5-氨基酮戊酸光动力疗法对宫颈癌细胞株凋亡的诱导作用

背景与目的:5-氨基酮戊酸-光动力疗法(5-aminolevulinic acidmediated photodynamic therapy,ALA-PDT)是近年来兴起的一种肿瘤消融新技术,其作用于宫颈癌细胞的机制尚不清楚.本课题探讨ALA-PDT对宫颈癌细胞凋亡的影响及其相关机制.方法:用MTT法检测ALA-PDT对8种人宫颈癌细胞增殖的影响,并筛选出ALA-PDT对宫颈癌细胞增殖影响的最佳作用参数.采用Annexin V-FITC/PI双染法、Hoechst 33342染色法和May-Grunwald-Farbstoff Giemsa染色法检测ALA-PDT对Me180细胞凋亡的影响.采用PI染色结合流式细胞术分析ALA-PDT对Me180细胞周期的影响.采用实时荧光定量RT-PCR法检测ALA-PDT对Me180细胞survivin等基因mRNA的影响,Western blot检测对Survivin蛋白表达的影响.结果:8种人宫颈癌细胞株中,Me180细胞株对ALA-PDT最敏感,与其它细胞株比较差异有统计学意义.ALA 2 mmol/L、10 J/cm2激光剂量孵育3 h为ALA-PDT体外杀伤Me180细胞较理想的实验条件,在该条件下 ALA的IC50为7.28×10-4mmol/L.ALA-PDT可显著诱导Me180细胞凋亡,并可将细胞阻滞于G0/C1期.ALA-PDT可显著抑制Me180细胞survivin基因的mRNA和蛋白表达.结论:ALA-PDT在体外对人宫颈癌Me180细胞有明显的增殖抑制作用,能诱导Me180细胞发生凋亡,其机理可能与抑制survivin基因表达有关.     

氨基酮戊酸光动力疗法联合手术治疗皮肤基底细胞癌的疗效观察

目的:分析手术联合5-氨基酮戊酸光动力疗法（ALA-PDT)治疗皮肤基底细胞癌的临床疗效。方法:皮肤基底细胞癌患者30例,按抛硬币法分为观察组和对照组,每组15例。观察组患者于术前开始进行ALA-PDT治疗,每周1次,共3-7次,然后进行手术治疗,对照组患者只采取手术切除,随访16-25个月。比较两组治疗一期愈合率、有效率,并评价疗效。结果:观察组患者总有效率为100％（15/15）,对照组总有效率为86.7％（13/15),差异无统计学意义（P>0.05）。两组治疗一期愈合率、不良反应率差异具有统计学意义（P<0.05）,观察组优于对照组。结论:手术联合ALA-PDT治疗皮肤基底细胞癌具有有效、安全等优点。     

5-氨基酮戊酸光动力治疗表浅基底细胞癌患者的疗效研究

目的探讨5-氨基酮戊酸(ALA)光动力疗法(PDT)治疗表浅基底细胞癌(BCC)患者的疗效。方法选取BCC患者65例,随机分为试验组(33例)和对照组(32例),两组患者均行ALA-PDT治疗,试验组在此基础上联合咪喹莫特治疗。对患者的治疗效果、美容效果和不良反应等跟踪随访记录。结果 ALA-PDT联合咪喹莫特治疗BCC后完全缓解率和部分缓解率显著高于对照组(P<0.05),而总有效率高于试验组,但差异无显著性(P>0.05)。随访1年后,对照组复发5例(15.63%),试验组2例复发(6.06%),试验组患者复发率显著低于对照组(P<0.05)。试验组美容效果优秀、良好及良好以上比例显著高于对照组(P<0.05),美容效果差比例显著低于对照组(P<0.05)。两组均未出现重度不良反应。结论 ALA-PDT联合咪喹莫特作为治疗BCC一种新治疗技术,兼顾了疗效和美容的效果,且疗效显著,安全有效,值得临床推荐。     

5-氨基酮戊酸乳膏光动力疗法治疗皮肤癌前病变和浅表皮肤肿瘤疗效观察

目的:观察5-氨基酮戊酸光动力疗法（5-ALA-PDT）治疗皮肤癌前病变和浅表皮肤肿瘤的疗效。方法:对7例日光性角化病、5例基底细胞癌、2例鲍温病、1例乳房外paget病患者进行5-氨基酮戊酸光动力疗法（5-ALA-PDT）治疗。结果:随访6个月,7例日光性角化病患者中,6例患者皮损获得完全缓解,1例部分缓解;5例基底细胞癌患者中,2例完全缓解,2例部分缓解,1例无反应。2例鲍温病患者均获得完全缓解;1例乳房外paget病患者部分缓解。结论:使用5-ALA-PDT治疗皮肤肿瘤,疗效好、痛苦小、美容效果满意,特别适合一般情况差,难以耐受手术的特殊患者,可在临床工作中进一步推广。     

5-氨基酮戊酸在光动力和声动力治疗肿瘤中的实验与临床研究进展

5-氨基酮戊酸（5-aminolevulinic acid,5-ALA）作为光敏剂、声敏剂已广泛应用于光动力疗法和声动力疗法中。5-ALA通过细胞内代谢产生原卟啉Ⅸ（Protoporphyrin Ⅸ,PpⅨ）,其具有的光敏性和声敏性可以在激光和超声作用下激活,产生氧自由基或活性氧物质,诱导细胞凋亡、坏死。本文就近年来5-ALA在光、声动力疗法治疗肿瘤中的实验与临床研究进展作一综述。     

5-氨基酮戊酸光动力疗法对CNE细胞迁移和侵袭的影响

 【摘要】　目的　观察5-氨基酮戊酸光动力疗法（5-ALA-PDT）作用于人类鼻咽癌细胞株CNE细胞后细胞生长抑制、迁移和侵袭性的改变。方法　通过MTT法检测5-ALA-PDT CNE细胞后,不同参数（5-ALA浓度及激光器能量密度）下的细胞生长抑制率变化。并观察不同参数下5-ALA-PDT后细胞划痕实验及Transwell侵袭小室试验。结果　5-ALA-PDT能明显抑制鼻咽癌高分化CNE细胞株的生长。5-ALA-PDT后,当药物浓度和能量密度达到一定水平时CNE细胞的迁移被抑制。并且药物浓度在饱和量1 mmol／L时,细胞迁移距离与光照时间呈线性负相关（P＜0.05）。光动力作用后,CNE细胞的侵袭能力也被抑制,与5-ALA浓度及激光的能量密度相关。在浓度相同的情况下,细胞侵袭数与照光时间呈线性负相关（r＞0.8,P＜0.0001）。结论　5-ALA-PDT作用于CNE细胞后,细胞的迁移和侵袭能力被抑制,抑制作用与能量密度和药物浓度相关。     

5-氨基酮戊酸光动力疗法联合重组人干扰素α2b软膏治疗老年患者表浅基底细胞癌的临床观察

目的 探讨5-氨基酮戊酸光动力疗法(5-Aminolevulinic acid mediated photodynamic therapy,ALA-PDT)联合重组人干扰素α2b软膏治疗老年表浅基底细胞癌(Basal cell carcinoma,BCC)患者的疗效。方法 选取143例BCC老年患者,随机分为PDT组(72例)和手术组(71例),PDT组采用ALA-PDT疗法联合干扰素治疗,手术组采取常规外科手术治疗,对两组患者的疗效、手术创伤面积、病变面积、复发情况、安全性等进行随访比较。结果 PDT组治疗后完全缓解率显著高于手术组(81.94% vs.57.75%,P＜0.05),而两组总有效率比较无统计学差异(91.66% vs.81.73%,P＞0.05);随访1年后,PDT组患者复发率显著低于手术组(9.72% vs.22.54%,P＜0.05);此外,PDT组平均创伤面积、治疗后病变面积也显著小于手术组(P＜0.05)。结论 ALA-PDT联合干扰素是一种临床疗效好,复发率低,创伤性小的治疗表浅基底细胞癌的有效方法。     

氨基酮戊酸光动力疗法治疗90例皮肤癌及癌前病变的临床观察

目的探讨氨基酮戊酸光动力疗法（ALA—PDT）治疗皮肤癌及癌前病变的临床价值。方法选取收治的90例皮肤癌患者行ALA—PDT治疗,观察评估其治疗效果和不良反应。结果治疗6个月后,完全反应67例（74．4％）,部分反应17例（18．9％）,无效6例（6．7％）,复发13例（14．4％）。基底细胞癌、鳞状细胞癌、疣状癌、日光性角化、Bowen病、Paget病等皮肤癌及癌前病变的有效率和复发率分别为92．9％和14．3％、92．0％和16．0％、100％和14．3％、100％和12．5％、90．0％和10．0％、91．7％和16．7％,各疾病类型之间的有效率和复发率差异均无统计学意义（P〉0．05）,且均未出现严重不良反应。结论ALA—PDT治疗皮肤癌及其癌前病变的效果满意、不良反应轻。     

光动力对人子宫颈癌Hela细胞的杀伤作用及光动力剂量的研究

目的 观察氨基酮戊酸(5-Aminolevulinic acid,ALA)介导的光动力疗法(photodynamic therapy,PDT)对人子宫颈癌Hela细胞株的杀伤效应,探讨最佳光动力剂量.方法 实验分为空白对照组、单纯激光组、单纯光敏剂组和实验组(照光+光敏剂).对体外培养的人子宫颈癌Hela细胞株进行光动力实验,应用MTT法检测其光密度值(OD570),观察不同光动力剂量下对人子宫颈癌Hela细胞在体外的抑制作用,并绘制其抑制曲线和观察其形态学变化.结果 实验组于治疗后明显抑制体外培养的人子宫颈癌Hela细胞,而空白对照组、单纯使用光敏剂组及单纯激光照射组对细胞增殖均无明显影响.光动力治疗后12 h,镜下细胞变形,细胞内出现大量的核碎裂、核融解、核消失等坏死征象,少部分表现为凋亡.结论 ALA介导的光动力疗法(ALA-PDT)可明显抑制体外培养的人子宫颈癌Hela细胞株的生长,ALA浓度为 0.5 mmol/L、激光剂量为5J/cm2是杀伤Hela 细胞的最佳剂量.     

激光光纤介入的光动力疗法治疗皮肤鳞状细胞癌的实验研究

目的：评价激光光纤介入内照射5-氨基酮戊酸盐酸盐-光动力疗法(OFI-ALA-PDT)和传统外照射5-氨基酮戊酸盐酸盐-光动力疗法(ALA-PDT)治疗皮肤鳞状细胞癌的临床疗效及不良反应。方法：收集我院门诊48例鳞状细胞癌患者,随机分为光纤内照射组和传统外照射组,光纤内照射组在外用5-氨基酮戊酸盐酸盐后运用光纤插入靶皮损内,进行光动力治疗,而传统外照射组外用5-氨基酮戊酸盐酸盐后进行光动力治疗,观察两组患者的临床疗效、复发率以及不良反应。结果：光纤内照射组的临床疗效明显高于传统外照射组,差异有统计学意义（P<0.05）,复发率与传统外照射组相比差异无统计学意义（P>0.05）,不良反应发生率低于传统外照射组,两者差异有统计学意义（P<0.05）。结论：OFI-ALA-PDT治疗皮肤鳞状细胞癌疗效确切,美容效果佳,治疗高分化鳞状细胞癌尤其是年老体弱、美容要求高的患者亦有一定的疗效,且治疗过程患者耐受性好,与ALA-PDT相比有一定优势。     

ALA-PDT诱导白血病细胞株HL-60凋亡

背景与目的:基于5-氨基乙酰丙酸的光动力疗法(ALA-PDT)利用肿瘤细胞对ALA产生的内源性光敏剂原卟啉IX(PpIX)的优先摄取,使肿瘤细胞在受到一定的光照后被选择性地杀伤。到目前为止,ALA-PDT引起肿瘤细胞破坏的确切机制尚未完全阐明。本研究探讨ALA-PDT对白血病细胞株HL-60的凋亡诱导作用。方法:以白血病细胞株HL-60为实验模型。实验分为4组,对照组、单纯ALA组、单纯光照组及ALA PDT组。用MTT法检测细胞的存活率,瑞氏染色观察细胞形态学改变,用Annexin V-FITC/PI双染法检测细胞凋亡率,并用共聚焦激光显微镜(LSCM)观察凋亡细胞的特征。结果:ALA PDT组光照后细胞形态学可见凋亡改变;MTT法显示细胞存活率明显下降,24 h为(46±9)%,48 h为(26±8)%,两者比较差异有显著性(P<0.05);流式细胞仪检测显示光照后4、5和24 h细胞凋亡率分别为(26±9)%、(29±11)%和(51±6)%,与对照组相比差异有显著性(P<0.05);LSCM观察AnnexinV-FITC单阳性及AnnexinV-FITC/PI双阳性细胞均具有典型的凋亡特征,而单纯ALA组、单纯光照组及对照组则无上述改变。结论:ALA-PDT能杀伤白血病细胞株HL-60,主要通过诱导凋亡的方式实现的,并呈一定的时间依赖性。     

Enhanced effects of aminolaevulinic acid-based photodynamic therapy through local hyperthermia in rat tumours

The possibility of enhancing aminolaevulinic acid (ALA)-based photodynamic therapy (PDT) by simultaneous application of localised hyperthermia (HT) was evaluated. Treatments of rat DS-sarcomas included: (i) control, (ii) ALA administration (375 mg kg(-1), i.p.), no illumination, (iii) 'nonthermal' illumination, (iv) ALA-PDT: that is, ALA administration, 'nonthermal' illumination, (v) localised HT, 43 degrees C, 60 min (vi) ALA-PDT+HT: ALA administration with full spectrum irradiation resulting in ALA-PDT and HT. Tumour volume was monitored for 90 days or until a target volume (3.5 ml) was reached. No differences were seen between the first three groups, with all tumours reaching the target volume by 8-11 days. A total of 13 and 15% of tumours did not reach the target volume by day 90 following HT or ALA-PDT treatment, respectively. ALA-PDT+HT showed the greatest antitumour effect (P=0.0001), with 61% of the tumours not reaching the target volume. Viability and in vitro growth were also assessed in cells from tumours excised after treatment. ALA-PDT+HT reduced the fraction of viable tumour cells by 85%, and in vitro culture showed pronounced growth delay compared to control cells. These results demonstrate an enhanced antitumour effect upon ALA+HT, which appears to involve direct cell toxicity rather than solely vascular damage.     

Silencing of ferrochelatase enhances 5-aminolevulinic acid-based fluorescence and photodynamic therapy efficacy

Background:

Recurrence of glioma frequently occurs within the marginal area of the surgical cavity due to invading residual cells. 5-Aminolevulinic acid (5-ALA) fluorescence-guided resection has been used as effective therapeutic modalities to improve discrimination of brain tumour margins and patient prognosis. However, the marginal areas of glioma usually show vague fluorescence, which makes tumour identification difficult, and the applicability of 5-ALA-based photodynamic therapy (PDT) is hampered by insufficient therapeutic efficacy in glioma tissues.

Methods:

To overcome these issues, we assessed the expression of ferrochelatase (FECH) gene, which encodes a key enzyme that catalyses the conversion of protoporphyrin IX (PpIX) to heme, in glioma surgical specimens and manipulated FECH in human glioma cell lines.

Results:

Prominent downregulation of FECH mRNA expression was found in glioblastoma tissues compared with normal brain tissues, suggesting that FECH is responsible for PpIX accumulation in glioblastoma cells. Depletion of FECH by small interference RNA enhanced PpIX fluorescence after exposure to 5-ALA concomitant with increased intracellular PpIX accumulation in glioma cells. Silencing of FECH caused marked growth inhibition and apoptosis induction by PDT in glioma cells.

Conclusion:

These results suggest that knockdown of FECH is a potential approach to enhance PpIX fluorescent quality for optimising the subjective discrimination of vague fluorescence and improving the effect of 5-ALA-PDT.     

Enhanced cytotoxic effects of 5-aminolevulinic acid-mediated photodynamic therapy by concurrent hyperthermia in glioma spheroids

During photodynamic therapy (PDT) both normal and pathological brain tissue, in close proximity to the light source, can experience significant temperature increases. The purpose of this study was to investigate the anti-tumor effects of concurrent 5-aminolevulinic acid (ALA)-mediated PDT and hyperthermia (HT) in human and rat glioma spheroids.Human or rat glioma spheroids were subjected to PDT, HT, or a combination of the two treatments. Therapies were given concurrently to simulate the conditions that will occur during patient PDT.Predictions of diffusion theory suggest that brain tissue immediately adjacent to a spherical light applicator may experience temperature increases approaching 8 °C for laser input powers of 2 W. In the in vitro model employed here, HT had no effect on spheroid survival at temperatures below 49 °C, while sub-threshold fluence PDT results in only modest decrease in survival. HT (40–46 °C) and PDT interact in a synergistic manner if the two treatments are given concurrently. The degree of synergism increases with increasing temperature and light fluence. Apoptosis is the primary mode of cell death following both low-fluence rate PDT and combined HT + PDT     

Improvement of DC vaccine with ALA-PDT induced immunogenic apoptotic cells for skin squamous cell carcinoma

Dendritic cell (DC) based vaccines have emerged as a promising immunotherapy for cancers. However, most DC vaccines so far have achieved only limited success in cancer treatment. Photodynamic therapy (PDT), an established cancer treatment strategy, can cause immunogenic apoptosis to induce an effective antitumor immune response. In this study, we developed a DC-based cancer vaccine using immunogenic apoptotic tumor cells induced by 5-aminolevulinic acid (ALA) mediated PDT. The maturation of DCs induced by PDT-treated apoptotic cells was evaluated using electron microscopy, FACS, and ELISA. The anti-tumor immunity of ALA-PDT-DC vaccine was tested with a mouse model. We observed the maturations of DCs potentiated by ALA-PDT treated tumor cells, including morphology maturation (enlargement of dendrites and increase of lysosomes), phenotypic maturation (upregulation of surface expression of MHC-II, DC80, and CD86), and functional maturation (enhanced capability to secrete IFN-γ and IL-12, and to induce T cell proliferation). Most interestingly, PDT-induced apoptotic tumor cells are more capable of potentiating maturation of DCs than PDT-treated or freeze/thaw treated necrotic tumor cells. ALA-PDT-DC vaccine mediated by apoptotic cells provided protection against tumors in mice, far stronger than that of DC vaccine obtained from freeze/thaw treated tumor cells. Our results indicate that immunogenic apoptotic tumor cells can be more effective in enhancing a DC-based cancer vaccine, which could improve the clinical application of PDT-DC vaccines.     

5-氨基乙酰丙酸介导的光动力对CNE2和CNE2/DDP细胞体外作用的观察

目的:观察5-氨基乙酰丙酸(5-ALA)介导的光动力对CNE2和CNE2/DDP的生物作用。方法:荧光分光光度计检测PpⅨ荧光强度;MTT法测定细胞存活率。结果:两种细胞内PpⅨ荧光强度与孵育时间(CNE2:F=65.56,P=0.000;CNE2/DDP:F=28.07,P=0.000)和5-ALA浓度(CNE2:F=6292.36,P=0.000;CNE2/DDP:F=914.36,P=0.000)成正相关,在相同孵育时间(t=17.28,P=0.01)及5-ALA浓度(t=36.07,P=0.000)条件下,CNE2胞内PpⅨ荧光强度均显著高于CNE2/DDP。CNE2或CNE2/DDP细胞存活率与孵育时间(CNE2:F=2245.98,P=0.000;CNE2/DDP:F=236.92,P=0.000)、5-ALA浓度(CNE2:F=55.26,P=0.000;CNE2/DDP:F=10.68,P=0.000)和激光能量(CNE2:F=3294.21,P=0.000;CNE2/DDP:F=285.65,P=0.000)成负相关,相同孵育时间(t=31.35,P=0.000)、5-ALA浓度(t=37.16,P=0.001)及激光能量(t=12.84,P=0.001)条件下,CNE2细胞存活率显著高于CNE2/DDP。结论:5-ALA-PDT对CNE2和CNE2/DDP细胞均具有杀伤作用,但在相同条件下,5-ALA-PDT对CNE2的杀伤作用大于CNE2/DDP。     

Photosensitization and mechanism of cytotoxicity induced by the use of ALA derivatives in photodynamic therapy.

The use of more lipophilic derivatives of 5-aminolevulinic acid (ALA) is expected to have better diffusing properties, and after conversion into the parent ALA, to reach a higher protoporphyrin IX (PPIX) formation rate, thus improving the efficacy of topical photodynamic therapy (PDT). Here we have analysed the behaviour of 3 ALA derivatives (ALA methyl-ester, hexyl ester and a 2-sided derivative) regarding PPIX formation, efficiency in photosensitizing cells and mechanism of cellular death. The maximum amount of porphyrins synthesized from 0.6 mM ALA was 47 +/- 8 ng/10(5)cells. The same amount was formed by a concentration 60-fold lower of hexyl-ALA and 2-fold higher of methyl-ALA. The 2-sided derivative failed to produce PPIX accumulation. Applying a 0.6 J cm(-2)light dose, cell viability decreased to 50%. With the 1.5 J cm(-2) light dose, less than 20% of the cells survive, and higher light doses produced nearly total cell killing. Comparing the PPIX production and the induced phototoxicity, the more the amount of porphyrins, the greater the cellular killing, and PPIX formed from either ALA or ALA-esters equally sensitize the cells to photoinactivation. ALA-PDT treated cells exhibited features of apoptosis, independently on the pro-photosensitizer employed. ALA-PDT can be improved with the use of ALA derivatives, reducing the amount of ALA necessary to induce efficient photosensitization.     

5-Aminolevulinic Acid -Mediated Photodynamic Therapy of Human Glioma Cells In Vitro

OBJECTIVE To investigate the effect of 5-aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT) on U251 human glioma cells in vitro.treated with ALA, a typelioma cells were routinely cultured and then of photosensitizer, at various concentrationsfollowed by light irradiation. The PDT-induced phototoxicity of the cells was determined by a MTT assay. In addition, cells were treated with ALA at a fixed concentration and subjected to various doses of light irradiation.RESULTS With the same light dosage (25.0 J/cm^2), the cell survival rates were 70.16% 5.02%, 50.19% 4.79%, 34.97% 5.34%, 27.04% 4.34%, and 24.26% 2.76% at ALA concentrations of 0.25, 0.5, 1.0, 2.0, and 4.0 mM,respectively (F =279.88, P =0.0000). But the survival rates of the cells incubated with 2.0 mM ALA compared to those with 4.0 mM ALA (27.04% 4.34% vs 24.26% 2.76%) showed no significant difference (P=0.611). At a single ALA concentration, the cell survival rates were 83.48% 6.79%,68.09% 6.02%, 33.75% 6.70%, 23.34% 5.08% and 15.14% 3.60% for light doses of 6.25, 12.5, 25.0, 50.0, and 100 J/cm2, respectively (F=422.03,P=0.0000). Without exposure to light, however, the cell survival rates were 96.64% 6.56%, 97.71% 5.48%, 98.10% 6.25%, 99.44% 7.02%, and 95.86% 7.80% for ALA concentrations at 0.25, 0.5, 1.0, 2.0, and 4.0 mM,respectively (F =0.68, P=0.6085). Without ALA in the medium, the cells urvival rates were 98.74% 6.20%, 96.49% 7.13%, 97.60% 5.94%,95.70% 4.86%, 98.08% 6.26% for light doses of 6.25, 12.5, 25.0, 50.0, and 100 J/cm2, respectively (F=0.6400, P=0.6368).CONCLUSION The PDT damage to the U251 cells increased with ALAconcentration within a relative lower range, but then plateaued at higherconcentrations. PDT damage was proportional to the doses of irradiatedlight. Without ALA, the light alone caused no photodynamic damage andALA itself was nontoxic. The ALA-induced PDT appears to be a promisingtherapy for glioma.