超声激活血卟啉对S180瘤细胞杀伤作用及形态学研究
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摘要
目前全球研究最热门的疾病有三种：肿瘤，心血管疾病，免疫系统疾病，但是随着人类平均寿命的延长及心脑血管系统疾病发生和死亡率的逐步下降，人类肿瘤的发生及死亡率将跃居首位。目前，治疗肿瘤的方法很多，而本世纪初利用超声(Ultrasound，US)能够穿透生物组织，激活癌细胞内富集并长时间潴留的光敏性物质血卟啉(Hematoporphyrin derivatives，HpD)，从而产生协同的抗癌作用的抗肿瘤方法即声动力疗法(SDT)日益受到人们的关注。
日本学者梅村晋一郎等于1989年提出了声动力疗法，该方法与目前国内外另外两种有应用前景的超声疗法： 高强度聚焦超声(HIFU)外科和超声热疗法相比，克服了它们应用于临床的缺陷，因而在抗肿瘤方面倍受人们的关注，成为极具应用前景的抗肿瘤方法，并在近年来得到了迅速发展。国内外关于SDT方法已进行了初步的研究，在离体人肿瘤细胞，动物移植瘤方面积累了一些资料，但是其机制的探讨，方法的实际应用尚需进行大量的研究工作。而且由于不同的超声频率、强度及血卟啉浓度在杀伤肿瘤的同时，对正常组织会造成一定程度的损伤，所以筛选合适的声强及血卟啉浓度对确定杀伤在体肿瘤组织的敏感阈值和触发位点，从而获得理想达到最佳的抗癌效果，减少对正常组织的损伤具有重要的意义。本文在国内外学者前期研究的基础上，依托国家自然科学基金项目“超声激活血卟啉抗肿瘤的细胞分子生物学机制”的资助，采用超声频率为1.1MHz，血卟啉浓度为2.5mg／ml及不同超声强度等实验参数，利用普通光学显微镜，荧光显微镜，透射电镜，扫描电镜，酶组织化学等方法，对腹水型S180肿瘤的形态结构及诱发的在体肉瘤组织的生长体积大小、组织细胞形态结构、线粒体酶活性等方面的动态变化进行观察分析，研究超声激活血卟啉对S180肉瘤组织及腹水瘤细胞的杀伤抑制作用所造成损伤在体内的动态变化发展过程，为进一步探讨SDT的杀伤作用机制及其临床应用积累丰富资料。
本实验得出以下主要结论：
1．通过超声激活血吟琳对5180肉瘤生长状况进行研究，筛选出了作用
于5180实体瘤有效的最低超声强度及处理时间；研究结果表明，血叶琳对
荷瘤小鼠肿瘤组织生长的抑制作用甚微，超声有一定的抑制肿瘤生长效应，
而超声激活血吟琳能够有效的抑制肿瘤组织的生长。
2．利用先镜及透射电镜技术观察超声激活血吟林处理后肿瘤组织细胞形
态结构的变化。发现血吟淋对肿瘤细胞影响较小；而单纯超声和超声激活血
吟淋均具有破坏细胞结构的作用，其破坏作用首先表现在生物膜系统和遗传
信息表达系统受损。
3．利用酶组织化学技术检测超声激活血吟琳对肿瘤细胞的细胞色素 C
氧化酶活性的影响。分析结果表明，细胞受损的原因除了超声激活血叶淋对
细胞内线粒体形态结构造成的直接损伤外，对其重要标记酶活性的影响亦是
造成细胞最终死亡的重要困素之一。
4．利用普通光镜、扫描电镜技术及m（m333叼）、N（碘化丙嚏）双
重染色等方法观察同一频率，不同强度的超声激活血吟琳处理小鼠后腹水型
5180肿瘤细胞形态学的变化。结果表明血吟琳使腹水瘤细胞表面微绒毛轻度
减少；不同强度超声对细胞形态和表面结构有不同程度的损伤，而超声激活
血吟淋对肿瘤细胞的损伤最大；当声强为 0．4 w／cm‘时超声激活血吟淋处理
的腹水瘤细胞表现出明显的细胞凋亡特征。
以上研究获得的实验数据和资料为 SDT法抗肿瘤机理研究提供了重要的
实验参数和依据，对声动力学疗法理论的完善及该疗法的早日临床应用积累
了丰富的资料。
There are three kinds of diseases studied hotly nowadays, that is cancer, cardiovascular disease, immune system disease. However, the study of cancer is noticed more and more with its mortality ranked the first. At present, many treatments have been invented in order to be antitumor. These methods include an application of ultrasound including high intensity focused ultrasound (HIFU) surgery, ultrasound thermal treatment and sonodynamic therapy (SDT). There are some deficiencies and difficulties to apply the first two to clinic, but the third treatment has its advantages. It has coordination effect combined used with the photosensitive drugs (such as hematoporphyrin) and can penetrating tissue deeply and being easier to focus, not need expensive equipment and avoiding thermal side-effect, so the application of sonodynamic treatment is a promising way on treatment of tumors in deep tissue. In order to apply this method to clinic, and treat tumors in human's body with effectively and conveniently activate photosensitive drugs, based on the internal and external studies, this paper is focused on the killing effect and developing process of SI80 ascitic tumor and solid tumor in order to accumulate lots of datas on the study of SDT mechanism.
This paper has designed to solve two significant problems. One is to define the optimum hematoporphyrin concentration, the optimum timing of the irradiation and the optimum ultrasonic intensity under firmed ultrasonic frequency, the other is to study whether or not SDT can cause cancer cells apoptosis phenomena in vivo .
Main conclusion as follow:
1.The effective lower intensity and time of treatment on SI80 are screened. HpD has little killing effect. US has certain killing effect, US with HpD can inhibit the tumor growth effectively.
2.The tumor structure by lighter microscopy and transelectric microscopy is observed. The destroy on tumor can be seen first on membrane system and genetic information system.
3.The activity of cytochromeC is detected. The paper makes conclusion from the changes of the activity of cytochromeC that the destroy on enzyme system is one of the most important factors to the death of tumor cells.
4.The structure of SI80 ascitic tumor after treatment is studied on the microvillus and other superficial changes. Most of cells have the characters of apoptosis.
日本学者梅村晋一郎等于1989年提出了声动力疗法，该方法与目前国内外另外两种有应用前景的超声疗法： 高强度聚焦超声(HIFU)外科和超声热疗法相比，克服了它们应用于临床的缺陷，因而在抗肿瘤方面倍受人们的关注，成为极具应用前景的抗肿瘤方法，并在近年来得到了迅速发展。国内外关于SDT方法已进行了初步的研究，在离体人肿瘤细胞，动物移植瘤方面积累了一些资料，但是其机制的探讨，方法的实际应用尚需进行大量的研究工作。而且由于不同的超声频率、强度及血卟啉浓度在杀伤肿瘤的同时，对正常组织会造成一定程度的损伤，所以筛选合适的声强及血卟啉浓度对确定杀伤在体肿瘤组织的敏感阈值和触发位点，从而获得理想达到最佳的抗癌效果，减少对正常组织的损伤具有重要的意义。本文在国内外学者前期研究的基础上，依托国家自然科学基金项目“超声激活血卟啉抗肿瘤的细胞分子生物学机制”的资助，采用超声频率为1.1MHz，血卟啉浓度为2.5mg／ml及不同超声强度等实验参数，利用普通光学显微镜，荧光显微镜，透射电镜，扫描电镜，酶组织化学等方法，对腹水型S180肿瘤的形态结构及诱发的在体肉瘤组织的生长体积大小、组织细胞形态结构、线粒体酶活性等方面的动态变化进行观察分析，研究超声激活血卟啉对S180肉瘤组织及腹水瘤细胞的杀伤抑制作用所造成损伤在体内的动态变化发展过程，为进一步探讨SDT的杀伤作用机制及其临床应用积累丰富资料。
本实验得出以下主要结论：
1．通过超声激活血吟琳对5180肉瘤生长状况进行研究，筛选出了作用
于5180实体瘤有效的最低超声强度及处理时间；研究结果表明，血叶琳对
荷瘤小鼠肿瘤组织生长的抑制作用甚微，超声有一定的抑制肿瘤生长效应，
而超声激活血吟琳能够有效的抑制肿瘤组织的生长。
2．利用先镜及透射电镜技术观察超声激活血吟林处理后肿瘤组织细胞形
态结构的变化。发现血吟淋对肿瘤细胞影响较小；而单纯超声和超声激活血
吟淋均具有破坏细胞结构的作用，其破坏作用首先表现在生物膜系统和遗传
信息表达系统受损。
3．利用酶组织化学技术检测超声激活血吟琳对肿瘤细胞的细胞色素 C
氧化酶活性的影响。分析结果表明，细胞受损的原因除了超声激活血叶淋对
细胞内线粒体形态结构造成的直接损伤外，对其重要标记酶活性的影响亦是
造成细胞最终死亡的重要困素之一。
4．利用普通光镜、扫描电镜技术及m（m333叼）、N（碘化丙嚏）双
重染色等方法观察同一频率，不同强度的超声激活血吟琳处理小鼠后腹水型
5180肿瘤细胞形态学的变化。结果表明血吟琳使腹水瘤细胞表面微绒毛轻度
减少；不同强度超声对细胞形态和表面结构有不同程度的损伤，而超声激活
血吟淋对肿瘤细胞的损伤最大；当声强为 0．4 w／cm‘时超声激活血吟淋处理
的腹水瘤细胞表现出明显的细胞凋亡特征。
以上研究获得的实验数据和资料为 SDT法抗肿瘤机理研究提供了重要的
实验参数和依据，对声动力学疗法理论的完善及该疗法的早日临床应用积累
了丰富的资料。
There are three kinds of diseases studied hotly nowadays, that is cancer, cardiovascular disease, immune system disease. However, the study of cancer is noticed more and more with its mortality ranked the first. At present, many treatments have been invented in order to be antitumor. These methods include an application of ultrasound including high intensity focused ultrasound (HIFU) surgery, ultrasound thermal treatment and sonodynamic therapy (SDT). There are some deficiencies and difficulties to apply the first two to clinic, but the third treatment has its advantages. It has coordination effect combined used with the photosensitive drugs (such as hematoporphyrin) and can penetrating tissue deeply and being easier to focus, not need expensive equipment and avoiding thermal side-effect, so the application of sonodynamic treatment is a promising way on treatment of tumors in deep tissue. In order to apply this method to clinic, and treat tumors in human's body with effectively and conveniently activate photosensitive drugs, based on the internal and external studies, this paper is focused on the killing effect and developing process of SI80 ascitic tumor and solid tumor in order to accumulate lots of datas on the study of SDT mechanism.
This paper has designed to solve two significant problems. One is to define the optimum hematoporphyrin concentration, the optimum timing of the irradiation and the optimum ultrasonic intensity under firmed ultrasonic frequency, the other is to study whether or not SDT can cause cancer cells apoptosis phenomena in vivo .
Main conclusion as follow:
1.The effective lower intensity and time of treatment on SI80 are screened. HpD has little killing effect. US has certain killing effect, US with HpD can inhibit the tumor growth effectively.
2.The tumor structure by lighter microscopy and transelectric microscopy is observed. The destroy on tumor can be seen first on membrane system and genetic information system.
3.The activity of cytochromeC is detected. The paper makes conclusion from the changes of the activity of cytochromeC that the destroy on enzyme system is one of the most important factors to the death of tumor cells.
4.The structure of SI80 ascitic tumor after treatment is studied on the microvillus and other superficial changes. Most of cells have the characters of apoptosis.
引文
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[64]Jin ZH, Miyoshi N, et al, J Dermotol 2000 May;27(5):294-306 Related Articles, Books,LinkOut
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