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1、 5-氨基酮戊酸论文：化疗药物联合光动力疗法对人宫颈癌Hela细胞杀伤作用的研究【中文摘要】宫颈癌是女性常见的恶性肿瘤。在我国,由于患者自我保健意识的缺失和早期诊断方法普及难度大,很多患者治疗时已经到了晚期,同时近年呈现年龄趋于年轻化的特点。光动力疗法(photodynamic therapy, PDT)是近年来逐渐兴起的一种有效、安全、副作用小、可协同性和重复性高,而且成本相对较小的一种浅表肿瘤治疗方法,为部分宫颈癌及癌前病变患者提供了另外一种可选择的治疗手段。5-氨基酮戊酸(5-ALA)是第二代的光敏剂,克服了第一代光敏剂在体内存留时间长的缺点,适合应用于宫颈癌的临床治疗。但光毒作用限制了
2、光敏剂的用量,使得光动力疗法的效果受到限制。顺铂(cisplatin)是宫颈癌临床上最常用的化疗药物。拓扑替康(topotecan)是一种广谱的抗肿瘤药物,和顺铂联合应用已有实验证实可以增强对癌细胞的细胞毒作用。但是由于肿瘤细胞耐药性和毒副作用,化疗药物对宫颈癌的治疗效果有待进一步提高。血红素氧合酶(HO)是生物体内催化血红素分解的限速酶。近年来发现血红素氧合酶-1(HO-1)与肿瘤的发生发展密切相关,并有研究证明HO-1有抗凋亡的功能。本实验旨在研究联合应用化疗药物和光动力疗法能否提高对宫颈癌Hela细胞的杀伤作用及其作用机理。探讨顺铂、拓扑替康联合光动力治疗对人宫颈癌细胞株Hela的杀伤作
3、用及细胞凋亡的机理。方法：将人宫颈癌细胞株Hela细胞分为空白对照组(control)、单纯光动力治疗组(PDT组)、顺铂加拓扑替康组(DDP-TPT组)、顺铂加光动力治疗组(DDP-PDT组)、拓扑替康加光动力治疗组(TPT-PDT组)以及顺铂和拓扑替康加光动力治疗组(DDP-TPT-PDT组)。5-ALA的实验浓度为lmmol/L,激光的强度为5J/cm2。空白组仅接受5J/cm2的激光照射,DDP组顺铂的剂量为10mg/L, TPT组拓扑替康的剂量0.4mg/L。DDP-TPT组,DDP-TPT-PDT组顺铂的剂量为5mg/L,拓扑替康的剂量为0.2mg/L。各组均避光孵育,力求其他实验
4、条件相同。噻唑蓝MTT法检测六组Hela细胞的增殖抑制情况。流式细胞仪(FCM)检测PDT组、DDP-PDT组、TPT-PDT组和DDP-TPT-PDT组Hela细胞的凋亡情况。免疫细胞化学(ICC)法检测PDT组、DDP-PDT组、TPT-PDT组和DDP-TPT-PDT组Hela细胞内HO-1的表达情况。金氏法检测单纯化疗组(DDP-TPT组)和光动力疗法(PDT组)对Hela细胞的增殖抑制情况是否有协同效应。结果：PDT组、DDP-PDT组、TPT-PDT组以及DDP-TPT-PDT组对Hela细胞均有明显的增殖抑制作用和诱导凋亡作用(P0.01); DDP-PDT组、TPT-PDT组和
5、DDP-TPT-PDT组对Hela细胞的增殖抑制(P0.01)和Hela细胞凋亡的程度(P0.05)明显强于PDT组；PDT组,DDP-PDT组,TPT-PDT组和DDP-TPT-PDT组均可显著抑制Hela细胞中HO-1的表达(P0.01),细胞中HO-1表达的下降趋势也与处理组对Hela细胞的诱导凋亡趋势相一致。PDT组和DDP-TPT组对Hela细胞的抑制作用没有协同效应(q=0.8591.023)。结论：体外光动力疗法、单一化疗药物联合光动力治疗和联合化疗药物与光动力疗法应用对人宫颈癌Hela细胞均有明显的杀伤作用；联合应用强于单纯光动力疗法的杀伤作用；细胞凋亡的机理可能与抑制HO-1
6、的基因表达有关。光动力治疗和联合化疗药物治疗没有协同效应；【英文摘要】Cervical carcinoma is a common malignant tumor in women.Many patients are suffering advanced stage carcinoma because of the deficiency of ego health care consciousnesses and the less widespread of early diagnosis methods.In recent years,the onset age tends to be yo
7、unger.Photodynamic therapy(PDT) with its effectiveness,safety and less side effects can be synergistic,repeatable,and relatively low costs treatment.PDT provides an opportunity for patients who have advanced cancer and refuse treatment with traditional cancer therapy. Cisplatin is considered as the
8、effective drug of cervical cancer treatment and most widely used in clinic.The main reason for failure of chemotherapy is the drug resistance to cancer cells.Topotecan is a broad spectrum anti-neoplastic drug.The combined use with cisplatin is confirmed.How to overcome drug resistance and ease the s
9、ide effect will be the key to increase 5-year survival rate for cervical cancer.Heme oxygenase(HO) is the rate-limiting enzyme of heme catabolism.The subtype,HO-1 has a close relationship with the occurrence and development of tumor.Restrain the expression of HO-1 can obviously induce the apoptosis
10、of tumor cells.This experiment will study if or not chemotherapy drugs combined with photodynamic therapy enhance the effect of photodynamic therapy to tumors.This study also explores its mechanism.:To study the effect and mechanism of cisplatin and/or topotecan combined with the 5-ALA photodynamic
11、therapy and its apoptosis mechanism in cervical carcinoma Hela cells in vitro.Methods:The Hela cell lines are divided into six groups (control group;PDT only group;cisplatin and topotecan group;cisplatin and PDT group;topotecan and PDT group;cisplatin,topotecan and PDT group).The concentration of 5-
12、ALA is 1mmol/L, the laser intensity is 5J/cm2.The dose of cisplatin is 10mg/L,the dose of topotecan is 0.4mg/L. The dose of cisplatin and topotecan in DDP-PDT group and DDP-TPT-PDT group is 5mg/L and 0.2mg/L. Avoid light incubation,under otherwise identical conditions.MTT assay was used to examine t
13、he proliferation effects of the six groups.The apoptosis is examined by the flow cytometry in the five groups(control group;PDT group;DDP-PDT group;TPT-PDT group and DDP-TPT-PDT group).The expressions of HO-1 in PDT group,DDP-PDT group,TPT-PDT group and DDP-TPT-PDT group were detected by immunocytoc
14、hemistry.The kim method is used to calculate the combination index,the value q.Results:The proliferation of Hela cells in vitro is restrained obviously in the four groups(PDT group;DDP-PDT group;TPT-PDT group;DDP-TPT-PDT group).The four groups remarkably induced apoptosis of Hela cells and inhibited
15、 expression of HO-1 in the cell line.The degree of apoptosis caused by the joint application of chemotherapeutics and photodynamic therapy was greater than that caused by photodynamic therapy alone.The expression of HO-1 in cells declined correspondingly. PDT group and DDP-TPT group arent synergism.
16、Conclusion:The effect to hela cells is obvious in PDT and chemotherapeutics combined with PDT.The effect to Hela cells of chemotherapeutics combined with photodynamic therapy is greater than that of photodynamic therapy only.The mechanism of apoptosis may be related with the suppression of HO-1 gene
17、.It is not apparent synergism in photodynamic therapy and chemotherapeutics.【关键词】5-氨基酮戊酸 顺铂 拓扑替康 光动力疗法 宫颈癌细胞株【英文关键词】5-aminolevulinic acid Cisplatin Topotecan Photodynaic therapy Apoptosis Cervical neoplasms【目录】化疗药物联合光动力疗法对人宫颈癌Hela细胞杀伤作用的研究中文摘要6-8ABSTRACT8-10符号说明11-12前言12-15一、材料和方法15-25二、结果25-28三、讨论28-31四、小结31-32结论32-33附图和附表33-35参考文献35-39致谢39-40攻读硕士学位期间发表论文40-41学位论文评阅及答辩情况表41