本文選題：DAD1　切入點：腎癌　出處：《南華大學》2015年碩士論文

【摘要】：背景：腎癌又稱腎細胞癌(renal cell carcinooma RCC)是成人腎臟最常見的惡性腫瘤,占成人惡性腫瘤2%-3%,在男性和女性惡性腫瘤中分別排第7位和第9位。在全球范圍內(nèi),每年大約新發(fā)病例27萬,其中12萬死亡,在我國,腎癌的發(fā)病率在泌尿系腫瘤中僅次于膀胱癌,并在過去20年中以每年6%速度遞增。病理上腎癌可分為：透明細胞癌、腎乳頭狀癌、嫌色細胞癌、集合管癌及未分化癌。其中透明細胞癌(clear cell renal cell carcinoma ccRCC)是最常見的病理類型,起源于近曲小管上皮細胞,且易發(fā)生轉(zhuǎn)移,占轉(zhuǎn)移性腎癌的80%-85%。腎癌對放療及化療均不敏感,手術(shù)切除仍是目前最佳的治療手段,但術(shù)后復(fù)發(fā)率約為20%-40%。由于腎癌的化療及放療抵抗性,因此臨床上沒有有效的術(shù)后治療手段。腎癌作為泌尿科常見疾病,尤其是早期腎癌無特殊臨床表現(xiàn),待出現(xiàn)血尿、腰痛或腹部包塊時多已是晚期。腎癌對放療、化療均不敏感,臨床上一般予行腎癌根治性切除術(shù),但是一旦出現(xiàn)淋巴結(jié)轉(zhuǎn)移,即使行根治性淋巴結(jié)清掃術(shù),患者生存期也極少超過5年,若出現(xiàn)肝、肺轉(zhuǎn)移或臨近器官浸潤則預(yù)后更差,因此摸索出一條新的治療方案變的尤為重要。目前靶向沉默目的基因來治療腫瘤正在被廣大研究者所關(guān)注,相關(guān)研究顯示沉默腫瘤細胞中抗凋亡的基因可以促使腫瘤細胞大幅度凋亡,起到治療腫瘤的作用。目的：合成寡糖基轉(zhuǎn)移酶亞基DAD1的siRNA序列,觀察其對人A498腎癌細胞增殖和侵襲能力的影響。方法：設(shè)計合成三條DAD1-siRNA干擾片段,采用脂質(zhì)體法轉(zhuǎn)染人A498腎癌細胞后采用定量PCR和免疫印跡法檢測轉(zhuǎn)染前后人A498腎癌細胞中DAD1 mRNA和蛋白的表達變化；MTT檢測轉(zhuǎn)染前后A498細胞增殖能力變化；AV-PI流式細胞術(shù)檢測各組人A498腎癌細胞轉(zhuǎn)染48h后的凋亡率變化；Transwell檢測細胞侵襲能力的變化。結(jié)果：定量PCR及免疫印跡法檢測顯示3條siRNA均能有效抑制DAD1 mRNA及蛋白表達(P0.05),以DAD1-siRNA 1作用效果最顯著(P0.05)。 MTT檢測顯示DAD1-siRNA轉(zhuǎn)染24、48和72h后的人A498腎癌細胞的增殖受到顯著抑制(P0.05)；流式細胞術(shù)檢測結(jié)果顯不DAD1-siRNA轉(zhuǎn)染人A498腎癌細胞后凋亡率增加(p0.05)。Transwell檢測結(jié)果顯示沉默人A498腎癌細胞的DAD1 mRNA表達后,其侵襲能力顯著降低(p0.05)。結(jié)論：DAD1-siRNA可明顯抑制人A498腎癌細胞的增殖,促進人A498腎癌細胞的凋亡,降低其侵襲能力。
[Abstract]:Background: renal cell carcinoma (RCC), also known as renal cell carcinoma (RCC), is the most common malignant tumor in adult kidney, accounting for 2-3% of adult malignant tumors. It ranks 7th and 9th in male and female malignant tumors respectively. Globally, about 270000 new cases occur every year. Among them, 120000 died. In our country, the incidence of renal cell carcinoma is second only to that of bladder cancer, and has been increasing at an annual rate of 6% in the past 20 years. Pathologically, renal cell carcinoma can be classified as clear cell carcinoma, papillary carcinoma of kidney, chromophobe cell carcinoma. Clear cell renal cell carcinoma ccRCC is the most common pathological type, originated from proximal convoluted tubule epithelial cells, and easily metastasized, accounting for 80% -85% of metastatic RCC. RCC is not sensitive to radiotherapy and chemotherapy. Surgical resection is still the best treatment method at present, but the recurrence rate is about 20% -40%. Because of the resistance to chemotherapy and radiotherapy of renal cell carcinoma, there is no effective postoperative treatment in clinic. Renal cell carcinoma is a common disease in urology. In particular, early renal cell carcinoma has no special clinical manifestation, and it is usually late when hematuria, low back pain or abdominal mass appears. Renal cell carcinoma is not sensitive to radiotherapy or chemotherapy, and is usually treated with radical resection of renal cell carcinoma clinically, but once lymph node metastasis occurs, Even with radical lymphadenectomy, the survival time of the patient is rarely more than 5 years. If liver, lung metastasis or adjacent organ infiltration occur, the prognosis is even worse. Therefore, it is very important to explore a new therapeutic scheme. At present, the target silencing target gene to treat tumor is being paid attention to by many researchers. Related studies have shown that silencing the anti-apoptotic genes in tumor cells can promote the large apoptosis of tumor cells and play a role in the treatment of tumor. Objective: to synthesize the siRNA sequence of oligosyltransferase subunit DAD1. To observe its effect on proliferation and invasion of human renal carcinoma cell A498. Methods: three interfering fragments of DAD1-siRNA were designed and synthesized. The expression of DAD1 mRNA and protein in human A498 renal carcinoma cells before and after transfection were detected by quantitative PCR and Western blotting after transfection with liposome. The apoptotic rate of human A498 renal cancer cell line was detected by RT-PCR. Transwell was used to detect the cell invasion ability. Results: quantitative PCR and Western blot analysis showed that three siRNA could effectively inhibit the expression of DAD1 mRNA and protein, and DAD1-siRNA 1 was used to detect the invasion ability of A498 renal cancer cells. MTT assay showed that the proliferation of human A498 renal cancer cells transfected with DAD1-siRNA for 24 h and 72 h was significantly inhibited, and flow cytometry showed that the apoptosis rate increased after transfection of DAD1-siRNA into human A498 renal cancer cells by flow cytometry. After silencing the expression of DAD1 mRNA in human A498 renal cancer cells, Conclusion the cell proliferation of human renal cancer cell line A498 can be inhibited, the apoptosis of human renal cancer cell line A498 can be promoted, and the invasiveness of human renal cancer cell line A498 can be decreased.
【學位授予單位】：南華大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R737.11

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