSD-t test was used for the post 22 comparison; Brown-Forsythe analysis was used for the data that did not meet the homogeneity of variance test, and 22 compared with Dunnett's T3 test. [fruit]: 1. HCT116 and HCT116-CRR cells in nude mice were 100%. There was no significant difference in weight of nude mice in each group. The activity of the radiotherapy group and the combined group of nude mice decreased significantly during the treatment. There was no significant difference in the tumor volume of nude mice in the.2. HCT116 cells. The volume of tumor volume in the combined group was less than that of the radiotherapy group (P0.05). The tumor volume inhibition rate of the combined group was 91.66% significantly higher than that of the radiotherapy group 88.13%, artemether. The EF of HCT116 cell xenografts in nude mice was 3.20, and there was no significant difference in the tumor volume of nude mice in HCT116-CRR cells. The volume of tumor volume in the combined group was less than that of the radiotherapy group (P0.05). The tumor volume inhibition rate of the combined group was 80.84% higher than that of the radiotherapy group (65.04%). The EF of artemether to HCT116-CRR cell nude mice was 2.43.3. HCT116 cells. The average tumor weight of nude mice in the combined group was less than that of the radiotherapy group (P0.05), the tumor inhibition rate in the combined group was 91.20% significantly higher than that in the radiotherapy group (88.83%), and the average tumor weight of nude mice in the HCT116-CRR cell combined group was less than that of the radiotherapy group (P0.05), and the tumor inhibition rate of 83.84% in the combined group was significantly higher than that in the radiotherapy group (41.353 + 2.628)% of the 67.24%.4. HCT116 cell group (41.353 + 2.628). Group (30.47 + 0.738)% (P0.05), the necrosis rate of tumor tissue in nude mice was no significant difference, the apoptosis rate of tumor tissue in nude mice of HCT116-CRR cell group was (24.280 + 1.883)% greater than that of radiotherapy group (12.803 + 0.788)% (P0.05). There was no significant difference in the necrosis rate of tumor tissue in each group of nude mice. The tumor tissue of each group was observed under the light microscope after HE staining, and the nude mouse tumor of HCT116 cell control group was nude mice. The tissue Center showed obvious area of hemorrhagic necrotic liquefaction, blood cell infiltration in the necrotic region, the necrotic area of tumor tissue in nude mice was obviously enlarged in the treatment group, and the most significant in the combined group. The tumor tissue of the nude mice of the HCT116-CRR cell control group was partly tumor necrosis, the tumor interstitial hyperplasia was obvious, and the necrotic region of the nude mice was necrotic area in the treatment group. The expression of EMT related factors in tumor tissue of nude mice was detected by.6. immunohistochemistry in different groups. The epithelial markers, E-cadherin, and beta -Catenin were strongly positive in the tumor tissues of each group of nude mice of each group of.HCT116 cells. Compared with the radiotherapy group, the expression of the combined group was up regulated, and the interstitial marker N-cadherin was negative in each group. The expression of Vimentin, Snail, Slug, Twist showed weak positive expression. Compared with the radiotherapy group, the joint group expressed the negative expression of the epithelial marker E-cadherin in the tumor tissue of.HCT116-CRR cells in the radiotherapy group, and was weakly positive in the combined group. The expression of beta -Catenin in each group was weakly positive, and the expression of the combined group was up regulated compared with the radiotherapy group. The interstitial marker N-cadherin and Slug were weakly positive in each group, Vimentin, Snail, and Twist were strongly positive. Compared with the radiotherapy group, the expression of.7. RT-PCR in the joint group was down regulated to detect the relative expression of EMT related factors in the tumor tissues of the nude mice. The tumor tissue of the group of.HCT116 cells in the combination group of the group of.HCT116 cells was compared with the radiotherapy group, and the gene table of the beta -Catenin was expressed. Compared with radiotherapy group, the expression of Vimentin was lower than radiotherapy group (P0.05). Compared with radiotherapy group, the gene expression of E-cadherin and beta -Catenin was higher than that of radiotherapy group (P0.05), N-cadherin, Vimentin, Snail, Slug, and the gene expression of Twist was lower than that of radiotherapy group. The protein expression level of EMT related factors in the tumor tissues of nude mice by blot was compared with the radiotherapy group. The protein expression of beta -Catenin was higher than that of the radiotherapy group (P0.05), and the protein expression of Vimentin was lower than that of the radiotherapy group (P0.05) the tumor tissue of the nude mice was compared with the radiotherapy group, E-cadhe, E-cadhe. Rin, the protein expression of beta -Catenin was higher than that of radiotherapy group (P0.05), the protein expression of N-cadherin, Vimentin, Snail, Slug, Twist decreased (P0.05). [Conclusion]: 1. ARE combined radiotherapy could significantly inhibit the growth of human colorectal carcinoma HCT116 cell xenografts, and enhance the effect of radiotherapy; ARE combined radiotherapy can significantly inhibit human knot. The growth of HCT116-CRR cell xenografts in rectal carcinoma of rectal cancer can enhance the effect of radiotherapy and reverse its chemoradiotherapy. 2. ARE combined with radiotherapy can promote the apoptosis and necrosis of human colorectal HCT116 cell xenografts in nude mice and enhance the effect of radiotherapy. ARE combined with radiotherapy can promote the withering of human colorectal HCT116-CRR cells in nude mice. Death and necrosis can enhance the effect of radiotherapy and reverse its chemoradiotherapy. 3. ARE combined with radiotherapy can regulate the expression of the epithelial marker of the tumor HCT116 cells in colorectal cancer and down regulate the expression of the interstitial marker Vimentin, suggesting that ARE may increase the efficacy of ARE by blocking EMT in nude mice transplanted with HCT116 cells in colorectal cancer. 4. ARE combined radiotherapy can regulate the expression of E-cadherin, the expression of beta -Catenin, the expression of N-cadherin, Vimentin, Snail, Slug, Twist of the tumor HCT116-CRR cells in nude mice of colorectal cancer, suggesting that ARE may reverse the effect of radiotherapy on the xenografts in nude mice of human colorectal cancer by reversing EMT. Chemoradiotherapy, 5. ARE can enhance the effect of radiotherapy for human colorectal cancer in nude mice, and may be a low toxic and efficient radiosensitizer used in the radiotherapy of colorectal cancer.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.34

【參考文獻(xiàn)】

相關(guān)期刊論文 前7條

1 王艷俊;蔣永新;劉珊;王紅;;同期放化療誘導(dǎo)人結(jié)直腸癌細(xì)胞發(fā)生上皮-間質(zhì)轉(zhuǎn)化[J];臨床與病理雜志;2017年01期

2 熊偉;蔣永新;劉珊;王紅;謝青;;蒿甲醚對人結(jié)直腸癌細(xì)胞的毒性和放療增敏作用的研究[J];現(xiàn)代預(yù)防醫(yī)學(xué);2015年17期

3 劉珊;蔣永新;熊偉;付鳳蓮;;腫瘤放療抵抗機制研究進(jìn)展[J];國際腫瘤學(xué)雜志;2014年10期

4 張曉紅;;青蒿素類藥物抗腫瘤作用機制研究概況[J];中醫(yī)學(xué)報;2014年01期

5 李斌;周紅;;青蒿素及其衍生物藥理作用研究有關(guān)進(jìn)展[J];中國臨床藥理學(xué)與治療學(xué);2010年05期

6 董俊清;趙妍妍;姚琦;蔣為薇;李軍;李斌;龐學(xué)利;周紅;;青蒿琥酯增加人腦膠質(zhì)瘤細(xì)胞CHG-5對β射線的敏感性[J];第三軍醫(yī)大學(xué)學(xué)報;2010年06期

7 周厚清;腫瘤放射增敏劑及增敏機制研究進(jìn)展[J];國外醫(yī)學(xué)(腫瘤學(xué)分冊);2001年06期

相關(guān)博士學(xué)位論文 前3條

1 朱明章;中醫(yī)專方聯(lián)合化療治療非小細(xì)胞肺癌療效及組方研究[D];廣州中醫(yī)藥大學(xué);2013年

2 張棟棟;1、EMT相關(guān)的LncRNA篩選及功能和機制研究2、Gadd45α對自噬的調(diào)控及分子機制研究[D];北京協(xié)和醫(yī)學(xué)院;2013年

3 張弦;β-欖香烯逆轉(zhuǎn)乳腺癌上皮—間質(zhì)轉(zhuǎn)化作用機制的基礎(chǔ)研究[D];大連醫(yī)科大學(xué);2013年

相關(guān)碩士學(xué)位論文 前2條

1 王紅;蒿甲醚通過PPARγ信號通路抑制小鼠Lewis肺癌生長實驗研究[D];昆明醫(yī)科大學(xué);2016年

2 劉維帥;常山酮逆轉(zhuǎn)放射治療導(dǎo)致Lewis肺癌細(xì)胞上皮間質(zhì)轉(zhuǎn)化的機制研究[D];天津醫(yī)科大學(xué);2014年

，

本文編號：1958287