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(P<0.05),SF2值分别为0.609±0.018和0.260±0.012(P<0.05)。
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(P<0.05),24h后G2/M期细胞明显增加(P<0.05),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(P<0.05)。
【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(P<0.05)。
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(P<0.05)。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(P<0.05)。
【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。
NPC (nasopharyngeal carcinoma, NPC), also known as "Guang don g cancer" is one of the top ten cancer [1], the World Health Organization ro ugh statistics that there are about 80% of nasopharyngeal carcinoma in China all over the world. NPC is in the top of the head and neck cancer incidence, in south China, especially Guangdong and Guangxi region is the highest incidence (30-80/10 million), give people a tremendous threat for the health .The happen of nasopharyngeal carcinoma is closely related to factors such as genetic, EB virus, environmental pollution .Due to the nasopharynx in the deep base of the skull, the surgery is very difficult, so the combined therapy dominated by radiation therapy (including radiotherapy,concurent chemoradiotherapy (concurrent chemoradiotherapy, CCRT), neoadjuvant chemotherapy (neoadjuvant chemotherapy , NAC)+ radiotherapy and radiotherapy + adjuvant chemotherapy (adjuvant chemotherapy, AC), etc.) is the main treatment for nasopharyngeal carcinoma. In newly diagnosed patients, patients with locally advanced and the late region is about 70%. These patients in clinical T1 + T2 a nd T3 + T4 who received conventional radiotherapy ,the local control rate of that was 64% to 95% and 44% to 68%~( [2, 3]), the main reason for treatment failure is local recurrence or distant metastasis, local recurrence rate was about 8% to 20%, regional recurrence rate was about 10% to 21% [4]. In recent years, with the rise of treatment modalities such as IMRT (Intensity-modulated radiation therapy, IMRT) and concurrent chemotherapy to carry out, the 5-year survival rate of the distant metastasis naso pharyngeal carcinoma have achieved a new breakthrough, more than 80% [5-9].In particularly, the meta-analysis have showed chemotherapy had made the 5-year disease-free survival of no metastatic naso pharynxgeal cancer increased by 10%, 5-year survival rate increased 6%, HR value was 0.86 [10]. The position of chemotherapy in the treatment of nasopharyngeal carcinoma has become increasingly prominent. The radiation and chemotherapy is still the main treatment for the recurrence and metastasis after radical cure, however, poor treatment, median survival time after systemic chemotherapy was only 10-20 months [11-13].
In summary, the relapse and metastasis after radical radiotherapy and chemotherapy is still the major cause of the treatment failure of locally advanced nasopharyngeal carcinoma, and the tumor cells survive the constantly mutate may be the source of recurrence, then the residual tumor cells induce resistance to radiation [14]. What is the molecular basis of the radiation resistance for these residual cells? How to overcome these remaining cells’resistant to radiation? So many problems have yet to resolve. Although chemotherapy in NPC, especially in the treatment of locally advanced nasopharyngeal carcinoma have becoming more and m ore attention, but the combination of chemotherapy and how effectively,what is the best comprehensive treatment strategy? That has become a hot research field of nasopharyngeal carcinoma, and caused widespread controversy academia. Why the difference between effect of chemotherapy in the patients with Recurrence or metastasis of nasopharyngeal carcinoma after radical radiotherapy and chemotherapy, whether the remaining cells have got the radiation resistance, at the same time produced a multi drug resistance (multidrug resistance, MDR)?
Therefore, in order to solve these problems, this project is to apply theγ-ray to the poorly differentiated nasopharyngeal carcinoma cell CNE-2 with the largesplit interval of radiation, to induce the resistance cell line CNE-2R, to take the further study of the relationship between the radiation resistance of nasopharyngeal carcinoma and chemosensitivety.
【objective】To establish a stable radiation-induced cell model of radiation
resistance through irradiating the NPC cell line CNE-2 that is relatively sensitive to radiation, so as to provide a good contrast models for the study of the radiation resistance mechanisms and the relationship between radiation resistance and multidrug resistance.
【methods】
1.To irradiate the nasopharyngeal carcinoma cell line CNE-2 that is relative sensitivity for X-ray ,with intermittent high-doseγ-irradiation (4 Gy/f, 15f, total 60Gy), culture each cell after exposure , take the next exposure to cells that survival still after 3-4 weeks and entered the exponential growth phase, the exposure and training process lasted 12 months, the resulting progeny cells was called CNE-2R (CNE-2 radiation induction ).
2.Use colony formation assay and the linear quadratic model to fit the dose-survival curve of CNE-2R and CNE-2 cells and calculate the radiation biology parameters, to detect its radiation resistant. Culture cells for 8 days in a row and draw the cell growth curve of CNE-2 and CNE-2R. To detect the change of the cell cycle of CNE-2R and its parent cell CNE-2 after irradiation by flow cytometry.
3.While the two cells been frozen for three months and passaged 10 times,carry colony formation experiments again and refit dose survival curve, to test the stability of the radiation resistance.
【results】
1.Theα/βvalues of the CNE-2R generated from intermittent high dose irradiation and its parent cell CNE-2 were 3.947±0.314 and 22.3 33±4.786 (P <0.05), SF2 values were 0.609±0.018 and 0.260±0.012 ( P <0.05).
2.The doubling time of CNE-2 cell is 24.8h, CNE-2R is 40.67h. The cycle distribution of CNE-2 cell was that G0/G1 phase was 59.4%±1.00, S phase was 29.1%±1.23, G2/M phase was 12.7%±0.80; the cell cycle distribution of CNE-2R was that G0/G1 phase was 69.6%±1.96, S phase was 23.6%±1.91, G2 of 6.7%±0.78. The exponential doubling time of CNE-2 was 40.6%, CNE-2R was 30.4%. After 12h of 4Gy irradiation , S phase of CNE-2 have increased significantly (P<0.05), after 24h the G2/M phase cells increased significantly (P<0.05), after 48h returned to normal state. CNE-2R cell cycle is not change significantly over time.
3.After the two cells cryopreserved 3 months and continued passage 10 times,α/βvalues of CNE-2R and CNE-2 cells were 4.049±1.122 and 21.637±1.203 (P<0.05), SF2 values were 0.605±0.055 and 0.226±0.008 (P<0.05).
【conclusions】
1.Compared to parental cells CNE-2,α/βvalue of the CNE-2R induced with intermittent high-dose radiation was significantly smaller, SF2 was significantly increased, showing a significant resistance to radiation.
2.Cell cycle analysis results indicate that the proportion of CNE -2R strain on proliferation was less than the CNE-2, the doubling time was longer than the CNE-2 cell lines. After irradiation by a single 4Gy, CNE-2 cells showed a redistibution phenomenon,the irradiation has little effect on cell cycle distribution of CNE-2R cells.
3.After frozen 3 months and continued passageing 10 times,the CNE-2R line successfully maintained its resistance to radiation.
【objective】The study was to research the relationship between radiation resistance and chemosensitivity of nasopharyngeal carcinoma CNE-2R cell lines ,to explore changes in sensitivity to chemotherapy of the CNE-2 cells before and after hypofractionated radiationinduced, whether produce multidrug resistance.
【methods】
Take NPC radiation resistance cell lines CNE-2R induced in the early experiments as the experimental group,the parental cell line CNE-2 as the control group, use MTT assay to detect killing rate of the two cells from cisplatin, 5-fluorouracil, carboplatin, paclitaxel , gemcitabine, doxorubicin and other drugs,and find the half inhibitory concentration, then calculate the relative resistance index of the resistance cell line CNE-2R; take the same concentration of cisplatin and 5-fluorouracil to act on the two sets of cells, 48 hours treatment ,use flow cytometry to detect the apoptosis.
【results】
1.MTT assay to detect cytotoxicity results
The resistance index of the NPC radioresistance cell line CNE-2R induced by intermittent high-dose radiation to cisplatin, 5-fluorouracil, carboplatin, paclitaxel,gemcitabine,doxorubicin 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,compared with CNE-2 cells,half inhibitory concentr ation IC50 were significantly incresed the differences were statistically significant (P <0.05).
2.The apoptosis induced by cisplatin or 5-fluorouracil
The apoptosis of CNE-2 and CNE-2R cultured in normal after 48h were 5.827%±0.033 and 6.297%±0.045, the difference was not statistically significant.The apoptosis of CNE-2 and CNE-2R cultured in 10μg/ml 5-Fu after 48h were 31.735%±2.529 and 16.18%±1.281, the difference was significant (P<0.05). The apoptosis of CNE-2 and CNE -2R cultured in 1μg/ml DDP after 48h were 56.98%±3.738 and 36. 897%±3.290, the difference was significant (P <0.05).
【conclusions】In this study, NPC radiation resistance cell lines CNE -2R induced in the early experiments, have produced radiation resistant, also have got multidrug resistance.
引文
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