西妥昔单抗与放化疗联用对喉鳞癌细胞的协同杀伤效应的实验研究

发布时间：2018-06-25 04:51

本文选题：喉肿瘤 + 西妥昔单抗　；参考：《山西医科大学》2013年硕士论文

【摘要】：目的：西妥昔单抗（Cetuximab)是一种人鼠嵌合型IgG单克隆抗体，可与细胞表面的表皮生长因子受体(epidermal growth factor receptor, EGFR)特异性结合，竞争性地阻断表皮生长因子和其配体的结合，从而阻断肿瘤细胞增殖、转移、侵袭以及血管生成等生物学效应。Cetuximab与部分化疗药物或放射线对不同种类的肿瘤细胞具有协同杀伤的效应，降低不同种类肿瘤细胞对Cetuximab的抵抗性，提高对Cetuximab的敏感性。本研究拟通过观察西妥昔单抗联合顺铂、放射线诱导人喉鳞癌细胞株H印-2的增殖、凋亡及细胞周期的影响，探讨西妥昔单抗与顺铂、放射线联合应用对Hep-2细胞的杀伤效应及对其调控机制的初步探讨。方法：1、体外培养人喉鳞状细胞癌Hep-2细胞株，取对数生长期的细胞进行试验。 2、采用CCK-8试剂盒（cell counting kit-8)分别检测不同剂量的西妥昔单抗、顺钼、放射线对人喉鳞癌Hep-2细胞的12h、24h、48h、72h生长抑制率。 3、实验组分别给予西妥昔单抗1036ug/ml，顺销3ug/ml,放射线4Gy,顺钼3μg/ml+放射线4Gy,西妥昔单抗1036u g/ml’+顺钼3μg/ml，西妥昔单抗1036μg/ml+放射线4Gy，西妥昔单抗1036μg/ml+顺柏3μg/m丨+放射线4Gy,同时设立阴性对照组（不添加任何药物），分别作用于人喉鳞癌Hep-2细胞株24h，倒置显微镜观察细胞形态，CCK-8试剂盒检测细胞生长抑制率，流式细胞仪检测不同干预方案对Hep-2凋亡率及细胞周期分布情况。结果：1、不同剂量的西妥昔单抗、顺铀、放射线对Hep-2细胞均有抑制作用，并在一定浓度范围内呈时间-剂量依赖性，24h半数抑制浓度（halfmaximal inhibitory concentration, IC50)分别为两妥昔单抗1036.84ii g/L、顺钼3.08μ g/ml、放射线4.18Gy; H印-2细胞对西妥昔单抗的生长抑制作用较敏感。 2、顺铂，放射线分别与西妥昔单抗联合应用时对Hep-2细胞的生长抑制率显著高于顺铂、放射线单独或联合应用（P0.001），具有协同杀伤效应。 3、经西妥昔单抗与放射线、西妥昔单抗与顺铂联合作用24h后处于早期凋亡阶段的Hep-2细胞分别为（21.92±3.00）%、（18.44±2.57）%，显著高于阴性对照组与放射线、顺铂的单独或联合作用组（P0.001），提示西妥昔单抗对顺铂或放射线在体外诱导人喉癌Hep-2细胞株的凋亡具有显著的协同作用。 4、单药西妥昔单抗组中S期细胞所占比例较阴性对照组比例显著增高（P0.001）；西妥昔单抗+放射线组（P=0.005），西妥昔单抗+顺铂+放射线组（P=0.002）中，G_0/G_1期细胞所占比例明显高于单纯放射线组。结论：1、人喉鳞癌Hep-2细胞株对西妥昔单抗诱导的细胞凋亡敏感，在一定浓度范围内呈时间和剂量依赖性。 2、顺铂和/或放射线与西妥昔单抗联用能显著提高对Hep-2细胞的生长抑制作用，对Hep-2细胞的增殖具有协同抑制效应。 3、西妥昔单抗与顺铂或放射线联用能显著提高Hep-2的凋亡率，提示西妥昔单抗对顺铂或放射线在体外诱导人喉癌Hep-2细胞株的凋亡具有显著的协同作用。 4、西妥昔单抗可以通过将Hep-2细胞阻滞在细胞分裂的S期达到抑制细胞分裂阻止其增殖失控的作用。西妥昔单抗与放射线联用亦可使人喉癌Hep-2细胞株阻滞在对放射线敏感的G_0/G_1期，体外可显著诱导细胞凋亡，，从而对放射线具有较强的协同作用。这可能是顺铂、放射线与西妥昔单抗联合应用对Hep-2细胞发挥协同效应的机制之一。
[Abstract]:Objective : To investigate the effect of cetuximab and cisplatin and radiation on the proliferation , apoptosis and cell cycle of human laryngeal squamous cell carcinoma cell line H - 2 , and to investigate the effect of cetuximab and cisplatin on the proliferation , apoptosis and cell cycle of human laryngeal squamous cell carcinoma cell line H - 2 .

Methods : 1 . Human laryngeal squamous cell carcinoma Hep2 cell line was cultured in vitro , and the cells with logarithmic growth were tested .

2 . CCK - 8 kit ( cell counting kit - 8 ) was used to detect the growth inhibition rate of cetuximab , cisplatin and radiation at 12 h , 24 h , 48 h and 72 h in human laryngeal squamous cell carcinoma .

3 . In the experimental group , 1036ug / ml , 3ug / ml , 4Gy , 1036u g / ml + radiation 4Gy , 1036u g / ml + irradiation 4Gy , 1036u g / ml + irradiation 4Gy , 1036u g / ml + cisplatin 3渭g / ml + radiation 4Gy were given .

Results : 1 . The inhibitory effect of different doses of cetuximab , UO2 and radiation on Hep2 cells was observed . The IC50 values were 106.84ii g / L , 3.08 渭g / ml , 4.18Gy and 4.18Gy , respectively , and the growth inhibition of cetuximab was more sensitive .

2 . The growth inhibition rate of cisplatin and radiation in combination with cetuximab was significantly higher than that of cisplatin , radiation alone or in combination ( P0.001 ) , which had synergistic killing effect .

3 . After the combined action of cetuximab and radiation , the combined action of cetuximab and cisplatin for 24 h was ( 21.92 卤 3.00 ) % , ( 18.44 卤 2.57 ) % , which was significantly higher than that of the negative control group ( P < 0.01 ) .

4 . The proportion of S - phase cells in the single - dose cetuximab group was significantly higher than that of the negative control group ( P0.001 ) .
In the cetuximab plus radiation group ( P = 0.005 ) , the percentage of G _ 0 / G _ 1 cells was significantly higher in the cetuximab plus cisplatin + radiation group ( P = 0.002 ) than in the simple radiation group .

Conclusion : 1 . Human laryngeal squamous cell carcinoma 2 2 cell line is sensitive to apoptosis induced by cetuximab , which is time and dose dependent within a certain concentration range .

2 , the combination of cisplatin and / or radiation and cetuximab can remarkably improve the growth inhibition effect on the Hep2 cells , and has a synergistic inhibitory effect on the proliferation of Hep2 cells .

3 . Combination of cetuximab and cisplatin or radiation could significantly increase the rate of apoptosis , suggesting that cetuximab had a significant synergistic effect on the apoptosis induced by cisplatin or radiation in vitro .

4 . The combination of cetuximab and radiation could arrest the cell division and prevent the cell division from being out of control .
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R739.65

【参考文献】