华卟啉钠介导的光动力疗法对裸鼠人舌鳞癌移植瘤杀伤作用的实验研究

目的:探讨华卟啉钠（sinoporphyrin sodium,DVDMS）介导的光动力疗法（photodynamic therapy,PDT）对舌鳞状细胞癌（tongue squamous cell carcinoma,TSCC）移植瘤治疗的疗效,为今后的临床治疗方案提供理论依据。方法:通过采用BALB/c-nu裸鼠体内移植瘤实验评估DVDMS-PDT对舌鳞癌的杀伤作用。治疗后定期测量小鼠体重、肿瘤体积,计算抑瘤率、生命延长率以评估杀伤作用;通过TUNEL分析和抗Bax、Bcl-2抗体的免疫组织化学法检测细胞凋亡情况。结果:本实验显示,各处理组肿瘤体积均小于对照组,DVDMS-PDT组肿瘤体积减小最明显（P＜0.01）;DVDMS-PDT组抑瘤率为53.57%,抑瘤效果明显优于其他组（P＜0.000 1）;DVDMS-PDT组生命延长率为61.51%,生存期最长（P＜0.05）;DVDMS-PDT组Bax蛋白表达明显上调、Bcl-2蛋白表达明显下降（P＜0.000 1）;DVDMS-PDT组TUNEL分析绿色荧光亮点最多（P＜0.000 1）。结论:DVDMS-PDT对舌鳞癌移植瘤具有明显的杀伤作用,可促进细胞凋亡,抑制细胞增殖。     

光动力疗法的抗肿瘤机制研究进展

光动力作用是指由光敏剂介导,在光的作用下,使机体细胞或生物分子发生功能或形态变化,累积而致细胞的损伤和坏死,又称光敏化－氧化作用。光动力疗法(PDT)是利用光敏剂介导的光动力作用进行疾病诊断和治疗的技术,由于光敏剂选择性地在肿瘤部位高浓度积聚,在激光照射下杀伤肿瘤细胞,目前PDT在临床上主要被用于治疗恶性实体瘤,也可用于一些癌前病变的治疗以及肿瘤诊断。本文对光动力疗法的特点以及抗肿瘤机制的研究进展做一综述。     

酞菁类光敏剂对小鼠肝癌的光动力治疗和作用机理的研究

“光敏疗法”现多用血卟琳衍生物(HPD)加激光,在治疗某些肿瘤上已收到确实疗效,有希望成为一种新的治疗方法。但是,HPD具有以下一些缺点:它是一混合物,其有效成分至今还未确定;在不同的制备条件下,不同批号HPD所含各组分的比例有所差别,使效果不一致;HPD主要光吸收峰在400nm左右,而临床应用630nm红光激发,未能充分发挥其光敏效     

光动力作用对膀胱癌细胞生长影响的体外实验研究

目的观察光动力治疗(PDT)对体外培养的人膀胱癌细胞的杀伤效应以及形态改变,同时探讨光敏剂(PhtofrinⅡ)浓度与杀伤效应的关系。方法将膀胱癌细胞(T-24)分组进行培养,扩增至一定数目后,加入光敏剂,然后用激光照射,绘制细胞生长曲线,观察PDT对肿瘤细胞生长的抑制情况,同时进行细胞形态学观察。结果癌细胞生长随光敏剂用药剂量不同而受到不同程度抑制;显微镜下见治疗后癌细胞结构受到破坏。结论体外光动力治疗可明显抑制膀胱癌细胞的增长,其效应与光敏剂浓度呈正相关。     

光动力治疗消化道和头颈部肿瘤的临床观察

目的　探讨光动力疗法治疗消化道和头颈部肿瘤的疗效和安全性。方法　选取消化道肿瘤９例、头颈部复发转移癌７例行光动力治疗,其中早期４例,晚期１２例。具体治疗：静脉输注血卟啉３～５ｍｇ／ｋｇ,４８～７２ｈ在肿瘤表面或瘤体内行激光照射（输出波长６３０ｎｍ,输出功率６００～８００ｍＷ,能量密度为２００～３００Ｊ／ｃｍ２）。根据治疗后肉眼及内镜观察、Ｂ超和ＣＴ检查肿瘤缩小及症状缓解情况评价疗效。结果　所有患者治疗后随访１０～３９个月。４例早期患者均获ＣＲ,随访期间无复发;１２例晚期患者,获ＳＲ７例,ＭＲ４例,ＮＲ１例。全组患者的总有效率为９３.７％。主要毒副反应为暴露部位皮肤肤色加深,无严重并发症。结论　光动力疗法对于早期病灶局限的恶性肿瘤可达到局部根治的作用,对于晚期病例也能缓解症状并改善生存质量。     

新型靶向光敏剂Ⅰ对Hep-2细胞的光动力抑瘤活性

 目的　评价新型靶向性光敏剂Ⅰ（PSⅠ）对喉癌Hep-2细胞的靶向性和光动力活性。方法　采用bleaching实验评价PSⅠ的稳定性; 荧光测定法观察Hep-2细胞对PSⅠ的摄取能力; 四甲基偶氮唑蓝（MTT）比色法检测PSⅠ浓度、光照剂量及光照时间点对Hep-2细胞存活率的影响。结果　在光照50 min后,PSⅠ的吸光度（A）值仅降低了11 %,表明其稳定性可满足光动力治疗的需要;Hep-2细胞对PSⅠ的摄取随PSⅠ浓度的增加而增加,但过量叶酸的存在可明显抑制Hep-2细胞对PSⅠ的摄取（t值分别为5.96,4.89,均P＜0.01）;MTT实验结果表明Hep-2细胞的存活率与PSⅠ浓度和光照剂量呈负相关（r＝-0.763,P＝0.017; r＝-0.946,P＝0.001）。当PSⅠ浓度为14 μmol／L、光照剂量为18 J／cm2时,Hep-2细胞的存活率仅为34 %,而在无光照时即使光敏剂浓度升至110 μmol／L,Hep-2细胞的存活率仍为100 %;给予PSⅠ 24 h后再进行光动力治疗（PDT）可有效地抑制Hep-2细胞的生长,其存活率只有32 %。结论　PSⅠ拥有满意的光稳定性和低的暗毒性,对Hep-2细胞有很好的靶向性和光动力活性。     

HMME介导的光动力疗法对骨肉瘤细胞的杀伤效应及机制研究

目的探讨血卟啉单甲醚介导的光动力疗法（HMME-PDT）对骨肉瘤LM-8细胞增殖与凋亡的影响及其机制。方法将LM-8细胞与不同浓度的HMME（10、20、30gg／m1）共同孵育,4h后,给予不同能量密度的激光（3、6、9J／cm2）照射,同时设空白组（无光敏剂也无光照）、暗毒组（无光照但加入光敏剂）、激光组（不加光敏剂但给予光照）。MTT法检测细胞存活率,采用AnnexinV-FITC／PI染色的流式细胞分析法检测LM-8细胞凋亡和周期情况,Hoechst33342染色法观察细胞凋亡,实时定量PCR（QRT-PCR）分别检测Caspase-3、Bcl-x、p53、RelAmRNA表达水平,免疫印迹（Westernblotting）分别检测caspase-3、Bcl-X、p53、RelA蛋白的相对表达量。结果HMME-PDT能显著抑制骨肉瘤LM-8细胞增殖,且杀伤效应随着HMME浓度及能量密度的增高而增强。然而单独光照或单独给予光敏剂孵育,对骨肉瘤LM-8细胞均无杀伤作用。流式细胞术检测发现凋亡率显著增高,10μg/mlHMME组为（12．3±2．82）％,20μg/mlHMME组为（20．67±5．58）％,30gg／mlHMME组为（42．53±4．641％,凋亡率与空白组比较,结果分别为（f=-2．889,P=0．045）、（f=-4．418,P=0．014）、（t=-5．780,P=0．04）。周期结果显示：G0／G1期细胞明显增高、s期细胞明显降低,301ag／ml组G0／G1期为（50．09±3．151％、S期为（28．17±1．10）％,与空白对照组比较,结果分别为（t=-6．081,P=0．004）、（t=5．852,P=0．004）。HMME-PDT处理LM-8细胞后径Hoeehst33342染色后呈现出典型的凋亡改变（如：核固缩、细胞变圆等）。QRT-PCR和Westernb10ring结果显示：HMME-PDT可显著上调Caspase-3mRNA和蛋白相对表达量且表达量随着HMME浓度增高而增高。而HMME-PDT可明显下调Bcl-x、p53、RelAmRNA和蛋白相对表达量且表达量随着HMME浓度增高而降低。结论在体外HMME-PDT对骨肉瘤LM-8细胞有显著的杀伤效应,其杀伤效应与Caspase-3、Bcl-x、p53和RelA密切相关。     

CHL对小鼠结肠肿瘤的预防作用及对COX-2的选择性抑制作用

背景与目的:研究表明叶绿酸铜钠(chlorophyllin,CHL)对多种诱变剂和致癌物都有较强的抗诱变作用,但有关CHL预防肿瘤的确切效果有待进一步研究.本研究探讨CHL对二甲肼(dimethylhydrazine,DMH)诱导的小鼠结肠肿瘤的预防作用及对环氧化酶2(cyclooxygenase2,COX-2)的选择性抑制作用.方法:(1)以DMH诱导小鼠结肠肿瘤,诱癌不同阶段予以不同浓度CHL,观察CHL对结肠肿瘤的化学预防作用;(2)MTT法测CHL对HT29肠癌细胞的半数抑制浓度(IC50)及生长曲线;(3)RT-PCR法检测CHL对HT29细胞COX-2和COX-1的影响;(4)Western blot和免疫组化法检测CHL对COX-2蛋白表达的影响;(5)免疫组化法检测CHL对NF-κB蛋白的表达影响.结果:(1)CHL组结肠癌发生率少于阳性对照组(P＜0.05),平均肿瘤数及腺癌百分率均低于阳性对照组(P＜0.05);(2)CHL能抑制HT29细胞生长且呈剂量依赖关系;(3)CHL能够选择性抑制HT29细胞COX-2 mRNA的表达;(4)CHL能够抑制肿瘤组织和HT29细胞COX-2蛋白的表达;(5)CHL能够抑制NF-κB蛋白的表达.结论:CHL能够抑制DMH诱导的小鼠结肠肿瘤的发生,这种预防作用与CHL对COX-2的选择性抑制作用有关,而CHL对COX-2的抑制作用可能通过对NF-κB的抑制来实现.     

脉冲激光和连续激光对肿瘤治疗光动力疗效的影响

光动力疗法（PDT）是治疗肿瘤的主要方法之一。以光与生物组织的相互作用原理及生物效应理论为依据,对不同运转方式的激光在肿瘤治疗过程中所产生的光动力效应、热效应以及生物刺激作用等方面进行分析,并对激光作用时间不同和总能量的差异对疗效的影响进行了分析。从发展的角度讲,为使PDT达到最佳疗效,同时有益于新光源的开发和利用,PDT采用脉冲激光无疑是一种最佳的选择。     

芦荟大黄素介导的光动力疗法对人口腔黏膜癌KB细胞杀伤效果的研究

目的 探讨芦荟大黄素(Aloe-emodin,AE)介导的光动力疗法(Photodynamic therapy,PDT)对人口腔黏膜癌KB细胞的杀伤效果。方法 通过MTT法检测不同实验条件对KB细胞的杀伤作用。实验分为空白对照组(Control)、单纯药物组(AE)、单纯光照组(Laser)和光动力组(PDT)。分别比较AE组不同浓度(10μM、20μM、30μM和40μM)、Laser组不同光照时间(10s、20s、40s、80s)与Control组对KB细胞的杀伤作用,分析PDT组AE浓度和时间对KB细胞杀伤作用的影响。结果 与Control组相比,AE组和Laser组对KB细胞的杀伤作用无统计学意义(P＞0.05)。PDT组对KB细胞的杀伤作用呈浓度和时间依赖性,当AE的浓度为40μM,照射时间为80s时,细胞存活率降低至56.7%。结论 AE介导的PDT对KB细胞有显著的杀伤作用。     

静脉注射福大赛因对昆明小鼠的皮肤光毒性

背景与目的:研究福大赛因对小鼠的皮肤光毒性.材料与方法:将昆明小鼠随机分为5组,分别为生理盐水组(给予生理盐水0.02 ml/g),溶剂对照组[给予溶剂0.02 ml/g).福大赛因低剂量组(2 mg/kg)和高剂量组(5 mg/kg)以及阳性对照组(给予血卟啉15 mg/kg).均采用静脉注射1次性给药.小鼠于给药后的不同时间接受不同强度模拟太阳光照射60 min.用6 mm打孔器取左、右耳廓各1耳片,并称重,通过小鼠耳片重量和肿胀率的变化评价皮肤光毒性.结果:福大赛因高、低剂量组于给药后1、3及24h接受模拟太阳光(40 mW/cm2)照射后,耳片重量与对照组相比显著增加(P<0.05),而低剂量组于给药后48 h照射后无明显异常,高剂量组则给药后7 d经照射无明显异常.给药后1、3 h不同强度模拟太阳光照射(10、20、30、40 mW/cm2),各组小鼠均出现毒性反应;10 mW/cm2组耳片重量与20、30、40 mW/cm2组间差异具有统计学意义(P<0.01).结论:福大赛因2 mg/kg体重给药48 h后光照无明显光毒性反应;5 mg/kg给药7 d后光照无明显光毒性反应.福大赛因产生光毒性的饱和光强度约为20 mW/cm2     

二氢卟吩类光敏剂在光动力疗法治疗恶性肿瘤中的研究进展

肿瘤的治疗是目前医学领域最为棘手的难题之一,患者多采用手术、放化疗相结合的综合治疗方式。与传统抗癌疗法相比,光动力疗法最突出的优点是能选择性杀灭局部肿瘤而不危及正常组织。光敏剂作为光动力疗法中至关重要的因素,它的研究受到越来越多的重视。本文就近年来二氢卟吩类光敏剂在光动力疗法治疗恶性肿瘤中的研究进展作一简要综述。     

Efficacy of antitumoral photodynamic therapy with hypericin: relationship between biodistribution and photodynamic effects in the RIF-1 mouse tumor model.

We investigated the hypericin-mediated PDT effects on the tumor and normal skin and in correlation with its biodistribution. These studies were carried out on C3H mice bearing RIF-1 tumors. The hypericin distribution and PDT effects were recorded at different intervals (0.5-24 hr) after intravenous injection of a 5-mg/kg dose of hypericin. After administration, rapid biphasic exponential decay was observed in the plasma drug concentration. It was found that hypericin was preferentially bound to the plasma lipoproteins. The tumor drug levels increased rapidly over the first few hours and reached a maximum around 6 hr after injection. In contrast, PDT efficacy was maximal when irradiation was performed at 0.5 hr after hypericin administration, which led to 100% cure. The PDT efficacy decreased rapidly as the administration-irradiation interval was prolonged. No tumor cure was obtained at the 6-hr interval, even though it was at this time that the tumor drug level peaked. Fluorescence microscopic studies showed that hypericin was mainly confined within the tumor vasculature at 0.5 hr after injection, whereupon it rapidly diffused to the surrounding tumor tissue. At 6 hr, a strong hypericin fluorescence was observed in the tumor tissue with only faint fluorescence within the vasculature, whereas at 24 hr the fluorescence in the tumor also decreased and became more diffused, and no fluorescence could be seen in the tumor vasculature. Like the tumor response, skin reactions were also found to be much more dramatic at short administration-irradiation intervals. Hypericin distribution and PDT response studies revealed a close correlation between the plasma drug level and the PDT effects, which suggests that vascular damage is the primary effect of hypericin-mediated PDT in this tumor model.     

选择性环氧化酶-2抑制剂对卵巢癌细胞抑制作用的体内体外研究

目的:研究选择性环氧化酶-2抑制剂美洛昔康在体内体外对卵巢癌生长侵袭的抑制作用。方法:使用2种卵巢癌细胞株,研究不同浓度的美洛昔康对于卵巢癌细胞增殖的抑制作用。利用移植卵巢癌细胞株小鼠模型,研究美洛昔康对小鼠背部皮下移植瘤生长以及肿瘤中VEGF表达、微血管密度以及细胞凋亡的影响,并将之与顺铂的作用相比较。结果:在体外实验中,美洛昔康对2种卵巢癌细胞的增殖均有抑制作用,且呈剂量依赖性。体内实验证明美洛昔康明显抑制小鼠皮下移植瘤的生长,其抑制率为30.79%（P＜ 0.0001）。各组肿瘤组织中VEGF的表达经半定量测定分别为:每高倍视野对照组5.17±0.52、、顺铂组4.32±0.73及美洛昔康组4.81±0.58。与对照组相比,美洛昔康饲养的小鼠肿瘤中VEGF的表达受到明显抑制（P＜0.0001）并伴随微血管密度的明显减低。TUNEL法对肿瘤组织中凋亡细胞计数,结果分别为:对照组12.8±1.3、顺铂组11.5±1.8及美洛昔康组23.3±2.7。与对照组比较,美洛昔康饲养的小鼠肿瘤中,细胞凋亡的数量明显增多（P＜0.001）。美洛昔康的抗肿瘤效果在某些方面优于顺铂。结论:美洛昔康在体内体外实验中表现出抗卵巢肿瘤生长和侵袭的作用。选择性环氧化酶-2抑制剂或许可以作为潜在的新型抗卵巢癌药物应用于临床。     

Photodynamic therapy in dermatology: current concepts in the treatment of skin cancer

Photodynamic therapy is a treatment modality that is developing rapidly and increasing in utilization within various medical specialties, including dermatology. This technique requires the presence of a photosensitizer, light energy and molecular oxygen to selectively destroy pathologic cells. A thorough understanding of photobiology and tissue optics is necessary to correctly and effectively utilize photodynamic therapy in dermatology. Photodynamic therapy has been approved by the US Food and Drug Administration to treat actinic keratoses. In Europe, photodynamic therapy is currently being used in the treatment of actinic keratoses and basal cell carcinoma. Other off-label uses of photodynamic therapy have included cutaneous lesions of Bowen’s disease, psoriasis, cutaneous T-cell lymphoma and acne. Most recently, photodynamic therapy has been employed in photorejuvenation. The advantages of photodynamic therapy include the capacity for noninvasive targeted therapy via topical application of the drug and local irradiation of involved areas, as well as the ability to generate excellent cosmetic results with minimal discomfort. This review summarizes the fundamentals of photodynamic therapy and its role in the treatment of cutaneous disorders, particularly skin malignancies.     

新型靶向光敏剂诱导Hep-2细胞光氧化行为的研究

[目的]研究新型光敏剂Ⅰ诱导Hep-2细胞的光氧化行为。[方法]利用MTT法检测光敏剂Ⅰ对Hep-2细胞的细胞毒性;采用活性氧特异性探针H2DCFDA通过激光共聚焦成像观察Hep-2细胞中活性氧的生成;通过测定超氧化物歧化酶(SOD)、谷胱甘肽(GSH)和丙二醛(MDA)水平及乳酸脱氢酶(LDH)渗漏检测观察Hep-2细胞的氧化应激反应。[结果]无光照时,光敏剂Ⅰ对Hep-2细胞的毒性为零,但光照后可明显抑制该细胞的生长,且其光毒性随光照剂量的增加而加强(r=-0.962,P=0.001)。光动力治疗后,细胞内DCFDA的荧光强度逐渐增强,在4h时达到高峰,随后又逐渐降低;细胞内SOD和GSH水平逐渐降低,3h后分别降低42.5%(P<0.01)和35.0%(P<0.01),而MDA含量却随时间延长逐渐增加,3h后增加54%(P<0.01)。LDH的渗出与光照剂量呈正相关(r=0.966,P=0.007)。[结论]新型光敏剂Ⅰ可有效光诱导Hep-2细胞死亡,而细胞内氧化应激反应可能是其光诱导Hep-2细胞死亡的重要作用机制。     

Extracellular vesicles from human liver stem cells inhibit renal cancer stem cell-derived tumor growth in vitro and in vivo

Cancer stem cells (CSCs) are considered as responsible for initiation, maintenance and recurrence of solid tumors, thus representing the key for tumor eradication. The antitumor activity of extracellular vesicles (EVs) derived from different stem cell sources has been investigated with conflicting results. In our study, we evaluated, both in vitro and in vivo, the effect of EVs derived from human bone marrow mesenchymal stromal cells (MSCs) and from a population of human liver stem cells (HLSCs) of mesenchymal origin on renal CSCs. In vitro, both EV sources displayed pro-apoptotic, anti-proliferative and anti-invasive effects on renal CSCs, but not on differentiated tumor cells. Pre-treatment of renal CSCs with EVs, before subcutaneous injection in SCID mice, delayed tumor onset. We subsequently investigated the in vivo effect of MSC- and HLSC-EVs systemic administration on progression of CSC-generated renal tumors. Tumor bio-distribution analysis identified intravenous treatment as best route of administration. HLSC-EVs, but not MSC-EVs, significantly impaired subcutaneous tumor growth by reducing tumor vascularization and inducing tumor cell apoptosis. Moreover, intravenous treatment with HLSC-EVs improved metastasis-free survival. In EV treated tumor explants, we observed both the transfer and the induction of miR-145 and of miR-200 family members. In transfected CSCs, the same miRNAs affected cell growth, invasion and survival. In conclusion, our results showed a specific antitumor effect of HLSC-EVs on CSC-derived renal tumors in vivo, possibly ascribed to the transfer and induction of specific antitumor miRNAs. Our study provides further evidence for a possible clinical application of stem cell-EVs in tumor treatment.     

体外MTT法预测卵巢上皮癌化疗药敏性与体内疗效相关性的评估

目的：预测卵巢上皮癌体外化疗敏感性与体内疗效的关系。方法：运用MTT法检测7种临床常用化疗药物及6种经验组合方案对卵巢上皮癌的体外抑制率进行了检测并与体内疗效结果进行比较。结果：7种药物抑制率范围为6．25％～51．43％其中PTX为51．43％最高，TXT为20．29％，DDP为18．57％，TPT为11．63％，ADM为11．43％．GEM8．82％及VP166．25％敏感性最差；6种联合方案的抑制率范围在10．00％～35．00％．TPT＋PTX＋DDP（35．00％）及PTX＋DDP（30．00％）较好，其余4种TXT＋DDP（20．59％），TPT＋DDP（17．65％），GEM＋DDP（11．76％）及VP16＋ADM（10．00％）均较差。本组药物抑制率与试验前是否用过化疗药无关体内、外敏感及耐药的总符合率为72．86％（51／70），不符合率27．14％（19／70），统计学有显著性差异（P〈0．05）。结论：MTT法对卵巢上皮癌的个体化治疗计划有参考价值．能在一定程度上指导临床用药。     

Effective treatment of chemoresistant breast cancer in vitro and in vivo by a factor VII-targeted photodynamic therapy

Background:

The purpose of this study was to test a novel, dual tumour vascular endothelial cell (VEC)- and tumour cell-targeting factor VII-targeted Sn(IV) chlorin e6 photodynamic therapy (fVII-tPDT) by targeting a receptor tissue factor (TF) as an alternative treatment for chemoresistant breast cancer using a multidrug resistant (MDR) breast cancer line MCF-7/MDR.

Methods:

The TF expression by the MCF-7/MDR breast cancer cells and tumour VECs in MCF-7/MDR tumours from mice was determined separately by flow cytometry and immunohistochemistry using anti-human or anti-murine TF antibodies. The efficacy of fVII-tPDT was tested in vitro and in vivo and was compared with non-targeted PDT for treatment of chemoresistant breast cancer. The in vitro efficacy was determined by a non-clonogenic assay using crystal violet staining for monolayers, and apoptosis and necrosis were assayed to elucidate the underlying mechanisms. The in vivo efficacy of fVII-tPDT was determined in a nude mouse model of subcutaneous MCF-7/MDR tumour xenograft by measuring tumour volume.

Results:

To our knowledge, this is the first presentation showing that TF was expressed on tumour VECs in chemoresistant breast tumours from mice. The in vitro efficacy of fVII-tPDT was 12-fold stronger than that of ntPDT for MCF-7/MDR cancer cells, and the mechanism of action involved induction of apoptosis and necrosis. Moreover, fVII-tPDT was effective and safe for the treatment of chemoresistant breast tumours in the nude mouse model.

Conclusions:

We conclude that fVII-tPDT is effective and safe for the treatment of chemoresistant breast cancer, presumably by simultaneously targeting both the tumour neovasculature and chemoresistant cancer cells. Thus, this dual-targeting fVII-tPDT could also have therapeutic potential for the treatment of other chemoresistant cancers.