本文選題：TMPyP4 + 光動力治療�。� 參考：《山東大學(xué)》2014年博士論文

【摘要】：在發(fā)展中國家,宮頸癌(Cervical Cancer)是女性癌癥導(dǎo)致的死亡中最常見的惡性腫瘤,全世界范圍內(nèi)大約每年有500,000新發(fā)病例,其中一半以上的新發(fā)病例及死亡病例是發(fā)生在亞洲-太平洋地區(qū),而在發(fā)達國家僅僅占到15%左右。我國是宮頸癌的高發(fā)地區(qū),其發(fā)病率位居世界第二位。目前宮頸癌的治療方式主要有手術(shù)治療、放射治療以及化學(xué)治療。隨著宮頸癌篩查項目的廣泛開展以及職業(yè)女性生育年齡的推遲,有生育要求的年輕浸潤性宮頸癌患者的比例逐年升高,因此保留宮頸癌患者生育功能的治療變得越來越重要。而對于那些合并多種內(nèi)科疾病、不能耐受放化療及手術(shù)的老年宮頸癌患者,也迫切的需要一種創(chuàng)傷小、效果好的治療方式。 光動力療法(photodynamic therapy, PDT)是近年來備受矚目的一種新興的治療技術(shù),它交叉融合了臨床醫(yī)學(xué)、光學(xué)及光電子學(xué)等學(xué)科發(fā)展起來。其作用原理是基于光敏劑(photosensitizer, PS)能優(yōu)先積聚在增長旺盛的細胞和組織中,通過相應(yīng)波長的激光照射,潴留在靶組織中的基態(tài)光敏劑吸收光子能量能夠從基態(tài)躍遷到激發(fā)單線態(tài),然后通過體系間竄躍躍遷到激發(fā)三線態(tài),處在激發(fā)三線態(tài)的光敏劑能夠激發(fā)兩類互相競爭的光氧化反應(yīng),Type Ⅰ是光子直接從光敏劑轉(zhuǎn)移至細胞內(nèi)的細胞器底物,產(chǎn)生自由基,這些自由基能夠與分子氧反應(yīng)產(chǎn)生多種活性氧物質(zhì)(radical oxygen species, ROS),如過氧化氫,超氧化物和羥自由基。Type Ⅱ是三線態(tài)光敏劑直接與氧分子反應(yīng)產(chǎn)生另一些ROS,單線態(tài)氧(1O2)。這兩種類型的光氧化反應(yīng)能夠?qū)е录毎麅?nèi)多種分子的氧化反應(yīng),觸發(fā)細胞的凋亡、壞死或者自噬等多種途徑導(dǎo)致細胞死亡。其中,單線態(tài)氧是光動力作用誘導(dǎo)腫瘤的主要損傷形式。與常規(guī)的手術(shù)、放化療等療法相比,光動力療法具有創(chuàng)傷小,毒性小、能夠重復(fù)使用,可保護器官的完整性、保留婦女的生育功能等優(yōu)點。目前已開始應(yīng)用于多種良惡性腫瘤的治療。 四甲基吡啶卟啉(5,10,15,20-tetra-(N-methyl-4-pyridyl) porphyrin,TMPyP4)是一種水溶性光敏劑,為四價陽離子的卟啉類衍生物。TMPyP4是一種高效的光敏劑,能選擇性的進入到癌細胞的細胞核中,而對正常的上皮細胞幾乎沒有毒性作用,在PDT過程中并能產(chǎn)生大量的1O2對癌細胞的DNA產(chǎn)生切割作用而殺傷腫瘤細胞。PDT的光損傷效果有賴于光敏劑的種類以及相應(yīng)波長的激光的穿透能力。TMPyP4的最長激發(fā)波長在420nm左右,因吸收系數(shù)小常導(dǎo)致光動力效應(yīng)深度不能滿足較大腫瘤的治療需要,影響了光動力治療的療效。適配體(aptamer)是一種簡單的、人工合成的、小分子的DNA或RNA寡聚核苷酸。它作為一種非免疫源性的抗體替代物,可以折疊成三維空間結(jié)構(gòu),高親合性、高特異性地與靶蛋白結(jié)合。AS1411適配體(AS1411aptamer)是基于26個堿基的富含鳥嘌呤的寡聚核苷酸。它可以形成穩(wěn)定的G-四聯(lián)體二聚物,高親和力、高特異性地與細胞表面的核仁素結(jié)合,通過核仁素的穿梭作用進入細胞核內(nèi)干擾DNA的正常復(fù)制和抑制細胞增殖。AS1411已經(jīng)證實幾乎在所有的腫瘤細胞中均能發(fā)揮明顯增殖抑制的作用,但是在相同的濃度時對正常的上皮細胞作用微弱。而且與其他的化療藥物不同的是,AS1411的作用緩慢,當(dāng)加入到細胞中的時候,并不是立即發(fā)揮細胞毒作用,而是先引起細胞分裂的停滯及細胞周期的阻滯,而明顯的細胞凋亡要在給藥后7天左右才能出現(xiàn)。目前,AS1411作為一種新型的抗癌藥物已經(jīng)在路易斯維爾大學(xué)的James Graham Brown癌癥研究中心完成了一期臨床試驗,2007年Antisoma公司宣布開始AS1411用于治療急性骨髓細胞白血病和腎細胞癌治療的二期臨床試驗。試驗結(jié)果顯示AS1411的安全性及對各種實體癌特別是腎細胞癌具有很好的抗癌效果,而后期的臨床試驗也顯示：AS1411對腫瘤細胞的抑制作用有時間尺度效應(yīng)(單一劑量的AS1411給予后數(shù)月才能出現(xiàn)腫瘤體積的縮小)和非常罕見的臨床獲益持續(xù)時間(許多患者能夠獲得長期的病情穩(wěn)定期)。 本研究旨在探討TMPyP4光動力治療(TMPyP4/PDT)聯(lián)合適配體AS1411對宮頸癌Caski細胞增殖抑制作用,并進一步探討TMPyP4/PDT聯(lián)合AS1411協(xié)同促進宮頸癌Caski細胞凋亡過程中細胞內(nèi)與凋亡相關(guān)的蛋白表達的變化,闡明TMPyP4/PDT聯(lián)合AS1411促進宮頸癌細胞凋亡的分子機制。以期兩者的聯(lián)合治療在臨床宮頸癌的光動力治療中能夠延長宮頸癌的光動力治療時效、減少光敏劑的用量。本課題進行了兩部分的研究,第一部分：TMPyP4/PDT及AS1411對宮頸癌Caski細胞株增殖抑制作用。第二部分：TMPyP4光動力治療聯(lián)合AS1411協(xié)同促進宮頸癌Caski細胞凋亡及其分子機制研究。 第一部分TMPyP4/PDT及AS1411對宮頸癌細胞株Caski的增殖抑制作用 研究目的 研究TMPyP4/PDT及AS1411對宮頸癌Caski細胞的增殖抑制作用。 研究方法 1.免疫熒光染色及流式細胞儀檢測宮頸癌Caski細胞的核仁素的表達。 2.應(yīng)用熒光顯微鏡檢測(?)TMPyP4進入到宮頸癌Caski細胞中的熒光定位實驗。 3.MTT法檢鋇(?)TMPyP4/PDT、AS1411及AS1411+TMPyP4/PDT對宮頸癌Caski細胞的增殖抑制作用。 結(jié)果： 1.宮頸癌Caski細胞膜表面、細胞漿及細胞核內(nèi)均有核仁素的表達。 2.TMPyP4能夠進入到宮頸癌Caski細胞的細胞質(zhì)及細胞核中。 3.在3J/cm2激光能量密度的照射下,TMPyP4/PDT對宮頸癌Caski細胞的增殖抑制率隨著TMPyP4濃度的增加而顯著增高。 4.MTT結(jié)果顯示TMPyP4/PDT (TMPyP4濃度為9μM,激光能量密度為3J/cm2)與5μM AS1411分別及聯(lián)合作用對宮頸癌Caski細胞均有增殖抑制作用。單純TMPyP4/PDT組能明顯抑制宮頸癌Caski細胞的增生,其增殖抑制作用在PDT后24h最高,隨著PDT后時間的延長,其對宮頸癌Caski細胞的增殖抑制率有所下降。單純AS1411治療組對宮頸癌Caski細胞在治療后24h無明顯增殖抑制作用,而在36h及48h顯示對宮頸癌Caski細胞逐漸增強的增殖抑制作用。AS1411+TMPyP4/PDT聯(lián)合治療組對宮頸癌Caski細胞的增殖抑制作用具有時間依賴性,隨著作用時間的延長,聯(lián)合組對細胞生長的抑制作用越明顯,聯(lián)合治療后48h達到高峰。 結(jié)論 1.TMPyP4/PDT中,TMPyP4能進入到宮頸癌Caski細胞的細胞漿及細胞核中,并呈劑量依賴的方式抑制宮頸癌細胞的增殖。 2.宮頸癌Caski細胞膜表面有核仁素的表達,AS1411通過細胞膜表面的核仁素抑制宮頸癌細胞的增殖,但是其抑制作用要在用藥后36-48h方能起效。 3. TMPyP4/PDT中,隨PDT后時間的延長,部分細胞活性恢復(fù),細胞的增殖抑制率降低,TMPyP4/PDT聯(lián)合AS1411治療中,AS1411作用后24h就有明顯增強的抑制細胞增殖的作用,且能夠持續(xù)至用藥后48h。 第二部分TMPy/PDT聯(lián)合AS1411協(xié)同促進宮頸癌Caski細胞凋亡及分子機制的研究 研究目的 探討TMPyP4/PDT聯(lián)合AS1411協(xié)同促進宮頸癌Caski細胞凋亡及其分子機制。 研究方法 1.應(yīng)用膜聯(lián)蛋白V-硫氰酸熒光素(AnnexinV-FITC)/碘化丙啶(PI)雙染色結(jié)合流式細胞術(shù)檢測單純AS1411組、單純TMPyP4/PDT組、TMPyP4/PDT+AS1411組后24h、36h及48h細胞的凋亡率。 2.應(yīng)用熒光顯微鏡觀察AO/EB染色不同處理后的宮頸癌Caski細胞的形態(tài)學(xué)變化。 3.應(yīng)用Westen-blot檢測單純AS1411組、單純TMPyP4/PDT組、AS1411+TMPyP4/PDT組后24h、36h及48h細胞內(nèi)Nf-kb、C23、Bax和Bcl-2蛋白的表達的變化。 結(jié)果： 1.TMPyP4/PDT能夠誘導(dǎo)宮頸癌Caski細胞凋亡,其凋亡率在PDT作用后24h、36h及48h均顯著高于正常對照組(p0.05)。AS1411作用后24h、36h對宮頸癌Caski細胞凋亡沒有明顯影響(p0.05)；48h單純AS1411可以輕度誘導(dǎo)宮頸癌Caski細胞凋亡(p0.05)。 AS1411+TMPyP4/PDT聯(lián)合治療能明顯誘導(dǎo)宮頸癌Caski細胞凋亡,隨著作用時間的延長24h、36h、48h,均能顯示出增強的誘導(dǎo)細胞凋亡的能力,與正常對照組相比,具有統(tǒng)計學(xué)差異p0.05),宮頸癌Caski細胞凋亡在聯(lián)合作用24h時,其凋亡率與單純TMPyP4/PDT之間無顯著性差異,隨著聯(lián)合作用時間延長至36h、48h,聯(lián)合治療組所致的細胞凋亡明顯高于單純TMPyP4/PDT,兩組間凋亡率比較有顯著性差異p0.05),聯(lián)合治療組48h細胞凋亡率顯著高于單純TMPyP4/PDT24h的細胞凋亡率(p0.05)。 2. AO/EB染色結(jié)果顯示,宮頸癌Caski細胞TMPyP4/PDT治療后24h、36h、48h均有顯著的晚期凋亡細胞出現(xiàn)。AS1411作用后48h有少量凋亡細胞的出現(xiàn),表現(xiàn)為早期凋亡。聯(lián)合治療組24h、36h、48h均有顯著的晚期凋亡細胞的出現(xiàn)。 3.單純AS1411在24h對宮頸癌Caski細胞內(nèi)促凋亡蛋白Bax、抗凋亡蛋白Bcl-2及細胞內(nèi)NF-κB和C23蛋白影響不大,隨著作用時間的延長,其明顯下調(diào)C23蛋白、NF-κB蛋白及抗凋亡蛋白Bcl-2的表達,在48h達到高峰。TMPyP4/PDT在24h可明顯下調(diào)宮頸癌Caski細胞內(nèi)抗凋亡蛋白Bcl-2、NF-κB及C23蛋白表達,但是隨著作用時間的延長至48h,Bcl-2蛋白的表達逐漸恢復(fù)。其對促凋亡蛋白Bax的表達影響不大。AS1411+TMPyP4/PDT聯(lián)合治療組中,聯(lián)合治療明顯下調(diào)宮頸癌Caski細胞內(nèi)抗凋亡蛋白Bcl-2、NF-κB及C23蛋白表達,其下調(diào)作用可延長至治療48h,并顯示出輕度增強促凋亡蛋白Bax的表達的作用。 結(jié)論 TMPyP4/PDT聯(lián)合AS1411有協(xié)同促進宮頸癌Caski細胞凋亡的作用,能夠增強光動力治療效果,減少光敏劑的用量,縮短AS1411發(fā)揮抗腫瘤作用的時間。其作用機制可能與調(diào)節(jié)宮頸癌Caski細胞內(nèi)抗凋亡蛋白Bcl-2、NF-κB、C23蛋白及Bax蛋白的表達有關(guān)。
[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錛，

本文編號：1884836