人胃癌相关基因的克隆及胃癌混合组织cDNA文库构建
|
摘要
研究背景：胃癌是最常见的恶性肿瘤之一，在我国居于恶性肿瘤发
病之首。胃粘膜细胞癌变过程中存在多基因的变化及积累，在这个过程
中，原癌基因的激活以及抑癌基因的失活起到了关键的作用。虽然现在
已经肯定了很多基因在胃癌中的异常变化，对胃癌的诊断和治疗有一定
的参考意义，但它们最初发现时均非通过研究胃癌所得，所以，对胃癌的
特异性较差，无法用于早期诊断，且在目前发现的癌症相关基因中，任何
一个单基因都不能从分子水平阐明胃癌发病的分子机制，因此，将胃癌
相关基因的克隆鉴定与基因组研究相结合，以基因的差异表达为出发点，
充分利用生物信息学的资料数据，确定在正常胄粘膜细胞、胃癌细胞和
肿瘤发展中间阶段细胞中表达的基因，将会为揭示胃癌发生和发展过程
中的分子机制并为胃癌和癌前病变的基困诊断及治疗提供理论依据。
研究目的：从人胃癌组织中克隆新的相关易感基因，探讨胃癌癌变
的分子机理，为胃癌的早诊早治提供理论依据。
研究方法：1、采用简单高效的互补DNA(cDNA)合成技术构建人胃
癌cDNA基因文库。以纯化的 poly(A)＋－RNA为模板，含 NotⅠ切点的
ol1go-(dT)15为引物，利用改良的cDNA快速合成方法，反转录合成cDNA
第一、二链，对双链cDNA末瑞修饰，连接连接子，经层析枉纯化去除小
片段和多余连接子后，重组于噬菌体载体。行体外包装后以大肠杆菌
Y1090行滴度测试及扩增，建成cDNA文库。
2、利用差异显示PCR技木获得的一条在胃癌癌旁和正常组织表达
量高的EST－W123，通过与Genebank的dbest库进行电子杂交，选取与
W123同源度高的若干EST，在它们共有的保守序列设计用于扩增的寡聚
4
军医进修学院 博士学仕论文 中又摘要
核普酸引物，用设计的 3’引物和 RACE试剂盒提供的 5’公用引物，进
行 3’cDNA末瑞扩增。扩增产物克隆入 ＂GEM T－Easy载体，重组质粒用
ECOR酶切和载体引物T7／SP6扩增鉴定，然后测序分析。对所得CDNA
进行生物信息学分析并提交Genbank。
3、用 Northern blot和C－PRINS枝术分析上达cDNAs在胃癌、癌
前病变x正常胃组织的表达，以多组织 Northern blot和虚拟 Northern
（SAGE）分析CDNAS在多种正常组织及肿瘤组织的表达情况。图象分析
方法量化 cDNA的表达，vX Mean OD为量化指标。
结呆：1、构建的 CDNA文库，含 3 2 X 10‘重组子，重组率为 96儿
扩增后文库的滴度达12 x 10“pfu／ml。cDNA片段大＊在0 5一3 skb之间，
平均13kb。
2、3’RACE扩增得到 7条带有卯 l yA尾的 CDNA片段，pci yA尾包
含20－30个’k”不等，有的片段可见力。尾信号AATAAA，将这些序列在计
算机软件上进行比对hll即）发现 它们除靠近5’瑞的共有序列外，
在 3’瑞明显不同。将t 7条CDNA分别命名为 W13、W25、W31、W41、W51、
W6 3和W74，大小分别为们3 223hp，W25 295hp，W31 4llbp，W41 556hp，W51
606hp，W63 1206hp，W74 1234hp。与Genbank nr和dbEST数据库同源比
较显示，各CDNA与编码蛋白的基因序列同源性低，表明可能代表未知的
基因，已登录Genbank dbEST数据库，登录号分别为 AF332603、AF332604、
AF 3 32605、AF325202、AF332606、AY032615、AY032616。
3、Northern blot显示7条cDNA中有 6条在胃癌及对应正常胃组
织表达，但是WZ 5探针未见阳性结果。W31、W41、W63探针各可见3个
转录本，大，J、IAIZ－6 okb不等，WI3有 2个转录本，大，J、为 2 okb、
14kb，W51片段有一个转录本，约3．skb，W74探针显影转录本大，J、主要
集中在 3 0－5 okb之间。4＆＃灰度扫描 ImageJ软件分析结果，WI3、W31、
5 军医进修学院 博士学仁论文 中文摘要
W4、WSI、W74在胃癌组织的表达强度高于正常组织，WO则与之相反在
胃癌组织表达降低。多组织Northern显示W41在白血病细胞、林巴瘤细
胞和正常肝脏组织表达丰度较高，在黑色素瘤细胞、胎盘和脑组织表达丰
度低。而 Wu在结肠组织表达丰度最高，前列腺和胃组织表达丰度较低。
4、原位卜叫ms检测显示W1的阳性表达主要位于胃癌组织、肠
化生上皮，正常胃组织的表达明显咸弱，三者组织表达的量化结果为
06054tO19们、04358t0n82、00685t0054，统?
Background:
Gastnc cancer is a kind of high malignant tumor At present, its incidence and mortality rate is still high in the world Especially in our country, gastric cancer is still a major killer Gastric carcinogenesis results from the accumulation of a series of genetic and biochemical changes in normal mucosal cells, and it is important that the protooncogenes are activated and tumor-suppressor genes lose functions in this process Despite the increase in our knowledge of oncogenes and tumor suppressor genes associated with gastric cancer over the past decades, the specificity of these genes are poor for gastric cancer, the molecular events leadings to the formation of gastric cancer are still not well understood Therefore, identication of genes that are involved in the transition of non-tumorigemc cells to malignant cells can be expected to help us understand the molecular mechanism underlying tumor formation and provide insight into gene diagnosis and treatment of gastric cancer and precancerous conditions
Objectives:
To search for new human gastric cancer-associated susceptible gene for early diagnosis and treatment of gastric cancer
Methods:
1 Construction of cDNA Library Total RNA and mRNA was extracted and converted to blunt-ended, double-stranded cDNA by Oligo(dT)-mediated reverse transciption followed by adapter addition Column chromotography isolates residual adapters and small bands Ligation of cDNA to the vector
7
and in vitro packaging of ligated cDNA and introduction into E ccli Y1090 Titer and amplify the phage
2 Cloning gastric cancer related candidated homologenes Previously, W123, a EST fragment derived from normal gastric and paracarcinoma tissue, was cloned using differentiated-display PCR technique A few homologous EST sequences were captured when processing similarity search in human EST database To search for additional W123 homologous genes in gastric tissue, we designed a primer for 3'-RACE from highly conserved region among the above ESTs Using this 3'primer and another 5' primer provided by 3'-RACE kit, we do rapid amplification of eDNA 3'end The cDNA bands got from 3'RACE were cloned into pGEM T-Easy vector, and the positive clones were confirmed by EcoR I enzyme digestion and T7/SP6 PCR amphcation Then ,the sequences of inserted fragments were analyzed The bioinformatics source was utilized to get more information of these cDNAs, and submitted them to Genbank
3 Testifying cDNAs expression in different gastric and other tissues Northern blot and C-PRJNS technology are applied to detect the expression of these bands in gastric cancer and normal tissues, combination of virtual Northern and multiple tissues Northern blot, expression of cDNAs in multiple normal and carcinoma tissues were analyzed Image analysis sofeware was used to quantitate the expression of cDNA bands, and mean OD was used as the quantitative index
Results:
1 The cDNA library has 3 2 X 1 O6recombinants,the recombining ratio is
96% The titer of amplified library is 12 Xl 012p~ml Identification of the
insert fragments of the recombinant phage DNA are between 0 5---3 5kb,
average size is 1 3kb
2 Seven cDNA fragments with polyA tail were cloned, polyA contains 20-30 "A" , sometimes AATAAA is found among cDNAs Comparison analysis with Genbank demonstrated all these ESTs may represent 7 unknown genes
8
contaimng a fragment of common sequence They were named W13 ~ W25.~ W3 1 ... W4 1~ W5 1. W63 and W74, respectively These bands were submitted to Genbank dbEST database and already admitted, and Accession Number
were AF332603~ AF332604~. AF332605N AF325202~. AF332606.. AY0326l5~, AY032616
3 Northern blot revealed Wi 3 W3 1 .~. W4 1-. W5 land W74 presented higher expression in gastric cancer tissue than normal tissue, but W63 expressed reversely W3 1, W4 1 and W63 have three transcnpts, the estimate mRNA full length is 1 2-~6 0kb, W51 is 3 5kb, the length of W74 is between 3 0-5 0kb Multiple tissue Northern blot revealed W4 1 presented a high
病之首。胃粘膜细胞癌变过程中存在多基因的变化及积累，在这个过程
中，原癌基因的激活以及抑癌基因的失活起到了关键的作用。虽然现在
已经肯定了很多基因在胃癌中的异常变化，对胃癌的诊断和治疗有一定
的参考意义，但它们最初发现时均非通过研究胃癌所得，所以，对胃癌的
特异性较差，无法用于早期诊断，且在目前发现的癌症相关基因中，任何
一个单基因都不能从分子水平阐明胃癌发病的分子机制，因此，将胃癌
相关基因的克隆鉴定与基因组研究相结合，以基因的差异表达为出发点，
充分利用生物信息学的资料数据，确定在正常胄粘膜细胞、胃癌细胞和
肿瘤发展中间阶段细胞中表达的基因，将会为揭示胃癌发生和发展过程
中的分子机制并为胃癌和癌前病变的基困诊断及治疗提供理论依据。
研究目的：从人胃癌组织中克隆新的相关易感基因，探讨胃癌癌变
的分子机理，为胃癌的早诊早治提供理论依据。
研究方法：1、采用简单高效的互补DNA(cDNA)合成技术构建人胃
癌cDNA基因文库。以纯化的 poly(A)＋－RNA为模板，含 NotⅠ切点的
ol1go-(dT)15为引物，利用改良的cDNA快速合成方法，反转录合成cDNA
第一、二链，对双链cDNA末瑞修饰，连接连接子，经层析枉纯化去除小
片段和多余连接子后，重组于噬菌体载体。行体外包装后以大肠杆菌
Y1090行滴度测试及扩增，建成cDNA文库。
2、利用差异显示PCR技木获得的一条在胃癌癌旁和正常组织表达
量高的EST－W123，通过与Genebank的dbest库进行电子杂交，选取与
W123同源度高的若干EST，在它们共有的保守序列设计用于扩增的寡聚
4
军医进修学院 博士学仕论文 中又摘要
核普酸引物，用设计的 3’引物和 RACE试剂盒提供的 5’公用引物，进
行 3’cDNA末瑞扩增。扩增产物克隆入 ＂GEM T－Easy载体，重组质粒用
ECOR酶切和载体引物T7／SP6扩增鉴定，然后测序分析。对所得CDNA
进行生物信息学分析并提交Genbank。
3、用 Northern blot和C－PRINS枝术分析上达cDNAs在胃癌、癌
前病变x正常胃组织的表达，以多组织 Northern blot和虚拟 Northern
（SAGE）分析CDNAS在多种正常组织及肿瘤组织的表达情况。图象分析
方法量化 cDNA的表达，vX Mean OD为量化指标。
结呆：1、构建的 CDNA文库，含 3 2 X 10‘重组子，重组率为 96儿
扩增后文库的滴度达12 x 10“pfu／ml。cDNA片段大＊在0 5一3 skb之间，
平均13kb。
2、3’RACE扩增得到 7条带有卯 l yA尾的 CDNA片段，pci yA尾包
含20－30个’k”不等，有的片段可见力。尾信号AATAAA，将这些序列在计
算机软件上进行比对hll即）发现 它们除靠近5’瑞的共有序列外，
在 3’瑞明显不同。将t 7条CDNA分别命名为 W13、W25、W31、W41、W51、
W6 3和W74，大小分别为们3 223hp，W25 295hp，W31 4llbp，W41 556hp，W51
606hp，W63 1206hp，W74 1234hp。与Genbank nr和dbEST数据库同源比
较显示，各CDNA与编码蛋白的基因序列同源性低，表明可能代表未知的
基因，已登录Genbank dbEST数据库，登录号分别为 AF332603、AF332604、
AF 3 32605、AF325202、AF332606、AY032615、AY032616。
3、Northern blot显示7条cDNA中有 6条在胃癌及对应正常胃组
织表达，但是WZ 5探针未见阳性结果。W31、W41、W63探针各可见3个
转录本，大，J、IAIZ－6 okb不等，WI3有 2个转录本，大，J、为 2 okb、
14kb，W51片段有一个转录本，约3．skb，W74探针显影转录本大，J、主要
集中在 3 0－5 okb之间。4＆＃灰度扫描 ImageJ软件分析结果，WI3、W31、
5 军医进修学院 博士学仁论文 中文摘要
W4、WSI、W74在胃癌组织的表达强度高于正常组织，WO则与之相反在
胃癌组织表达降低。多组织Northern显示W41在白血病细胞、林巴瘤细
胞和正常肝脏组织表达丰度较高，在黑色素瘤细胞、胎盘和脑组织表达丰
度低。而 Wu在结肠组织表达丰度最高，前列腺和胃组织表达丰度较低。
4、原位卜叫ms检测显示W1的阳性表达主要位于胃癌组织、肠
化生上皮，正常胃组织的表达明显咸弱，三者组织表达的量化结果为
06054tO19们、04358t0n82、00685t0054，统?
Background:
Gastnc cancer is a kind of high malignant tumor At present, its incidence and mortality rate is still high in the world Especially in our country, gastric cancer is still a major killer Gastric carcinogenesis results from the accumulation of a series of genetic and biochemical changes in normal mucosal cells, and it is important that the protooncogenes are activated and tumor-suppressor genes lose functions in this process Despite the increase in our knowledge of oncogenes and tumor suppressor genes associated with gastric cancer over the past decades, the specificity of these genes are poor for gastric cancer, the molecular events leadings to the formation of gastric cancer are still not well understood Therefore, identication of genes that are involved in the transition of non-tumorigemc cells to malignant cells can be expected to help us understand the molecular mechanism underlying tumor formation and provide insight into gene diagnosis and treatment of gastric cancer and precancerous conditions
Objectives:
To search for new human gastric cancer-associated susceptible gene for early diagnosis and treatment of gastric cancer
Methods:
1 Construction of cDNA Library Total RNA and mRNA was extracted and converted to blunt-ended, double-stranded cDNA by Oligo(dT)-mediated reverse transciption followed by adapter addition Column chromotography isolates residual adapters and small bands Ligation of cDNA to the vector
7
and in vitro packaging of ligated cDNA and introduction into E ccli Y1090 Titer and amplify the phage
2 Cloning gastric cancer related candidated homologenes Previously, W123, a EST fragment derived from normal gastric and paracarcinoma tissue, was cloned using differentiated-display PCR technique A few homologous EST sequences were captured when processing similarity search in human EST database To search for additional W123 homologous genes in gastric tissue, we designed a primer for 3'-RACE from highly conserved region among the above ESTs Using this 3'primer and another 5' primer provided by 3'-RACE kit, we do rapid amplification of eDNA 3'end The cDNA bands got from 3'RACE were cloned into pGEM T-Easy vector, and the positive clones were confirmed by EcoR I enzyme digestion and T7/SP6 PCR amphcation Then ,the sequences of inserted fragments were analyzed The bioinformatics source was utilized to get more information of these cDNAs, and submitted them to Genbank
3 Testifying cDNAs expression in different gastric and other tissues Northern blot and C-PRJNS technology are applied to detect the expression of these bands in gastric cancer and normal tissues, combination of virtual Northern and multiple tissues Northern blot, expression of cDNAs in multiple normal and carcinoma tissues were analyzed Image analysis sofeware was used to quantitate the expression of cDNA bands, and mean OD was used as the quantitative index
Results:
1 The cDNA library has 3 2 X 1 O6recombinants,the recombining ratio is
96% The titer of amplified library is 12 Xl 012p~ml Identification of the
insert fragments of the recombinant phage DNA are between 0 5---3 5kb,
average size is 1 3kb
2 Seven cDNA fragments with polyA tail were cloned, polyA contains 20-30 "A" , sometimes AATAAA is found among cDNAs Comparison analysis with Genbank demonstrated all these ESTs may represent 7 unknown genes
8
contaimng a fragment of common sequence They were named W13 ~ W25.~ W3 1 ... W4 1~ W5 1. W63 and W74, respectively These bands were submitted to Genbank dbEST database and already admitted, and Accession Number
were AF332603~ AF332604~. AF332605N AF325202~. AF332606.. AY0326l5~, AY032616
3 Northern blot revealed Wi 3 W3 1 .~. W4 1-. W5 land W74 presented higher expression in gastric cancer tissue than normal tissue, but W63 expressed reversely W3 1, W4 1 and W63 have three transcnpts, the estimate mRNA full length is 1 2-~6 0kb, W51 is 3 5kb, the length of W74 is between 3 0-5 0kb Multiple tissue Northern blot revealed W4 1 presented a high
引文
[1] 王刚石、王孟薇、尢纬缔等人胃癌差异表达cDNA片段的克隆与初步 表达分析中华医学遗传学杂志，2001，1。
[2] Galaud JP,Carriere M,Pauly N,et al Construction of two ordered cDNA libraries enriched in genes encoding plasmalemma and tonoplast proteins from a high-efficiency expression library Plant J 1999, 17(1) 111-118
[3] Starkey MP,Umrania Y,Mundy CR,et al Reference cDNA library facilities available from European Sources Mol Biotechnol,1998,9(1) 35-57
[4] Sambrook J,Fntsch E F,Mamatis T Molecular cloning-a laboratory manual [M] Second edition New York Cold spring harbor laboratory press, 1989 396-431
[5] Chomczynski P,Sacchi N Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extration Analy Biochem, 1987,162(1) 156-159
[6] Verma I M Reverse transcripatase In Boyer PD ed The enzymes 3rd New York Academic Press 1981 14 87-95
[7] Christian E Gruber,Berns A,et al Construction of SuperScriptTM Human cDNA Libraries Focus, 17(2) 43-44
[8] Lisa A Peterson An Improved Method for Construction of Directionally Cloned cDNA Libraries from Microdissected Cells Cancer Research, 1998,58 5326-5328
[9] Gubler U,Hohman BJ A sample and very efficient method for generating cDNA library Gene,1983,25(2-3) 263-269
[10]卢圣栋主编 现代分子生物学实验技术[M] 第二版 北京 中国协和医科大学出版社，1999 327
[11]James M D'Alessio LAMBDA ZIPLOX~(TM) Automatic Subcloning of cDNA Focus,14(3) 76-79
[12]Clarke L,Cardon J A colony bank containing sythetlc Col E,hybrid plasmids represemative of the entire E Coli genome Cell, 1976, 9(1) 91-95
[13]Maniatis T, Kee SG, Efstratladis A,et al Amplification and characterization of a beta-globm gene synthesized in vitro et al Cell 1976, 8 163
[14]Adams MD,Dubrick M,Anthony R,et al Sequence identification of 2,375 human brain genes Nature, 1992,355(6359～6363) 632～634
[15]Adams S,Kerlavage A R, Fleischmann RD, et al Initial assessment of human gene diversity and expression patterns based upon 83 million ncletides of eDNA sequence[J] Nature, 1995, 377 3-174
[16]唐冬生，禹宽平，汤熙翔等 生物信息学技术克隆人类神经髓鞘蛋白零家族基因ACTA BIOCHNICAet BIOPHYSICA SINICA 2000,32(4)364-368
[17]Rossi D1,Vicai AP,Franz-Baccon K,et al Identification through biomformatics of two new macrophage proiflammatory human chemokines MIP-3 alpha and MIP-3 beta J Immunol, 1997,158 1033-
[18]Komeev S,Blackshaw S, Davies JA cDNA libraries from a few neural cells[J] Prog Neurobiol 1994,42 339-346
[19]Krizman DB Chuaqm R F Meitzer P S Constrctionn of a Representative cDNA library from Prostatic Intraepithelial Neoplasia[J] Can Res, 1996, 56 5380-5383
[2] Galaud JP,Carriere M,Pauly N,et al Construction of two ordered cDNA libraries enriched in genes encoding plasmalemma and tonoplast proteins from a high-efficiency expression library Plant J 1999, 17(1) 111-118
[3] Starkey MP,Umrania Y,Mundy CR,et al Reference cDNA library facilities available from European Sources Mol Biotechnol,1998,9(1) 35-57
[4] Sambrook J,Fntsch E F,Mamatis T Molecular cloning-a laboratory manual [M] Second edition New York Cold spring harbor laboratory press, 1989 396-431
[5] Chomczynski P,Sacchi N Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extration Analy Biochem, 1987,162(1) 156-159
[6] Verma I M Reverse transcripatase In Boyer PD ed The enzymes 3rd New York Academic Press 1981 14 87-95
[7] Christian E Gruber,Berns A,et al Construction of SuperScriptTM Human cDNA Libraries Focus, 17(2) 43-44
[8] Lisa A Peterson An Improved Method for Construction of Directionally Cloned cDNA Libraries from Microdissected Cells Cancer Research, 1998,58 5326-5328
[9] Gubler U,Hohman BJ A sample and very efficient method for generating cDNA library Gene,1983,25(2-3) 263-269
[10]卢圣栋主编 现代分子生物学实验技术[M] 第二版 北京 中国协和医科大学出版社，1999 327
[11]James M D'Alessio LAMBDA ZIPLOX~(TM) Automatic Subcloning of cDNA Focus,14(3) 76-79
[12]Clarke L,Cardon J A colony bank containing sythetlc Col E,hybrid plasmids represemative of the entire E Coli genome Cell, 1976, 9(1) 91-95
[13]Maniatis T, Kee SG, Efstratladis A,et al Amplification and characterization of a beta-globm gene synthesized in vitro et al Cell 1976, 8 163
[14]Adams MD,Dubrick M,Anthony R,et al Sequence identification of 2,375 human brain genes Nature, 1992,355(6359～6363) 632～634
[15]Adams S,Kerlavage A R, Fleischmann RD, et al Initial assessment of human gene diversity and expression patterns based upon 83 million ncletides of eDNA sequence[J] Nature, 1995, 377 3-174
[16]唐冬生，禹宽平，汤熙翔等 生物信息学技术克隆人类神经髓鞘蛋白零家族基因ACTA BIOCHNICAet BIOPHYSICA SINICA 2000,32(4)364-368
[17]Rossi D1,Vicai AP,Franz-Baccon K,et al Identification through biomformatics of two new macrophage proiflammatory human chemokines MIP-3 alpha and MIP-3 beta J Immunol, 1997,158 1033-
[18]Komeev S,Blackshaw S, Davies JA cDNA libraries from a few neural cells[J] Prog Neurobiol 1994,42 339-346
[19]Krizman DB Chuaqm R F Meitzer P S Constrctionn of a Representative cDNA library from Prostatic Intraepithelial Neoplasia[J] Can Res, 1996, 56 5380-5383