鼻咽癌PCDGF表达及siRNA对HNE-1细胞株增殖的影响

发布时间：2018-07-09 14:49

本文选题：鼻咽肿瘤 + 畸胎瘤细胞源性生长因子　；参考：《重庆医科大学》2012年硕士论文

【摘要】：背景：鼻咽癌(nasopharyngeal carcinoma，NPC)是中国南方常见的一种头颈部恶性肿瘤，其恶性程度高，具有强大的侵袭转移能力，且发病部位隐蔽，早期诊断难。畸胎瘤细胞源性生长因子（PC cell-derivedgrowth factor，PCDGF）又名progranulin，acrogranin，属于生长因子家族:颗粒蛋白(granulin，GRN)中的一员，其在多种肿瘤中高表达，如乳腺癌[2][3]，卵巢癌[4]，食管鳞状细胞癌[5]，非小细胞肺癌[6]，多发性骨髓细胞瘤[7]等，参与肿瘤的发生、增殖、浸润、转移。因此沉默肿瘤细胞中PCDGF表达有助于防治肿瘤的增殖转移。RNAi是一种抑制特定基因产物表达的有效方法，是由双链RNA（dsRNA）介导的特异性降解相应序列的mRNA，导致靶基因的沉默[8]。本研究采用RNA干扰技术，，沉默PCDGF表达，探讨其对鼻咽癌细胞株HNE-1生物学行为的影响，以期为鼻咽癌的诊断与治疗提供实验室依据。目的：探讨畸胎瘤细胞源性生长因子（PC cell-derived growthfactor，PCDGF）在人鼻咽癌中的表达，并研究其表达与患者临床病理特征之间的关系。构建针对PCDGF特异性的小干扰RNA（smallinterferirng RNA，siRNA），研究其对鼻咽癌HNE-1细胞增殖的影响。方法：采用免疫组织化学的方法检测53例鼻咽癌，28例鼻咽慢性炎症组织中PCDGF蛋白的表达情况，并结合患者的临床病理资料对结果进行统计分析；同时采用荧光免疫细胞化学法检测人鼻咽癌细胞株HNE-1细胞PCDGF表达。根据PCDGF基因设计合成两条siRNA，利用lipofectamineTM2000转染鼻咽癌HNE-1细胞，并在荧光显微镜下检测转染效率。通过real-time PCR、Western blot检测转染后细胞PCDGFmRNA和蛋白表达，采用MTT检测转染后细胞的生长增殖情况，FCM法检测细胞周期分布。结果：53例鼻咽癌组织中PCDGF阳性表达率为82.02%（44/53），28例鼻咽部慢性炎症组织中阳性率为17.86%（5/28），两组间差异有统计学意义（P0.01）。鼻咽癌组织中PCDGF的表达与肿瘤浸润深度、淋巴结转移及病理分期密切相关（P0.05），而与性别无关（P＞0.05）。2条重组质粒均能特异性的抑制PCDGF mRNA和蛋白的表达，其中siRNA-1的沉默效率最高。转染48h后，HNE-1细胞中PCDGF mRNA和蛋白表达水平分别下调59.3%和57.6%，细胞增殖抑制率为（37.07±12.4）%，siRNA-1组G0/G1期细胞增多, S期细胞数量明显减少（P0.05），细胞周期被阻滞于G1期。结论：特异性siRNA能够有效沉默PCDGF基因表达，并显著抑制鼻咽癌HNE-1细胞增殖。
[Abstract]:Background: nasopharyngeal carcinoma (nasopharyngeal) is a common malignant tumor of head and neck in southern China. Its malignant degree is high, it has strong ability of invasion and metastasis, and its location is hidden, so it is difficult to diagnose early. Teratoma cell cell-derivedgrowth factor-derived growth factor (PCDGF), also known as progranulinacrogranin, is a member of the growth factor family, granulinomatous GRN, which is highly expressed in many kinds of tumors. For example, breast cancer [2] [3], ovarian cancer [4], esophageal squamous cell carcinoma [5], non-small cell lung cancer [6], multiple myelocytoma [7], are involved in tumorigenesis, proliferation, invasion and metastasis. Therefore, silencing the expression of PCDGF in tumor cells is an effective method to inhibit the expression of specific gene products. It is a specific mRNAs mediated by double-stranded RNA (dsRNA), which leads to the silencing of target genes [8]. In this study, RNA interference technique was used to silence the expression of PCDGF in nasopharyngeal carcinoma cell line HNE-1 in order to provide laboratory basis for the diagnosis and treatment of nasopharyngeal carcinoma. Objective: to investigate the expression of PC cell-derived growth factor (PCDGF) in human nasopharyngeal carcinoma (NPC) and its relationship with clinicopathological features. PCDGF-specific small interfering RNA (smallinterferirng RNA-siRNA) was constructed to study its effect on the proliferation of nasopharyngeal carcinoma (NPC) HNE-1 cells. Methods: the expression of PCDGF protein was detected by immunohistochemical method in 28 cases of nasopharyngeal carcinoma (NPC) with chronic inflammation, and the results were statistically analyzed in combination with the clinicopathological data of the patients. The expression of PCDGF in human nasopharyngeal carcinoma cell line HNE-1 was detected by fluorescence immunocytochemistry. Two siRNAs were designed and synthesized according to PCDGF gene. The HNE-1 cells were transfected with lipofectamine TM2000 and the transfection efficiency was detected by fluorescence microscope. The expression of PCDGF mRNA and protein in transfected cells was detected by real-time PCR Western blot, and the cell cycle distribution was detected by FCM method. Results the positive rate of PCDGF was 82.02% (44 / 53) in 53 cases of nasopharyngeal carcinoma and 17.86% (5 / 28) in 28 cases of chronic inflammation of nasopharynx (P0.01). The expression of PCDGF in nasopharyngeal carcinoma was closely related to the depth of tumor invasion, lymph node metastasis and pathological stage (P0.05), but not to sex (P > 0.05). The expression of PCDGF mRNA and protein was specifically inhibited by the recombinant plasmid (P > 0.05). The silencing efficiency of siRNA-1 was the highest. After 48 hours of transfection, the expression of PCDGF mRNA and protein in HNE-1 cells decreased by 59.3% and 57.6%, respectively. The cell proliferation inhibition rate was (37.07 卤12.4)% in the G _ 0 / G _ 1 phase, and the number of S phase cells in the G _ 0 / G _ 1 phase increased significantly (P0.05), and the cell cycle was blocked in G _ 1 phase. Conclusion: specific siRNA can effectively inhibit the expression of PCDGF gene and inhibit the proliferation of nasopharyngeal carcinoma HNE-1 cells.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R739.63

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