本文關(guān)鍵詞： 鼻咽癌 細(xì)胞株 輻射抗拒 線性二次模型 細(xì)胞周期 鼻咽癌 輻射抗拒 多藥耐藥 細(xì)胞凋亡　出處：《廣西醫(yī)科大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：【目的】通過對放療相對敏感的鼻咽癌細(xì)胞系CNE-2進(jìn)行輻射誘導(dǎo)建立穩(wěn)定的輻射抗拒細(xì)胞模型,從而為研究輻射抗拒機制及輻射抗拒與多藥耐藥之間關(guān)系提供良好的對比模型。 【方法】 1、選取對X射線相對敏感的鼻咽癌細(xì)胞株CNE-2進(jìn)行間歇性大劑量γ射線照射(每次4Gy,共15次,總量60Gy),每次照射后的細(xì)胞繼續(xù)培養(yǎng),待3-4周存活的細(xì)胞進(jìn)入指數(shù)生長期后進(jìn)行下一次照射,整個照射及培養(yǎng)過程歷時12個月,最終得到的后代細(xì)胞命名為CNE-2R(CNE-2 radiation induction)。 2、采用克隆形成實驗及線性二次模型擬合CNE-2R和CNE-2細(xì)胞的劑量存活曲線并計算相關(guān)放射生物學(xué)參數(shù),檢測其輻射抗拒性。連續(xù)8天培養(yǎng)細(xì)胞,繪制CNE-2和CNE-2R細(xì)胞生長曲線。流式細(xì)胞技術(shù)檢測CNE-2R及其親本細(xì)胞CNE-2經(jīng)單次照射后細(xì)胞周期的變化。 3、兩種細(xì)胞同時凍存三個月并傳代10次后,重新進(jìn)行集落形成實驗擬合劑量存活曲線,檢測其輻射抗拒穩(wěn)定性。 【結(jié)果】 1、經(jīng)大劑量間歇性照射得到的CNE-2R和其親代細(xì)胞CNE-2的α/β值分別為3.947±0.314和22.333±4.786(P0.05),SF2值分別為0.609±0.018和0.260±0.012(P0.05)。 2、CNE-2細(xì)胞的倍增時間為24.8h,CNE-2R為40.67h。CNE-2細(xì)胞周期分布為G0/G1期為59.4%±1.00,S期為29.1%±1.23,G2/M期為12.7%±0.80;CNE-2R細(xì)胞周期分布為G0/G1期為69.6%±1.96,S期為23.6%±1.91,G2期6.7%±0.78。CNE-2增值指數(shù)為40.6%,CNE-2R增值指數(shù)為30.4%。經(jīng)4Gy照射后12hCNE-2細(xì)胞S期細(xì)胞明顯增加(P0.05),24h后G2/M期細(xì)胞明顯增加(P0.05),48h后基本恢復(fù)正常狀態(tài),CNE-2R細(xì)胞周期未隨時間的變化而明顯改變。 3、兩種細(xì)胞同時凍存3個月并繼續(xù)傳代10次后CNE-2R和CNE-2細(xì)胞的α/β值分別為4.049±1.122和21.637±1.203(P 0. 05),SF2值分別為0.605±0.055和0.226±0.008(P0.05)。 【結(jié)論】 1、與親本CNE-2細(xì)胞相比較,經(jīng)間歇性大劑量照射誘導(dǎo)得到的CNE-2R細(xì)胞α/β值明顯變小,SF2值明顯增大,顯示出了明顯的輻射抗拒性。 2、細(xì)胞周期檢測結(jié)果說明CNE-2R株處于增殖期的細(xì)胞比例較CNE-2少,其倍增時間長于CNE-2細(xì)胞株。經(jīng)單次4Gy照射后CNE-2細(xì)胞株表現(xiàn)出了再分布現(xiàn)象,而照射對CNE-2R細(xì)胞的細(xì)胞周期分布影響很小。 3、CNE-2R細(xì)胞株經(jīng)過凍存3個月并繼續(xù)傳代10次后成功保持了其輻射抗拒性。 【目的】 研究鼻咽癌CNE-2R輻射抗拒與化療藥物敏感性之間的關(guān)系,探討CNE-2細(xì)胞株大分割輻射誘導(dǎo)前后化療藥物敏感性的改變,驗證是否產(chǎn)生多藥耐藥性。 【方法】 以前期實驗誘導(dǎo)出的鼻咽癌輻射抗拒細(xì)胞株CNE-2R為實驗組,以親代細(xì)胞株CNE-2為對照組,應(yīng)用MTT法檢測兩組細(xì)胞對順鉑、5-氟尿嘧啶、卡鉑、紫杉醇、吉西他濱、阿霉素等藥物的殺傷率,并求出半數(shù)抑制濃度,進(jìn)而計算抗拒細(xì)胞株CNE-2R的相對耐藥指數(shù);以相同濃度的順鉑和5-氟尿嘧啶作用于兩組細(xì)胞,處理48小時后,應(yīng)用流式細(xì)胞儀技術(shù)檢測其凋亡率。 【結(jié)果】 1)MTT法檢測細(xì)胞殺傷結(jié)果: 經(jīng)間歇性大劑量放療誘導(dǎo)出的鼻咽癌輻射抗拒細(xì)胞系CNE-2R對順鉑、5-氟尿嘧啶、卡鉑、紫杉醇、吉西他濱、阿霉素的耐藥指數(shù)分別為3.26±0.31、3.65±0.09、10.04±1.34、1.51±0.03、8.95±0.20、3.16±0.39,半數(shù)抑制濃度IC50較CNE-2細(xì)胞均有明顯升高,差異均具有統(tǒng)計學(xué)意義(P0.05)。 2)順鉑或5-氟尿嘧啶誘導(dǎo)的細(xì)胞凋亡: 正常培養(yǎng)48h的CNE-2和CNE-2R細(xì)胞凋亡率分別為5.827%±0.033和6.297%±0.045,差異無統(tǒng)計學(xué)意義。經(jīng)10цg/ml 5-Fu處理48h后的CNE-2和CNE-2R細(xì)胞凋亡率分別為31.735%±2.529和16.18%±1.281,差異具有統(tǒng)計學(xué)意義(P0.05)。經(jīng)1цg/ml DDP處理48h后的CNE-2和CNE-2R細(xì)胞凋亡率分別為56.98%±3.738和36.897%±3.290,差異具有統(tǒng)計學(xué)意義(P0.05)。 【結(jié)論】 本實驗前期誘導(dǎo)出的輻射抗拒細(xì)胞株CNE-2R,在產(chǎn)生輻射抗拒性的同時,產(chǎn)生了多藥耐藥性。
[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.

【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R739.63

【引證文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 林戀竹;溪黃草有效成分分離純化、結(jié)構(gòu)鑒定及活性評價[D];華南理工大學(xué);2013年

，

本文編號：1493886