NSCLC放射抗拒细胞实验平台的建立及初步研究
发布时间：2018-11-11 19:33
【摘要】：目的： 1.在mRNA水平和蛋白水平上检测非小细胞肺癌常规分割放疗抵抗细胞系和大分割放疗抵抗细胞系DNA损伤修复能力的变化。 2.检测放疗抵抗细胞的细胞周期分布、凋亡水平的变化情况及分析放疗抵抗产生的机制。 方法： 1.体外培养A549, A549-2GyR, A549-4GyR细胞,给予三种细胞不同剂量射线照射,克隆形成实验检测细胞增值能力。 2.根据全基因组表达谱芯片结果,分析筛选出与DNA损伤修复相关且有表达量差异的蛋白。体外培养A549, A549-2GyR, A549-4GyR细胞,通过RT-PCR获取目的蛋白DNA模板,运用Real-time PCR精确定量分析抵抗细胞中修复相关蛋白在mRNA水平上的差异。 3. Western Blot检测抵抗细胞中基础状态下Ku70、Ku80、DNA-PKcs、LIG4、 Rad50、XRCC4、Mrel1等修复相关蛋白和p53、p21、CylinD1等周期相关蛋白的表达水平。 4.流式细胞术检测抵抗细胞的细胞周期分布(PI单染)和凋亡水平(Annexin V/PI双染)。 结果： 1.放疗后,A549, A549-2GyR, A549-4GyR细胞增值能力依次增强。 2.通过对全基因组芯片结果进行分析筛选,选择KKu70、Ku80、DNA-PKcs、LIG4、 Rad50、XRCC4作为目的分子,Real-time PCR发现XRCC4、Ku80、DNA-PKcs和Rad50的mRNA表达水平在A549,A549-2GyR和A549-4GyR依次递减,而Ku70和LIG4也是在A549-4GyR细胞中表达量最低。 3. Western Blot检测到修复相关蛋白DNA-PKcs、XRCC4、LIG4、Ku80、Ku70、 Rad50、Mrel1、NBN、XLF的蛋白表达在A549, A549-2GyR和A549-4GyR中依次递减；细胞周期相关蛋白p53、Phospho-p53(Ser20)、Phospho-p53(Ser37)、Cyclin D1、CDK2表达量也依次递减,而p21表达量则逐渐增多。 4.流式细胞术检测到基础状态下A549细胞周期分布,G1期62.63%,G2期10.04%,S期27.33%; A549-2GyR为G1期64.45%,G2期11.77%,S期23.79%；A549-4GyR为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%。 结论： 1.通过多次放疗筛选,可获得放疗抵抗细胞。 2.与对照组细胞A549和常规分割抵抗细胞A549-2GyR相比,大分割抵抗细胞A549-4GyR细胞中DNA损伤修复相关蛋白存在表达量上的差异。实验结果证实损伤修复分子Ku70、Ku80、DNA-PKcs、LIG4、Rad50、XRCC4在mRNA转录水平上存在差异,在大分割放疗抵抗细胞中表达量最低。 3.在蛋白翻译水平上,大分割抵抗细胞与对照组和常规分割抵抗细胞相比,蛋白表达量也降低,与mRNA水平检测结果基本一致。 4.基础状态下,A549、A549-2GyR和A549-4GyR在凋亡水平上无明显差异。 5.流式结果发现大分割抵抗细胞较对照组和常规分割抵抗细胞有更多的细胞阻滞在G1/S期,并且与蛋白表达检测结果相一致,而DNA损伤修复蛋白的表达水平却降低,因此,细胞周期发生阻滞,细胞有更充分的时间应对放射造成的DNA损伤可能是抵抗产生的主要原因。
[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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R734.2;R730.55
本文编号：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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R734.2;R730.55
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