【作者】 李斌；

【导师】 陈刚； 李如昌； 方杰； 贾国金；

【作者基本信息】 复旦大学 ， 外科学， 2012， 博士

【摘要】 研究目的：光动力学疗法(Photodynamic Therapy, PDT)的核心是光敏剂。叶绿酸e4是由本研究组研制开发的具有自主知识产权的最新的新型光敏剂,其光动力学特性以及对膀胱癌的光动力杀伤效果和机制尚未明确。为此,本研究旨在探讨叶绿酸e4的制备及其光动力学特征,明确其体外、体内对膀胱癌光动力杀伤作用和作用机制,为其临床应用治疗膀胱癌提供基础资料。研究方法：1、以中药蚕砂为原料,提取得到叶绿素粗制品,经过碱水解、分离、提取、纯化等步骤得到叶绿酸e6,再由叶绿酸e6转化为叶绿酸e4。2、采用质谱仪分析叶绿酸e4分子量,高压液相色谱分析样品的纯度,使用荧光分光光度计分析其吸收光谱和吸收峰等光学特性。3、体外培养膀胱癌T24和5637细胞到对数生长期,应用MTT法检测不同浓度的叶绿酸e4结合不同能量650nm波长激光对膀胱癌细胞的生长抑制作用,探讨叶绿酸e4光动力学治疗对T24和5637细胞的体外杀伤作用。4、采用流式细胞仪观察叶绿酸e4光动力学处理T24和5637细胞后死亡情况；透射电镜技术观察T24和5637细胞超微结构的变化,判断细胞器形态变化和有无发生凋亡。5、将叶绿酸e4与T24和5637细胞孵育后,再用线粒体特异性探针标记线粒体,以激光共聚焦显微镜为成象工具,研究叶绿酸e4在细胞内的亚细胞分布。6、采用细胞凋亡蛋白抗体芯片技术,检测T24和5637细胞光动力处理前后,包括Bcl-2、Bax、Bad、Caspase-3、Caspase-9和细胞色素C、SMAC等凋亡相关蛋白的表达情况,探讨叶绿酸e4光动力学作用诱导T24和5637细胞凋亡的可能机理。7、建立大鼠原位膀胱癌模型,初步观察叶绿酸e4介导的光动力学作用对大鼠膀胱癌的体内杀伤效果。研究结果：1、制备得到的叶绿酸e4,样品纯度为94.28%,相对分子量为553,在可见光的红光区域均有很强的吸收峰,吸收峰为661.5nm、606.0nm、501.5nm、403.5nm。2、叶绿酸e4各浓度2.5、5和10μ g/m1对T24细胞生长抑制率在4J/cm2下为20.03%、49.77%和82.43%,在1J/cm2为6.1%、77.38%和79.32%,各浓度500ng/ml、1μ g/m1和2μ g/ml对5637细胞生长抑制率在4J/cm2下为16.61%、29.11%和85.06%,在1J/cm2为13.39%、57.94%和71.20%。3、T24细胞在10μ g/ml叶绿酸e4和4J/cm2激光条件下,光动力处理后,行流式细胞检测,细胞凋亡率为32.04±1.3%、坏死率为7.12±0.7%,细胞对照组凋亡率为3.77±0.3%、坏死率为2.61±0.4%；5637细胞在2μ g/m1叶绿酸e4和4J/cm2激光条件下,光动力处理后,行流式细胞检测,细胞凋亡率为45.33±1.7%、坏死率为3.64±0.7%,细胞对照组凋亡率为5.64±0.7%、坏死率为1.57±0.4%。4、T24和5637细胞光动力学处理后,透射电镜检测显示线粒体明显肿胀变性呈空泡样变,细胞核内染色质核染色质固缩,边集在核膜下,呈境界分明的颗粒状或新月形,凋亡小体形成。对照组无相应改变。5、T24和5637细胞与叶绿酸e4和线粒体探针孵育后,行激光共聚焦显微镜检测显示504nm激发叶绿酸e4发出的红光与488nm激发线粒体探针产生的绿光分布基本一致。6、在T24细胞处理组中促凋亡蛋白Bid、Bax、Bad均较对照组有明显的升高；而抗凋亡蛋白Bcl-2明显下降,Bcl-w蛋白相较于对照组也有下降;处理组细胞色素C为对照组的1.8倍；Caspase9和Caspase3处理组有不同程度的升高；促凋亡蛋白SMAC处理组高与对照组；凋亡抑制蛋白XIAP处理组明显低于对照组；在5637细胞PDT处理组中促凋亡蛋白Bid、Bax、Bad较对照组有明显的升高；而抗凋亡蛋白Bcl-2明显下降,Bcl-w蛋白对照组也有下降；处理组细胞色素C水平高于对照组;Caspase9和Caspase3处理组有不同程度的升高；促凋亡蛋白SMAC处理组高与对照组。7、叶绿酸e4在20J/cm2激光能量下对大鼠膀胱癌光动力处理后,观察到明显成片的肿瘤组织的坏死(4/4)。研究结论：1、制备的叶绿酸e4纯度高,光学特性符合理想光敏剂要求。2、叶绿酸e4结合650nm激光,体外光动力学杀伤膀胱癌T24和5637细胞显著,具有剂量和能量依赖性。3、叶绿酸e4主要定位在肿瘤细胞线粒体中,通过诱导线粒体途径的快速凋亡实现杀伤肿瘤细胞作用的。4、T24和5637细胞经PDT处理后,促凋亡蛋白Bid、Bad、Bax较抑制凋亡蛋白Bcl-2、Bcl-w等占优势,线粒体中细胞色素C、SMAC及凋亡诱导因子等释放入细胞质,激活了Caspase-9,随后再激活其他Caspase,共同作用诱导细胞发生凋亡。5、叶绿酸e4介导的光动力学作用对大鼠膀胱癌,有明确的杀伤效果。更多还原

【Abstract】 Objective:The key factor of photodynamic therapy was photosensitizer (PS). Our research group has recently developed a novel photosensitizer with complete proprietary intellectual property rights named chlorophyllin e4, but the photodynamic characters as well as the photodynamic effect and mechanism against bladder cancer cells are still undefined. In our experiments, we aimed to investigate the preparation and the photodynamic characters of chlorophyllin e4, and clarify the photodynamic effect and phototoxicity mechanism. The information we obtained in this paper would provide a scientific foundation for chlorophyllin e4mediated photodynamic therapy against bladder tumor in clinical pratice.Methods:1.We first obtained crude chlorophyll materials extracted from traditional Chinese medicine excrementum bombycis. Then the crude chlorophyll was undergone basic hydrolysis, extraction and purification, and we got chlorophyllin e6. Chlorophyllin e6was then converted into chlorophyllin e4under certain procedures.2. The relative molecular weight of chlorophyllin e4was tested by spectrumanalyzer, the purity of the sample was detected by high pressure liquid chromatograph, and the absorption spectrum and peak was analysed by fluorescence spectrophotometer3.T24and5637bladder cancer cells were incubated to exponential phase in vitro. Afterwards the cells were cultivated with different concentrations of chlorophyllin e4and then irradiated with650nm laser light. The controls included cells treated with chlorophyllin e4but without light as well as cells exposed to laser light without chlorophyllin e4. Photocytotoxicity was monitored with MTT assay.4. Flow cytometry was used to analysis the apoptosis of T24and5637cells induced by chlorophyllin e4-based photodynamic therapy. And Transmission Electron Microscope (TEM) was used to evaluate the morphological and ultra-structural changes of the cells.5. T24and5637cells were first co-incubated with chlorophyllin e4and then the Mito-Tracker Green probe was used to label mitochondrion, the confocal laser scanning microscopy (CLSM) excited by488nm and405nm dual channels was applied to reveal chlorophyllin e4intracellular localization.6.The Apoptosis Antibody Array was used to detect the expression of apoptosis related proteins after the cells were treated, including Bcl-2, Bax, Caspase-3, Caspase-9and cytochrome c, and so that we clarify the possible mechanism of T24and5637cells apoptosis.7. We established orthotopic SD rad bladder cancer model, and preliminary observed the effect of chlorophyllin e4mediated PDT against rat bladder cancer in vivo.Results:1. The purity of chlorophyllin e4was94.28%, the relative molecular weight was553and the absorption peak were661.5nm,606.Onm,501.5nm,403.5nm.2. Chlorophyllin e4exhibited significant photocytotoxicity in both T24and5637cells. In T24cells, with4J/cm2light dose, the growth inhibition rates of2.5,5and10μg/ml chlorophyllin e4mediated photodynamic effect were20.03%、49.77%and82.43%, while with1J/cm2light dose, the growth inhibition rates were6.1%、77.38%and79.32%respectively. In5637cells, with4J/cm2light dose, the growth inhibition rates of500ng/ml、1μg/ml and2μg/ml chlorophyllin e4mediated photodynamic effect were16.61%、29.11%and85.06%, while with1J/cm2light dose, the growth inhibition rates were13.39%、57.94%and71.20%respectively. Treatment with chlorophyllin e4or laser light alone did not induce cytotoxicity.3. With10μg/ml and4J/cm2light dose, the apoptosis rate of T24cells after chlorophyllin e4mediated PDT was32.04±1.3%, the necrosis rate was7.12±0.7%; the apoptosis and necrosis rate of control group were3.77±0.3%and2.61±0.4%. With2μg/ml and4J/cm2light dose, the apoptosis rate of5637cells after chlorophyllin e4mediated PDT was45.33±1.7%, the necrosis rate was the necrosis rate was3.64±0.4%; the apoptosis and necrosis rate of control group were5.64±0.7%and1.57±0.4%. Both apoptosis rates were significantly higher than control groups.4. T24and5637cells treated by chlorophyllin e4mediated PDT, were then observed by TEM, the results showed the mitochondria were obviously swelling as vacuolar degeneration, and nuclear chromatin were dense and in lump at different size, which located mainly in the nuclear membrane. But the control groups did not showed the same change.5. The confocal laser scanning microscopy (CLSM) revealed that, the red fluorescence stimulated by504nm laser light emitted by chlorophyllin e4and green fluorescence stimulated by488nm emitted by Mito-Tracker Green probe distributed almost the same.6. In T24cells, the expression of Bid, Bax, Bad, cytochrome, Caspase-3, Caspase-9and SMAC were rised in different degree in PDT treated group, while Bcl-2, Bcl-w and XIAP protein decreased. And in5637cells, the situation was similar, the expressions of Bid, Bax, Bad, cytochrome, Caspase-3, Caspase-9and SMAC were rised in different degree in PDT treated group, while Bcl-2, Bcl-w decreased.7. The rat bladder tumor was observed obviously sections of tissue necrosis after chlorophyllin e4mediated PDT with20J/cm2.Conclusion:1.The purity of chlorophyllin e4that we prepared was high, and the optical properties meet ideal photosensitizer requirements.2. The photodynamic effect of chlorophyllin e4combine with650nm laser light on T24and5637cells is significant, and depends on the PS concentration and laser dose.3. Chlorophyllin e4mainly locates and functions at mitochondria, and induces apoptosis, which may be one of the mechanisms against T24and5637cells.4. In PDT treated T24and5637cells, the expression of Bid、Bad、Bax were higher than Bcl-2、Bcl-w, which triggering cytochrome, SMAC and AIF were released into cytoplasm, then Caspase-9and other Caspase were activated. The cooperative action was induced cell apoptosis.5. Chlorophyllin e4mediated PDT showed clear effect against rat bladder cancer in vivo.更多还原