拓扑替康对鼻咽癌移植瘤时辰放射增敏作用的机制研究
发布时间：2018-08-09 08:33
【摘要】：目的：通过构建人低分化鼻咽癌裸鼠移植瘤模型,从细胞水平和分子水平两方面探讨拓扑替康(TPT)时辰放射增敏的作用机制。 方法：240只BALB/c (nu/nu)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。随后,在裸鼠大腿外侧皮下接种人低分化鼻咽癌细胞(CNE2),建立裸鼠鼻咽癌移植瘤模型。成瘤并达到入组标准后,按照随机分组的原则,将达标的裸鼠随机分为4组：空白对照组、TPT治疗组(单化组)、RT组(单放组)、TPT+RT组,每组48只。观察四个时辰亚组3HAL0(光照后小时,hours after light onset)、9HAL0、15HAL0、21HALO治疗后的情况。其中,拓扑替康给药采用单次腹腔注射给药法,10mg/kg,在各时辰点前30分钟给入；RT组、TPT+RT组在4个时辰点分别进行单次放疗,剂量为18Gy；空白对照组在移植瘤达标后不给予任何治疗。时辰放疗结束后1小时处死各组的一半裸鼠,剩余裸鼠观察生长曲线,并测定肿瘤再生长延缓时间(TGD)。按要求获取肿瘤标本后,一部分采用免疫组化法检测各组肿瘤标本中HP-1、γ-H2AX的表达,用免疫组化图像分析软件进行半定量分析；一部分用DNA琼脂糖凝胶电泳法检测拓扑异构酶Ⅰ的表达；一部分采用流式细胞技术检测细胞周期DNA含量及凋亡率。本实验采用完全随机单因素方差分析法(ANOVA法)对各组的检测结果进行统计学分析；并采用q检验进行组间两两比较,检验其差异性。 结果：1.各处理因素均对鼻咽癌移植瘤产生不同程度的抑制作用。通过鼻咽癌移植瘤的时辰放射增敏的整体动物试验,发现：相同时辰点治疗时,以TPT+RT组对肿瘤的对肿瘤的抑制效果最好。不同时辰亚组治疗时,在放射增敏组疗效有较明显差异,15HAL021HAL09HAL03HAL0,以15HAL0整体疗效最好。2.免疫组化检查结果显示：在对照组中,HP-1的表达呈15HAL021HAL09HAL03HAL0,3HAL0与15HAL0时辰亚组比较有统计学的差异,提示鼻咽癌组织中乏氧状况具有时间节律性,肿瘤组织乏氧状况为3HAL0最明显,15HAL0较低。同一时辰亚组,TPT+RT组、RT组与对照组之间HP-1及γ-H2AX的表达差异均有统计学意义(P0.01)。不同时辰亚组,在TPT+RT组和RT组相同组别中HP-1的表达：3HAL09HAL021HAL015HAL0,而γ-H2AX的表达：15HAL021HAL09HAL03HAL0,两者在15HAL0与3HAL0的表达水平比较均有显著性差异(P0.01)。3. DNA琼脂糖凝胶电泳法检测DNA拓扑异构酶Ⅰ的表达,呈15HAL021HAL09HAL03HAL0趋势,15HAL0与3HAL0组间比较有统计学意义。4.流式细胞技术检测细胞周期含量及凋亡率,各处理组与对照组比较,S期细胞比例下降和凋亡指数增加,与对照组比较有统计学意义。 结论：本实验证实了拓扑替康对鼻咽癌移植瘤具有时辰放射增敏作用,对肿瘤的时辰放射增敏作用呈现昼夜节律性,以15HAL0的TPT+RT组对肿瘤的治疗效果最好。探讨拓扑替康时辰放射增敏的机制,可能与肿瘤乏氧的时间节律性,细胞周期的改变及凋亡,DNA双链的损伤,拓扑异构酶Ⅰ的表达变化等相关。
[Abstract]:Objective: to construct a nude mouse model of nude mice with low differentiated nasopharyngeal carcinoma (TPT), and to explore the mechanism of radioactivity sensitization of topotecan (TPT) from the level of cell and molecular level.
Methods: 240 BALB/c (nu/nu) nude mice were raised synchronously under unified light conditions to establish a unified biological rhythm for at least 3 weeks. Then, a human low differentiated nasopharyngeal carcinoma cell (CNE2) was inoculated subcutaneously on the lateral thigh of nude mice, and a nude mice model of nasopharyngeal carcinoma was established. Rats were randomly divided into 4 groups: blank control group, TPT treatment group (single group), group RT (single play group), group TPT+RT, 48 in each group. The four hour subgroup 3HAL0 (hours after light onset) and 9HAL0,15HAL0,21HALO treatment were observed. Among them, topotecan was given a single intraperitoneal injection, 10mg/kg, before 30 hours. In group RT and group TPT+RT, group RT and group TPT+RT were treated with a single radiotherapy at 4 hour points, and the dosage was 18Gy. The blank control group did not give any treatment after the transplant tumor reached the standard. After the end of the radiotherapy, half of the nude mice were killed in each group. The growth curve of the remaining nude mice was observed and the tumor regrowth delay time (TGD) was measured. The tumor markers were obtained according to the requirement. After this, part of the immunohistochemical method was used to detect the expression of HP-1, gamma -H2AX in the tumor specimens of each group. Semi quantitative analysis was carried out by immunohistochemical image analysis software. A part of the expression of topoisomerase I was detected by DNA agarose gel electrophoresis, and a part of the cell cycle DNA content and apoptosis rate were detected by flow cytometry. A complete random single factor analysis of variance (ANOVA) was used to analyze the results of each group, and the difference between the 22 groups was tested by Q test.
Results: 1. all the treatment factors have different inhibitory effects on the transplanted tumor of nasopharyngeal carcinoma. Through the whole animal test of the time radiosensitization of the nasopharyngeal carcinoma, it is found that the TPT+RT group has the best effect on the tumor in the same time point treatment. The results of 15HAL021HAL09HAL03HAL0, the best.2. immunization test with the overall effect of 15HAL0 showed that in the control group, the expression of HP-1 showed a statistically significant difference between the 15HAL021HAL09HAL03HAL0,3HAL0 and the 15HAL0 hour subgroup, suggesting that the hypoxia in the nasopharyngeal carcinoma tissues has a time rhythm and the hypoxic state of the tumor tissue is 3HAL0 The expression of HP-1 and gamma -H2AX between the same time subgroup, the TPT+RT group, the RT group and the control group was statistically significant (P0.01). The expression of HP-1 in the same group of the TPT+RT group and the RT group in the TPT+RT group and the RT group: 3HAL09HAL021HAL015HAL0, and the expression of gamma -H2AX: 15HAL021HAL09HAL03HAL0, both in the TPT+RT and in the RT. The expression level was significantly different (P0.01).3. DNA agarose gel electrophoresis was used to detect the expression of DNA topoisomerase I, showing a trend of 15HAL021HAL09HAL03HAL0. There was a significant difference between 15HAL0 and 3HAL0 group, and.4. flow cytometry was used to detect the cell cycle content and apoptosis rate. Each treatment group was compared with the control group, and the proportion of S phase cells was under the proportion of the control group. The decrease and apoptosis index increased significantly compared with the control group.
Conclusion: this experiment confirmed that topotecan has the time radiosensitizing effect on nasopharyngeal carcinoma transplantation tumor, and has the circadian rhythm of the time radiosensitization of the tumor. The effect of 15HAL0 TPT+RT group on the tumor is the best. Changes and apoptosis, DNA double strand damage, expression of topoisomerase I and so on.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R739.63
[Abstract]:Objective: to construct a nude mouse model of nude mice with low differentiated nasopharyngeal carcinoma (TPT), and to explore the mechanism of radioactivity sensitization of topotecan (TPT) from the level of cell and molecular level.
Methods: 240 BALB/c (nu/nu) nude mice were raised synchronously under unified light conditions to establish a unified biological rhythm for at least 3 weeks. Then, a human low differentiated nasopharyngeal carcinoma cell (CNE2) was inoculated subcutaneously on the lateral thigh of nude mice, and a nude mice model of nasopharyngeal carcinoma was established. Rats were randomly divided into 4 groups: blank control group, TPT treatment group (single group), group RT (single play group), group TPT+RT, 48 in each group. The four hour subgroup 3HAL0 (hours after light onset) and 9HAL0,15HAL0,21HALO treatment were observed. Among them, topotecan was given a single intraperitoneal injection, 10mg/kg, before 30 hours. In group RT and group TPT+RT, group RT and group TPT+RT were treated with a single radiotherapy at 4 hour points, and the dosage was 18Gy. The blank control group did not give any treatment after the transplant tumor reached the standard. After the end of the radiotherapy, half of the nude mice were killed in each group. The growth curve of the remaining nude mice was observed and the tumor regrowth delay time (TGD) was measured. The tumor markers were obtained according to the requirement. After this, part of the immunohistochemical method was used to detect the expression of HP-1, gamma -H2AX in the tumor specimens of each group. Semi quantitative analysis was carried out by immunohistochemical image analysis software. A part of the expression of topoisomerase I was detected by DNA agarose gel electrophoresis, and a part of the cell cycle DNA content and apoptosis rate were detected by flow cytometry. A complete random single factor analysis of variance (ANOVA) was used to analyze the results of each group, and the difference between the 22 groups was tested by Q test.
Results: 1. all the treatment factors have different inhibitory effects on the transplanted tumor of nasopharyngeal carcinoma. Through the whole animal test of the time radiosensitization of the nasopharyngeal carcinoma, it is found that the TPT+RT group has the best effect on the tumor in the same time point treatment. The results of 15HAL021HAL09HAL03HAL0, the best.2. immunization test with the overall effect of 15HAL0 showed that in the control group, the expression of HP-1 showed a statistically significant difference between the 15HAL021HAL09HAL03HAL0,3HAL0 and the 15HAL0 hour subgroup, suggesting that the hypoxia in the nasopharyngeal carcinoma tissues has a time rhythm and the hypoxic state of the tumor tissue is 3HAL0 The expression of HP-1 and gamma -H2AX between the same time subgroup, the TPT+RT group, the RT group and the control group was statistically significant (P0.01). The expression of HP-1 in the same group of the TPT+RT group and the RT group in the TPT+RT group and the RT group: 3HAL09HAL021HAL015HAL0, and the expression of gamma -H2AX: 15HAL021HAL09HAL03HAL0, both in the TPT+RT and in the RT. The expression level was significantly different (P0.01).3. DNA agarose gel electrophoresis was used to detect the expression of DNA topoisomerase I, showing a trend of 15HAL021HAL09HAL03HAL0. There was a significant difference between 15HAL0 and 3HAL0 group, and.4. flow cytometry was used to detect the cell cycle content and apoptosis rate. Each treatment group was compared with the control group, and the proportion of S phase cells was under the proportion of the control group. The decrease and apoptosis index increased significantly compared with the control group.
Conclusion: this experiment confirmed that topotecan has the time radiosensitizing effect on nasopharyngeal carcinoma transplantation tumor, and has the circadian rhythm of the time radiosensitization of the tumor. The effect of 15HAL0 TPT+RT group on the tumor is the best. Changes and apoptosis, DNA double strand damage, expression of topoisomerase I and so on.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R739.63
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