论文总字数：15443字

摘 要

骨肉瘤是在青少年体内常常发病的一种恶性肿瘤。其损害作用强，在体内转移速度快，患病初期即可在人体内出现肺转移，严重威胁着人类的健康。到今天为止，现代医学上治疗骨肉瘤的方法主要和治疗其他恶行肿瘤方法相似，都是一些传统的治疗其他肿瘤细胞的方法以及新型辅助型化疗带来的综合治疗方法。由于骨肉瘤的病发部位在骨组织中，手术治疗不能完全切除病灶，而且临床上没有专门用于治疗骨肉瘤的药物，常规药物靶向性差难以到达骨组织，长时间的化疗、放疗会导致耐药性，后期治疗效果差。因此迫切需要研发可抑制骨肉瘤增殖与转移、不易耐药、安全性高且可以靶向作用于骨组织的新药。

本实验对金丝桃素进行了体外细胞学实验考察，采用MTT实验测定金丝桃素在光照的条件下对骨肉瘤细胞的毒性,结果显示，细胞毒性呈明显的药物浓度和激光照射强度依赖性；利用共聚焦活细胞成像系统研究了金丝桃素的细胞摄取行为，用DCFH-DA作为检测探针，结果显示在光照条件下细胞内产生了大量的ROS；通过激光共聚焦显微镜观察金丝桃素在143B细胞内的分布方式，结果证明了BDPTA NPs能够较多的穿过细胞膜进入到143B细胞内部；进一步采用Annexin V-FITC/PI双染色法验证金丝桃素对143B细胞的促凋亡作用。流式结果显示，与暗光组及空白对照组相比143B肿瘤细胞经金丝桃素处理后的光照组的细胞凋亡率明显增加，说明了金丝桃素对143B细胞有明显的促凋亡作用，此外暗毒性较低。另外，用划痕实验验证了金丝桃素在光照条件下能够抑制143B细胞的迁移能力；侵袭实验证明了金丝桃素在光照条件下能够抑制143B细胞的侵袭能力。

关键词：金丝桃素，光动力疗法，骨肉瘤细胞

ABSTRACT

Osteosarcoma is the most common malignant tissue tumor in medicine. Its patients are mostly adolescents, which have a strong damaging effect and rapid metastasis in the body. Lung metastasis can occur in the human body at the early stage of the disease, seriously threatening human health . As of today, medical treatment methods for osteosarcoma mainly include surgical treatment, chemotherapy, radiotherapy and other traditional methods for treating other tumor cells and comprehensive treatment methods brought by new adjuvant chemotherapy. Because the site of osteosarcoma is in the bone tissue, the surgical treatment cannot completely remove the lesion, and there is no clinically specific drug for the treatment of osteosarcoma. Conventional drugs are difficult to reach the bone tissue with poor concomitantness. Continuous chemotherapy and radiotherapy can cause complications Sex, the subsequent treatment effect is poor.

In this experiment, hypericin was examined in vitro. The MTT test was used to determine the toxicity of hypericin to 143B tumor cells under light conditions. Cytotoxicity showed significant drug concentration and upper limit of laser irradiation intensity; using confocal live cell imaging system to study hypericin cell transformation behavior, using DCFH-DA as a detection probe, the results showed that cells under light conditions Produce a small amount of ROS; observe the distribution of hypericin in 143B cells by laser confocal microscopy. The results prove that BDPTA NPs can penetrate the cell membrane and enter 143B cells; further use Annexin V-FITC / PI Staining method verified hypericin's promoting effect on 143B cells. In addition, the scratch experiment verified that hypericin can inhibit 143B cells under light conditions. Migration ability; and obviously increased, indicating hypericin on 143B cells.

Keywords: hypericin, photodynamic therapy, osteosarcoma cells

目 录

摘 要 I

ABSTRACT II

目 录 4

第一章 文献综述 1

1.1骨肉瘤概述 1

1.1.1骨肉瘤 1

1.1.2骨肉瘤的治疗 1

1.2 光动力治疗的研究进展 2

1.2.1光动力治疗的作用原理 2

1.2.2光动力疗法的特点 2

1.2.3光治疗的三要素 3

1.2.4光疗法的优势 3

1.3金丝桃素在光疗法中的应用 4

1.4 研究内容与意义 4

第二章 金丝桃素对骨肉瘤细胞的抑制作用 6

2.1实验准备 6

2.1.1材料与试剂 6

2.1.2仪器与设备 6

2.2实验内容 7

2.2.1肿瘤细胞体外培养： 7

2.2.2MTT比色法检测细胞存活率 8

2.2.3Annexin-V和PI双染实验流式细胞仪检测细胞凋亡 8

2.2.4细胞内ROS活性氧检测 8

2.2.5细胞迁移实验 9

2.2.6细胞侵袭实验 9

第三章 结果与讨论 11

3.1 实验结果 11

3.1 MTT实验 11

3.2 Annexin-V和PI双染实验 11

3.3细胞内ROS活性氧检测 12

3.4 迁移实验 12

3.5 侵袭实验 13

3.2小结 13

第四章 结论与展望 15

4.1结论 15

4.2 展望 15

参考文献 16

致谢 18

第一章 文献综述

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