鼻咽癌辐射抗拒细胞株CNE-2R模型的构建及其化疗药物敏感性初步研究

发布时间：2018-02-06 07:13

本文关键词： 鼻咽癌细胞株辐射抗拒线性二次模型细胞周期鼻咽癌辐射抗拒多药耐药细胞凋亡　出处：《广西医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：【目的】通过对放疗相对敏感的鼻咽癌细胞系CNE-2进行辐射诱导建立稳定的辐射抗拒细胞模型,从而为研究辐射抗拒机制及辐射抗拒与多药耐药之间关系提供良好的对比模型。【方法】 1、选取对X射线相对敏感的鼻咽癌细胞株CNE-2进行间歇性大剂量γ射线照射(每次4Gy,共15次,总量60Gy),每次照射后的细胞继续培养,待3-4周存活的细胞进入指数生长期后进行下一次照射,整个照射及培养过程历时12个月,最终得到的后代细胞命名为CNE-2R(CNE-2 radiation induction)。 2、采用克隆形成实验及线性二次模型拟合CNE-2R和CNE-2细胞的剂量存活曲线并计算相关放射生物学参数,检测其辐射抗拒性。连续8天培养细胞,绘制CNE-2和CNE-2R细胞生长曲线。流式细胞技术检测CNE-2R及其亲本细胞CNE-2经单次照射后细胞周期的变化。 3、两种细胞同时冻存三个月并传代10次后,重新进行集落形成实验拟合剂量存活曲线,检测其辐射抗拒稳定性。【结果】 1、经大剂量间歇性照射得到的CNE-2R和其亲代细胞CNE-2的α/β值分别为3.947±0.314和22.333±4.786(P0.05),SF2值分别为0.609±0.018和0.260±0.012(P0.05)。 2、CNE-2细胞的倍增时间为24.8h,CNE-2R为40.67h。CNE-2细胞周期分布为G0/G1期为59.4%±1.00,S期为29.1%±1.23,G2/M期为12.7%±0.80;CNE-2R细胞周期分布为G0/G1期为69.6%±1.96,S期为23.6%±1.91,G2期6.7%±0.78。CNE-2增值指数为40.6%,CNE-2R增值指数为30.4%。经4Gy照射后12hCNE-2细胞S期细胞明显增加(P0.05),24h后G2/M期细胞明显增加(P0.05),48h后基本恢复正常状态,CNE-2R细胞周期未随时间的变化而明显改变。 3、两种细胞同时冻存3个月并继续传代10次后CNE-2R和CNE-2细胞的α/β值分别为4.049±1.122和21.637±1.203(P 0. 05),SF2值分别为0.605±0.055和0.226±0.008(P0.05)。【结论】 1、与亲本CNE-2细胞相比较,经间歇性大剂量照射诱导得到的CNE-2R细胞α/β值明显变小,SF2值明显增大,显示出了明显的辐射抗拒性。 2、细胞周期检测结果说明CNE-2R株处于增殖期的细胞比例较CNE-2少,其倍增时间长于CNE-2细胞株。经单次4Gy照射后CNE-2细胞株表现出了再分布现象,而照射对CNE-2R细胞的细胞周期分布影响很小。 3、CNE-2R细胞株经过冻存3个月并继续传代10次后成功保持了其辐射抗拒性。【目的】研究鼻咽癌CNE-2R辐射抗拒与化疗药物敏感性之间的关系,探讨CNE-2细胞株大分割辐射诱导前后化疗药物敏感性的改变,验证是否产生多药耐药性。【方法】以前期实验诱导出的鼻咽癌辐射抗拒细胞株CNE-2R为实验组,以亲代细胞株CNE-2为对照组,应用MTT法检测两组细胞对顺铂、5-氟尿嘧啶、卡铂、紫杉醇、吉西他滨、阿霉素等药物的杀伤率,并求出半数抑制浓度,进而计算抗拒细胞株CNE-2R的相对耐药指数;以相同浓度的顺铂和5-氟尿嘧啶作用于两组细胞,处理48小时后,应用流式细胞仪技术检测其凋亡率。【结果】 1)MTT法检测细胞杀伤结果: 经间歇性大剂量放疗诱导出的鼻咽癌辐射抗拒细胞系CNE-2R对顺铂、5-氟尿嘧啶、卡铂、紫杉醇、吉西他滨、阿霉素的耐药指数分别为3.26±0.31、3.65±0.09、10.04±1.34、1.51±0.03、8.95±0.20、3.16±0.39,半数抑制浓度IC50较CNE-2细胞均有明显升高,差异均具有统计学意义(P0.05)。 2)顺铂或5-氟尿嘧啶诱导的细胞凋亡: 正常培养48h的CNE-2和CNE-2R细胞凋亡率分别为5.827%±0.033和6.297%±0.045,差异无统计学意义。经10цg/ml 5-Fu处理48h后的CNE-2和CNE-2R细胞凋亡率分别为31.735%±2.529和16.18%±1.281,差异具有统计学意义(P0.05)。经1цg/ml DDP处理48h后的CNE-2和CNE-2R细胞凋亡率分别为56.98%±3.738和36.897%±3.290,差异具有统计学意义(P0.05)。【结论】本实验前期诱导出的辐射抗拒细胞株CNE-2R,在产生辐射抗拒性的同时,产生了多药耐药性。
[Abstract]:[Objective] to establish a stable radioresistance cell model by radiating the nasopharyngeal carcinoma cell line CNE-2, which is relatively sensitive to radiotherapy, so as to provide a good contrast model for studying the relationship between radiation resistance mechanism and the relationship between radiation resistance and multidrug resistance.
[method]
1, the relative sensitivity to X radiation nasopharyngeal carcinoma cell line CNE-2 by intermittent high dose gamma irradiation (4Gy each time, 15 times in total, total 60Gy), each time after irradiation the cells were cultured for 3-4 weeks, the survival of the cells entered exponential growth phase after the next irradiation, irradiation and the whole training process lasted 12 month, the final cell lines named CNE-2R (CNE-2 radiation induction).
2, the clone formation experiment and linear two model fitting CNE-2R and CNE-2 cell dose survival curve and calculate the radiobiological parameters, the detection of resistant to radiation. 8 consecutive days of cultured cells, CNE-2 and draw the growth curve of CNE-2R cells. Flow cytometry CNE-2R and its parental cell line CNE-2 by cell cycle changes of single irradiation after.
3, two kinds of cells were stored for three months at the same time and passed for 10 times, then the colony formation experiment was re conducted to fit the dose survival curve, and the stability of radiation resistance was detected.
[results]
1, the alpha / beta values of CNE-2R and parental cells CNE-2 obtained by high-dose intermittent irradiation were 3.947 + 0.314 and 22.333 + 4.786 (P0.05) respectively, and SF2 values were 0.609 0.609, 0.018 and 0.260 + 0.012 (P0.05), respectively.
2, the doubling time of CNE-2 cells for 24.8h, CNE-2R for the cell cycle distribution of 40.67h.CNE-2 for G0/G1 was 59.4% + 1, 29.1% + 1.23 S period, G2/M period of 12.7% + 0.80; CNE-2R cell cycle distribution of the G0/G1 for a period of 69.6% + 1.96, 23.6% + 1.91 S, G2 6.7% + 0.78.CNE-2 value index 40.6%, the CNE-2R value index of 30.4%. irradiated by 4Gy 12hCNE-2 cells in S cells were significantly increased (P0.05), G2/M phase cells increased significantly after 24h (P0.05), returned to normal state after 48h, CNE-2R cell cycle did not change with time and change.
3, two cells were cryopreserved for 3 months and continued for 10 times. The CNE-2R / CNE-2 cell's alpha / beta values were 4.049 + 1.122 and 21.637 + 1.203 (P 0.05) respectively, and SF2 values were 0.605, 0.055, and 0.055 + P0.05 respectively.
[Conclusion]
1, compared with parental CNE-2 cells, the CNE-2R / beta value of CNE-2R cells induced by intermittent high-dose irradiation decreased significantly, and the value of SF2 increased significantly, showing a significant radiant resistance.
2, cell cycle detection showed that the proportion of CNE-2R cells in proliferative phase was less than that of CNE-2, and its doubling time was longer than that of CNE-2 cell line. After single 4Gy irradiation, CNE-2 cell lines showed redistribution phenomenon, while irradiation had little effect on the cell cycle distribution of CNE-2R cells.
3, the CNE-2R cell line kept its radiation resistance after 3 months of cryopreservation and continued to be passaged for 10 times.
[Objective]
Objective to study the relationship between CNE-2R radiation resistance and chemosensitivity of nasopharyngeal carcinoma (NPC), and to explore the sensitivity of CNE-2 cell line to chemotherapeutic drugs before and after irradiation.
[method]
In the previous experiment induced nasopharyngeal carcinoma radioresistant cell line CNE-2R as the experimental group, with the parental cell line CNE-2 as the control group, MTT was used to detect two groups of 5- cells to cisplatin, fluorouracil, carboplatin, paclitaxel, gemcitabine, the killing rate of adriamycin and other drugs, and calculate the half inhibitory concentration, and the relative resistance index of resistance CNE-2R cells were calculated; with the same concentration of cisplatin and 5- fluorouracil in two groups of cells, after 48 hours of treatment, the rate of apoptosis detected by flow cytometry.
[results]
1) MTT assay was used to detect cell killing results:
After intermittent high dose radiotherapy induced radioresistance of nasopharyngeal carcinoma cell line CNE-2R to cisplatin, fluorouracil 5-, carboplatin, paclitaxel, gemcitabine, drug resistance index of adriamycin were 3.26 + 0.31,3.65 + 0.09,10.04 + 1.34,1.51 + 0.03,8.95 + 0.20,3.16 + 0.39, half inhibitory concentration IC50 than CNE-2 cells were significantly increased, the differences were statistically significant (P0.05).
2) apoptosis induced by cisplatin or 5- fluorouracil:
The normal cultured 48h CNE-2 and apoptosis rate of CNE-2R cells were 5.827% + 0.033 and 6.297% + 0.045, the difference was not statistically significant. The CNE-2 10 tse.png g/ml 5-Fu after 48h treatment and apoptosis rate of CNE-2R cells were 31.735% + 2.529 and 16.18% + 1.281, the difference was statistically significant (P0.05). After 1 CNE-2 a g/ml DDP after 48h treatment and apoptosis rate of CNE-2R cells were 56.98% + 3.738 and 36.897% + 3.290, the difference was statistically significant (P0.05).
[Conclusion]
The radiation resistance cell strain CNE-2R induced in the early stage of the experiment produces multidrug resistance at the same time that the radiation resistance is produced.

【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R739.63

【引证文献】