【作者】 李华涛；

【导师】 刘云；

【作者基本信息】 东北农业大学 ， 临床兽医学， 2011， 硕士

【摘要】 光动力学疗法是激光技术、光导技术、光信息处理技术、生物光化学技术和现代医学技术有机结合的产物,是肿瘤治疗的新技术。它与传统手术、放疗和化疗三大治疗肿瘤疗法相比,有极大的优越性。犬乳腺肿瘤是兽医临床常见的一种肿瘤性疾病。犬乳腺肿瘤与人类乳腺癌有很多相似之处,是作为人乳腺肿瘤研究的出色的模型。HMME联合632.8 nm的He-Ne激光诱导体外犬乳腺肿瘤细胞凋亡,并取得了明显效果。但凋亡的机理仍存在许多问题有待深入研究。线粒体途径是细胞凋亡的重要途径之一,光动力学疗法通过光敏剂产生活性氧诱导细胞凋亡,HMME-PDT是否造成犬乳腺肿瘤细胞线粒体损伤至今尚未报道,线粒体在HMME-PDT治疗中损伤程度及在诱导凋亡中所起的作用还不清楚。本实验选用自然发生乳腺肿瘤病例的转移灶分离培养的细胞系CHMm,用HMME联合632.8 nm的He-Ne激光体外作用CHMm细胞。采用倒置显微镜观察HMME-PDT处理后犬乳腺肿瘤细胞形态学变化;用TUNEL末位法检测细胞凋亡;考马斯亮蓝检测细胞内总蛋白的含量;采用荧光显微镜检测HMME在细胞内的分布;采用Regand染色法对线粒体进行染色,光镜下观察HMME-PDT对犬乳腺肿瘤细胞线粒体损伤情况;通过提取线粒体DNA与琼脂糖凝胶电泳观察HMME-PDT对犬乳腺肿瘤细胞线粒体DNA损伤情况;通过线粒体膜电位变化检测、线粒体Ca²⁺转运能力的测定及线粒体ATP酶活性的检测,观察线粒体结构和功能的损伤情况。实验结果显示:HMME-PDT能诱导犬乳腺肿瘤细胞出现空泡化,使细胞脱落死亡,并具有时间依赖性。单独光敏剂和单独激光在不同程度的抑制细胞的增殖,呈现增殖缓慢,密度增加较小。HMME-PDT实验组处理后,犬乳腺肿瘤细胞出现明显的凋亡形态;细胞核浓染,固缩,出现致密浓染的颗粒状强荧光,3h后出现统计学差异（P < 0.05）,6 h后差异极显著（P < 0.01）。HMME主要分布在细胞质中,细胞核周围区域荧光较强,光敏剂分布较多。细胞质中线粒体被染成蓝色颗粒,线粒体聚集越多染色越深,HMME-PDT实验组从3 h开始细胞质染色逐渐变浅,细胞质被染色的颗粒成分随时间增加,越来越少,整个细胞着色变浅,细胞数目不断降低。经HMME-PDT处理后线粒体DNA损伤出现断裂,在100～200bp之间出现断裂的条带,实验组从3 h开始亮度开始增强,条带变宽,并在24到最亮。流式细胞仪检测线粒体膜电位降低;实验组Ca²⁺摄取能力逐渐降低,Na⁺-K⁺-ATPase和Ca²⁺-ATPase活性都显著降低。通过本实验的研究得出以下结论:HMME-PDT能诱导犬乳腺肿瘤细胞质内出现空泡,细胞停止增殖并死亡脱落;单独光敏剂和单独激光对犬乳腺肿瘤的杀伤作用较弱。HMME-PDT诱导犬乳腺肿瘤细胞发生凋亡,随处理后时间延长杀伤效应增加。HMME在犬乳腺肿瘤细胞系中主要分布在细胞质中,在细胞核周围分布较多;实验组细胞代谢在逐渐减弱;HMME-PDT损伤肿瘤细胞线粒体,使线粒体数目下降,线粒体膜电位降低,线粒体Ca²⁺转运能力下降,线粒体Na⁺-K⁺-ATP酶和Ca²⁺-ATP酶活性都显著降低,导致线粒体功能损伤。更多还原

【Abstract】 Photodynamic therapy （PDT） was a new treatment for cancer patients, owing to combine the medical application of laser, light guiding and biological photochemical technologies, as well as the modern optical medicine. There a great advantage compared with conventional surgery, radiotherapy and chemotherapy treatment. PDT had been study in human health research and clinical trials on various antitumor. PDT was still in the basic theory research and exploratory stage on canine breast cancer. Canine breast cancer is a common neoplastic disease in clinical veterinary. Canine breast cancer has many likenesses with human breast cancer, as an excellent model for human breast cancer. CHMm cells apoptosis induced by HMME-PDT. Therefore, many issues would to be further study in the apoptosis induced by HMME-PDT. Mitochondrial pathway is one important way of apoptosis. Mitochondrial damage has not yet been report in CHMm cells apoptosis caused by HMME-PDT. Role of mitochondrial damage in CHMm cells apoptosis with HMME-PDT treatment was unclear.CHMm cell line collected and isolated from the clinical metastatic breast cancer, treated with HMME and a He-Ne laser at a wavelength of 632.8 nm in vitro. Changes of cell morphology determined following HMME-PDT treatment. Apoptosis was analysed using TUNEL. Cell total protein was detected by Coomassie Brilliant Blue G-250. Intracellular distribution of HMME was detected by fluorescence microscopy. Mitochondrial damage was detected by Regand staining. Mitochondrial DNA damage was determined by agarose gel electrophoresis. Changes of mitochondrial membrane potential were detected by flow cytometry. Changes of Ca²⁺ transport ability in cytoplasm and mitochondrial detected. Na⁺-K⁺-ATPase and Ca²⁺-ATPase detected by spectrophotometry.The results showed that CHMm cells vacuolated and died off at a time-dependent. Photosensitizer alone and separate laser inhibited cell proliferation at different levels, showing a slow proliferation, the smaller increase in density. We have examined the apoptosis using TUNEL assay, the nuclei of the experimental group cells at later time points showing high levels of （or bright） staining and pyknosis. The intensity and number of brightly stained cells increased as the toxicity or apoptosis progresses, significant different after 6 h （P<0.01）. HMME was mainly distributed in the cytoplasm, displaying strong fluorescence around the nucleus. Mitochondria was stained blue cytoplasm granules, mitochondria stained gathered deeper. Cytoplasmic components stained shallow and particles decreased along with the time. The whole cell colouring shallow and reduce the number of cells. Mitochondrial DNA was fracture after HMME-PDT treatment, the fragment between 100²00bp bands in the experimental group, bands broadening and brightness after 3h. Mitochondrial membrane potential disappeared; Uptake ability of Ca²⁺ decreased within 4～12min significantly different （P<0.01）. Activities of Na⁺-K⁺-ATPase and Ca²⁺-ATPase significantly reduced.Conclusions of all studies: CHMm cells vacuolated stop proliferating and died off after treated by HMME-PDT. Effects of separate photosensitizer and laser irradiation on cells were weak. HMME-PDT induced apoptosis and the killing effects increased prolong treatment time. HMME distributed in cytoplasm and mainly around the nucleus in CHMm cells. Cell mitochondrial damaged by HMME-PDT, mitochondrial density and membrane potential decreased. Mitochondrial Ca²⁺transport ability and Activities of Na⁺-K⁺-ATPase and Ca²⁺-ATPase decreased. Mitochondria’s structure and function damaged by HMME-PDT.更多还原