顺铂耐药及非耐药肺癌细胞对竹红菌乙素的吸收及其光动力杀伤效应的比较

目的对比研究耐药及非耐药肺癌细胞株对竹红菌乙素(HB)-PDT的反应性,对比研究细胞耐药对光敏剂竹红菌素的吸收是否造成影响,探讨肿瘤细胞耐药的相关机制及HB-PDT对耐药肿瘤杀伤的可能性。方法(1)以不同浓度的竹红菌乙素(HB)分别孵育人肺腺癌细胞系A549细胞及经顺铂诱导耐药的多药耐药细胞系A549/DDP细胞,然后分别以铜蒸气激光混合光在饱和光剂量条件下进行照光处理,照光后置于37℃含5%CO2的孵箱中继续孵育24h,然后用MTT法分别测定不同HB浓度、不同细胞的存活率,分别绘制HB对2种细胞杀伤曲线并拟合曲线方程,根据方程求出HB对2种细胞的半数杀伤浓度(IC50)。根据曲线、拟合曲线方程及IC50比较经顺铂诱导耐药及非耐药细胞系HB-PDT杀伤效应有无差别。(2)对人肺腺癌细胞系A549细胞以及顺铂诱导的多耐药细胞系A549/DDP细胞进行光敏剂吸收实验,分别测定并绘制光敏剂被2种细胞吸收的标准曲线、浓度-含量关系曲线、时间-含量关系曲线,根据曲线及拟合方程比较分析2种细胞对光敏剂竹红菌素的吸收差异。结果HB-PDT对经顺铂诱导耐药及非耐药细胞系均具有很强的杀伤效应,二者无统计学差异(P>0·05):竹红菌乙素-PDT对A549细胞及A549/DDP细胞的半数杀伤浓度(IC50)分别为33·82ng/ml和34·19ng/ml。A549/DDP细胞与A549细胞对光敏剂吸收的标准曲线、浓度-含量关系曲线、时间-含量关系曲线均极为相似,顺铂诱导耐药的细胞系A549/DDP细胞与亲代细胞系A549细胞间对光敏剂的吸收无统计学差异(P>0·05)。2种细胞吸收曲线对比后未见到耐药细胞在光敏剂吸收过程中将光敏剂排出细胞外所引起的细胞内光敏剂含量降低,证实A549/DDP细胞中不存在将光敏剂“泵”出细胞外的作用。结论HB-PDT对经顺铂诱导耐药及非耐药细胞系均具有很强的杀伤作用。对顺铂产生多药耐药的肿瘤细胞与非耐药肿瘤细胞对HB的吸收无明显差别,因而对顺铂耐药的肿瘤细胞对于PDT仍然非常敏感。     

五种竹红菌素衍生物对A549细胞的光动力效应研究

目的比较五种新型竹红菌素衍生物分别为竹红菌素乙素（hypocrellin,HB）的二位ω-氨基磺酸衍生物THB、3HB和4HB,及十七位ω-氨基磺酸衍生物3SB和4SB对体外培养的人肺腺癌上皮细胞（A549）的光动力（photodynamic therapy,PDT）效应,筛选光动力活性和安全性较好的竹红菌素衍生物。方法 （1）杀伤效应。将0.94 nmol/ml的5种新型竹红菌素衍生物和HB分别与A549细胞孵育4 h后,分别以波长630和532 nm激光照射,功率密度20 mW/cm2,照射时间1 000 s,能量密度20 J/cm2,照光后继续避光孵育24 h后采用MTT法测定细胞存活率。（2）安全系数。分别以波长532和630 nm激光照射,以血卟啉（hematoporph-yrin derivative,HpD）为对照光敏剂,研究17-4-amino-1-butane-sulfonic acid-hypocrellin B（4SB）对A549细胞的光动力效应及和暗毒性,并比较安全系数（暗毒性IC50/光毒性IC50）。结果 （1）杀伤效应。五种竹红菌素衍生物中,4SB在630和532 nm激光照射下对A549的光动力杀伤作用强于其它衍生物,接近HB。（2）安全系数。波长532 nm激光照射,4SB的光毒性分别为103.86和84.16 ng/ml是HpD 960.14 ng/ml的10.53和11.4倍,但前两者之间差异无显著意义（P〉0.05）;波长630 nm激光照射下,4SB光毒性的IC50为50.7 ng/ml,HpDIC50为1 069.88 ng/ml,暗毒性HpD、4SB分别为7.84、21.93μg/ml,安全系数4SB（432.5）〉HpD（7.3）。HpD在532和630 nm两波长下的光毒性IC50差异无显著意义（P〉0.05）,而4SB在532和630 nm两波长下的光毒性差异有显著意义（P〈0.05）。结论 5种衍生物可能成为有价值的光敏剂,值得进一步深入研究。     

痂囊腔菌素A与竹红菌乙素介导的光动力反应对ECV304细胞杀伤效应的比较研究

目的探讨痂囊腔菌素A光动力学疗法(EA-PDT)对ECV304细胞的杀伤效应,并与竹红菌乙素光动力学疗法(HB-PDT)进行比较。方法以不同浓度的痂囊腔菌素A(elsinochromeA,EA)及竹红菌乙素(hypocrellinB,HB)分别孵育人脐静脉血管内皮ECV304细胞4h,然后分别用波长532nmKTP激光,以20mW/cm2功率密度照射1000s。照光后置于37℃、5%CO2的孵育箱中继续孵育24h后,用MTT法分别测定不同浓度下的细胞存活率,绘制杀伤曲线并拟合曲线方程,根据方程求出不同光敏剂对细胞的半数杀伤浓度(IC50)。结果EA-PDT和HB-PDT对ECV304细胞的IC50分别为50·97ng/ml和85·20ng/ml,二者比较差异有统计学意义(P<0·05),EA-PDT对ECV304细胞有较强的杀伤效应。结论EA-PDT对ECV304细胞株的杀伤效应强于HB-PDT,在血管性病变治疗方面可能有广阔的应用前景。     

竹红菌乙素及其衍生物对人胃腺癌细胞光动力杀伤效应的比较

目的测定两种新型光敏剂5-氨基-1-戊磺酸取代竹红菌乙素衍生物（PENSHB）和15位脱乙酰基13位3-氨基-1-丙磺酸取代的竹红菌乙素衍生物（DPROHB）的细胞毒性及其光动力杀伤效应,并与其母体竹红菌乙素（hypocrellin B,HB）的细胞毒性、光动力杀伤效应进行比较。初步了解两种新型光敏剂的光动力作用效果。方法三种光敏剂分别孵育人胃腺癌BGC-823细胞,光动力组：采用波长532 nm、功率密度20 mW/cm2 KTP激光照射1 000 s。光敏剂组：仅孵育24 h。用MTT法分别测定各组不同光敏剂浓度下的癌细胞存活率。分别绘制药物对细胞光毒及暗毒的杀伤曲线,并拟合曲线方程,计算光动力组半数杀伤浓度（50%inhibition concentration,IC50-PDT）及光敏剂组半数杀伤浓度（50%inhibition concentration,IC50-DT）。并由此计算光敏剂的安全系数。结果 PENSHB、DPROHB及HB对BGC-823细胞的半数杀伤浓度（IC50-PDT）分别为26.22、28.14和48.03 nM;PENSHB、DPROHB的IC50-PDT与HB的IC50-PDT比较差异有显著意义（P〈0.01）。PENSHB的IC50-PDT与DPROHB的IC50-PDT比较差异无显著意义。光敏剂组PENSHB、DPROHB及HB半数杀伤浓度（IC50-DT）,分别为3.42、4.20和4.48μM,三者比较差异无显著意义（P〉0.05）。PENSHB、DPROHB及HB的安全系数依次为130,173,93。结论 PENSHB和DPROHBP对BGC-823细胞株的PDT杀伤效应无明显区别,但均强于HB,三种光敏剂对细胞暗毒性相当,PENSHB和DPROHBP较HB安全性有所提高。     

铜蒸气激光照射下11种光敏剂杀伤强度的比较

目的　检测目前国内常用的和中国科学院化学研究所合成的共 11种光敏剂 ,比较它们对细胞杀伤效应的强弱 ,筛选出适于临床应用的新型光敏剂。方法　将乳腺癌MDAMB 5 4 3细胞接种于 96孔培养板 ,然后加入用RPMI16 4 0培养基配置的不同浓度的不同光敏剂 ,4h后照光 ,2 4h后用四唑盐比色法测定细胞存活率。以血卟啉衍生物 (HpD)作为标准对照。结果　在 5 10 6nm和 5 78 2nm的混合光照射下 ,竹红菌素A(HA)、竹红菌素B(HB)和 5 ,15 二芳基 2 ,3 二羟基卟吩 (DPCOH)比HpD有更强的杀伤效应。血啉甲醚 (HMME)的杀伤效应是HpD的 1 2左右。其他的一些化合物在此波长条件下杀伤效应较差 ,其中一些是因其溶解性不好而不能完全发挥其杀伤效能。结论　竹红菌素类光敏剂和DPCOH是很有应用开发前景的新型光敏剂     

铜蒸气激光照射下三种光敏剂杀伤效应的研究

目的 测定血卟啉衍生物(HpD)和竹红菌素A(HA)、竹红菌素B(HB)在铜蒸气激光照射下的半数杀伤浓度和半数杀伤光剂量，了解HA、HB与HpD杀伤特性的差异。方法 将小鼠肺血管内皮细胞接种于96孔培养板，然后加入用DMEM(低糖)培养基配制的光敏剂，4h后应用铜蒸气激光510．6nm和578.2nm单色光分别照射，24h后用四唑蓝比色法测定细胞存活率。结果 在510.6nm和578.2nm波长饱和光剂量条件下HpD的半数杀伤浓度分别是HA的63、51和70.96倍，是HB的27．08和25．03倍。在饱和光敏剂剂量条件下510.6nm和578．2nm的半数杀伤光剂量HA分别是HpD的6．77和6．59倍，HB则分别是HpD的5．45和4．91倍。结论 HA、HB在510.6nm和578．2nm波长的杀伤效应都较HpD强并和光剂量、波长密切相关。     

光动力疗法中竹红菌乙素在细胞中含量的检测

目的 研究光敏剂孵育时ECV304细胞和培养液中竹红菌乙素(HB)的含量,以及HB在细胞和培养液中的光漂白.方法 体外培养ECV304细胞,HB的孵育浓度分别为10、30 μg/ml,每个浓度组再分为照光组和未照光组.未照光组HB孵育2 h后,用荧光显微镜观察细胞中HB的荧光分布;然后分别用乙酸乙酯萃取细胞和培养液中的HB后用吸收光谱测定HB的含量.照光组用532 nm激光照射,采用上述方法测量细胞和培养液中HB的含量,并进行HPLC图谱分析.结果 HB的孵育浓度为10、30 μg/ml,孵育浓度增加细胞中HB浓度并不随之成比例增加,激光照射使细胞和培养液中的HB都发生了光漂白.结论 利用HB的浓度吸收光度值的标准曲线,可以估算出细胞中以及光照后HB的含量.     

亚苄基环戊酮介导的光动力对多重耐药铜绿假单胞菌的体外杀伤效应

目的探讨新型光敏剂亚苄基环戊酮化合物P3介导的光动力对多重耐药铜绿假单胞菌的体外杀伤效应。方法实验对象为铜绿假单胞标准菌(ATCC27853)1株和临床多重耐药菌(PA1、PA2、PA3)3株。(1)检测实验菌株与光敏剂P3的结合特性:以荧光光谱检测法检测孵育时间和孵育浓度对实验菌株与光敏剂P3结合的影响,先将4株铜绿假单胞菌与10μM光敏剂分别孵育不同时间(5、15、30、60、120和150 min),根据前期的检测结果再选择不同浓度(2. 5、5、10、25和50μM)的光敏剂P3孵育30 min。(2)观察光敏剂P3介导的PDT对铜绿假单胞菌的体外光动力抗菌效应,即PDT组(B组),按照不同浓度的光敏剂P3分为4组分别为2. 5μM(B1组)、5μM(B2组)、10μM(B3组)和25μM(B4组),药物与4种菌株的孵育时间30 min后,进行PDT处理,激光波长532 nm,功率密度40 mW/cm2,照射时间600 s,同时设立3个对照组(A组):空白对照组(A1组)、单纯照光组(A2组)和单纯光敏剂组(A3组),用稀释平板法培养24 h进行菌落计数。结果孵育时间5～30 min时,四株铜绿假单胞菌与光敏剂P3的结合量随孵育时间延长而逐渐增加;30 min后趋于饱和。浓度梯度实验结果显示,四株铜绿假单胞菌与光敏剂P3的结合量呈孵育浓度剂量依赖性增加。在相同孵育浓度和相同孵育时间条件下,四株铜绿假单胞菌与光敏剂P3的结合量未见显著差异。光敏剂P3对四株铜绿假单胞菌的PDT杀伤作用随着光敏剂P3浓度增高逐渐增强,当光敏剂P3浓度为25μM时,PDT对4株铜绿假单胞菌株均达到有效杀伤,即活性下降均4Log;光敏剂P3对铜绿假单胞标准菌和临床耐药菌的PDT杀伤效应比较,差异无统计学意义(P0. 05);单纯照光和单纯光敏剂对细菌的存活无影响。结论光敏剂P3介导的PDT对铜绿假单胞菌有良好的体外杀伤作用,其作用不受细菌耐药性的影响。     

铜蒸气激光照射下光敏剂血卟啉衍生物和血卟啉单甲醚杀伤特性研究

目的 测定血卟啉衍生物(HpD)和血卟啉单甲醚(HMME)在铜蒸气激光照射下的半数杀伤浓度(IC50)和半数杀伤光剂量(IED50),了解HpD与HMME杀伤特性的差异.方法 将小鼠肺血管内皮细胞接种于96孔培养板,然后加入用DMEM(低糖)培养基配置的光敏剂中,4h后应用铜蒸气激光(510.6nm和578.2nm)单色光分别照射,24h后用MTT法测定细胞存活率.结果 在510.6nm和578.2nm波长下,饱和光剂量条件下HMME的IC50分别是HpD的1.31和1.24倍,在饱和光敏剂剂量条件下,HpD的IED50分别是HMME的1.18和1.17倍.结论 HpD在510.6nm和578.2nm波长的杀伤效应均较HMME强并和光剂量、波长密切相关.     

血啉甲醚对体外培养的类风湿关节炎滑膜细胞光动力杀伤作用的初步研究

探讨血啉甲醚（hematoporphyrin monomethyl ether,HMME)对体外培养的类风湿关节炎患者滑膜细胞的光动力杀伤作用。方法：用四唑盐化色法（MTT）检测了不同能量的铜蒸气激光,不同浓度的HMME对体外培养的类风湿关节炎滑膜细胞的杀伤效应,并与血卟啉衍生物（hematoporphyrin derivative,HpD)进行比较。结果：（1）单纯照光及单加HMME,HpD对细胞存活率均无明显抑制作用。（2）各种浓度的HMME在不同剂理的激光作用下对细胞均有杀伤作用,浓度越高其杀伤作用越明显;HMME浓度一定,随着照光剂量的增大杀伤作用增强;不同浓度HMME介导的光动力杀伤作用所能产生的最大抑制率不同,此时所需的光剂量也不同。（3）HMME介导的光动力效应对滑膜细胞的杀伤作用受照光前孵育时间的影响。（4）相同浓度的HMME介导的光动力杀伤效应较HpD更强。结论：HMME介导的光动力效应对滑膜细胞的杀伤作用受光敏剂的浓度及照光能量密度的影响,且明显强于HpD,临床应用于滑膜切除术可能具有更大的优势。     

治疗视网膜脉络膜新生血管的竹红菌乙素光动力学疗法实验系统的建立

目的 建立一种治疗视网膜脉络膜新生血管的竹红菌乙素光动力学疗法实验系统。方法 1.在裂隙灯显微镜下开创激光窗口，光纤连接激光器和裂隙灯显微镜，使激光和裂隙灯照明同光路到达眼底，作为照射系统；2．通过四甲偶氮唑盐(MTT)法检测光敏剂竹红菌乙素(HB)体外光敏性，和苯并卟啉衍生物单酸A环(BPD-MA)相比，初步判断其光敏性；3．以光敏剂HB为例，青紫蓝兔为实验对象，进行PDT治疗，通过眼底观察，荧光眼底造影、光学显微镜和电子显微镜检查照光部位的生物学效应，检测建立的实验系统的实用性，可靠性。结果 照射系统输出稳定，由激光器发出的功率和裂隙灯显微镜的激光窗口末端的输出的功率的耦合率为60％。国产HB体外光敏性和BPD-MA相似。HB-PDT仅对脉络膜毛细血管造成损伤而对周围正常组织损伤小，达到选择性治疗目的，有进一步研究价值。结论 新建立的照射系统和光敏剂光敏性体外评价系统，以及体内观察生物学效应的方法，可作为初步判定光敏剂是否适合治疗视网膜或脉络膜新生血管的方法。     

Mitoxantrone as a prospective photosensitizer for photodynamic therapy of breast cancer

Photodynamic therapy has the potential to become an effective alternate to surgery for the treatment of cancer. In recent years, there has been a focus on identifying more effective and less toxic photosentisizers for use in photodynamic therapy. The purpose of this study was to assess the effectiveness of mitoxantrone, a chemotherapeutic agent, as a photosensitizer for photodynamic therapy in the MCF-7 human breast cancer cell line. Cytotoxicity was evaluated for different concentrations of mitoxantrone, and photosensitivity was assessed using a non-coherent light source. The percentage of the cell survival after 24 h was investigated using the MTT assay. Overall, the results showed that mitoxantrone is a remarkably efficient photosensitizer that could mediate MCF-7 cell death at a low concentration (5 μM) with modest exposure to light. It is surprising to find that a chemotherapeutic agent can be an effective photosensitizer for PDT in vitro.     

Tumor therapy with hematoporphyrin derivative and lasers via a percutaneous fiberoptic technique: preclinical experiments

Photodynamic therapy relies on uptake of a photosensitizer (hematoporphyrin derivative [HpD]) by tumor cells and subsequent interaction of the photosensitizer with penetrating light. This technique has been applied in multiple animal systems and several clinical trials. The therapeutic results in large, deep tumors are limited by poor uptake and distribution of the HpD and limited penetration of tumors by light, even at high wavelengths. In various experiments with mice, HpD was injected into tumors, and light was applied via laser fiberoptics inserted through a sheath catheter. Preliminary findings indicated that intratumoral injection enables excellent distribution of HpD in high concentrations, thus optimizing the sensitivity of the tumor cells. The sheath catheter and fiberoptics enable excellent distribution of light. Experiments with T-cell lymphomas demonstrated significant response of the tumors to the combination of intratumoral HpD and interstitial light application.     

Microfluidic system with light intensity filters facilitating the application of photodynamic therapy for high-throughput drug screening

Background: Photodynamic therapy utilizes light energy with a photosensitizer (a light-sensitive drug) to kill cancer cells through creation of singlet oxygen via light activation. When a photosensitizer is injected into the bloodstream and exposed to a specific wavelength of light, it generates oxygen to destroy or damage nearby cancer cells, while minimizing side effects on normal cells. Although photodynamic therapy is effective for treating cancer, various parameters, such as the optimum light intensity and photosensitizer dose, are currently poorly understood due to the complexity of conventional experimental schemes.Methods: To effectively perform a simultaneous single parallel test for several different light irradiation conditions on each cell, a microfluidic device was developed to generate eight different intensities from a single light-emitting diode source, through eight different color dye concentrations functioning as light intensity filters. To show that this novel high-throughput microfluidic system can analyze the effects of various light intensities during photodynamic therapy, the optimum light intensities and photosensitizer doses were determined for two different cancer cell lines.Results: Optimum light intensities and photosensitizer were determined for all cell lines. The photodynamic therapy effects in response to different irradiated light intensities were characterized by analyzing cell viability after photosensitizer treatmentConclusions: The developed platform is capable of being used as a photodynamic therapy screening tool. The proposed platform provides a simple and robust way to optimize the combined parameters of light intensity and dosage for diverse types of cancer cells.     

Photodynamic Therapy with PLGA-Lipid Hybrid Nanoparticles to Overcome the Diverse Mechanisms of Multidrug Resistance in Lung Cancer Cells

Multidrug resistance (MDR) continues to be a critical hurdle to cancer therapy. Two main drug resistance mechanisms have been attributed to MDR, drug-selected MDR can be induced after receiving chemotherapy, and metastasis-associated MDR can be developed by adaptation of the cell in response to changing microenvironmental conditions during metastasis. The objective of the present study was to evaluate the efficacy of nanoparticle-mediated photodynamic therapy (PDT) to overcome MDR of lung cancer cells. The nanoparticles used consist of biodegradable poly(D,L-lactide-co-glycolide) (PLGA) as polymeric core encapsulating a photosensitizer, 5,10,15,20-Tetrakis(4-hydroxy-phenyl)-21H,23H-porphine (pTHPP), wrapped with a mixture of lecithin and PEGylated phospholipids as lipid shell. The resulting nanoparticles had an average particle size of 70.4 ± 1.4 nm and zeta potential value of -39.2 mV with monodisperse distribution. The PDT effect of the nanoparticles was evaluated using two different MDR models established from the same cell line, the A549 human lung adenocarcinoma. An MDR cell line, A549RT-eto, established by continuous exposure to Etoposide, was used a model of drug-selected MDR. Metastasis-associated MDR cells were obtained by culturing A549 cells as floating cells under non-adherent conditions, which simulate metastatic floating cells in the blood or lymphatic circulations, without experience of drug exposure. Compared to the A549 parental cells, the A549RT-eto cells showed 17.4- and 1.8-fold increased resistance to Etoposide and Paclitaxel, respectively. In contrast, the A549 floating cells exhibited resistance to Etoposide (11.6-fold) and Paclitaxel (57.8-fold) compared to A549 attached cells. Both MDR cells were equally sensitive to the photocytotoxic effect of the PDT with pTHPP-loaded nanoparticles. The cellular uptake of pTHPP and light-induced superoxide anion generation were observed at similar levels in both MDR and parental cells. The PDT treatment with nanoparticles induced apoptosis in the two cell lines as detected by flow cytometry. This work suggests that PLGA-lipid hybrid nanoparticles are a potentially effective drug delivery system for using PDT to overcome MDR lung cancer, independent of the mechanisms for MDR.     

激光光动力学疗法治疗恶性黑色素瘤6例

目的 探讨激光光动力学疗法治疗恶性黑色素瘤的临床疗效.方法 恶性黑色素瘤患者6例,静脉滴注血卟啉衍生物(HpD)5 me/kg后6～72 h内以金蒸气激光或半导体激光照光,功率密度120～150 mW/cm2,能量密度120～200 J/cm2.治疗前后观察病灶情况,1个月后评价疗效.结果 6例患者中,完全效应4例,部分效应2例,总有效率达100%.1例患者随访6年未见复发.所有病例均未出现严重毒副反应.结论 光动力学疗法是治疗恶性黑色素瘤的有效方法,值得进一步深入研究.