【作者】 苏小睿；

【导师】 王梦亮；

【作者基本信息】 山西大学 ， 微生物学， 2007， 硕士

【摘要】 光动力疗法（PDT）是国际上新兴起的一种治疗手段，目前已广泛用于对各类癌症进行早期诊断和治疗。光敏剂是影响PDT的重要因素，其光学特性直接影响到治疗效果及其副作用，目前国际上所使用的第二代光敏剂的吸收峰为670nm或690nm，由于对生物组织穿透性不够，同时，由于其在可见光区有较宽的吸收谱，治疗后可见光光敏副作用大等不足，使得长波激发的光敏剂及与其相适应的光源成为该疗法所追求的目标。本文的主要目的就是寻找一种具有光动力学活性，在400-700nm的可见光区域没有吸收，吸收峰在750nm以上的光敏剂。若用于PDT治疗，可直接用于体内组织的诊断和治疗，并可大大增加了在组织中的穿透能力。同时，减少了治疗后的可见光光敏反应。并对其光动力学治疗方面的机理及其生物效应进行深入的研究。（1）以类球红杆菌（Rhodobacter sphaeroides）为材料，采用乙醇超声提取、滑石粉吸附去除杂质，并用DEAE-sepharose CL-6B（2.7×6.5）和sepharose CL-6B（2.7×18）柱层析分离，获得较纯的细菌叶绿素。在对前期工作总结的基础上，筛选出锌菌绿素做为后期的研究材料，以细菌叶绿素为配体，合成锌细菌叶绿素配合物（Zn-BCA），并采用紫外可见光谱、¹H NMR和¹³C NMR核磁共振法对其结构进行了表征。细菌脱镁叶绿素与金属锌配位后，其特征吸收峰出现相应的红移和蓝移，¹H NMR和¹³C NMR核磁共振数据与文献报道一致，分子式为C₃₅H₃₆N₄O₆Zn-R’。（2）金属细菌叶绿素极性较小，采用脂质体作为药物载体，是提高靶向，减小副作用的最有效方法之一。Zn-BCA脂质体包裹的研究中，通过100nm滤膜挤压法获得粒径＜100nm脂质体，并与Zn-BCA进行孵化。该脂质体粒径均匀，相变温度达到50℃，回收率＞95.00％；日内和日间精密度RSD＜5.84％，包封率＞53.00％，且实验重现性良好。（3）采用MTT比色法研究了Zn-BCA-PDT灭杀HL-60细胞的浓度、暗孵育时间、照光时间和光波长参数，初步总结了对HL-60白血病肿瘤细胞进行Zn-BCA-PDT的较佳处理方案为：细胞密度为1×10⁵个／mL，Zn-BCA浓度10μg／mL，暗室孵育3h，照光时间50min，光源波长为800nm。（4） Zn-BCA作为PDT光敏剂，其治疗效果除了与它本身的光动力学特性有关以外，能否高度选择性的聚集在肿瘤组织和细胞上也是影响其疗效的关键因素，因此光敏剂在动物不同组织中的分布，不仅会影响其治疗效果，而且会影响其对正常组织的副作用。通过检测Zn-BCA的自发荧光来了解Zn-BCA在小鼠体内的代谢，其在体内的代谢速度很快，5h后各个组织中的荧光强度已经很低。（5）在此基础上观察了Zn-BCA光敏剂对荷瘤小鼠的光动力疗效及光动力治疗后的光敏副作用。采用昆明种小鼠腹腔接种S180细胞制作裸鼠腹水瘤模型，分0.25mg／kg PDT组和1.0mg／kg PDT组，以存活时间为指标，探讨Zn-BCA光敏剂的光动力疗效。并以是否出现眼结膜水肿和皮肤红斑，及生存状态和存活时间为指标观察Zn-BCA光敏剂对正常裸鼠和荷瘤小鼠PDT前后的影响。结果发现，PDT后，荷瘤小鼠的存活时间与对照组相比明显延长，其中1.0mg／kg的Zn-BCA脂质体治疗组荷瘤小鼠的生存时间比对照组生存时间平均延长8.1d。裸鼠经尾静脉注射Zn-BCA后，不经过暗孵育，立即到光反应室照光，那么光照组与其对照组相比表现出强烈的皮肤光敏作用，并且生存时间很短，提示Zn-BCA在PDT中具有较强疗效，而荷瘤小鼠经过4h的避光再进行PDT治疗，没有发现明显的皮肤光敏作用。更多还原

【Abstract】 Nowadays, photodynamic therapy (PDT) has become a hot Point whichhas been broadly employed to diagnoses and therapy of tumorous andnontumorous diseases. Photo sensitizer plays an important role in PDT. Theoptical characteristics of photosensitizer have the directly influences oncurative effect of PDT. The characteristic absorption of the second-generationphoto sensitizer locates in 670 nm or 690nm. However, there are manydisadvantages of the second-generation photo sensitizer to therapy cancerssuch as the poor penetrable ability in tissues, the wide absorption spectrum invisible range and the obvious side-effects. In our study, we have synthesizedsome new photo sensitizers which have characteristic absorption in 750 nm.Because of their great penetrable ability, the new photo sensitizer can bedirectly used to diagnoses and therapy tumorous and nontumorous diseases.Moreover, the side-effects have decreased greatly. The mechanism of PDTand the bioactivities of the new photo sensitizers have been studied in theresearch as well.(1) Bacterialchlorophyll a was extracted by supersonic in ethanol fromRhodobacter sphaeroides, absorbed by French chalk, separated withDEAE-sepharose CL-6B(2.7×6.5)and Sepharose CL-6B(2.7×18). Bchla50mg/100g (wet weight) is obtained. The synthesis of transition metal (Cu,Zn, Co, Fe) was investigated by using bacteriochlorophyll a as ligand. Theultraviolet-visual spectrum and the fluorescence spectrum of thesecompounds were analyzed respectively. The conclusions showed that thecharacteristic absorption of different metal-bacteriochlorophyll a appearedbiggish red and blue shift. The most remarkable Cu-BCA’ characteristic absorption had blue shift 97 nm and red shift 29 nm. In addition, allmetal-bacteriochlorophyll a but Cu-BCA emitted fluorescence, and Zn-BCAhad red shift 57 nm and blue shift 80nm, the biggest emission wavelength ofZn-BCA was 770 nm.(2) Liposomes were introduced to be a drug carrier because of smallpolarity’s metal-bacteriochlorophyll. It could be one of the most effectivemethods that could improve drug targer and minish the side effects of body.In the experiment, liposomes were sized by extrusion through 100nm porepolycarbonate membrane and then incubated with Zn-BCA. The procedureprovided unilamellar liposomes with a mean diameter and high temperatureof phase shift at 50℃. The recovery rates of the liposomes were high(＞95.00%). The relative standard deviations (RSD) of intra-day and inter-daywere less than 5.84%. The envelop rates were more than 53.00%.(3) MTT colorimetry was used to investigate parameters ofZn-BCA-PDT to HL-60 leukaemic cells, which contained Zn-BCA dose,dark incubating time, photoreaction time and light wavelength, Theoptimized comditions of experiment of light sensitization of Zn-BCA-PDT toHL-60 were observed, cell density was 1×10~5/mL; Zn-BCA dose was 10μg/mL; dark incubating time and photoreaction time were 3 h and 50 min;light wavelength was 800 nm, respectively.(4) As a photosensitizer in PDT, the key factor of effecting the curativeeffect of Zn-BCA were high selective in tumor tissue and cell. Therefore,It’distributing in animal tissues would influence not only the curative effectbut also the side effect on common tissue. In this article, spontaneityfluorescence of Zn-BCA was used to observed metabolism of Zn-BCA inmice tissues. The results showed that metabolizing of Zn-BCA in tissue werevery fast, and it’ fluorescence intensity were very low after 5 hours.(5) Kunming mice were inoculated to execute tumor mice modelthrough injected into HL-60 leukaemic cells, and treated with 0.25mg/Kg and1.0 mg/Kg Zn-BCA, respectively. The photodynamic effect and side effect ofZn-BCA were remarkably observed. The results showed that the survive time of tumor mice which were treated with PDT were significantly prolongedcompared with the control group. Injected with 1.0 mg/kg Zn-BCA liposomeand immediately photoreacted without dark incubation, mice presentedstrong skin photosensitive effect and short survive time compared withcontrol group. It’s suggested that the curative effect of Zn-BCA-PDT wasprominent. No remarkable skin photosensitive effects were observed in tumormice which were carried out PDT after 4 hours’ dark incubation.更多还原