拓撲替康對鼻咽癌移植瘤時辰放射增敏作用的機制研究
發(fā)布時間：2018-08-09 08:33
【摘要】：目的：通過構建人低分化鼻咽癌裸鼠移植瘤模型,從細胞水平和分子水平兩方面探討拓撲替康(TPT)時辰放射增敏的作用機制。 方法：240只BALB/c (nu/nu)裸鼠在統(tǒng)一光照條件下同步化飼養(yǎng),建立統(tǒng)一的生物節(jié)律至少3周。隨后,在裸鼠大腿外側皮下接種人低分化鼻咽癌細胞(CNE2),建立裸鼠鼻咽癌移植瘤模型。成瘤并達到入組標準后,按照隨機分組的原則,將達標的裸鼠隨機分為4組：空白對照組、TPT治療組(單化組)、RT組(單放組)、TPT+RT組,每組48只。觀察四個時辰亞組3HAL0(光照后小時,hours after light onset)、9HAL0、15HAL0、21HALO治療后的情況。其中,拓撲替康給藥采用單次腹腔注射給藥法,10mg/kg,在各時辰點前30分鐘給入；RT組、TPT+RT組在4個時辰點分別進行單次放療,劑量為18Gy；空白對照組在移植瘤達標后不給予任何治療。時辰放療結束后1小時處死各組的一半裸鼠,剩余裸鼠觀察生長曲線,并測定腫瘤再生長延緩時間(TGD)。按要求獲取腫瘤標本后,一部分采用免疫組化法檢測各組腫瘤標本中HP-1、γ-H2AX的表達,用免疫組化圖像分析軟件進行半定量分析；一部分用DNA瓊脂糖凝膠電泳法檢測拓撲異構酶Ⅰ的表達；一部分采用流式細胞技術檢測細胞周期DNA含量及凋亡率。本實驗采用完全隨機單因素方差分析法(ANOVA法)對各組的檢測結果進行統(tǒng)計學分析；并采用q檢驗進行組間兩兩比較,檢驗其差異性。 結果：1.各處理因素均對鼻咽癌移植瘤產(chǎn)生不同程度的抑制作用。通過鼻咽癌移植瘤的時辰放射增敏的整體動物試驗,發(fā)現(xiàn)：相同時辰點治療時,以TPT+RT組對腫瘤的對腫瘤的抑制效果最好。不同時辰亞組治療時,在放射增敏組療效有較明顯差異,15HAL021HAL09HAL03HAL0,以15HAL0整體療效最好。2.免疫組化檢查結果顯示：在對照組中,HP-1的表達呈15HAL021HAL09HAL03HAL0,3HAL0與15HAL0時辰亞組比較有統(tǒng)計學的差異,提示鼻咽癌組織中乏氧狀況具有時間節(jié)律性,腫瘤組織乏氧狀況為3HAL0最明顯,15HAL0較低。同一時辰亞組,TPT+RT組、RT組與對照組之間HP-1及γ-H2AX的表達差異均有統(tǒng)計學意義(P0.01)。不同時辰亞組,在TPT+RT組和RT組相同組別中HP-1的表達：3HAL09HAL021HAL015HAL0,而γ-H2AX的表達：15HAL021HAL09HAL03HAL0,兩者在15HAL0與3HAL0的表達水平比較均有顯著性差異(P0.01)。3. DNA瓊脂糖凝膠電泳法檢測DNA拓撲異構酶Ⅰ的表達,呈15HAL021HAL09HAL03HAL0趨勢,15HAL0與3HAL0組間比較有統(tǒng)計學意義。4.流式細胞技術檢測細胞周期含量及凋亡率,各處理組與對照組比較,S期細胞比例下降和凋亡指數(shù)增加,與對照組比較有統(tǒng)計學意義。 結論：本實驗證實了拓撲替康對鼻咽癌移植瘤具有時辰放射增敏作用,對腫瘤的時辰放射增敏作用呈現(xiàn)晝夜節(jié)律性,以15HAL0的TPT+RT組對腫瘤的治療效果最好。探討拓撲替康時辰放射增敏的機制,可能與腫瘤乏氧的時間節(jié)律性,細胞周期的改變及凋亡,DNA雙鏈的損傷,拓撲異構酶Ⅰ的表達變化等相關。
[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.
【學位授予單位】：瀘州醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：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.
【學位授予單位】：瀘州醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R739.63
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