放射对乳腺癌细胞侵袭转移潜能的影响及相关机制研究

发布时间：2018-02-13 02:53

  本文关键词： 乳腺癌 放射治疗 循环肿瘤细胞 侵袭转移 基质金属蛋白酶-2　出处：《上海交通大学》2014年硕士论文　论文类型：学位论文

【摘要】：背景和目的放射治疗可降低乳腺癌患者保乳和乳房切除后的局部复发风险,提高生存率,在早期和局部进展期乳腺癌治疗中发挥重要作用。然而,近年来研究发现放射治疗还可能通过改变肿瘤微环境,增加肿瘤细胞的侵袭转移潜能。这里我们探讨放射后乳腺癌细胞侵袭及转移潜能的变化及其机制。材料和方法1、在体外以不同剂量(0、4、8Gy)X线照射人乳腺癌MDA-MB-435细胞。明胶酶谱法检测放射后24h、48h细胞培养上清液中基质金属蛋白酶MMP-2活性,Western bloting检测放射后24h、48h细胞内MMP-2蛋白表达水平,Transwell法测定放射后24h、48h细胞的侵袭能力,评价放射治疗对乳腺癌侵袭潜能的影响及其机制。2、构建同时表达增强型绿色荧光蛋白(Enhanced Green Fluorecence Protein,e GFP)和荧光素酶(Luciferase,Luc)的慢病毒载体,将GFP-Luciferase融合蛋白载体转染到人乳腺癌MDA-MB-435细胞株。将稳定表达GPF和1uciferase的MDA-MB-435细胞(MDA-MB-435-e GFP-Luc)接种裸鼠,建立原位乳腺癌转移动物模型并在接种后第二周分组,放射治疗组采用8Gy单次剂量照射原位肿瘤,采用在体流式细胞仪(In Vivo Flow Cytometry,IVFC)每周检测裸鼠耳根部动脉循环肿瘤细胞(Circulating tumour cells,CTCs),同步采用小动物活体成像系统(In Vivo Image System,IVIS)监测肿瘤生长及全身转移情况。接种后第6周处死两组裸鼠,取出肺组织进行活组织成像及病理组织切片HE染色,比较两组裸鼠肺组织转移灶数目,并采用免疫组织化学染色检测原发肿瘤组织内MMP-2表达,评价放射治疗对乳腺癌转移潜能的影响及其机制。结果1、放射后乳腺癌MDA-MB-435细胞侵袭能力、MMP-2表达及活性显著提高并均呈现时间及剂量依赖性,单次8Gy照射48h达到最高。2、成功建立了稳定表达GFP/Luciferase的乳腺癌细胞株:绿色荧光表达强,GFP转染效率达到99.58%,体外发光能力与细胞数目呈正相关(R2=0.998)。肿瘤细胞接种后第二周IVFC探测到体内CTCs信号峰,IVIS成像检查检测到肺内微小转移灶。肿瘤细胞的形态学特征、生长速度、侵袭及迁移特性与转染前无明显变化。3、照射组接受放射后肿瘤体积、CTCs数目、肿瘤部位荧光强度呈现先减小后增加趋势。放疗后第4周(即肿瘤接种后第6周)对照组和实验组肿瘤体积分别为519.66±32.89mm3,497.28±45.86mm3(p0.05)无统计学差异,每小时CTCs数目分别为7.8±1.9,11.8±2.3(p0.05)有统计学差异,提示放疗后第4周肿瘤细胞侵袭转移能力获得进一步增强。放射治疗组肿瘤组织MMP-2表达及肺转移灶明显多于对照组。结论1、结合IVFC和IVIS技术可以实现对MDA-MB-435-GFP-Luc细胞系原位乳腺癌模型的CTCs、肿瘤生长及微小转移灶进行早期、实时动态监测。2、放射线照射能够通过增强乳腺癌细胞MMP-2的表达或活性而增强其侵袭转移潜能。
[Abstract]:Background and objective radiotherapy can reduce the risk of breast conserving and local recurrence after mastectomy in breast cancer patients, improve survival rate and play an important role in early and locally advanced breast cancer treatment. In recent years, studies have found that radiotherapy may also change the tumor's microenvironment, To investigate the changes and mechanisms of invasion and metastasis potential of breast cancer cells after radiation. Materials and methods 1. In vitro, human breast cancer MDA-MB-435 cells were irradiated with different doses of 0, 4 and 8 Gy ~ (-1) rays. The activity of matrix metalloproteinase (MMP-2) in the supernatant of cell culture at 24 h after radiation was detected by gel enzyme assay and the expression level of MMP-2 protein in the cell at 24 h or 48 h after radiation was detected by Western bloting. The invasion ability of cells was measured by Transwell method at 24 h or 48 h after irradiation. To evaluate the effect of radiotherapy on the invasive potential of breast cancer and its mechanism. 2. To construct a lentivirus vector expressing enhanced Green Fluorecence protein (GFP) and luciferase luciferase (Lucc) simultaneously. GFP-Luciferase fusion protein vector was transfected into human breast cancer MDA-MB-435 cell line. MDA-MB-435-e GFP-Lucc cells, which expressed GPF and 1uciferase stably, were inoculated into nude mice to establish the animal model of breast cancer metastasis in situ and divided into groups at the second week after inoculation. In the radiotherapy group, the tumor was irradiated with a single dose of 8 Gy. In vivo flow cytometry (in Vivo Flow Cytometry) was used to detect the circulating tumour tumour cells CTCs in the ear root artery of nude mice every week. The tumor growth and metastasis were monitored by the small animal in vivo imaging system (in Vivo Image system IVISs). The nude mice were killed 6 weeks after inoculation, and the two groups were killed at 6 weeks after inoculation. Lung tissue was taken out for biopsy and HE staining. The number of lung metastatic foci in the two groups was compared, and the expression of MMP-2 in the primary tumor tissue was detected by immunohistochemical staining. To evaluate the effect of radiotherapy on the metastatic potential of breast cancer and its mechanism. Results 1. The expression and activity of MMP-2 in MDA-MB-435 cells were significantly increased in a time and dose-dependent manner. The breast cancer cell line expressing GFP/Luciferase stably was successfully established. The transfection efficiency of GFP was 99.58, and the luminescence ability in vitro was positively correlated with the number of cells. The tumor cells were inoculated at the second week after inoculation. IVFC detects in vivo CTCs signal peaks and IVIS imaging detects tiny metastases in the lung. Morphological features of tumor cells, The growth rate, invasion and migration characteristics were not significantly different from those before transfection. The fluorescence intensity of tumor site decreased first and then increased. There was no significant difference in tumor volume between the control group and the experimental group at the 4th week after radiotherapy (that is, the 6th week after tumor inoculation), the tumor volume of the control group and the experimental group were 519.66 卤32.89mm3n3c497.28 卤45.86mm3p0.05, respectively, and the number of CTCs per hour was 7.8 卤1.9nil 卤2.3p0.05, respectively. The results suggest that the ability of invasion and metastasis of tumor cells is further enhanced at the 4th week after radiotherapy. The expression of MMP-2 and lung metastasis in the radiotherapy group is significantly higher than that in the control group. Conclusion 1. Combined with IVFC and IVIS technique, the tumor cell line of MDA-MB-435-GFP-Luc can be achieved. In situ breast cancer model, CTCs, tumor growth and micrometastasis were performed early. Real-time dynamic monitoring. 2. Radiation irradiation can enhance the invasion and metastasis potential of breast cancer cells by enhancing the expression or activity of MMP-2.
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.9;R730.55
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本文编号：1507158