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短发夹RNA特异及交叉抑制胰腺癌细胞PANC-1和Miapaca-2突变型K-ras基因表达的实验研究
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摘要
胰腺癌(pancreatic carcinoma)是严重危害人类生命健康的恶性肿瘤之一,其恶性程度极高而且发病隐匿,难以早期诊断和治疗,临床确诊者大多属于晚期癌。胰腺癌的预后极差,5年生存率总体低于5%。除早期手术外,尚无其他有效治疗措施,而且由于很难早期诊断,只有大约14%的患者可以选择手术治疗。近年来胰腺癌发生率在国内外均呈逐年上升趋势,据报道,近十年发病率上升了3-7倍。1991年,Hrudan等统计,胰腺癌是美国地区肿瘤死亡原因的第四位,仅次于肺癌,结肠癌和乳腺癌。在我国,随着人们生活水平的提高,饮食结构的变化,胰腺癌的发病率从1980年的第二十上升到第五位。上海疾控中心2000年统计的胰腺癌发病率为每十万人有6人发病,其中5.5人死亡,发病率几乎接近于死亡率,这一数字触目惊心。一般认为,肿瘤发生是由于细胞生长调控机制紊乱造成的。正常细胞生长增殖是由两大类基因调控的,一类是正调控信号,促进细胞生长和增殖,并阻止其发生终末分化,现知多数癌基因(oncogene)属此类。另一类为负调控信号,促进细胞成熟,向终末分化,抗癌基因(anti-oncogene)可能发挥这方面的作用。这两种信号保持着动态平衡,对正常细胞生长、增殖和死亡进行精确的调控。一旦两者之间的正常关系被破坏,则会使细胞的调控紊乱从而造成细胞恶变。Ras基因定位于1lq13,其基因家族包括H—ras、K-ras和N—ras。K-ras基因编码的蛋白是位于细胞膜上的鸟苷酸结合蛋白,负责将外部信号转导到细胞内部。其编码的K-ras p21 ras蛋白具有GTP酶活性,突变可导致ras蛋白失去GTP酶活性,GTP处于持续激活状态,细胞在持续激活的信号传导刺激下不断增殖,发生癌变。胰腺癌K-ras基因的第一外显子第12密码子点突变已被证实是胰腺肿瘤发生的早期事件,是早期诊断胰腺癌的重要指标。K-ras基因在胰腺癌中的突变率平均在80%左右,其中以12密码子突变最为常见。第12位密码子的点突变又以GAT (46%), GTT(32%),和CGT (13%)三种形式最为多见。有研究表明抑制突变K-ras的表达,可有效抑制胰腺癌细胞的生长。RNA干扰(RNA interference)技术是一项近年来发现的抑制基因表达的新技术,由于其具有特异性强,效率高等优点而成为在肿瘤研究中的热点。本实验根据突变的K-ras基因序列构建针对两种胰腺癌细胞株的特异性的shRNA质粒载体pGenesil-1-P1, pGenesil-1-M2及阴性对照pGenesil-1-HK。观察RNA干扰用于K-ras基因突变型胰腺癌的治疗效果,并验证两种非特异性的shRNA序列对不同胰腺癌细胞是否有交叉抑制作用,为胰腺癌的基因治疗寻找新的途径。
     目的:研究短发夹RNA(shRNA)对人胰腺癌细胞PANC-1和Miapaca-2中突变型K-ras基因表达的抑制作用,以及两shRNA片段对非特异胰腺癌细胞是否具有交叉抑制作用。
     方法:1构建针对PANC-1细胞中突变型K-ras基因的重组质粒载体pGenesil-1-P1,以及针对Miapaca-2细胞中突变型K-ras基因的重组质粒载体pGenesil-1-M2,阴性对照质粒pGenesil-1-HK。2细胞培养,并应用脂质体2000将重组质粒pGenesil-1-P1转染到胰腺癌细胞PANC-1,重组质粒载体pGenesil-1-M2转染到胰腺癌细胞Miapaca-2中。以及将两重组质粒载体交叉转染两胰腺癌细胞。3提取转染48小时后的细胞RNA,总蛋白,应用RT-PCR,免疫印迹(western blotting )法检测RNA干扰对胰腺癌细胞突变型K-ras基因表达的影响。4应用CCK-8法测量细胞生长曲线。
     结果:测序证明质粒载体构建成功,通过荧光显微镜观察到重组质粒转染成功转染到胰腺癌细胞,RT-PCR产物的琼脂糖电泳结果以及western结果显示特异性转染组的mRNA及蛋白的表达均减少(p<0.05),而非特异性转染组的K-ras基因的mRNA及蛋白的表达均未见明显变化。应用CCK-8方法检测细胞生长曲线显示特异性转染组细胞的生长受到明显抑制(p<0.05),而非特异性转染组的细胞的生长未受到明显抑制。
     结论:特异性shRNA重组质粒转染胰腺癌细胞后,胰腺癌细胞突变型K-ras基因的mRNA及蛋白的表达以及细胞的生长受到抑制,而非特异性转染和阴性对照质粒的胰腺癌细胞的mRNA及蛋白的表达以及细胞的生长未受到抑制。
Pancreatic carcinoma is one of the high malignant tumors that threaten human health,Its high malignancy and difficulty in early diagnosis lead to poor prognosis,The survival rate of five years is poor than 5%.Except for early operation, there is not other effectiven treat measures. And because early diagnose is difficult,only 14% of the patients can be treated by operation. In recent years, the incidence of pancreatic carcinoma has been on the rise with a 3~7-fold increase reportedly, both at home and abroad, within the past decade. According to statistics by Hrudan et al In 1991, pancreatic carcinoma was the fourth leading cause of death in the United States, preceded only by lung cancer, colon cancer, and breast cancer. In China, accompanied with improvement in quality of life and change of diets, the incidence of pancreatic carcinoma has increased from No. 20 in 1980 to No. 5 in mortality. Based on the statistics by Center for Disease Control in Shanghai in 2000, there were 6/100000 new patients per year, with a morbidity of 5.5 that was almost equaled to mortality.
     In tumorigenesis and progression of pancreatic cancer. K-ras mutation might be the most important factor. In the patients with pancreatic carcinoma, about 80% of their genes have mutations, and most of these mutations are found to be located in codon 12 of exon one. GAT(46%),GTT(32%) and CGT(13%) are the most common form of K-ras mutation at codon 12.
     Research has shown that restraining the expression of K-ras mutation can restrain the progression of pancreatic cancer cells effectively.RNA interference is a new technology found in recent years to restrain gene expression in recent years, and because of the idiosyncracy and high performance, it becomes the focus in tumor research field. Basing on choppy K-ras gene order, this recearch will build shRNA plasmid carrier:
     pGenesil-1-P1 and pGenesil-1-M2 Which aim at the idiosyncracy of tow kinds of pancreatic cancer cells. And to observe the treatment of RNA interference for pancreatice carcinoma of K-ras mutation and to validate that if two kinds of nonspecific shRNA order have crossed restraining effect on pancreatic cancer cells,and find a new way for gene theraty of pancreatic cancer.
     Aim:
     To investigate the inhibition efficacy of the growth of pancreatic carcinoma cell lines after K-ras shRNA transduction by plasmid and the effect of two shRNA
     Methord:
     1 To structure the specific recombination plasmids pGenesil-1-P1 pGenesil-1-M2 for human pancreastic cancer and negative plasmids-----pGenesil-1-HK.
     2 Culture pancreatic carcinoma cell line. and transduce pGenesil-1-P1,Genesil-1-M2 and Genesil-1-HK into pancreatic carcinoma cell line,
     3 Etracted the total RNA and protein use the RT-PCR and westernblotting methods to detective the effective of RNAi for pancreatic carcinoma which the k-ras gene mutation
     4 The cell growth activities were estimated by CCK-8 cell proliferation assay.
     RESULTS:
     Order-checking proved the sequence of plasmid was right, the transfection of plasmid was successfully through fluorescence microscope. Results of RT-PCR revealed that cells treated with specific shRNA transfection had lower transcription of K-ras mRNA as compared with the notspecific and control, Western blots indicated a decreased expression, as compared with the notspecific and controls of K-ras protein in the cells transducted by shRNA. Specific transfection plasmid exhibits an anti- proliferative effect in cell proliferation assay.
     Conclusions:
     After transfect into the pancreatic cancer cell line , The specific shRNA recombination plasmids can inhibit the mutant k-ras gene mRNA and protein expression, exhibits cell proliferation.but the notspecific transfection can not.
引文
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