[1]董起杭,万里新,张　凯,等.安罗替尼联合放射治疗对食管鳞状细胞癌细胞增殖、凋亡及放射治疗敏感性的影响[J].新乡医学院学报,2022,39(2):106-112.[doi:10.7683/xxyxyxb.2022.02.002]
　DONG Qihang,WAN Lixin,ZHANG Kai,et al.Effect of anlotinib combined with radiotherapy on the proliferation,apoptosis and radiotherapy sensitivity of esophageal squamous cell carcinoma cells[J].Journal of Xinxiang Medical University,2022,39(2):106-112.[doi:10.7683/xxyxyxb.2022.02.002]
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安罗替尼联合放射治疗对食管鳞状细胞癌细胞增殖、凋亡及放射治疗敏感性的影响
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《新乡医学院学报》[ISSN:1004-7239/CN:41-1186/R]

卷:
39
期数:
2022年2
页码:
106-112
栏目:
基础研究
出版日期:
2022-02-05

文章信息/Info

Title:
Effect of anlotinib combined with radiotherapy on the proliferation,apoptosis and radiotherapy sensitivity of esophageal squamous cell carcinoma cells
作者:
董起杭1; 万里新2; 张　凯3; 王　卓4; 陶海云2; 杜云辉2; 高社干5; 6; 兰子君5; 6; 原　翔5; 6
（1.新乡医学院,河南　新乡　453003;2.南阳市中心医院肿瘤内科，河南　南阳　473000；3.南阳市中心医院放射治疗科，河南　南阳　473000；4.南阳市中心医院药学科，河南　南阳　473000； 5.河南科技大学第一附属医院肿瘤内科，河南　洛阳　471003；6.河南省肿瘤表观遗传学重点实验室，河南　洛阳　471003）
Author(s):
DONG Qihang1; WAN Lixin2; ZHANG Kai3; WANG Zhuo4; TAO Haiyun2; DU Yunhui2; GAO Shegan5; 6; LAN Zijun5; 6; YUAN Xiang5; 6
(1.Xinxiang Medical University,Xinxiang 453003;2.Department of Oncology,Nanyang Central Hospital,Nanyang 473000;3.Department of Radiotherapy,Nanyang Central Hospital,Nanyang 473000;4.Department of Pharmacy,Nanyang Central Hospital,Nanyang 473000;5.Department of Oncology,the First Affiliated Hospital of Henan University of Science and Technology,Luoyang 471003；6.Henan Provincial Key Laboratory of Tumor Epigenetics，Luoyang 471003)
关键词:
安罗替尼; 食管鳞状细胞癌; 放射治疗敏感性
Keywords:
anlotinib; esophageal squamous cell carcinoma; radiotherapy sensitivity
分类号:
R735.1
DOI:
10.7683/xxyxyxb.2022.02.002
文献标志码:
A
摘要:
目的　探讨安罗替尼联合放射治疗对食管鳞状细胞癌细胞的增殖、凋亡及放射治疗敏感性的影响。方法　将对数生长期的食管鳞状细胞癌KYSE-150细胞随机分为0 μmol·L-1安罗替尼组、2 μmol·L-1安罗替尼组、4 μmol·L-1安罗替尼组、8 μmol·L-1安罗替尼组、16 μmol·L-1安罗替尼组、32 μmol·L-1安罗替尼组、64 μmol·L-1安罗替尼组、128 μmol·L-1安罗替尼组，分别用含终浓度为0、2、4、8、16、32、64、128 μmol·L-1安罗替尼的培养基进行培养。采用细胞计数试剂盒-8法检测8组食管鳞状细胞癌KYSE-150细胞的增殖能力，并计算细胞增殖抑制率。应用Graphpad Prism8软件绘制细胞增殖抑制曲线并计算半抑制浓度（IC50），选择IC50值最小时的安罗替尼浓度及处理时间作为后续实验的药物处理条件。另收集对数生长期的食管鳞状细胞癌KYSE-150细胞，随机分为0 Gy单独照射组、2 Gy单独照射组、4 Gy单独照射组、6 Gy单独照射组、0 Gy联合照射组、2 Gy联合照射组、4 Gy联合照射组、6 Gy联合照射组。0 Gy单独照射组、2 Gy单独照射组、4 Gy单独照射组、6 Gy单独照射组细胞分别用不含安罗替尼的培养液培养48 h后，更换不含安罗替尼的培养液，并分别给予0、2、4、6 Gy的X射线照射；0 Gy联合照射组、2 Gy联合照射组、4 Gy联合照射组、6 Gy联合照射组使用含终浓度4 μmol·L-1安罗替尼的培养液培养48 h后，更换为不含安罗替尼的培养液，同时分别给予0、2、4、6 Gy的X射线照射。采用平板克隆形成实验检测不同X射线照射剂量的单独照射组和联合照射组细胞的克隆形成能力，并计算各组的存活率。将 0 Gy单独照射组、2 Gy单独照射组、4 Gy单独照射组、6 Gy单独照射组归为单独照射组，将0 Gy联合照射组、2 Gy联合照射组、4 Gy联合照射组、6 Gy联合照射组细胞归为联合照射组，采用单击多靶模型检测安罗替尼联合放射治疗对KYSE-150细胞放射治疗的敏感性。采用流式细胞术检测0 Gy单独照射组、4 Gy单独照射组、0 Gy联合照射组和4 Gy联合照射组细胞凋亡情况。结果　安罗替尼干预24 h时，2 μmol·L-1安罗替尼组、4 μmol·L-1安罗替尼组、8 μmol·L-1安罗替尼组、16 μmol·L-1 安罗替尼组、32 μmol·L-1安罗替尼组、64 μmol·L-1安罗替尼组、128 μmol·L-1安罗替尼组细胞的增殖抑制率均显著高于0 μmol·L-1安罗替尼组（P<0.05），4 μmol·L-1安罗替尼组、8 μmol·L-1安罗替尼组、16 μmol·L-1安罗替尼组、32 μmol·L-1安罗替尼组、64 μmol·L-1安罗替尼组、128 μmol·L-1安罗替尼组细胞的增殖抑制率随安罗替尼干预浓度的升高而升高（P<0.05）；安罗替尼干预48、72 h时，0 μmol·L-1安罗替尼组、2 μmol·L-1安罗替尼组、4 μmol·L-1安罗替尼组、8 μmol·L-1安罗替尼组、16 μmol·L-1安罗替尼组、32 μmol·L-1安罗替尼组、64 μmol·L-1安罗替尼组、128 μmol·L-1安罗替尼组细胞的增殖抑制率随安罗替尼干预浓度的升高而升高（P<0.05）；0 μmol·L-1安罗替尼组细胞安罗替尼干预24、48、72 h时的增殖抑制率比较差异无统计学意义（P>0.05）；安罗替尼干预48 h时，4 μmol·L-1安罗替尼组、 8 μmol·L-1安罗替尼组、16 μmol·L-1安罗替尼组、32 μmol·L-1安罗替尼组、64 μmol·L-1安罗替尼组细胞的增殖抑制率显著高于安罗替尼干预24 h时同药物处理浓度的安罗替尼组（P<0.05）；安罗替尼干预72 h时，8 μmol·L-1安罗替尼组、16 μmol·L-1安罗替尼组、32 μmol·L-1安罗替尼组、64 μmol·L-1安罗替尼组、128 μmol·L-1安罗替尼组细胞的增殖抑制率显著高于安罗替尼干预24 h时同药物处理浓度的安罗替尼组（P<0.05）；安罗替尼干预72 h时，32 μmol·L-1安罗替尼组、64 μmol·L-1安罗替尼组细胞的增殖抑制率显著高于安罗替尼干预48 h后同药物处理浓度的安罗替尼组（P<0.05）。安罗替尼处理24、48、72 h时食管鳞状细胞癌KYSE-150细胞的IC50值分别为（12.7±0.5）、（3.7±1.0）、（6.8±0.7） μmol·L-1。2 Gy联合照射组、4 Gy联合照射组、6 Gy联合照射组细胞的存活率均显著低于相同照射剂量的单独照射组（P<0.05）； 4 Gy单独照射组、6 Gy单独照射组细胞的存活率均显著低于2 Gy单独照射组（P<0.05）；4 Gy联合照射组、6 Gy联合照射组细胞的存活率均显著低于 2 Gy联合照射组（P<0.05）；6 Gy单独照射组细胞的存活率显著低于4 Gy单独照射组（P<0.05）；6 Gy联合照射组细胞的存活率显著低于4 Gy联合照射组（P<0.05）。单独照射组和联合照射组的D0值分别为5.84、4.11 Gy,放射增敏比为 1.42。4 Gy单独照射组、0 Gy联合照射组、4 Gy联合照射组细胞凋亡率显著高于0 Gy单独照射组（P<0.05）；4 Gy联合照射组细胞凋亡率显著高于4 Gy单独照射组、0 Gy联合照射组（P<0.05）；4 Gy单独照射组与 0 Gy联合照射组细胞凋亡率比较差异无统计学意义（P>0.05）。结论　安罗替尼联合放射治疗可抑制食管鳞状细胞癌KYSE-150细胞的增殖能力，并增加食管鳞状细胞癌细胞对放射治疗的敏感性。
Abstract:
Objective　To explore the effects of anlotinib combined with radiotherapy on the proliferation,apoptosis and radiotherapy sensitivity of esophageal squamous cell carcinoma cells.Methods　The esophageal squamous cell carcinoma KYSE-150 cells in the logarithmic growth phase were randomly divided into 0,2,4,8,16,32,64,128 μmol·L-1 anlotinib group.The proliferation ability of esophageal squamous cell carcinoma KYSE-150 cells in the eight groups was detected by the cell counting kit-8 method.The cell proliferation inhibition curve was drawed by Graphpad Prism 8 software and the half-inhibitory concentration (IC50) was obtained.In addition,the esophageal squamous cell carcinoma KYSE-150 cells in the logarithmic growth phase were collected and randomly divided into 0 Gy single irradiation group,2 Gy single irradiation group,4 Gy single irradiation group,6 Gy single irradiation group,0 Gy combined irradiation group,2 Gy combined irradiation group,4 Gy combined irradiation group,6 Gy combined irradiation group.The clonogenic ability of cells in the single irradiation group and the combined irra- diation group with different X-ray irradiation doses were detected by the plate clone formation experiment,and the survival rate of cells in the each group was calculated.The sensitivity of anlotinib combined with radiotherapy to KYSE-150 cell radiotherapy was detected by the single-click multi-target model.The apoptosis rate of cells was detected by flow cytometry.Results　The inhibition rate of cell proliferation in the 2 μmol·L-1 anlotinib group,4 μmol·L-1 anlotinib group,8 μmol·L-1 anlotinib group,16 μmol·L-1 anlotinib group,32 μmol·L-1 anlotinib group,64 μmol·L-1 anlotinib group,128 μmol·L-1 anlotinib group was significantly higher than that in the 0 μmol·L-1 anlotinib group at 24 hours after anlotinib intervention (P<0.05),and the inhibition rate of cell proliferation increased with the increase of anlotinib concentration in the 4 μmol·L-1 anlotinib group,8 μmol·L-1 anlotinib group,16 μmol·L-1 anlotinib group,32 μmol·L-1 anlotinib group,64 μmol·L-1 anlotinib group and 128 μmol·L-1 anlotinib group (P<0.05);the inhibition rate of cell proliferation increased with the increase of anlotinib concentration in the 0 μmol·L-1 anlotinib group,2 μmol·L-1 anlotinib group,4 μmol·L-1 anlotinib group,8 μmol·L-1 anlotinib group,16 μmol·L-1 anlotinib group,32 μmol·L-1 anlotinib group,64 μmol·L-1 anlotinib group and 128 μmol·L-1 anlotinib group at 48,72 hours after anlotinib intervention (P<0.05) ;there was no significant difference in the inhibition rate of cell proliferation in the 0 μmol·L-1 anlotinib group at 24,48,72 hours after ahlotinib intervention (P>0.05);the inhibition rate of cell proliferation in the 4 μmol·L-1 anlotinib group,8 μmol·L-1 anlotinib group,16 μmol·L-1 anlotinib group,32 μmol·L-1 anlotinib group at 48 hours after anlotinib intervention was significantly higher than that in the groups of treatment with the same concentration of anlotinib for 24 hours (P<0.05);the innibition rate of cell proliferation in the 8 μmol·L-1 anlotinib group,16 μmol·L-1 anlotinib group,32 μmol·L-1 anlotinib group,64 μmol·L-1 anlotinib group and 128 μmol·L-1 anlotinib group at 72 hours after anlotinib intervention was significantly higher than that in the groups of treatment with the same concentration anlotinib for 24 hours (P<0.05);the inhibition rate of cell proliferation in the 32 μmol·L-1 anlotinib group,64 μmol·L-1 anlotinib group at 72 hours after anlotinib intervention was significantly higher than that in the groups of treatment with the same concentration anlotinib for 48 hours (P<0.05).The IC50 value of anlotinib treatment in esophageal squamous cell carcinoma KYSE-150 cells for 24,48,72 hours were (12.7±0.5),(3.7±1.0),(6.8±0.7) μmol·L-1,respectively.The survival rate of cells in the 2 Gy combined irradiation group,4 Gy combined irradiation group,6 Gy combined irradiation group was significantly lower than that in the single irradiation group with the same dose (P<0.05);the survival rate of the cells in the 4 Gy single irradiation group,6 Gy single irradiation group was significantly lower than that in the 2 Gy single irradiation group (P<0.05);the survival rate of the cells in the 4 Gy combined irradiation group and the 6 Gy combined irradiation group was significantly lower than that in the 2 Gy combined irradiation group (P<0.05);the survival rate of cells in the 6 Gy single irradiation group was significantly lower than that in the 4 Gy single irradiation group (P<0.05);the survival rate of the cells in the 6 Gy combined irradiation group was significantly lower than that in the 4 Gy combined irradiation group (P<0.05).The D0 value in the single irradiation group and the combined irradiation group were 5.84 and 4.11 Gy,respectively,and the sensitizer enhancement ratio was 1.42.The apoptosis rate of cells in the 4 Gy single irradiation group,0 Gy combined irradiation group and 4 Gy combined irradiation group was significantly higher than that in the 0 Gy single irradiation group (P<0.05);the apoptosis rate of cells in the 4 Gy combined irradiation group was significantly higher than that in the 4 Gy single irradiation group and the 0 Gy combined irradiation group (P<0.05);there was no significant difference in the apoptosis rate of cells between the 4 Gy single irradiation group and the 0 Gy combined irradiation group (P>0.05).Conclusion　Anlotinib combined with radiotherapy can inhibit the proliferation of esophageal squamous cell carcinoma KYSE-150 cells and increase the sensitivity of esophageal squamous cell carcinoma cells to radiotherapy.

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更新日期/Last Update: 2022-02-05