【作者】 练兵；

【导师】 王继群；

【作者基本信息】 暨南大学 ， 眼科学， 2009， 博士

【摘要】 目的:1、临床研究的目的在于探讨鼻咽癌眼部症状的临床表现和影像学及病理学诊断及相互对应的关系,意义在于鼻咽癌眼部的症状虽不多见,但也占一定比例,在临床工作中容易被忽视造成误诊和漏诊,在鼻咽癌高发区应对眼部症状进行鼻咽癌的排查,为鼻咽癌的早期诊断提供依据。2、实验研究在于探讨单独和联合转染siRNA(hTERTsiRNA,PCNAsiRNA)技术对人鼻咽癌CNE2细胞相关基因表达以及细胞增殖的抑制作用,同时探讨此技术对CNE2细胞的细胞周期和凋亡的影响,以及对相关蛋白表达的抑制作用,旨在为鼻咽癌的治疗提供新思路和方法。方法:1、临床研究部分:选择2003-2008年6年间562名鼻咽癌患者,对其中82名有眼部症状患者的临床表现,病理诊断和CT表现进行回顾性分析。2、实验研究部分:1)hTERT和PCNAsiRNA的序列设计、合成和筛选。2)分别单独转染hTERTsiRNA、PCNAsiRNA,以及联合转染hTERTsiRNA和PCNAsiRNA:通过MMT法观察细胞增殖情况,流式细胞仪检测细胞周期的变化,实时定量PCR检测细胞相关基因的沉默效应,倒置相差显微镜和免疫组织化学检测细胞生长及相关基因的蛋白表达。结果:1、临床研究显示眼部症状的NPC患者占全部NPC患者的14.6%(82/562)。男性64人(70眼),女性18人(21眼)。患者平均年龄为50.5岁(26-78岁)。累及双眼者9人。来自广东省65例,湖南省6例,江西省4例,广西省4例,海南省2例,湖北省1例。视朦32例(37眼),复视30例(31眼),眼球运动受限25例(26眼),视力下降12例(14眼),眼睑下垂8例,失明6例,眼眶肿胀3例,眼球突出3例,眼痛1例。累及左眼36例,右眼37例,双眼9例。颅底骨质破坏及颅内侵犯40例(包括卵圆孔、棘孔、岩尖、斜坡、蝶窦基底、蝶骨大翼、翼内外板、枕骨大孔、颈静脉孔、海绵窦);侵犯眼眶6例;眶内转移2例(左眼);累及蝶窦6例,上颌窦3例,筛窦3例。咽旁间隙68例,茎突内侧软组织49例。(同一病例可有多种表现,数据有重叠)2、实验研究显示(1)siRNA单独和联合转染都能在不同程度有效抑制hTERT、PCNA及RB1相关基因的表达及细胞增殖:(2)转染48h内未观察到细胞凋亡,但细胞静止于G0/G1期,肿瘤细胞的增殖受到抑制;(3)单独和联合转染后CNE2细胞中hTERT、PCNA及RB1蛋白表达明显受到抑制。(4)联合转染在对CNE2细胞的hTERT、PCNA及RB1基因表达和细胞增殖的抑制作用以及对CNE2细胞的周期影响以及相关蛋白表达的抑制作用优于单独转染,差异有统计学意义。结论:1、鼻咽癌眼部症状具有多样化和复杂性,在临床工作中应引起足够重视。2、siRNA(hTERT、PCNA)单独和联合转染对CNE2细胞的相关基因和蛋白及细胞增殖、细胞周期都有干扰效应,联合转染的siRNA干扰作用优于单独转染。为鼻咽癌的基因治疗提供新的思路和方法。更多还原

【Abstract】 Objective:1.The purpose of clinical study is to explore the eye complication of nasopharyngeral carcinoma(NPC),to analyses the clinical manifestation,CT characteristics and pathological diagnosis of eye complications of NPC,to provide the base for early diagnosis of NPC.2.The purpose of experiment study is to explore the effect of separate and combining hTERTsiRNA,PCNAsiRNA on NPC CNE2 cell;to explore whether targeting siRNA(hTERT, PCNA separate and combining) could inhibit the expressing of related gene and related protein of NPC CNE2 cell,and tumor cells growth,meantime to explore the effect of targeting siRNA (hTERT,PCNA separate and combining ) to cell cycle and apoptosis of NPC CNE2 cell,and to discuss the future application on siRNA in the gene therapy of NPC.Methods:1.The part of clinical study:to retrospective study of 82 cases eye complications in 562 cases NPC,to study their clinical manifestation,CT characteristics and pathological diagnosis.2. The part of experiment study:l) to designing,synthesis and screen effective siRNA ofhTERT and PCNA.2) Effective hTERTsiRNA,PCNAsiRNA separate and combining was transfected into NPC CNE2 cell line by using Lip2000TM siRNA transfection reagent.The relate mRNA and protein were detected by Real-time polymerase chain reaction(Real-time PCR) and immunohistochemistry respectively.MTT assay was used to determine the inhibitory effect of siRNA on tumor cells growth and inverted phase contrast microscope and flow cytometry were used to observe the cell growth,cell cycle,and the apoptosis induced by siRNA.Results:1.The clinical studies showed that eye complication cases were 82cases of 562 NPC cases(14.6%),there were male 64 cases(70eyes) and female 18cases(21 eyes),The mean age at diagnosis was 50.5 years.The bilateral eyes were involved in 9cases.65cases came from Guangdong,6cases from Hunan,4cases from Jiangxi,4cases from Guangxi,2cases from Hainan, 1 cases from Hubei.32cases(37eyes) with blurred vision,30cases(31 eyes) with diplopia, 25cases(26eyes) with ocular movement disorder,12cases(14eyes)with decrease of visual acuity, 8cases with ptosis,6cases with acroisa,3cases with fossa orbitalis swelling,3cases with exophthalmos,1case with ophthalmalgia,36 cases in left and 37 cases in right eyes,9 cases in bilateral eyes.CT appearances:40cases suffered from skull base distracted,6cases with orbit involved,2cases(in left eyes) with orbit metastasis,6cases with sphenoidal sinus involved, 3cases maxillary sinus and 3cases ethmoidal sinus,68case with parapharyngeal space involved, 49cases with soft issue in wall of styloid process involved(there were many kind of shows in th same case,so the data were repeated in these cases).2.The experimental studies showed that 1) Separate and combining siRNA(hTERT,PCNA) transfected could inhibit the expression of hTERT,PCNA and RB1 gene,and cell growth effectually.2) Apoptosis were not observed in transfected CNE2 cells after 48h,but cell cycle stasis in G0/G1 stage.3) Separate and combining siRNA transfected could inhibit the expressio of hTERT,PCNA and RB1 protein obviously.4) Combining siRNA transfected was better than separate siRNA transfected in inhibiting the expression of related gene,protein and cell cycle growth,the difference was significant.Conclusion:1.As there were multiplicity and complexity in eye complication of NPC,so clinician should think highly of these cases.2.Separate and combining siRNA transfected (hTERT,PCNA) could inhibit the expression of related gene and protein of CNE2 cell,and interfere cell growth and cell cycle effectually,combining siRNA transfected was better than separate siRNA transfected in interfering effects to NPC CNE2 cell.This study provides new thoughts and methods for NPC gene therapy.更多还原