【作者】 任新瑜；

【导师】 刘彤华； 梁智勇；

【作者基本信息】 中国协和医科大学 ， 病理学与病理生理学， 2007， 博士

【摘要】 胰腺癌由于其起病隐匿、易转移、解剖位置复杂、手术困难和对放化疗不敏感等原因,已经成为主要的致死性癌症之一。寻找有效的基因治疗方法是改善胰腺癌预后的重要措施之一。有研究发现在植物中局部转染外源无启动子DNA可以引起整个系统的序列特异性基因沉默。但至今无人将其应用于人的细胞,特别是人的癌细胞。本实验从两方面验证了运用克隆有目的基因cDNA全长片段的重组质粒载体能够特异地抑制人胰腺癌细胞基因的表达。(1):利用绿色荧光蛋白(Green FluorescentProtein,GFP)的可视性,观察到整合入胰腺癌细胞系Panc-1的外源基因GFP被抑制;(2)观察到胰腺癌最常见的内源性癌基因K-ras在三种胰腺癌细胞系(Panc-1、Miapaca-2、Aspc-1)中被特异性抑制。1.构建3种重组质粒:含有GFP基因cDNA全长片段、含有K-ras基因cDNA全长片段与含K-ras基因cDNA第一外显子的片段(1)获取GFP基因cDNA全长片段:由pEGFP-C1质粒图谱信息得到GFP全长cDNA序列。设计特异引物并在引物两端加入酶切位点,扩增出GFP基因的cDNA全长片段。(2)获取K-ras cDNA全长片段:质粒pOPRSVI/MCS含K-ras cDNA全长。在其多克隆位点两侧选取不同的酶切位点进行酶切。(3)获取K-ras第一外显子DNA片段:提取含野生型K-ras基因的胰腺癌细胞系Bxpc-3的基因组DNA,在K-ras第一外显子两侧的内含子里设计引物(两端加入酶切位点)扩增含第一外显子的基因片段。(4)构建3种重组质粒:将上述基因片段分别连接到复制型质粒puc19。重组质粒分别命名为puc-GFP、puc-K-ras、puc-exon。3种重组质粒分别在DH5a大肠杆菌中进行转化、扩增并且采用琼脂糖电泳及测序鉴定正确序列。2.将外源基因GFP整合入人胰腺癌细胞系Panc-1将表达GFP的质粒pEGFP-C1转染入Panc-1细胞,并进行G418筛选。挑取具有抗新霉素抗性的克隆进一步培养,得到基因组内整合有EGFP基因的克隆细胞。倒置显微镜及流式细胞仪检测细胞克隆纯度。3.验证puc-GFP对GFP蛋白表达的抑制效应(1)流式细胞术及蛋白印迹技术证实puc-GFP可抑制整合入人胰腺癌细胞系Panc-1中的外源基因GFP的表达。抑制效应和转染的puc-GFP呈剂量、时间依赖性效应关系。(2)流式细胞术及倒置显微镜观察证实,puc-GFP可抑制GFP表达质粒pEGFP-C1在Panc-1细胞中的瞬时转染效率。抑制效应与puc-GFP与pEGFP-C1共转染细胞时的比值呈正相关。4.验证puc-K-ras及puc-exon对细胞内同源癌基因表达的抑制效果(1)实时定量RT-PCR检测mRNA的表达:puc-K-ras转染细胞系Panc-1后,分别在下面三种情况下收获细胞并提取其细胞的总RNA:a.不同剂量、同一时间;b.同一剂量,不同时间;c.挑取puc-K-ras稳定转染细胞系。将上述总RNA,经逆转录酶合成cDNA,最后在实时定量PCR扩增仪上进行扩增并分析结果。实验a、c设有未处理组、空质粒puc19组作为对照,实验b除设有上述两种对照组外还有K-ras siRNA组作为阳性对照;结果表明:a.puc-K-ras可抑制Panc-1细胞内源性癌基因K-ras的表达,并且抑制强度与转染质粒剂量呈正比;b.puc-K-ras瞬时转染Panc-1细胞后前两天其K-ras mRNA明显上升,第三天开始下降,直到转染后第十天K-ras mRNA水平仍明显低于正常对照组及空质粒组;c.puc-K-ras稳定转染细胞系的K-ras mRNA远远低于正常对照组和puc19稳定转染细胞系。(2)蛋白印迹检测蛋白表达:提取转染细胞及其各对照组细胞的总蛋白,经SDS-PAGE电泳及免疫杂交,结果表明:a.转染puc-K-ras的细胞K-ras蛋白明显下降。抑制效果与转染质粒呈剂量依赖关系;b.转染后第四天,K-ras蛋白开始下降,直到转染后第十天仍明显低于正常对照组和空质粒组;c.puc-K-ras稳定转染细胞系的K-ras蛋白远远低于正常对照组和puc19稳定转染细胞系。(3) puc-K-ras引起的基因抑制具有序列特异性:第一,在GFP表达已经被puc-GFP抑制的细胞中添加GFP表达质粒可使减弱的绿色荧光部分恢复;第二,puc-GFP可抑制整合入Panc-1细胞内的GFP基因表达,但不影响K-ras基因的表达;同样,puc-K-ras转染整合有GFP基因的Panc-1细胞,K-ras蛋白下降的同时不影响GFP的蛋白表达。(4)无启动子DNA片段引起的K-ras基因表达抑制与第一外显子的关系:为验证最常发生点突变的第一外显子是否是导致基因抑制的效应区,我们构建了含K-ras cDNA第一外显子DNA序列的质粒,并将其转染Panc-1细胞。蛋白印迹发现,只含第一外显子序列的DNA片段并不能引发序列特异性基因抑制。(5)无启动子DNA片段引起的K-ras基因抑制与点突变的关系:为观察此基因抑制与点突变的关系。Puc-K-ras分别转染另两种胰腺癌细胞系Miapaca-2和Aspc-1,它们含有与Panc-1相同或不同的K-ras点突变。蛋白印迹及实时定量RT-PCR(reverse transcriptive polymerase chain reaction)结果表明,在这两种细胞系中puc-K-ras具有与在Pane-1细胞内类似的抑制K-ras基因表达的效果。这说明无启动子DNA片段引起的特异性基因抑制与点突变无明显相关性。我们的研究表明无启动子的基因cDNA全长片段可在人胰腺癌细胞内引发序列特异的基因表达抑制,可能成为一种新的有效的基因治疗方法,但导致这种基因抑制的具体机制有待于进一步研究。另外,本实验结果提示无启动子DNA片段引起的序列特异性基因抑制可能与外源DNA片段中所含基因的cDNA片段长度相关,但与细胞内同源基因是否有点突变没有关系。本研究的创新点是首次证实了在植物中有效的同源无启动子DNA片段能使特异性基因沉默的方法,同样亦可用于抑制人类癌细胞特异性癌基因的表达。更多还原

【Abstract】 Pancreatic cancer is exceptionally aggressive with high mortality,and up to now no effective therapeutic approaches has been found.So it is urgent to find more powerful method to diagnose the disease earlier and treat it more effectively.But even the new powerful research tool siRNAs has its own problem,i.e.the efficiency.In recent years,several researchers reported that delivery of promoterless double-strand DNA molecules into cells could induce sequence-specific gene silencing in plants.The plasmid having the target gene’s cDNA inserted alone was sufficient to induce silencing.This simple and efficient method of gene target silencing has not been used in human cells yet,especially in human cancer cells.On one hand,genetically encoded fluorescent proteins have become widely used as markers in living cells.The application of these fluorescent proteins as noninvasive tags revealed new aspects of protein dynamics and the biological processes they regulated.The modification of naturally occurring fluorescent proteins as well as the identification of new fluorescent proteins now provide researchers with a variety of useful fluorescent markers suitable for all kind of investigations in live-cell imaging studies.Enhanced jellyfish green fluorescent protein(EGFP) a mutant of green fluorescent protein(GFP) with reinforced green fluorescent was selected as a visible index.A plasmid containing GFP full-length cDNA was constructed.The silencing efficiency was observed through detecting the GFP expression by transfecting the plasmid into the steady-expressing GFP clone cell or by contransfecting it with GFP expressing plasmid pEGFP-C1.K-RAS is a member of the RAS family of GTP-binding proteins that mediate a wide variety of cellular functions including proliferation,differentiation,and survival.The activation of K-ras oncogene is the most important initial step in the genesis of human pancreatic cancer and most of human pancreatic cancers have high frequency of K-ras point mutation.All these together with the importance of ras in cell function and survival,highlight K-ras being an attractive target for the treatment of pancreatic cancer.K-ras was thus chosen as the target gene in the present study.Aim:The present study aimed to test the efficiency of the gene silencing effect by introducing promoterless double-strand DNA containing coding region of a gene in human pancreatic cancer cells.Material and method:First of all,several plasmids were constructed. Considering the low transfect efficiency of naked DNA,promoterless plasmid puc-19 was used as a vector of target gene’s full-length cDNA fragment.To construct plasmids containing promoterless DNA of the target gene,GFP and K-ras full length cDNA were obtained and ligated into puc-19.The constructs were named as puc-GFP and puc-K-ras respectively.Puc-exon,with DNA sequence of K-ras first exon integrated in puc-19,was also constructed to testify if the silencing efficiency had relationship with specific sequence in cDNA.Next,puc-GFP was transfected to GFP transformed human pancreatic cancer cell line Pane-1 cells to visualize the silencing efficiency.Flow cytometry,phase contrast fluorescence microscopy and Western blot were used to detect expression of GFP.Finaly,puc-K-ras and puc-exon were transfected to Panc-1 to observe whether K-ras gene expression could be inhibited.Puc-K-ras was also transfected to two other human pancreatic cancer cell lines Miapaca-2 and Aspc-1 to detect the K-ras gene expression.As compared with Panc-1,the two cell lines have similar or different point mutation of K-ras.Real-time transcriptive PCR and Western blot were used to detect the K-ras gene expression.Results:(1) Panc-1 exogenous integrated gene GFP could be silenced by plasmid puc-GFP,which containing promoterless DNA fragment of GFP full-length cDNA.Puc-GFP also inhibited the transient transfecting efficiency of GFP expressing plasmid.(2) Endogenous gene K-ras of Panc-1,Miapaca-2,Aspc-1 cells also could be silenced by promoterless plasmid having its full-length cDNA integrated.The silencing efficiency was correlated to the length of cDNA fragment.Different K-ras point mutant did not affect the silencing efficiency.Conclusion:The results of the present study indicate that the transfection of human pancreatic cancer cells with plasmids carrying a full length cDNA sequence of the target gene is sufficient to decrease the expression of the genes.It may take effect through reduction of the endogenous transcripts.The effective region of the promoterless DNA fragment is not clear yet and need to study further.The result of present study indicats that the whole length of cDNA fragment rather than the consensus sequences may account for the silencing effects.This study is for the first time to prove the sequence-specific gene silencing method used in plant, also is effective in silencing the target gene in human pancreatic cancer cells.更多还原