鼻咽癌Hedgehog信号通路表达及功能研究

发布时间：2019-06-06 03:37

【摘要】：鼻咽癌是一个多因素遗传性的头颈部常见肿瘤，其发生发展具有多阶段性、隐蔽性等特点，是我国常见的恶性肿瘤之一；高发区主要集中在南方五省(广东、广西、湖南、福建、江西)，有广东癌之称。尽管由于外科手术以及化疗手段地进步，，但由于其发病部位的隐蔽以及检测指标的有效性等问题，导致其诊断、治疗仍存在相当大的困难，多数患者发现病症进行诊断后已是癌症后期；因此，急切需要一种针对于鼻咽癌的比较合理、有效的诊断以及预后手段。 Hedgehog（Hh）信号通路发现于果蝇中的一个进化保守的信号通路，最初的功能是调节胚胎发育，随后发现其在人类的多种癌症中异常激活，参与肿瘤的发生、分化、侵袭和转移。然而，对于Hh信号通路是否在鼻咽癌中存在异常表达以及其在鼻咽癌中的功能性影响，目前并不明确。本研究首先运用荧光定量PCR检测了Hh信号通路的PTCH-1、SMO和GLI-1基因在鼻咽癌以及鼻咽炎临床组织中的表达情况。研究显示PTCH-1、SMO和GLI-1基因表达于全部的鼻咽组织中，而荧光定量PCR结果表明PTCH、GLI-1在鼻咽癌组织以及鼻咽炎组织中存在表达差异（P0.05），PTCH表达下调，GLI-1则表达上调，而SMO基因表达无差异。免疫组织化学检测进一步显示，GLI-1蛋白在鼻咽癌中存在明显上调，上述结果表明Hh信号通路在鼻咽癌中存在导常激活。通过特异性的信号通路抑制剂环巴胺（cyclopamine）对Hh鼻咽癌不同分化程度的细胞系CNE1、CNE2和HNE2的细胞株进行通路功能性干扰。CCK-8细胞增殖实验显示，CNE1、CNE2和HNE2在信号通路受抑制情况下，细胞生长和增殖受到一定程度的抑制。流式细胞仪检测发现，cyclopamine抑制通路导致CNE1、CNE2和HNE2细胞凋亡增加。荧光定量PCR检测通路干扰对目的基因的表达影响发现，下游基因PTCH1、Cyclin E表达下调。本研究结果显示，Hh信号通路在鼻咽癌中存在异常激活，通过对信号通路的干扰研究发现，Hh促进鼻咽癌细胞的增殖及凋亡，并对信号通路的靶标基因表达具有抑制作用。本研究推动实现鼻咽癌从Hh信号通路作为诊断和分子靶标的研究提供新的科学依据。
[Abstract]:Nasopharyngeal carcinoma (NPC) is a multifactorial inherited common head and neck tumor, its occurrence and development has the characteristics of multi-stage, concealment and so on, and it is one of the common malignant tumors in China. The high incidence area is mainly concentrated in five southern provinces (Guangdong, Guangxi, Hunan, Fujian, Jiangxi), known as Guangdong cancer. Although due to the progress of surgery and chemotherapy, due to the concealment of the location of the disease and the effectiveness of the detection index, there are still considerable difficulties in the diagnosis and treatment of the disease. Most patients found that the disease was diagnosed in the late stage of cancer. Therefore, there is an urgent need for a more reasonable and effective diagnosis and prognosis of nasopharyngeal carcinoma. Hedgehog (Hh) signaling pathway is found in an evolutionary conservative signaling pathway in Drosophila melanogaster. Its initial function is to regulate embryonic development, and then it is found that it is abnormally activated in a variety of human cancers and is involved in tumorigenesis, differentiation, invasion and metastasis. However, it is not clear whether there is abnormal expression of Hh signaling pathway in nasopharyngeal carcinoma and its functional effect in nasopharyngeal carcinoma. In this study, the expression of PTCH-1,SMO and GLI-1 genes in nasopharyngeal carcinoma (NPC) and nasopharyngitis (NPC) was detected by fluorescence quantitative PCR (FQ-PCR). The results showed that PTCH-1,SMO and GLI-1 genes were expressed in all nasopharyngeal tissues, while the results of fluorescence quantitative PCR showed that the expression of PTCH,GLI-1 was different in nasopharyngeal carcinoma tissues and nasopharyngeal inflammation tissues (P 0.05), PTCH expression was down-regulated. The expression of GLI-1 was up-regulated, but the expression of SMO gene was not different. Immunohistochemical analysis further showed that GLI-1 protein was up-regulated in nasopharyngeal carcinoma (NPC). These results indicated that Hh signaling pathway was often activated in nasopharyngeal carcinoma (NPC). Cyclopramine (cyclopamine), a specific signal pathway inhibitor, interfered with the pathway function of Hh nasopharyngeal carcinoma cell lines CNE1,CNE2 and HNE2 with different degrees of differentiation. CCK-8 cell proliferation test showed that CNE1, The growth and proliferation of CNE2 and HNE2 were inhibited to a certain extent under the condition of signal pathway inhibition. Flow cytometry showed that cyclopamine inhibitory pathway increased apoptosis of CNE1,CNE2 and HNE2 cells. Fluorescence quantitative PCR was used to detect the effect of pathway interference on the expression of target gene. It was found that the expression of downstream gene PTCH1,Cyclin E was down-regulated. The results showed that Hh signaling pathway was abnormally activated in nasopharyngeal carcinoma (NPC). Through the interference of signal pathway, it was found that Hh promoted the proliferation and apoptosis of nasopharyngeal carcinoma cells, and inhibited the expression of target gene in signal pathway. This study promotes the study of nasopharyngeal carcinoma from Hh signaling pathway as a diagnosis and molecular target to provide a new scientific basis.
【学位授予单位】：中山大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R739.63

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