NSCLC放射抗拒細胞實驗平臺的建立及初步研究
發(fā)布時間：2018-11-11 19:33
【摘要】：目的： 1.在mRNA水平和蛋白水平上檢測非小細胞肺癌常規(guī)分割放療抵抗細胞系和大分割放療抵抗細胞系DNA損傷修復(fù)能力的變化。 2.檢測放療抵抗細胞的細胞周期分布、凋亡水平的變化情況及分析放療抵抗產(chǎn)生的機制。 方法： 1.體外培養(yǎng)A549, A549-2GyR, A549-4GyR細胞,給予三種細胞不同劑量射線照射,克隆形成實驗檢測細胞增值能力。 2.根據(jù)全基因組表達譜芯片結(jié)果,分析篩選出與DNA損傷修復(fù)相關(guān)且有表達量差異的蛋白。體外培養(yǎng)A549, A549-2GyR, A549-4GyR細胞,通過RT-PCR獲取目的蛋白DNA模板,運用Real-time PCR精確定量分析抵抗細胞中修復(fù)相關(guān)蛋白在mRNA水平上的差異。 3. Western Blot檢測抵抗細胞中基礎(chǔ)狀態(tài)下Ku70、Ku80、DNA-PKcs、LIG4、 Rad50、XRCC4、Mrel1等修復(fù)相關(guān)蛋白和p53、p21、CylinD1等周期相關(guān)蛋白的表達水平。 4.流式細胞術(shù)檢測抵抗細胞的細胞周期分布(PI單染)和凋亡水平(Annexin V/PI雙染)。 結(jié)果： 1.放療后,A549, A549-2GyR, A549-4GyR細胞增值能力依次增強。 2.通過對全基因組芯片結(jié)果進行分析篩選,選擇KKu70、Ku80、DNA-PKcs、LIG4、 Rad50、XRCC4作為目的分子,Real-time PCR發(fā)現(xiàn)XRCC4、Ku80、DNA-PKcs和Rad50的mRNA表達水平在A549,A549-2GyR和A549-4GyR依次遞減,而Ku70和LIG4也是在A549-4GyR細胞中表達量最低。 3. Western Blot檢測到修復(fù)相關(guān)蛋白DNA-PKcs、XRCC4、LIG4、Ku80、Ku70、 Rad50、Mrel1、NBN、XLF的蛋白表達在A549, A549-2GyR和A549-4GyR中依次遞減；細胞周期相關(guān)蛋白p53、Phospho-p53(Ser20)、Phospho-p53(Ser37)、Cyclin D1、CDK2表達量也依次遞減,而p21表達量則逐漸增多。 4.流式細胞術(shù)檢測到基礎(chǔ)狀態(tài)下A549細胞周期分布,G1期62.63%,G2期10.04%,S期27.33%; A549-2GyR為G1期64.45%,G2期11.77%,S期23.79%；A549-4GyR為G1期72.37%,G2期8.7%,S期18.93%。凋亡水平分析,A549凋亡率0.7%, A549-2GyR凋亡率1.7%, A549-4GyR凋亡率0.3%。 結(jié)論： 1.通過多次放療篩選,可獲得放療抵抗細胞。 2.與對照組細胞A549和常規(guī)分割抵抗細胞A549-2GyR相比,大分割抵抗細胞A549-4GyR細胞中DNA損傷修復(fù)相關(guān)蛋白存在表達量上的差異。實驗結(jié)果證實損傷修復(fù)分子Ku70、Ku80、DNA-PKcs、LIG4、Rad50、XRCC4在mRNA轉(zhuǎn)錄水平上存在差異,在大分割放療抵抗細胞中表達量最低。 3.在蛋白翻譯水平上,大分割抵抗細胞與對照組和常規(guī)分割抵抗細胞相比,蛋白表達量也降低,與mRNA水平檢測結(jié)果基本一致。 4.基礎(chǔ)狀態(tài)下,A549、A549-2GyR和A549-4GyR在凋亡水平上無明顯差異。 5.流式結(jié)果發(fā)現(xiàn)大分割抵抗細胞較對照組和常規(guī)分割抵抗細胞有更多的細胞阻滯在G1/S期,并且與蛋白表達檢測結(jié)果相一致,而DNA損傷修復(fù)蛋白的表達水平卻降低,因此,細胞周期發(fā)生阻滯,細胞有更充分的時間應(yīng)對放射造成的DNA損傷可能是抵抗產(chǎn)生的主要原因。
[Abstract]:Objective: 1. The changes of DNA damage and repair ability of conventional fractionated radiotherapy resistant cell lines and large fraction radiotherapy resistant cell lines were detected at the level of mRNA and protein. 2. The cell cycle distribution, apoptosis level and the mechanism of radiation resistance were analyzed. Methods: 1. A549, A549-2Gy R, A549-4GyR cells were cultured in vitro. Three kinds of cells were irradiated with different doses of radiation. 2. According to the results of the whole genome expression microarray, the proteins associated with DNA damage repair and different expression levels were screened out. A549, A549-2Gy R, A549-4GyR cells were cultured in vitro. The target protein DNA template was obtained by RT-PCR, and the difference of repair related proteins in resistant cells at mRNA level was quantitatively analyzed by Real-time PCR. 3. Western Blot was used to detect the expression of repair related proteins such as Ku70,Ku80,DNA-PKcs,LIG4, Rad50,XRCC4,Mrel1 and p53 p21 and cyclin D1 in the basic state of resistant cells. 4. Cell cycle distribution (PI single staining) and apoptosis level (Annexin V/PI double staining) of resistant cells were detected by flow cytometry. Results: 1. After radiotherapy, A 549, A 549-2 Gy R, A549-4GyR cells increased in turn. 2. Through the analysis and screening of the whole genome microarray results, KKu70,Ku80,DNA-PKcs,LIG4, Rad50,XRCC4 was selected as the target molecule. Real-time PCR found that the mRNA expression levels of XRCC4,Ku80,DNA-PKcs and Rad50 decreased in turn at A549A549-2GyR and A549-4GyR, respectively. Ku70 and LIG4 were also the lowest in A549-4GyR cells. 3. The protein expression of repair related protein DNA-PKcs,XRCC4,LIG4,Ku80,Ku70, Rad50,Mrel1,NBN,XLF was detected by Western Blot in A549, A549-2GyR and A549-4GyR. The expression of cell cycle associated protein p53, phospho-p53 (Ser20) and Phospho-p53 (Ser37), Cyclin D1 + CDK2) decreased in turn, while the expression of p21 increased gradually. 4. The distribution of A549 cell cycle was detected by flow cytometry, and the distribution of A549 cell cycle was detected in G _ 1 phase (62.63) and G _ 2 phase (10.04) and S phase (27.33%), A549-2GyR was 64.45 G _ 2 phase 11.77 ~ (th) and 23.79% in G _ (1) phase and G _ (2) phase in G _ 1 phase. A549-4GyR was 72.37 in G1 and 8.7in G2, and 18.93 in S. The apoptotic rate of A549 was 0.7%, that of A549-2GyR was 1.7%, and that of A549-4GyR was 0.3%. Conclusion: 1. Radiation resistant cells can be obtained by multiple radiotherapy screening. 2. Compared with A549 cells and A549-2GyR cells, there were significant differences in the expression of DNA damage repair related proteins in A549-4GyR cells. The results showed that there was a difference in the mRNA transcription level of the damage repair molecule Ku70,Ku80,DNA-PKcs,LIG4,Rad50,XRCC4, and the lowest expression level was found in the large fraction radiotherapy resistant cells. 3. At the level of protein translation, the protein expression of the large segment resistant cells was lower than that of the control group and the conventional division resistant cells, which was consistent with the results of mRNA level detection. 4. There was no significant difference in apoptotic level between A549 and A549-2 Gy R and A549-4GyR. 5. The results of flow cytometry showed that the large segment resistance cells had more cell arrest in G 1 / S phase than those in the control group and the conventional division resistant cells, and the results were consistent with the results of protein expression test, but the expression level of DNA damage repair protein was decreased. Cell cycle arrest and more adequate time to respond to radiation-induced DNA damage may be the main cause of resistance.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R734.2;R730.55
本文編號：2325905
[Abstract]:Objective: 1. The changes of DNA damage and repair ability of conventional fractionated radiotherapy resistant cell lines and large fraction radiotherapy resistant cell lines were detected at the level of mRNA and protein. 2. The cell cycle distribution, apoptosis level and the mechanism of radiation resistance were analyzed. Methods: 1. A549, A549-2Gy R, A549-4GyR cells were cultured in vitro. Three kinds of cells were irradiated with different doses of radiation. 2. According to the results of the whole genome expression microarray, the proteins associated with DNA damage repair and different expression levels were screened out. A549, A549-2Gy R, A549-4GyR cells were cultured in vitro. The target protein DNA template was obtained by RT-PCR, and the difference of repair related proteins in resistant cells at mRNA level was quantitatively analyzed by Real-time PCR. 3. Western Blot was used to detect the expression of repair related proteins such as Ku70,Ku80,DNA-PKcs,LIG4, Rad50,XRCC4,Mrel1 and p53 p21 and cyclin D1 in the basic state of resistant cells. 4. Cell cycle distribution (PI single staining) and apoptosis level (Annexin V/PI double staining) of resistant cells were detected by flow cytometry. Results: 1. After radiotherapy, A 549, A 549-2 Gy R, A549-4GyR cells increased in turn. 2. Through the analysis and screening of the whole genome microarray results, KKu70,Ku80,DNA-PKcs,LIG4, Rad50,XRCC4 was selected as the target molecule. Real-time PCR found that the mRNA expression levels of XRCC4,Ku80,DNA-PKcs and Rad50 decreased in turn at A549A549-2GyR and A549-4GyR, respectively. Ku70 and LIG4 were also the lowest in A549-4GyR cells. 3. The protein expression of repair related protein DNA-PKcs,XRCC4,LIG4,Ku80,Ku70, Rad50,Mrel1,NBN,XLF was detected by Western Blot in A549, A549-2GyR and A549-4GyR. The expression of cell cycle associated protein p53, phospho-p53 (Ser20) and Phospho-p53 (Ser37), Cyclin D1 + CDK2) decreased in turn, while the expression of p21 increased gradually. 4. The distribution of A549 cell cycle was detected by flow cytometry, and the distribution of A549 cell cycle was detected in G _ 1 phase (62.63) and G _ 2 phase (10.04) and S phase (27.33%), A549-2GyR was 64.45 G _ 2 phase 11.77 ~ (th) and 23.79% in G _ (1) phase and G _ (2) phase in G _ 1 phase. A549-4GyR was 72.37 in G1 and 8.7in G2, and 18.93 in S. The apoptotic rate of A549 was 0.7%, that of A549-2GyR was 1.7%, and that of A549-4GyR was 0.3%. Conclusion: 1. Radiation resistant cells can be obtained by multiple radiotherapy screening. 2. Compared with A549 cells and A549-2GyR cells, there were significant differences in the expression of DNA damage repair related proteins in A549-4GyR cells. The results showed that there was a difference in the mRNA transcription level of the damage repair molecule Ku70,Ku80,DNA-PKcs,LIG4,Rad50,XRCC4, and the lowest expression level was found in the large fraction radiotherapy resistant cells. 3. At the level of protein translation, the protein expression of the large segment resistant cells was lower than that of the control group and the conventional division resistant cells, which was consistent with the results of mRNA level detection. 4. There was no significant difference in apoptotic level between A549 and A549-2 Gy R and A549-4GyR. 5. The results of flow cytometry showed that the large segment resistance cells had more cell arrest in G 1 / S phase than those in the control group and the conventional division resistant cells, and the results were consistent with the results of protein expression test, but the expression level of DNA damage repair protein was decreased. Cell cycle arrest and more adequate time to respond to radiation-induced DNA damage may be the main cause of resistance.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R734.2;R730.55
【參考文獻】
相關(guān)期刊論文 前5條
1 鄒劍;葉惠平;張懿;楊玉瓊;趙厚育;陳飛;劉世喜;;載ATM反義寡核苷酸納米粒對小鼠SCCⅦ細胞放射增敏實驗研究[J];中國耳鼻咽喉顱底外科雜志;2009年04期
2 任濤;陳曉品;;ATM基因與腫瘤細胞放射敏感性關(guān)系的研究進展[J];癌變.畸變.突變;2008年04期
3 李革;李林虎;尹玩熙;;直腸癌自發(fā)性細胞凋亡指數(shù)、ATM蛋白表達和放射敏感性的關(guān)系[J];廣東醫(yī)學;2008年03期
4 張梅光;王曉凌;徐世杰;沈云天;樓建林;鄭偉;;肺癌患者射線誘發(fā)的淋巴細胞遺傳損傷及ATM蛋白表達的研究[J];實用腫瘤雜志;2007年05期
5 李作生;李保慶;;p21和p27基因多態(tài)性與腫瘤的相關(guān)性[J];國際遺傳學雜志;2006年04期
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