甘氨双唑钠对鼻咽癌移植瘤的时辰放射增敏作用
摘要
目的:通过观察甘氨双唑钠(CMNa)对鼻咽癌荷瘤裸鼠时辰放射增敏后肿瘤生长的影响,检测肿瘤组织中HIF-1α、γ-H2AX和凋亡蛋白的表达水平,来探讨甘氨双唑钠对鼻咽癌移植瘤的时辰放射增敏机制。
方法:130只Balb/c裸鼠在统一光照条件下同步化饲养,建立统一的生物节律至少3周以上。建立统一的生物节律后,在裸鼠右侧大腿外侧处接种低分化鼻咽癌细胞(CNE-Ⅱ),建立裸鼠鼻咽癌移植瘤模型。成瘤并达到入组标准后,将112只移植瘤裸鼠随机分为3组:放疗(radiotherapy,RT)组、RT+CMNa组、空白对照组,并将每一组按4个时辰3HALO(光照后小时,hours after light onset)、9HALO、15HALO、21HALO分为4个亚组。其中RT+CMNa组分别在3HALO、9HALO、15HALO、21HALO四个时间点前1小时给予腹腔注射甘氨双唑钠20mg/kg,并且RT组、RT+CMNa组在四个时间点分别进行单次放疗,剂量为10Gy。时辰放疗结束后24小时处死各亚组的一半裸鼠,获取肿瘤标本;剩余裸鼠测定再生长延长时间(regrowth delay time,TGD),绘制生长曲线;用免疫组化法检测各组肿瘤标本中HIF-1α、γ-H2AX和凋亡蛋白的表达水平,并用免疫组化图像分析软件进行半定量分析。本实验采用完全随机单因素方差分析法对各组的免疫组化检测结果进行统计学分析。
结果:各处理因素均对鼻咽癌裸鼠移植瘤有不同程度的抑制作用。通过对各组TGD的比较,以RT+CMNa组对肿瘤的抑制效果最好。在该组中TGD:15HALO>21HALO>9HALO>3HALO,15HALO与3HALO的TGD比较有统计学意义(P<0.05)。各组肿瘤标本的免疫组化检测结果(PI值)显示:HIF-1α、γ-H2AX及凋亡蛋白的表达水平呈时间节律性,同时辰的放射增敏组和单纯放疗组之间HIF-1α、γ-H2AX及凋亡蛋白的表达差异均有统计学意义(P<0.05)。HIF-1α的表达在RT+CMNa组和RT组中:3HALO>9HALO>21HALO>15HALO,其中15HALO与3HALO的HIF-1α表达水平比较有统计学意义(P<0.05)。γ-H2AX的表达在RT+CMNa组和RT组中:15HALO>21HALO>9HALO>3HALO,其中15HALO与3HALO的γ-H2AX表达水平比较有统计学意义(P<0.05)。凋亡蛋白的表达在RT+CMNa组和RT组中:15HALO>21HALO>9HALO>3HALO,其中15HALO与3HALO,9HALO的凋亡蛋白表达水平比较有统计学意义(P<0.05)。
结论:甘氨双唑钠联合时辰放疗对鼻咽癌裸鼠移植瘤有明显的时辰放射增敏作用,以15HALO RT+CMNa组对肿瘤的抑制效果最佳。证实了甘氨双唑钠对肿瘤的时辰放射增敏作用具有时间节律性,其对肿瘤的抑制效果与相关免疫组化结果具有良好的一致性。其机制可能与HIF-1α的表达,DNA双链损伤,凋亡有关。
Objective: To investigate effect on tumor growth and the expressions of HIF-1α,γ-H2AX and apoptosis proteins after the chronomodulated radiosensitization by CMNa on human nasopharyngeal carcinoma(NPC) in nude mice, and to disclose the mechanism of chronomodulated radiosensitization on nasopharyngeal carcinoma.
Methods: 130 Balb/c mice were synchronized with an alternative lighting regimen with 12 hours for light and 12 hours for dark at least 3 weeks. Human nasopharyngeal poorly differentiated squamous cell carcinoma(CNE-II) cells were implanted into the right lateral thigh of each mouse. Three weeks after transplantation, 112 nude mice xenografts were divided into 3 groups: radiotherapy(RT) group, RT+CMNa group and control group. Each group was divided into 4 subgroups at four time points of 3HALO, 9HALO, 15HALO, 21HALO. CMNa (20mg/kg) were injected introperitoneally one hour before four time points in RT+CMNa group. RT group and RT+CMNa group were given radiotherapy which dose of 10Gy at four time points. Half of nude mice were exceuted 24 hours after radiotherapy. The remaining mice were determined regrowth delay time (TGD), and the growth curve was drawn. The expressions of HIF-1α,γ-H2AX and apoptosis proteins were detected by immunohistochemistry and were analyzed by Image-Pro Plus(IPP) software. Data were analyzed by completely randomized single-factor variance analysis method.
Results: The processing factors have different inhibition for NPC xenografts in nude mice. According to TGD, RT+CMNa group obtained the best inhibitory action of tumor. In this group, TGD had statistical difference between 15HALO and 3HALO (P<0.05). According to immunehistochemical results, the expressions HIF-1α,γ-H2AX and apoptosis proteins have circadian rhythm. At the same time point, the expressions of HIF-1α,γ-H2AX and apoptosis proteins had significant difference between RT+CMNa group and RT group (P<0.05). The expression of HIF-1αin RT+CMNa group and RT group: 3HALO>9HALO>21HALO>15HALO. There were statistical difference between 15HALO and 3HALO (P<0.05). The expression ofγ-H2AX in RT+CMNa group and RT group: 15HALO>21HALO>9HALO>3HALO. There were statistical difference between 15HALO and 3HALO (P<0.05). The expression of apoptosis proteins in RT+CMNa group and RT group: 15HALO>21HALO>9HALO>3HALO. There were statistical difference among 15HALO, 3HALO and 9HALO (P<0.05).
Conclusion: This study showed that CMNa combined with chronomodulated radiotherapy could obviously enhance the radiosentivity of NPC xenografts, and RT+CMNa group at 15HALO can get the greatest efficacy. The chronomodulated radiosensitization by CMNa have circadian rhythm. Inhibitory effect on tumor and the results of immunohistochemistry have good consistency. Its mechanism might be related to the expression of HIF-1α, the injury of DNA double strands and apoptosis.
方法:130只Balb/c裸鼠在统一光照条件下同步化饲养,建立统一的生物节律至少3周以上。建立统一的生物节律后,在裸鼠右侧大腿外侧处接种低分化鼻咽癌细胞(CNE-Ⅱ),建立裸鼠鼻咽癌移植瘤模型。成瘤并达到入组标准后,将112只移植瘤裸鼠随机分为3组:放疗(radiotherapy,RT)组、RT+CMNa组、空白对照组,并将每一组按4个时辰3HALO(光照后小时,hours after light onset)、9HALO、15HALO、21HALO分为4个亚组。其中RT+CMNa组分别在3HALO、9HALO、15HALO、21HALO四个时间点前1小时给予腹腔注射甘氨双唑钠20mg/kg,并且RT组、RT+CMNa组在四个时间点分别进行单次放疗,剂量为10Gy。时辰放疗结束后24小时处死各亚组的一半裸鼠,获取肿瘤标本;剩余裸鼠测定再生长延长时间(regrowth delay time,TGD),绘制生长曲线;用免疫组化法检测各组肿瘤标本中HIF-1α、γ-H2AX和凋亡蛋白的表达水平,并用免疫组化图像分析软件进行半定量分析。本实验采用完全随机单因素方差分析法对各组的免疫组化检测结果进行统计学分析。
结果:各处理因素均对鼻咽癌裸鼠移植瘤有不同程度的抑制作用。通过对各组TGD的比较,以RT+CMNa组对肿瘤的抑制效果最好。在该组中TGD:15HALO>21HALO>9HALO>3HALO,15HALO与3HALO的TGD比较有统计学意义(P<0.05)。各组肿瘤标本的免疫组化检测结果(PI值)显示:HIF-1α、γ-H2AX及凋亡蛋白的表达水平呈时间节律性,同时辰的放射增敏组和单纯放疗组之间HIF-1α、γ-H2AX及凋亡蛋白的表达差异均有统计学意义(P<0.05)。HIF-1α的表达在RT+CMNa组和RT组中:3HALO>9HALO>21HALO>15HALO,其中15HALO与3HALO的HIF-1α表达水平比较有统计学意义(P<0.05)。γ-H2AX的表达在RT+CMNa组和RT组中:15HALO>21HALO>9HALO>3HALO,其中15HALO与3HALO的γ-H2AX表达水平比较有统计学意义(P<0.05)。凋亡蛋白的表达在RT+CMNa组和RT组中:15HALO>21HALO>9HALO>3HALO,其中15HALO与3HALO,9HALO的凋亡蛋白表达水平比较有统计学意义(P<0.05)。
结论:甘氨双唑钠联合时辰放疗对鼻咽癌裸鼠移植瘤有明显的时辰放射增敏作用,以15HALO RT+CMNa组对肿瘤的抑制效果最佳。证实了甘氨双唑钠对肿瘤的时辰放射增敏作用具有时间节律性,其对肿瘤的抑制效果与相关免疫组化结果具有良好的一致性。其机制可能与HIF-1α的表达,DNA双链损伤,凋亡有关。
Objective: To investigate effect on tumor growth and the expressions of HIF-1α,γ-H2AX and apoptosis proteins after the chronomodulated radiosensitization by CMNa on human nasopharyngeal carcinoma(NPC) in nude mice, and to disclose the mechanism of chronomodulated radiosensitization on nasopharyngeal carcinoma.
Methods: 130 Balb/c mice were synchronized with an alternative lighting regimen with 12 hours for light and 12 hours for dark at least 3 weeks. Human nasopharyngeal poorly differentiated squamous cell carcinoma(CNE-II) cells were implanted into the right lateral thigh of each mouse. Three weeks after transplantation, 112 nude mice xenografts were divided into 3 groups: radiotherapy(RT) group, RT+CMNa group and control group. Each group was divided into 4 subgroups at four time points of 3HALO, 9HALO, 15HALO, 21HALO. CMNa (20mg/kg) were injected introperitoneally one hour before four time points in RT+CMNa group. RT group and RT+CMNa group were given radiotherapy which dose of 10Gy at four time points. Half of nude mice were exceuted 24 hours after radiotherapy. The remaining mice were determined regrowth delay time (TGD), and the growth curve was drawn. The expressions of HIF-1α,γ-H2AX and apoptosis proteins were detected by immunohistochemistry and were analyzed by Image-Pro Plus(IPP) software. Data were analyzed by completely randomized single-factor variance analysis method.
Results: The processing factors have different inhibition for NPC xenografts in nude mice. According to TGD, RT+CMNa group obtained the best inhibitory action of tumor. In this group, TGD had statistical difference between 15HALO and 3HALO (P<0.05). According to immunehistochemical results, the expressions HIF-1α,γ-H2AX and apoptosis proteins have circadian rhythm. At the same time point, the expressions of HIF-1α,γ-H2AX and apoptosis proteins had significant difference between RT+CMNa group and RT group (P<0.05). The expression of HIF-1αin RT+CMNa group and RT group: 3HALO>9HALO>21HALO>15HALO. There were statistical difference between 15HALO and 3HALO (P<0.05). The expression ofγ-H2AX in RT+CMNa group and RT group: 15HALO>21HALO>9HALO>3HALO. There were statistical difference between 15HALO and 3HALO (P<0.05). The expression of apoptosis proteins in RT+CMNa group and RT group: 15HALO>21HALO>9HALO>3HALO. There were statistical difference among 15HALO, 3HALO and 9HALO (P<0.05).
Conclusion: This study showed that CMNa combined with chronomodulated radiotherapy could obviously enhance the radiosentivity of NPC xenografts, and RT+CMNa group at 15HALO can get the greatest efficacy. The chronomodulated radiosensitization by CMNa have circadian rhythm. Inhibitory effect on tumor and the results of immunohistochemistry have good consistency. Its mechanism might be related to the expression of HIF-1α, the injury of DNA double strands and apoptosis.
引文
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