【摘要】：目的：光動力療法(Photodynamic Therapy, PDT)是借助某種特定的藥物(稱為光敏劑)進(jìn)入患者體內(nèi)后,富集于生長異常的組織(如腫瘤),光敏劑經(jīng)一定波長光的輻照后發(fā)生光動力敏化反應(yīng)而產(chǎn)生活性氧(reactive oxygen species, ROS),導(dǎo)致生物大分子光氧化失活,并由此造成細(xì)胞損傷以達(dá)到破壞目標(biāo)病變組織的目的。我們通過本實驗評估PDT對喉癌HEp-2細(xì)胞遷移與侵襲的影響,探討PDT誘導(dǎo)的ROS在抑制喉癌HEp-2細(xì)胞遷移與侵襲中的作用機制。 方法：不同濃度的光敏劑9-羥基脫鎂葉綠酸甲酯a(9-hydroxypheophorbide a,9-HPbD)與HEp-2細(xì)胞共培養(yǎng)6小時,隨之以激光照射15分鐘(波長664nm,能量密度2.0J/cm2),培養(yǎng)24小時后,MTT法測定Hep-2細(xì)胞的活性,確定亞致死劑量。利用還原型谷胱甘肽(GSH)抑制ROS的產(chǎn)生,將HEp-2細(xì)胞分為4組：正常對照組(無光動力療法干預(yù),無GSH干預(yù))、光動力組(有光動力療法干預(yù),無GSH干預(yù))、GSH組(無光動力療法干預(yù),有GSH干預(yù))、光動力加GSH組(有光動力療法干預(yù),有GSH干預(yù))。H2DCFDA探針檢測細(xì)胞內(nèi)活性氧水平；劃痕試驗、transwell小室侵襲試驗檢測亞致死劑量光動力療法處理24h后HEp-2細(xì)胞的遷移與侵襲能力；Western blotting對比分析MEKl/2和ERK1/2的磷酸化水平及MMP-2和MMP-9的表達(dá)情況。 結(jié)果：9-HPbD介導(dǎo)的PDT對人喉癌HEp-2細(xì)胞具有明顯殺傷效應(yīng),且與藥物濃度有關(guān)。PDT誘導(dǎo)喉癌HEp-2細(xì)胞產(chǎn)生ROS,而GSH抑制PDT介導(dǎo)的ROS的產(chǎn)生。細(xì)胞劃痕試驗、transwell小室遷移和侵襲試驗顯示PDT抑制喉癌HEp-2細(xì)胞的遷移與侵襲能力,而GSH使PDT抑制喉癌HEp-2細(xì)胞的遷移與侵襲的能力下降。Western顯示光動力抑制喉癌HEp-2細(xì)胞中MEK1/2和ERK1/2的磷酸化,下調(diào)喉癌HEp-2細(xì)胞MMP-2和MMP-9的表達(dá),而GSH可以逆轉(zhuǎn)這一結(jié)果。 結(jié)論：光動力產(chǎn)生的ROS抑制MEK1/2和ERKl/2的磷酸化,下調(diào)MMP-2和MMP-9的表達(dá),進(jìn)而抑制喉癌HEp-2細(xì)胞的遷移與侵襲。
[Abstract]:Objective: photodynamic therapy (Photodynamic Therapy, PDT) is the use of a specific drug (known as Guang Min) into the body of patients, enriched in abnormal growth tissue (such as tumor), The reactive oxygen species (reactive oxygen species, ROS),) produced by the photodynamic sensitization reaction of Guang Min after irradiation with a certain wavelength of light caused photooxidation inactivation of biomolecules and caused cell damage in order to destroy the target pathological tissues. We evaluated the effect of PDT on the migration and invasion of HEp-2 cells in laryngeal carcinoma and explored the mechanism of PDT induced ROS in inhibiting the migration and invasion of HEp-2 cells. Methods: HEp-2 cells were co-cultured with different concentrations of Guang Min (9-hydroxypheophorbide a 9-HPbD) for 6 hours and then irradiated with laser for 15 minutes (wavelength 664 nm, energy density 2.0J/cm2). After 24 hours of culture, the activity of Hep-2 cells was determined by MTT assay, and the sublethal dose was determined. Using reduced glutathione (GSH) to inhibit the production of ROS, HEp-2 cells were divided into four groups: normal control group (no photodynamic therapy intervention, no GSH intervention), photodynamic group (photodynamic therapy intervention, no GSH intervention). GSH group (no photodynamic therapy intervention, GSH intervention), photodynamic therapy plus GSH group (photodynamic therapy intervention, GSH intervention). H2DCFDA probe detection of intracellular reactive oxygen species; The migration and invasiveness of HEp-2 cells were detected by transwell chamber invasion test and sublethal photodynamic therapy for 24 hours. The phosphorylation levels of MEKl/2 and ERK1/2 and the expression of MMP-2 and MMP-9 were compared between MEKl/2 and ERK1/2 by; Western blotting. Results: 9-HPbD mediated PDT had a significant killing effect on human laryngeal cancer HEp-2 cells and was related to drug concentration. PDT induced ROS, production of HEp-2 cells and GSH inhibited PDT mediated ROS production. Cell scratch test, transwell chamber migration and invasion test showed that PDT inhibited the migration and invasion of laryngeal carcinoma HEp-2 cells. GSH decreased the ability of PDT to inhibit the migration and invasion of laryngeal carcinoma HEp-2 cells. Western showed photodynamic inhibition of MEK1/2 and ERK1/2 phosphorylation, and down-regulated the expression of MMP-2 and MMP-9 in HEp-2 cells. GSH can reverse this result. Conclusion: photodynamic ROS inhibits the phosphorylation of MEK1/2 and ERKl/2, down-regulates the expression of MMP-2 and MMP-9, and inhibits the migration and invasion of HEp-2 cells.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R739.65

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