本文選題：鼻咽腫瘤 + 畸胎瘤細胞源性生長因子��； 參考：《重慶醫(yī)科大學》2012年碩士論文

【摘要】：背景：鼻咽癌(nasopharyngeal carcinoma，NPC)是中國南方常見的一種頭頸部惡性腫瘤，其惡性程度高，具有強大的侵襲轉移能力，且發(fā)病部位隱蔽，早期診斷難。畸胎瘤細胞源性生長因子（PC cell-derivedgrowth factor，PCDGF）又名progranulin，acrogranin，屬于生長因子家族:顆粒蛋白(granulin，GRN)中的一員，其在多種腫瘤中高表達，如乳腺癌[2][3]，卵巢癌[4]，食管鱗狀細胞癌[5]，非小細胞肺癌[6]，多發(fā)性骨髓細胞瘤[7]等，參與腫瘤的發(fā)生、增殖、浸潤、轉移。因此沉默腫瘤細胞中PCDGF表達有助于防治腫瘤的增殖轉移。RNAi是一種抑制特定基因產(chǎn)物表達的有效方法，是由雙鏈RNA（dsRNA）介導的特異性降解相應序列的mRNA，導致靶基因的沉默[8]。本研究采用RNA干擾技術，，沉默PCDGF表達，探討其對鼻咽癌細胞株HNE-1生物學行為的影響，以期為鼻咽癌的診斷與治療提供實驗室依據(jù)。 目的：探討畸胎瘤細胞源性生長因子（PC cell-derived growthfactor，PCDGF）在人鼻咽癌中的表達，并研究其表達與患者臨床病理特征之間的關系。構建針對PCDGF特異性的小干擾RNA（smallinterferirng RNA，siRNA），研究其對鼻咽癌HNE-1細胞增殖的影響。 方法：采用免疫組織化學的方法檢測53例鼻咽癌，28例鼻咽慢性炎癥組織中PCDGF蛋白的表達情況，并結合患者的臨床病理資料對結果進行統(tǒng)計分析；同時采用熒光免疫細胞化學法檢測人鼻咽癌細胞株HNE-1細胞PCDGF表達。根據(jù)PCDGF基因設計合成兩條siRNA，利用lipofectamineTM2000轉染鼻咽癌HNE-1細胞，并在熒光顯微鏡下檢測轉染效率。通過real-time PCR、Western blot檢測轉染后細胞PCDGFmRNA和蛋白表達，采用MTT檢測轉染后細胞的生長增殖情況，F(xiàn)CM法檢測細胞周期分布。 結果：53例鼻咽癌組織中PCDGF陽性表達率為82.02%（44/53），28例鼻咽部慢性炎癥組織中陽性率為17.86%（5/28），兩組間差異有統(tǒng)計學意義（P0.01）。鼻咽癌組織中PCDGF的表達與腫瘤浸潤深度、淋巴結轉移及病理分期密切相關（P0.05），而與性別無關（P＞0.05）。2條重組質粒均能特異性的抑制PCDGF mRNA和蛋白的表達，其中siRNA-1的沉默效率最高。轉染48h后，HNE-1細胞中PCDGF mRNA和蛋白表達水平分別下調59.3%和57.6%，細胞增殖抑制率為（37.07±12.4）%，siRNA-1組G0/G1期細胞增多, S期細胞數(shù)量明顯減少（P0.05），細胞周期被阻滯于G1期。 結論：特異性siRNA能夠有效沉默PCDGF基因表達，并顯著抑制鼻咽癌HNE-1細胞增殖。
[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 鹵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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R739.63

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