TMPyP4光动力治疗联合AS1411协同促进宫颈癌Caski细胞凋亡机制的研究

发布时间：2018-05-13 21:17

  本文选题：TMPyP4 + 光动力治疗　； 参考：《山东大学》2014年博士论文

【摘要】：在发展中国家,宫颈癌(Cervical Cancer)是女性癌症导致的死亡中最常见的恶性肿瘤,全世界范围内大约每年有500,000新发病例,其中一半以上的新发病例及死亡病例是发生在亚洲-太平洋地区,而在发达国家仅仅占到15%左右。我国是宫颈癌的高发地区,其发病率位居世界第二位。目前宫颈癌的治疗方式主要有手术治疗、放射治疗以及化学治疗。随着宫颈癌筛查项目的广泛开展以及职业女性生育年龄的推迟,有生育要求的年轻浸润性宫颈癌患者的比例逐年升高,因此保留宫颈癌患者生育功能的治疗变得越来越重要。而对于那些合并多种内科疾病、不能耐受放化疗及手术的老年宫颈癌患者,也迫切的需要一种创伤小、效果好的治疗方式。 光动力疗法(photodynamic therapy, PDT)是近年来备受瞩目的一种新兴的治疗技术,它交叉融合了临床医学、光学及光电子学等学科发展起来。其作用原理是基于光敏剂(photosensitizer, PS)能优先积聚在增长旺盛的细胞和组织中,通过相应波长的激光照射,潴留在靶组织中的基态光敏剂吸收光子能量能够从基态跃迁到激发单线态,然后通过体系间窜跃跃迁到激发三线态,处在激发三线态的光敏剂能够激发两类互相竞争的光氧化反应,Type Ⅰ是光子直接从光敏剂转移至细胞内的细胞器底物,产生自由基,这些自由基能够与分子氧反应产生多种活性氧物质(radical oxygen species, ROS),如过氧化氢,超氧化物和羟自由基。Type Ⅱ是三线态光敏剂直接与氧分子反应产生另一些ROS,单线态氧(1O2)。这两种类型的光氧化反应能够导致细胞内多种分子的氧化反应,触发细胞的凋亡、坏死或者自噬等多种途径导致细胞死亡。其中,单线态氧是光动力作用诱导肿瘤的主要损伤形式。与常规的手术、放化疗等疗法相比,光动力疗法具有创伤小,毒性小、能够重复使用,可保护器官的完整性、保留妇女的生育功能等优点。目前已开始应用于多种良恶性肿瘤的治疗。 四甲基吡啶卟啉(5,10,15,20-tetra-(N-methyl-4-pyridyl) porphyrin,TMPyP4)是一种水溶性光敏剂,为四价阳离子的卟啉类衍生物。TMPyP4是一种高效的光敏剂,能选择性的进入到癌细胞的细胞核中,而对正常的上皮细胞几乎没有毒性作用,在PDT过程中并能产生大量的1O2对癌细胞的DNA产生切割作用而杀伤肿瘤细胞。PDT的光损伤效果有赖于光敏剂的种类以及相应波长的激光的穿透能力。TMPyP4的最长激发波长在420nm左右,因吸收系数小常导致光动力效应深度不能满足较大肿瘤的治疗需要,影响了光动力治疗的疗效。适配体(aptamer)是一种简单的、人工合成的、小分子的DNA或RNA寡聚核苷酸。它作为一种非免疫源性的抗体替代物,可以折叠成三维空间结构,高亲合性、高特异性地与靶蛋白结合。AS1411适配体(AS1411aptamer)是基于26个碱基的富含鸟嘌呤的寡聚核苷酸。它可以形成稳定的G-四联体二聚物,高亲和力、高特异性地与细胞表面的核仁素结合,通过核仁素的穿梭作用进入细胞核内干扰DNA的正常复制和抑制细胞增殖。AS1411已经证实几乎在所有的肿瘤细胞中均能发挥明显增殖抑制的作用,但是在相同的浓度时对正常的上皮细胞作用微弱。而且与其他的化疗药物不同的是,AS1411的作用缓慢,当加入到细胞中的时候,并不是立即发挥细胞毒作用,而是先引起细胞分裂的停滞及细胞周期的阻滞,而明显的细胞凋亡要在给药后7天左右才能出现。目前,AS1411作为一种新型的抗癌药物已经在路易斯维尔大学的James Graham Brown癌症研究中心完成了一期临床试验,2007年Antisoma公司宣布开始AS1411用于治疗急性骨髓细胞白血病和肾细胞癌治疗的二期临床试验。试验结果显示AS1411的安全性及对各种实体癌特别是肾细胞癌具有很好的抗癌效果,而后期的临床试验也显示：AS1411对肿瘤细胞的抑制作用有时间尺度效应(单一剂量的AS1411给予后数月才能出现肿瘤体积的缩小)和非常罕见的临床获益持续时间(许多患者能够获得长期的病情稳定期)。 本研究旨在探讨TMPyP4光动力治疗(TMPyP4/PDT)联合适配体AS1411对宫颈癌Caski细胞增殖抑制作用,并进一步探讨TMPyP4/PDT联合AS1411协同促进宫颈癌Caski细胞凋亡过程中细胞内与凋亡相关的蛋白表达的变化,阐明TMPyP4/PDT联合AS1411促进宫颈癌细胞凋亡的分子机制。以期两者的联合治疗在临床宫颈癌的光动力治疗中能够延长宫颈癌的光动力治疗时效、减少光敏剂的用量。本课题进行了两部分的研究,第一部分：TMPyP4/PDT及AS1411对宫颈癌Caski细胞株增殖抑制作用。第二部分：TMPyP4光动力治疗联合AS1411协同促进宫颈癌Caski细胞凋亡及其分子机制研究。 第一部分TMPyP4/PDT及AS1411对宫颈癌细胞株Caski的增殖抑制作用 研究目的 研究TMPyP4/PDT及AS1411对宫颈癌Caski细胞的增殖抑制作用。 研究方法 1.免疫荧光染色及流式细胞仪检测宫颈癌Caski细胞的核仁素的表达。 2.应用荧光显微镜检测(?)TMPyP4进入到宫颈癌Caski细胞中的荧光定位实验。 3.MTT法检钡(?)TMPyP4/PDT、AS1411及AS1411+TMPyP4/PDT对宫颈癌Caski细胞的增殖抑制作用。 结果： 1.宫颈癌Caski细胞膜表面、细胞浆及细胞核内均有核仁素的表达。 2.TMPyP4能够进入到宫颈癌Caski细胞的细胞质及细胞核中。 3.在3J/cm2激光能量密度的照射下,TMPyP4/PDT对宫颈癌Caski细胞的增殖抑制率随着TMPyP4浓度的增加而显著增高。 4.MTT结果显示TMPyP4/PDT (TMPyP4浓度为9μM,激光能量密度为3J/cm2)与5μM AS1411分别及联合作用对宫颈癌Caski细胞均有增殖抑制作用。单纯TMPyP4/PDT组能明显抑制宫颈癌Caski细胞的增生,其增殖抑制作用在PDT后24h最高,随着PDT后时间的延长,其对宫颈癌Caski细胞的增殖抑制率有所下降。单纯AS1411治疗组对宫颈癌Caski细胞在治疗后24h无明显增殖抑制作用,而在36h及48h显示对宫颈癌Caski细胞逐渐增强的增殖抑制作用。AS1411+TMPyP4/PDT联合治疗组对宫颈癌Caski细胞的增殖抑制作用具有时间依赖性,随着作用时间的延长,联合组对细胞生长的抑制作用越明显,联合治疗后48h达到高峰。 结论 1.TMPyP4/PDT中,TMPyP4能进入到宫颈癌Caski细胞的细胞浆及细胞核中,并呈剂量依赖的方式抑制宫颈癌细胞的增殖。 2.宫颈癌Caski细胞膜表面有核仁素的表达,AS1411通过细胞膜表面的核仁素抑制宫颈癌细胞的增殖,但是其抑制作用要在用药后36-48h方能起效。 3. TMPyP4/PDT中,随PDT后时间的延长,部分细胞活性恢复,细胞的增殖抑制率降低,TMPyP4/PDT联合AS1411治疗中,AS1411作用后24h就有明显增强的抑制细胞增殖的作用,且能够持续至用药后48h。 第二部分TMPy/PDT联合AS1411协同促进宫颈癌Caski细胞凋亡及分子机制的研究 研究目的 探讨TMPyP4/PDT联合AS1411协同促进宫颈癌Caski细胞凋亡及其分子机制。 研究方法 1.应用膜联蛋白V-硫氰酸荧光素(AnnexinV-FITC)/碘化丙啶(PI)双染色结合流式细胞术检测单纯AS1411组、单纯TMPyP4/PDT组、TMPyP4/PDT+AS1411组后24h、36h及48h细胞的凋亡率。 2.应用荧光显微镜观察AO/EB染色不同处理后的宫颈癌Caski细胞的形态学变化。 3.应用Westen-blot检测单纯AS1411组、单纯TMPyP4/PDT组、AS1411+TMPyP4/PDT组后24h、36h及48h细胞内Nf-kb、C23、Bax和Bcl-2蛋白的表达的变化。 结果： 1.TMPyP4/PDT能够诱导宫颈癌Caski细胞凋亡,其凋亡率在PDT作用后24h、36h及48h均显著高于正常对照组(p0.05)。AS1411作用后24h、36h对宫颈癌Caski细胞凋亡没有明显影响(p0.05)；48h单纯AS1411可以轻度诱导宫颈癌Caski细胞凋亡(p0.05)。 AS1411+TMPyP4/PDT联合治疗能明显诱导宫颈癌Caski细胞凋亡,随着作用时间的延长24h、36h、48h,均能显示出增强的诱导细胞凋亡的能力,与正常对照组相比,具有统计学差异p0.05),宫颈癌Caski细胞凋亡在联合作用24h时,其凋亡率与单纯TMPyP4/PDT之间无显著性差异,随着联合作用时间延长至36h、48h,联合治疗组所致的细胞凋亡明显高于单纯TMPyP4/PDT,两组间凋亡率比较有显著性差异p0.05),联合治疗组48h细胞凋亡率显著高于单纯TMPyP4/PDT24h的细胞凋亡率(p0.05)。 2. AO/EB染色结果显示,宫颈癌Caski细胞TMPyP4/PDT治疗后24h、36h、48h均有显著的晚期凋亡细胞出现。AS1411作用后48h有少量凋亡细胞的出现,表现为早期凋亡。联合治疗组24h、36h、48h均有显著的晚期凋亡细胞的出现。 3.单纯AS1411在24h对宫颈癌Caski细胞内促凋亡蛋白Bax、抗凋亡蛋白Bcl-2及细胞内NF-κB和C23蛋白影响不大,随着作用时间的延长,其明显下调C23蛋白、NF-κB蛋白及抗凋亡蛋白Bcl-2的表达,在48h达到高峰。TMPyP4/PDT在24h可明显下调宫颈癌Caski细胞内抗凋亡蛋白Bcl-2、NF-κB及C23蛋白表达,但是随着作用时间的延长至48h,Bcl-2蛋白的表达逐渐恢复。其对促凋亡蛋白Bax的表达影响不大。AS1411+TMPyP4/PDT联合治疗组中,联合治疗明显下调宫颈癌Caski细胞内抗凋亡蛋白Bcl-2、NF-κB及C23蛋白表达,其下调作用可延长至治疗48h,并显示出轻度增强促凋亡蛋白Bax的表达的作用。 结论 TMPyP4/PDT联合AS1411有协同促进宫颈癌Caski细胞凋亡的作用,能够增强光动力治疗效果,减少光敏剂的用量,缩短AS1411发挥抗肿瘤作用的时间。其作用机制可能与调节宫颈癌Caski细胞内抗凋亡蛋白Bcl-2、NF-κB、C23蛋白及Bax蛋白的表达有关。
[Abstract]:In developing countries, cervical cancer (Cervical Cancer) is the most common malignant tumor caused by female cancer. Around 500000 new cases are found worldwide. More than half of the new cases and deaths occur in the Asia Pacific region, and in the developed countries, only about 15%. China is a cervical cancer. The incidence of high incidence is the second largest in the world. The main treatment methods for cervical cancer are surgical treatment, radiation therapy and chemical treatment. With the extensive development of cervical cancer screening and the delay in the reproductive age of professional women, the proportion of young invasive cervical cancer patients with reproductive requirements is increasing year by year, so it is preserved. The treatment of the reproductive function of the patients with cervical cancer is becoming more and more important. For the elderly patients who are not tolerant to chemotherapy and surgery, there is an urgent need for a small, effective treatment.
Photodynamic therapy (PDT) is a newly emerging treatment technology in recent years. It has been intersecting the development of clinical medicine, optics and optoelectronics. Its principle is that the photosensitizer (photosensitizer, PS) can accumulate in the growing cells and tissues, through the corresponding wavelengths. Laser irradiation, the ground state photosensitizer retention in the target tissue absorbs photon energy from the ground state to the excited single state, and then leaps through the system to excite the three line state, and the photosensitizer in the three line state can stimulate the two kinds of competitive photooxidation reactions. Type I is the transfer of photons directly from the photosensitizer to the cell. The organelle substrates, producing free radicals, these free radicals can react with molecular oxygen to produce a variety of reactive oxygen species (radical oxygen species, ROS), such as hydrogen peroxide, superoxide and hydroxyl radical.Type II, which are three linear photosensitizers to react with oxygen molecules directly to produce another ROS, single state oxygen (1O2). These two types of photooxidation reaction It can lead to the oxidation of various molecules in cells, trigger cell apoptosis, necrosis or autophagy, and lead to cell death. Among them, single state oxygen is the main damage form of photodynamic induced tumor. Compared with conventional surgery, radiotherapy and chemotherapy, photodynamic therapy has small trauma, small toxicity and can be reused. It can protect the integrity of organs and preserve the fertility of women. Now it has been applied to many kinds of benign and malignant tumors.
Four methylpyridine porphyrin (5,10,15,20-tetra- (N-methyl-4-pyridyl) porphyrin, TMPyP4) is a water-soluble photosensitizer. The porphyrin derivative,.TMPyP4, a tetravalent cation, is a highly efficient photosensitizer. It can selectively enter the cell nucleus of cancer cells and has little toxic effect on the normal epithelial cells. In the PDT process, it can be used as a photosensitizer. The effect of a large number of 1O2 on the DNA production of cancer cells and the killing effect of.PDT on tumor cells depends on the type of photosensitizer and the penetration ability of the laser at the corresponding wavelength, the longest excitation wavelength of.TMPyP4 is around 420nm. Because of the small absorption coefficient, the photodynamic response depth can not meet the needs of the treatment of larger tumors. It affects the efficacy of photodynamic therapy. The aptamer (aptamer) is a simple, synthetic, small molecule DNA or RNA oligodeoxynucleotide. As a non immunogenic antibody substitute, it can be folded into three dimensional spatial structure, high affinity, highly specific and target protein binding.AS1411 aptamers (AS1411aptamer) are based on 26 A nucleotide rich in guanine - rich oligodeoxynucleotides. It can form a stable G- four conjoined two polymer, highly affinity, highly specific to the nucleolus on the cell surface, and through the shuttle action of nucleolus into the nucleus to interfere with normal replication of DNA and inhibit cell proliferation,.AS1411 has been confirmed almost in all tumor cells. Unlike other chemotherapeutic drugs, the effect of AS1411 is slow. When added to the cell, it does not play the cytotoxic effect immediately, but causes the stagnation of cell division and cell cycle arrest. AS1411, as a new type of anticancer drug, has completed a clinical trial at the James Graham Brown cancer research center in Luis Weil University. In 2007, Antisoma announced that AS1411 was used to treat acute myelocytic leukemia and renal cell cancer. The two phase of the clinical trial. The results showed that AS1411 was safe and had good anticancer effects for various solid cancers, especially renal cell carcinoma, and later clinical trials also showed that AS1411 had a time scale effect on tumor cells (a single dose of AS1411 was given months to reduce tumor volume) and Very rare clinical benefit duration (many patients are able to achieve long-term stability).
The purpose of this study is to explore the inhibitory effect of TMPyP4 photodynamic therapy (TMPyP4/PDT) combined with aptamer AS1411 on the proliferation of cervical cancer Caski cells, and to further explore the synergistic effect of TMPyP4/PDT combined with AS1411 to promote apoptosis related protein expression during the apoptosis of cervical cancer Caski cells, and to clarify that TMPyP4/PDT combined AS1411 promotes cervical cancer. In order to prolong the photodynamic therapy of cervical cancer and reduce the dosage of photosensitizer in the photodynamic therapy of cervical cancer, two parts have been done in this study, the first part: the inhibitory effect of TMPyP4/PDT and AS1411 on the proliferation of Caski cell lines of cervical cancer. The second part: TMPyP4 Photodynamic therapy combined with AS1411 promotes apoptosis of cervical cancer Caski cells and its molecular mechanism.
Part 1 the inhibitory effect of TMPyP4/PDT and AS1411 on the proliferation of cervical cancer cell line Caski
research objective
Objective to study the inhibitory effect of TMPyP4/PDT and AS1411 on the proliferation of cervical cancer Caski cells.
research method
1. the expression of nucleolus in cervical cancer Caski cells was detected by immunofluorescence staining and flow cytometry.
2. fluorescence microscopy (TMPyP4) was used to detect fluorescent localization in cervical cancer Caski cells.
The inhibitory effect of barium (TMPyP4/PDT), AS1411 and AS1411+TMPyP4/PDT on proliferation of cervical cancer Caski cells was detected by 3.MTT.
Result锛，

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