【作者】 邓秋媛；

【导师】 董稚明；

【作者基本信息】 河北医科大学 ， 病理学与病理生理学， 2008， 硕士

【摘要】 目的:我国是食管癌的高发国家之一,细胞周期素D1(CyclinD1)、血管内皮生长因子(vascular endothelial growth factor , VEG F)和E-钙粘蛋白(E-cadherin)分别在肿瘤增殖、侵袭及转移不同阶段发挥重要作用。全反式维甲酸(all-trans retinoic acid,ATRA)是一种理想的诱导分化剂,可以诱导多种肿瘤细胞分化、并逆转其恶性表型。单用ATRA毒副作用大,易产生继发性耐药。本实验研究了多种浓度、不同时间条件下ATRA对人食管癌细胞株TE13体外增殖情况的影响;单用ATRA和ATRA联合低剂量射线,探讨其对TE13凋亡、周期的影响以及是否具有协同增效作用。另外还探讨了单用ATRA和ATRA联合低剂量射线,对TE13细胞CyclinD1、VEGF和E-cadherin的表达改变的影响以及三者之间的相关性;为食管癌的临床治疗提供理论依据。方法:1体外培养TE13细胞。2以化疗药物ATRA和60Coγ射线为处理因素。3 MTT比色法测定不同浓度ATRA处理组分别作用12h、24h、48h和72h后,对TE13细胞的吸光度(OD值)和细胞增殖抑制率(Inhibition Rate,IR%)的影响。并以48h的IR%为标准,选取分别达到约25%、50%以及75%的药物浓度作为流式细胞术(flow cytometry,FCM)及免疫细胞化学(SP法)的检测浓度。4分组:设ATRA处理组(终浓度分别为0.78μmol/L、6.25μmol/L、12.5μmol/L)和ATRA联合γ射线处理组(0.78μmol/L+4Gy、6.25μmol/L+4Gy、12.5μmol/L+4Gy)以及正常对照组分别作用24h和48h,用FCM对TE13细胞进行凋亡率的检测以及周期分析、并检测CyclinD1、VEGF和E-cad三种蛋白表达的改变情况;SP法测各组CyclinD1、VEGF和E-cad表达变化情况。5实验数据经SPSS13.0统计软件处理,采用重复测量设计方差分析,方差齐性检验、单因素方差分析,两两比较SNK-q检验以及直线相关性分析,P<0.05为统计学上的显著性差异。结果:1在浓度为0.39μmol/L至12.5μmol/L浓度范围内,ATRA对TE13细胞的抑制作用呈现剂量依赖效应关系和时间依赖效应关系。2 TE13经ATRT及ATRT联合γ射线分别处理24h和48h,细胞增殖能力明显下降,更多的细胞被阻滞于G0/G1期,细胞凋亡明显增加,其中以两者联合应用更为显著。3 FCM检测结果表明,单用ATRA和联合γ射线分别作24h、48h,随剂量增大,CyclinD1、VEGF的平均荧光指数(FI)值递减,E-cad的FI值递增,且联合剂量和对应单药相比,效果更明显。4各处理组作用于TE13细胞24h,FCM测FI值示,CyclinD 1和VEGF呈显著正相关(r=0.95,P<0.05), CyclinD1和E-cad呈显著负相关(r=-0.885,P<0.05), VEGF和E-cad呈显著负相(r=-0.918,P<0.05)。5各处理组作用于TE13细胞48h,FCM测FI值示,CyclinD1和VEGF呈显著正相关(r=0.972,P<0.05), CyclinD和E-cad呈显著负相关(r=-0.953,P<0.05), VEGF和E- cad呈显著负相关(r=-0.962,P<0.05)。6单用ATRA及ATRA联合60Coγ射线作用48h,SP法检测TE13细胞CyclinD1、VEGF和E-cad蛋白表达结果。结果示随剂量增大,CyclinD1、VEGF阳性细胞百分率递减,E-cad阳性细胞百分率递增,且联合剂量和对应单药相比,效果显著。相关性分析示,CyclinD1和VEGF呈显著正相关(r=0.752, P<0.05), CyclinD1和E-cad呈显著负相关(r=-0.775, P<0.05), VEGF和E–cad呈显著负相关(r=-0.738,P<0.05)。结论:1 ATRA对TE13细胞具有明显的增殖抑制作用,呈现剂量以及时间依赖性。2 ATRA能诱导TE13细胞凋亡并将其阻断于G0/G1期,呈剂量依赖性;联合γ射线,二者能协同抑制食管癌TE13细胞的恶性增殖并促使其凋亡。3 ATRA能下调TE13细胞CyclinD1、VEGF蛋白表达,上调E-cad蛋白表达,且呈浓度依赖效应关系;ATRA联合γ射线应用能协同增强这一效应。4 CyclinD1、VEGF表达呈正相关,CyclinD1、E-cad表达呈负相关,VEGF、E-cad表达呈负相关。更多还原

【Abstract】 Objective: CyclinD1, VEGF and E-cad are important proteins to proliferation, invasion and metastasis of esophageal cancer. All-trans retinoic acid (ATRA) is an efficient inducing agent for cell division. This paper explores the inhibitory effect of ATRA or the combination of ATRA andγradial on cell division and apoptosis of esophageal carcinoma Cell TE13. This paper also explores the effect of ATRA or the combination of ATRA andγ-ray on CyclinD1,VEGF and E-cad expression changes .Methods:1 The human esophageal cancer cell line TE13 was cultured. TE13 cell in adherent growth was divided into control and experimental groups.2 The experimental groups were cultured with ATRA concentration of 0.39μmol/L, 0.78μmol/L, 1.56μmol/L, 3.13μmol/L, 6.25μmol/L and 12.50μmol/L.3 The differences of cell proliferation inhibition rate between difference groups were dectected by MTT assay after 12, 24, 48 and 72 hours.4 The experiment groups were divided into two sections. In the first section, the human esophageal cancer cell line TE13 was cultured conventionally with 0.78μmol/L, 6.25μmol/L and 12.5μmol/L ATRA. In the second section, the human esophageal cancer cell line TE13 was cultured with ATRA and 60Coγ-ray (0.78μmol/L+4Gy、6.25μmol/L+4Gy、12.5μmol/L+4Gy). The cell apoptosis and cell cycle distribution were detected by FCM after 24h and 48h. Expression change of CyclinD1, VEGF, E-cad were also detected by immunecytoche- mistry (method SP).5 Statistical analyses were performed using repeated measure ANOVA, test for equal variance, student-newman-keuls multiple-range test with SPSS 13.0 software.Results:1 The inhibition effects of ATRA on TE13 cells were significantly dose dependent and time dependent. The difference of OD value and inhibition ratio between experimental groups and control group was statistically significant (P<0.05). The difference of OD value and inhibition ratio between adjacent experimental groups was also statistically significant (P<0.05).2 Compared with control group, the cell proliferation was obviously inhibited and there was a significant cell cycle change in experimental groups after disposal by ATRA or the combination of ATRA andγ-ray. While the disposal dose increased, G0/G1 cells ratio increased and S+G2/M cells ratio decreased.3 While the disposal dose increased, the FI value of CyclinD1 and VEGF decreased and the FI value of E-cad increased. The results of FCM indicated that the effects of combined of ATRA andγ-ray are more manifest than those of single drug after either 24h or 48h.4 The results of FCM after 24h indicated that there was signific- ant positive correlation between the FI of CyclinD1 and VEGF(r=0.950, P<0.05). There was negative correlation between the CyclinD1 and E-cad(r=-0.885, P<0.05). The correlation betw- een VEGF and E-cad was also negative (r=-0.918, P<0.05).5 The results of FCM after 48h indicated that there was signific- ant positive correlation between the FI of CyclinD1 and VEGF(r=0.972, P<0.05). There was negative correlation between the CyclinD1 and E-cad(r=-0.953, P<0.05). And there was also negative correlation between VEGF and E-cad(r=-0.962, P< 0.05).6 The expression of CyclinD1,VEGF and E-cad were detected by SP method. While the disposal dose increased, the percentages of positive cell of CyclinD1 and VEGF decreased and the percentage of positive cell of E-cad increased. The combined of ATRA and andγ-ray are more effective on CyclinD1, VEGF and E-cad expression changes than single ATRA. The ananysis indicated the positive correlation between the expression of CyclinD1 and VEGF (r=0.752, P<0.05), the negative correlation between the expression of CyclinD1 and E-cad(r=-0.775, P<0.05)and the negative correlation between the expression of VEGF and E-cad(r=-0.738, P<0.05). Conclusions:1 The inhibition effects of ATRA on TE13 cell proliferation were signifcantly dose dependent and time dependent.2 ATRA can block up the cell cycle of TE13 line to G0/G1 period. ATRA can also induce the cell apoptosis.Both of the effects were dose dependent. The combination of ATRA andγ-ray can jointly inhibite the proliferation of TE13 cell and induce the apoptosis of TE13 cell.3 ATRA can not only down regulate the expression of CyclinD1 and VEGF protein in TE13 but also up regulate the expression of E-cad protein. The regulation role were dose depen- dent. The combination of ATRA andγ-ray can enhance this effect.4 The expression of CyclinD1 and VEGF is positive correlated The expression of CyclinD1 and E-cad is negative correlated. The expression of VEGF and E-cad is negative correlated.更多还原