应用基因芯片技术探讨口腔扁平苔藓发病机制的研究
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摘要
目的:口腔扁平苔藓是一种常见的非感染性口腔黏膜疾病,不易治愈,有一定的癌变倾向。该病好发于中年人,多见于40岁以上妇女,随年龄增长症状加重。该病病因不明,发病机制错综复杂。近年来,口腔扁平苔藓的癌变病例报道呈上升趋势,因此,对口腔扁平苔藓发病机制的研究成为口腔医学研究的热点。基因芯片技术是目前国际上兴起不久且发展迅速的一项生物技术,它在生命科学领域扮演着越来越重要的角色。基因芯片技术是目前基因研究方面最先进、最有效的方法之一,随着生物学技术的发展,基因芯片技术越来越广泛应用于疾病发病机制的研究,从而发现差异表达基因。本实验应用基因芯片技术筛选口腔扁平苔藓与正常口腔黏膜组织差异表达基因及其信号传导通路,分析口腔扁平苔藓发病相关的差异表达基因和信号传导通路,进而对口腔扁平苔藓的发病机制进行深入研究。
方法:
1样本采集
所有病变组织均来源于2008年5月至11月河北医科大学第四医院口腔科临床诊断为口腔扁平苔癣的患者,并经知情同意取病理时切取少量病变黏膜(组织病理诊断为口腔扁平苔藓的继续保留)。标本于切除后10 min内装入经无菌DEPC处理的冻存管中,然后储存于液氮罐中转运到低温冰箱中保存。对照正常组织来源于河北医科大学第四医院口腔科无菌条件下健康者拔智齿时切除的龈黏膜或口腔整复术中切除的口腔黏膜,保存方法同OLP组织标本。
2芯片的制作
由上海康成技术有限公司提供的含有32 050个基因的高密度基因组芯片(phalanx台湾)。取适量(50~100 mg)正确保存的OLP组织样本和正常组织,分别使用BioPulverize冰冻粉碎组织,加1ml的RNA抽提试剂Trizol(Invitrogen),使用Mini-Bead-Beater-16匀浆后抽提RNA。使用Nanodrop测定RNA在分光光度计230 nm、260 nm和280 nm的吸收值,以计算浓度并评估纯度。用甲醛电泳试剂进行变性琼脂糖凝胶电泳,检测RNA纯度及完整性。取得RNA QC报告。经检测的RNA样本是高质量的,完整的,没有RNase污染,没有DNA污染,继续实验。样本RNA进行逆转录反应合成cDNA,cDNA第二链合成,aRNA合成及纯化,荧光标记aRNA并纯化。使用Nanodrop检测荧光标记效率,标记效率合格以保证后续芯片实验结果的可靠性。使用Ambion的RNA Fragmentation Reagents对标记好的aRNA进行片段化处理。在标准条件下将标记好的探针和高密度基因组芯片进行杂交。
3芯片扫描和图像分析
使用GenePix 4000B芯片扫描仪扫描芯片的荧光强度,并将实验结果转换成数字型数据保存,使用配套软件(Genepix Pro V6.0和Genesring)对原始数据进行分析运算,采用FoldChang和T-test方法筛选的两组样本间的差异表达基因和通路,以表达差异2倍为阳性,为有统计学意义。
4 RT-PCR验证结果的可靠性
本实验为了排除实验结果中筛选差异基因的错误和假阳性,对实验结果部分标本的部分基因采用RT-PCR验证。实验随机选取7例标本(3例正常和4例OLP)并随机选取3个表达上调的基因(PHF19、DBH、CD72)进行RT-PCR验证,为校正此差异,将RNA反转录合成cDNA。PCR扩增。引物序列为: GAPDH :反义5’GGGAAACTGTGGCGTGAT3’,正义:5’GAGTGGGTGTCGCTGTTGA3’,片段长度为299 bp。PHF19::反义F:5’CGGGAAGATCAAGAGGGTCA3’,正义: R:5’ATGCAGCGTCGGCAGAAC3’,片段长度为276 bp。DBH:反义F:5'TGGTGATAGAAGGACGAAACG3',正义: R:5'GCAGTAGCCAGTG AGGATGAA3',片段长度为158bp。CD72:反义F:5'CCAAATGGTGGTTCA GGGA3',正义: R:5'GGCACAGGTTCTTGTTGGC3',片段长度为265bp。PCR反应条件: 95℃变性3min, 40个PCR循环(94℃,20s;59℃,20s;72℃,30s);72 oC延伸5 min。PCR反应同时扩增GAPDH作为内参照,复性温度及循环次数根据不同引物及模板进行调整PCR产物与100 bp DNA Ladder在2%琼脂糖凝胶电泳,溴化乙锭染色,检测PCR产物是否为单一特异性扩增条带并进行灰度扫描成像。RT-PCR验证结果由Rotor-Gene Real-Time Analysis Software 6.0(Build 14)软件处理。
5统计学方法
应用Spss 13.0统计软件进行统计学处理,实验数据各组间比较利用单样本均数比较T检验分析,以P<0.05为差异有统计学意义。
结果:
1经过杂交筛选并与正常组织比较,从32 050个基因中筛选出有统计学意义的差异表达基因142个,其中上调表达基因25个,下调表达基因117个。这些差异基因从分子生物学角度来说大部分是细胞突变、生理性改变免疫应答、增殖等。从细胞组成角度来讲大多是细胞器、细胞外基质、蛋白质的合成等。从分子功能角度讲大多是黏附、催化活性、信号传导功能、酶的活性等。
2有差异表达的通路8个,为感染类疾病相关通路,氨基酸、蛋白质、碳水化合物的合成和代谢类、聚糖的生物合成和代谢类及外源性化学物质的合成和代谢类,与OLP密切相关通路是与参与感染类疾病的幽门螺杆菌感染上皮细胞信号传导通路(Epithelial cell signaling in Helicobacter pylori infection )。
结论:
1.应用基因芯片可初筛选出口腔扁平苔藓差异表达基因和信号传导通路,
2.口腔扁平苔藓发生、发展过程中存在着多条不同基因的表达差异,是一种多基因变化的级联反应。
3.口腔扁平苔藓的发生、发展过程中存在着信号传导功能通路表达调控的改变。
Objective:Oral Lichen Planus(OLP) is one most non-infectious common diseases mucous membrane of mouth ,not easy to cure,there is a certain tendency to cancer.This disease predilection in the middle-aged,more common in woman over 40 years of age,the symptom increased with age. The disease of unknown etiology and pathogenesis of complex.
In recent years, the reported cases of oral lichen planus cancerous upward trend, therefore, the pathogenesis of oral lichen planus oral medical research studies to become hot spots. Gene chip technology is currently the international community will soon rise and rapid development of a biotechnology, life sciences, it is playing an increasingly important role. Gene chip technology is currently the most advanced genetic research, the most effective methods, with the development of biological technology, gene chip technology becomes more widely used in pathogenesis studies, to discover differentially expressed genes. In this study, cDNA microarray screening of oral lichen planus and normal oral mucosa of differentially expressed genes and their signal transduction pathways, analysis of oral Lichen Planus of the differentially expressed genes and signal transduction pathways, and then shudied the pathogenesis of oral lichen planus
Method:
1 Sample collection
All Samples were collected from May to November in 2008 in the fourth Hospital of Hebei Medical University, the clinical diagnosis of dental patients with oral lichen planus, and after informed consent to take a small amount of pathological lesions were cut when the mucosa ( histopathological diagnosis of oral lichen planus to reservations). Specimen was loaded in the frozen pipes which were sterile DEPC treated and then stored in liquid nitrogen tanks in transit to save the low-temperature refrigerator within 10 min after excision. The control normal tissues derived from the Fourth Hospital of Hebei Medical University, dental sterile conditions, healthy pulling wisdom teeth removed when the gingival or oral mucosa plasty in the removal of oral mucosa .the preservation method is identical with OLP tissue
2 Chips
The high-density arrays containing 32 050 genes in the genome were supplied by Shanghai Kangchen Technology Limited (phalanx Taiwan).To taked the appropriate amount tissue (50 ~ 100 mg) of the OLP tissue samples and normal tissues saved correctly,, using the BioPulverize frozen crushed organizations respectively, plus 1ml of RNA extraction reagent Trizol (Invitrogen), using the Mini-Bead-Beater-16 after the extraction of homogenized RNA. Determination of RNA using the Nanodrop spectrophotometer 230 nm, 260 nm and 280 nm absorption values to calculate the concentration and to assess the purity. Electrophoresis reagents with formaldehyde denaturing agarose gel electrophoresis to detect RNA purity and integrity. To obtain RNA QC report. The detection of high-quality RNA samples, complete, no RNase contamination, there is no DNA contamination, continue the experiment. RNA samples were reverse transcription reaction synthesis cDNA, cDNA second strand synthesis, aRNA synthesis and purification, fluorescent labeling and purification of aRNA. Nanodrop test the efficiency of the use of fluorescent marker, marking the efficiency of compliance in order to ensure the reliability of experimental results the follow-up chips. The use of Ambion RNA Fragmentation Reagents marked with a good aRNA for fragment processing. Under standard conditions will mark a good probe and high-density genomic hybridization chip.
3 Chip scanning and image analysis
Scaned the chip fluorescence intensity using the GenePix 4000B scanner and converted the results into numeric data retention, to analyze the raw data calculations using of software packages (Genepix Pro V6.0 and Genesring) and filt out the differentially expressed genes of two groups between the sample and channels by FoldChang and T-test method of screening, to express the difference between two times the positive, to have statistical significance.
4 RT-PCR verified the reliability of the results
In order to exclude errors and false positive results in gene selection in this experiment,we validated the results of some of the experimental samples part of the gene using RT-PCR validation.7 cases were randomly selected experimental samples (3 normal and 4 cases of OLP) and randomly selected three upregulated genes (PHF19, DBH, CD72) for RT-PCR validation, in order to correct this difference, the synthesis of RNA reverse transcription cDNA. PCR amplification. Primer sequence: GAPDH:antisense :5’GGGAAACTGTGGCGTGAT3’,Justice:5’GAGTGGGTGTCGCTGTTGA3’, fragment length 299 bp. PHF19:: anti-sense F: 5’CGGGAAGATCAAGA GGGTCA3’,Justice:R:5’ATGCAGCGTCGGCAGAAC3’, fragment length 276 bp. DBH: antisense F: 5’TGGTGATAGAAGGACGAAACG3’, Justice:R:5’GCAGTAGCCAGTG AGGATGAA3’, fragment length of 158bp. CD72: antisense F: 5’CCAAATGGTGGTTCAGGGA3’,Justice:R:5’GGCA CAGGTTCTTGTTGGC3’, fragment length of 265bp. PCR reaction conditions: 95℃denaturation 3min, 40 Ge PCR cycles (94℃, 20s; 59℃, 20s; 72℃, 30s); 72℃extension of 5 min. PCR reaction also amplified GAPDH as an internal reference, the annealing temperature and cycle times according to different primers and templates to adjust PCR product with 100 bp DNA Ladder 2% agarose gel electrophoresis, ethidium bromide staining to detect whether the PCR product a single specific amplification bands and make grayscale imaging scans. RT-PCR validation results from the Rotor-Gene Real-Time Analysis Software 6.0 (Build 14) software processing.
5 Statistical Methods
Using Spss 13.0 statistical software for statistical processing, the experimental data comparison between each group using one-sample mean comparison T test analysis to P <0.05 for the difference statistically significant.
Results: 1 There were 142genes exhibited significant changes in expression levels in the OLP in comparison with normal tissues. 25 genes were overexp ressed more than doubled and 117 genes were under 2 expressed to below of the control level.The differentially expressed genes have different functions,the biological process functions are cellular process,physiological process,regulation of biological process and development;the cellular component functions are cell,organell,extracellular region,protein complex and extracellular matrix;the molecular functions are binding,catalytic activity,single transducer activity and transpoter activity.
2 There were 8 pathways different in the expression,they were Epithelial cell signaling in Helicobacter pylori infection-Homo sapiens (human),Cysteine metabolism-Homo sapiens (human),ECM-receptor interaction-Homo sapiens(human),Pyruvate metabolism-Homo sapiens (human),Beta Oxidation of Unsaturated Fatty AcidsDrug metabolism -cytochrome P450-Homo sapiens (human) , Tyrosine metabolism - Homo sapiens (human) and Glycosaminoglycan egradation - Homo sapiens (human). The Epithelial cell signaling in Helicobacter pylori infection pathway is closely related to infection diseases and take part in OLP. The gene GIT1 and CCL5 play a momentous part in OLP .
Conclusion:
1The differentially expressed genes and signal transduction pathways can be selected by microarray
2 There are multiple differeeeence in the expression of different genes,is a multi-gene changes in cascade in OLP occurrence and development.
3 Differentially expressed genes and pathways with different functions were revealed in OLP, which may play some roles in the progression of OLP.
方法:
1样本采集
所有病变组织均来源于2008年5月至11月河北医科大学第四医院口腔科临床诊断为口腔扁平苔癣的患者,并经知情同意取病理时切取少量病变黏膜(组织病理诊断为口腔扁平苔藓的继续保留)。标本于切除后10 min内装入经无菌DEPC处理的冻存管中,然后储存于液氮罐中转运到低温冰箱中保存。对照正常组织来源于河北医科大学第四医院口腔科无菌条件下健康者拔智齿时切除的龈黏膜或口腔整复术中切除的口腔黏膜,保存方法同OLP组织标本。
2芯片的制作
由上海康成技术有限公司提供的含有32 050个基因的高密度基因组芯片(phalanx台湾)。取适量(50~100 mg)正确保存的OLP组织样本和正常组织,分别使用BioPulverize冰冻粉碎组织,加1ml的RNA抽提试剂Trizol(Invitrogen),使用Mini-Bead-Beater-16匀浆后抽提RNA。使用Nanodrop测定RNA在分光光度计230 nm、260 nm和280 nm的吸收值,以计算浓度并评估纯度。用甲醛电泳试剂进行变性琼脂糖凝胶电泳,检测RNA纯度及完整性。取得RNA QC报告。经检测的RNA样本是高质量的,完整的,没有RNase污染,没有DNA污染,继续实验。样本RNA进行逆转录反应合成cDNA,cDNA第二链合成,aRNA合成及纯化,荧光标记aRNA并纯化。使用Nanodrop检测荧光标记效率,标记效率合格以保证后续芯片实验结果的可靠性。使用Ambion的RNA Fragmentation Reagents对标记好的aRNA进行片段化处理。在标准条件下将标记好的探针和高密度基因组芯片进行杂交。
3芯片扫描和图像分析
使用GenePix 4000B芯片扫描仪扫描芯片的荧光强度,并将实验结果转换成数字型数据保存,使用配套软件(Genepix Pro V6.0和Genesring)对原始数据进行分析运算,采用FoldChang和T-test方法筛选的两组样本间的差异表达基因和通路,以表达差异2倍为阳性,为有统计学意义。
4 RT-PCR验证结果的可靠性
本实验为了排除实验结果中筛选差异基因的错误和假阳性,对实验结果部分标本的部分基因采用RT-PCR验证。实验随机选取7例标本(3例正常和4例OLP)并随机选取3个表达上调的基因(PHF19、DBH、CD72)进行RT-PCR验证,为校正此差异,将RNA反转录合成cDNA。PCR扩增。引物序列为: GAPDH :反义5’GGGAAACTGTGGCGTGAT3’,正义:5’GAGTGGGTGTCGCTGTTGA3’,片段长度为299 bp。PHF19::反义F:5’CGGGAAGATCAAGAGGGTCA3’,正义: R:5’ATGCAGCGTCGGCAGAAC3’,片段长度为276 bp。DBH:反义F:5'TGGTGATAGAAGGACGAAACG3',正义: R:5'GCAGTAGCCAGTG AGGATGAA3',片段长度为158bp。CD72:反义F:5'CCAAATGGTGGTTCA GGGA3',正义: R:5'GGCACAGGTTCTTGTTGGC3',片段长度为265bp。PCR反应条件: 95℃变性3min, 40个PCR循环(94℃,20s;59℃,20s;72℃,30s);72 oC延伸5 min。PCR反应同时扩增GAPDH作为内参照,复性温度及循环次数根据不同引物及模板进行调整PCR产物与100 bp DNA Ladder在2%琼脂糖凝胶电泳,溴化乙锭染色,检测PCR产物是否为单一特异性扩增条带并进行灰度扫描成像。RT-PCR验证结果由Rotor-Gene Real-Time Analysis Software 6.0(Build 14)软件处理。
5统计学方法
应用Spss 13.0统计软件进行统计学处理,实验数据各组间比较利用单样本均数比较T检验分析,以P<0.05为差异有统计学意义。
结果:
1经过杂交筛选并与正常组织比较,从32 050个基因中筛选出有统计学意义的差异表达基因142个,其中上调表达基因25个,下调表达基因117个。这些差异基因从分子生物学角度来说大部分是细胞突变、生理性改变免疫应答、增殖等。从细胞组成角度来讲大多是细胞器、细胞外基质、蛋白质的合成等。从分子功能角度讲大多是黏附、催化活性、信号传导功能、酶的活性等。
2有差异表达的通路8个,为感染类疾病相关通路,氨基酸、蛋白质、碳水化合物的合成和代谢类、聚糖的生物合成和代谢类及外源性化学物质的合成和代谢类,与OLP密切相关通路是与参与感染类疾病的幽门螺杆菌感染上皮细胞信号传导通路(Epithelial cell signaling in Helicobacter pylori infection )。
结论:
1.应用基因芯片可初筛选出口腔扁平苔藓差异表达基因和信号传导通路,
2.口腔扁平苔藓发生、发展过程中存在着多条不同基因的表达差异,是一种多基因变化的级联反应。
3.口腔扁平苔藓的发生、发展过程中存在着信号传导功能通路表达调控的改变。
Objective:Oral Lichen Planus(OLP) is one most non-infectious common diseases mucous membrane of mouth ,not easy to cure,there is a certain tendency to cancer.This disease predilection in the middle-aged,more common in woman over 40 years of age,the symptom increased with age. The disease of unknown etiology and pathogenesis of complex.
In recent years, the reported cases of oral lichen planus cancerous upward trend, therefore, the pathogenesis of oral lichen planus oral medical research studies to become hot spots. Gene chip technology is currently the international community will soon rise and rapid development of a biotechnology, life sciences, it is playing an increasingly important role. Gene chip technology is currently the most advanced genetic research, the most effective methods, with the development of biological technology, gene chip technology becomes more widely used in pathogenesis studies, to discover differentially expressed genes. In this study, cDNA microarray screening of oral lichen planus and normal oral mucosa of differentially expressed genes and their signal transduction pathways, analysis of oral Lichen Planus of the differentially expressed genes and signal transduction pathways, and then shudied the pathogenesis of oral lichen planus
Method:
1 Sample collection
All Samples were collected from May to November in 2008 in the fourth Hospital of Hebei Medical University, the clinical diagnosis of dental patients with oral lichen planus, and after informed consent to take a small amount of pathological lesions were cut when the mucosa ( histopathological diagnosis of oral lichen planus to reservations). Specimen was loaded in the frozen pipes which were sterile DEPC treated and then stored in liquid nitrogen tanks in transit to save the low-temperature refrigerator within 10 min after excision. The control normal tissues derived from the Fourth Hospital of Hebei Medical University, dental sterile conditions, healthy pulling wisdom teeth removed when the gingival or oral mucosa plasty in the removal of oral mucosa .the preservation method is identical with OLP tissue
2 Chips
The high-density arrays containing 32 050 genes in the genome were supplied by Shanghai Kangchen Technology Limited (phalanx Taiwan).To taked the appropriate amount tissue (50 ~ 100 mg) of the OLP tissue samples and normal tissues saved correctly,, using the BioPulverize frozen crushed organizations respectively, plus 1ml of RNA extraction reagent Trizol (Invitrogen), using the Mini-Bead-Beater-16 after the extraction of homogenized RNA. Determination of RNA using the Nanodrop spectrophotometer 230 nm, 260 nm and 280 nm absorption values to calculate the concentration and to assess the purity. Electrophoresis reagents with formaldehyde denaturing agarose gel electrophoresis to detect RNA purity and integrity. To obtain RNA QC report. The detection of high-quality RNA samples, complete, no RNase contamination, there is no DNA contamination, continue the experiment. RNA samples were reverse transcription reaction synthesis cDNA, cDNA second strand synthesis, aRNA synthesis and purification, fluorescent labeling and purification of aRNA. Nanodrop test the efficiency of the use of fluorescent marker, marking the efficiency of compliance in order to ensure the reliability of experimental results the follow-up chips. The use of Ambion RNA Fragmentation Reagents marked with a good aRNA for fragment processing. Under standard conditions will mark a good probe and high-density genomic hybridization chip.
3 Chip scanning and image analysis
Scaned the chip fluorescence intensity using the GenePix 4000B scanner and converted the results into numeric data retention, to analyze the raw data calculations using of software packages (Genepix Pro V6.0 and Genesring) and filt out the differentially expressed genes of two groups between the sample and channels by FoldChang and T-test method of screening, to express the difference between two times the positive, to have statistical significance.
4 RT-PCR verified the reliability of the results
In order to exclude errors and false positive results in gene selection in this experiment,we validated the results of some of the experimental samples part of the gene using RT-PCR validation.7 cases were randomly selected experimental samples (3 normal and 4 cases of OLP) and randomly selected three upregulated genes (PHF19, DBH, CD72) for RT-PCR validation, in order to correct this difference, the synthesis of RNA reverse transcription cDNA. PCR amplification. Primer sequence: GAPDH:antisense :5’GGGAAACTGTGGCGTGAT3’,Justice:5’GAGTGGGTGTCGCTGTTGA3’, fragment length 299 bp. PHF19:: anti-sense F: 5’CGGGAAGATCAAGA GGGTCA3’,Justice:R:5’ATGCAGCGTCGGCAGAAC3’, fragment length 276 bp. DBH: antisense F: 5’TGGTGATAGAAGGACGAAACG3’, Justice:R:5’GCAGTAGCCAGTG AGGATGAA3’, fragment length of 158bp. CD72: antisense F: 5’CCAAATGGTGGTTCAGGGA3’,Justice:R:5’GGCA CAGGTTCTTGTTGGC3’, fragment length of 265bp. PCR reaction conditions: 95℃denaturation 3min, 40 Ge PCR cycles (94℃, 20s; 59℃, 20s; 72℃, 30s); 72℃extension of 5 min. PCR reaction also amplified GAPDH as an internal reference, the annealing temperature and cycle times according to different primers and templates to adjust PCR product with 100 bp DNA Ladder 2% agarose gel electrophoresis, ethidium bromide staining to detect whether the PCR product a single specific amplification bands and make grayscale imaging scans. RT-PCR validation results from the Rotor-Gene Real-Time Analysis Software 6.0 (Build 14) software processing.
5 Statistical Methods
Using Spss 13.0 statistical software for statistical processing, the experimental data comparison between each group using one-sample mean comparison T test analysis to P <0.05 for the difference statistically significant.
Results: 1 There were 142genes exhibited significant changes in expression levels in the OLP in comparison with normal tissues. 25 genes were overexp ressed more than doubled and 117 genes were under 2 expressed to below of the control level.The differentially expressed genes have different functions,the biological process functions are cellular process,physiological process,regulation of biological process and development;the cellular component functions are cell,organell,extracellular region,protein complex and extracellular matrix;the molecular functions are binding,catalytic activity,single transducer activity and transpoter activity.
2 There were 8 pathways different in the expression,they were Epithelial cell signaling in Helicobacter pylori infection-Homo sapiens (human),Cysteine metabolism-Homo sapiens (human),ECM-receptor interaction-Homo sapiens(human),Pyruvate metabolism-Homo sapiens (human),Beta Oxidation of Unsaturated Fatty AcidsDrug metabolism -cytochrome P450-Homo sapiens (human) , Tyrosine metabolism - Homo sapiens (human) and Glycosaminoglycan egradation - Homo sapiens (human). The Epithelial cell signaling in Helicobacter pylori infection pathway is closely related to infection diseases and take part in OLP. The gene GIT1 and CCL5 play a momentous part in OLP .
Conclusion:
1The differentially expressed genes and signal transduction pathways can be selected by microarray
2 There are multiple differeeeence in the expression of different genes,is a multi-gene changes in cascade in OLP occurrence and development.
3 Differentially expressed genes and pathways with different functions were revealed in OLP, which may play some roles in the progression of OLP.
引文
1 LIN Mei. Discussion on evaluation of oral lichen planus′therapeutic effects[J]. Chin J Stomatol, 2005, 40(2):105- 107
2詹启敏.分子肿瘤学[M].北京:人民卫生出版社,2005,225-226,265-266
3 Bascones C,conzalez-Moles MA,Esparza G,et al.Apopotsis and cell cycle arrest in oral lichen planus Hypothesis on their possible influence on its malignant transformation[J].Arch oral Biol,2005,50(10):873-81
4程克斌,王乐民,部恒骏,等.肺栓塞一深静脉血栓形成相关基因群表达谱变化的研究.中华医学杂志,2007, 87(34):2420-2422
5 TOM P MONIE,ANDREW J PERRIN,JAMES R BIRTLEY,et al.Structural insights into the transcriptional and translational roles of Ebp[J].Eur Mole Biol Orga(EMBO) 2007,26:3936–3944
6 XUEWU ZHANG,KERRY A,PICKIN,RON BOSE,et al.Inhibition of the EGF receptor by binding of MIG6 to an activating kinase domain interface[J] . NATURE, 2007,11;450(29): 741-745
7 Lee HJ,Song JY,Kim JW,et al.Association study of polymorphisms in synaptic vesicle-associated genes SYN2 and CPLX2 with schizophrenia[J].Behav Brain Funct.2005,31(1):15
8林元珠,高顺强,徐世正,主编.现代儿童皮肤病学[M],北京:学苑出版社,2008,463
9 XU J,LU S,TAO J,et al.CD72 polymorphism associated with child-onset of idiopathic thrombocytopenic purpura in Chinese patients[J].J Clin Immunol.2008,3;28(3):214-9
10 WANG J,TABA Y,PANG J,et al. GIT1 mediates VEGF-induced podosome formation in endiothelial cells:critical role for PLC gamma Arterioscler[J]. Thromb Vasc Biol.2009.9;29(2):202-8
11 SATO H,SUZUKI-INOUE K,INOUE O,et al.Regualation of adaptor protein GIT1 in platelets,leading to the interaction between GIT1 andintegrin alpha(11b)beta3[j].Biochem Biophys Res Commun.2008,368(1):157-61
12 STOCKTON R,REUTERSHAN J,SCOTT D,et al.Induction of vascular permeability:beta PIX and GIT1 scaffold the activation of extracellular signal-regulated kinase by PAK[J].Mol Biol Cell.2007,18(6):2346-55
13 JONE NP,KATAN M.Role of phospholipase C gamma in cell spreading requires association with a beta_Pix/GIT1-containing complex,leading to activation of Cdc42 and Rac[J]Mol cell Biol 2007,27(16):5790-805
14 HONG SJ,CHOI HJ,HONG S,et al.Transcription factor GATA-3 regulates the transcriptional activity of dopamine Mol Immunol, 2007 ,44(12):3100-3111
15 20 Stehlik C, Hayashi H, Pio F, Godzik A, et al. CARD6 is a modulator of NF-kappa B activation by Nod beta-hydroxylase by interacting with Sp1 and AP4[J].neurochem Res.2008,33(9):1821-31
16 LASKY-SU J,NEALE BM,FRANKE B,et al. Genome-wide association scan of equantitative traits for attention deficit hyperactivity disorder indentifies novel associations and confirms candidate gene associations[J].Am J Med Genet B Neuropsychiatr Genet.2008, 147B(8): 1345-54
17 TOGSVERD M,WERGE TM,TANKO LB,et al.Cognitive performance in elderly women:significance of the 19bp insertion/deletion polymorphism in the 5' flank of the dopamine beta-hydroxylase gene, educational level,body fat measures,serum triglyceride,alcohol consumption and age[J]. J Geriatr Psychiatry.2007,22(9):883-9
18 Magalh?es A, Marcos NT, Carvalho AS, et al. Helicobacter pylori cag pathogenicity island-positive strains induce syndecan-4 expression in gastric epithelial cells.FEMS Immunol Med Microbiol, 2009 ,56(3): 223-232.
19 Weidinger S, Klopp N, Rummler L, et al.Association of NOD1 polymorphisms with atopic eczema and related phenotypes.J Allergy Clin Immunol, 2005 ,116(1):177-184.
20 Uehara A, Fujimoto Y, Fukase K, et al.Various human epithelial cells express functional Toll-like receptors, NOD1 and NOD2 to produce anti-microbial peptides, but not proinflammatory cytokines.1- and Cardiak-mediated pathways.J Biol Chem,2003 ,278(34):31941-31949
21 Chuang JY, Yang WH, Chen HT, et al. CCL5/CCR5 axis promotes the motility of human oral cancer cells. J Cell Physiol, 2009 ,220(2):418-426.
22 ALESSANDRA MARINI, MD,_MARTIN WAGENMANN, MD,et al. Squamous Cell Cancer and Human Papillomavirus Infection in Oral Lichen Planus[M]: the American Society for Dermatologic Surgery, Blackwell Publishing 2007,33(6):756–760
23程克斌,王乐民,部恒骏,等.肺栓塞一深静脉血栓形成相关基因群表达谱变化的研究[J].中华医学杂志.2007,87(34):2420-2422
1 Campbell AM , Fckdahl TT , Fowlks E , et al. Colabative programs : genome consortium for axctive teaching ( GCAT) [J ] . Science ,2006 ,311 :1103-1104
2詹启敏.主编.分子肿瘤学[M].北京:人民卫生出版社,2005:619
3周小玉,许进明,华岚,等.微柱凝胶法鉴定ABO血型正反定型不一致原因分析[J ] .临床输血与检验, 2007 , 9(4) :367
4 Zhou L, Lim QE, Wan G,et al. Normalization with genes encoding ribosomal proteins but not GAPDH provides an accurate quantification of gene expressions in neuronal differentiation of PC12 cellsal. [J ]BMC Genomics. 2010 ,29(11):75 [Epub ahead of print]
5胡玉崇,唐慧君.应用基因芯片筛查宫颈癌的相关基因.[J ]内蒙古医学杂志,2008,40(1)43-83
6宋健.胰腺癌甲基化异化基因的检测、芯片筛查和诊断研究第二军医大博士论文2008
7詹启敏.主编.分子肿瘤学[M].北京:人民卫生出版社,2005:225-226,265-266
8李秉琦.主编.口腔黏膜病学[M].第二版北京:人民卫生出版社.2003,83-85
9 Petruzzi M, De BenedittisM. Oral lichen planus: a preliminary clini-cal study on treatment with tazarotene. Oral Dis ,2002,8:291-295
10张陆壮.中国医科大学硕士学位论文2003年5月
11 Ran Ping,Yong Jun-guang,Shi Yong-de,et al.Analysis and significance of whole blood apparent viscosity,casson viscosity and yield stree in hemorheology.Chinese Journal of Clinical Rehabilitation. 2005, 15(9): 192-193
12何巍,季旭东,李龙江等.口腔鳞状细胞癌组织中Bax和CDK4的表达. [J ]郑州大学学报(医学版) 2003,39(6):1017-1019
13周刚.武汉大学博士学位论文2004年4月
14姚希,沈佳丽,殷操等.口腔扁平苔藓Caspase-3,TNF-α和TNFRΙ的表达及与细胞凋亡的关系. [J ]第四军医大学学报,2006,27(7):580-583
15张延琳,王嘉陵,丁蕾等.扁平苔藓患者血小板聚集和血液流变学研究. [J ]口腔临床医学杂志,2005,21(8):502-503
16刘青,周威,吴织芬等.口腔扁平苔藓中cyclinD1和E2F-1的表达及意义. [J ]临床口腔医学杂志2005,21(2):82-83
17章卫平,陈宇,耿宁等.口腔扁平苔藓中基质金属蛋白酶及其组织抑制剂的表达. [J ]中华口腔医学杂志,2006,41(7):420-421
18尚云峰,柳志文,付本燕等.口腔黏膜扁平苔藓患者口腔黏膜组织中cyclin D_1,ki-67及凋亡的表达研究. [J ]口腔医学,2005,25(1):19-21
19耿玉兰,李增宁,康琼英等.口腔扁平苔藓患者唾液bFGF检测及意义. [J ]实用口腔医学杂志,2005,18(3):355-356
20付洁,陈伟,孙正等.口腔扁平苔藓组织中表皮生长因子及其受体表达变化的研究[J ].中华口腔医学杂志,2005,40(6):455-458
21邸萍,高岩. CD44在口腔扁平苔藓中的表达及意义. [J ]现代口腔医学杂志, 2003,17(5):410-412
22许彦枝,尚丽乔,刘铁军.藓化饮对口腔黏膜癌前病变的逆转作用. [J ]肿瘤防治研究,2008,35(12):874-877
23 Ichimura M, Hiratsuka K, Ogura N,et al. Expression profile of chemokines and chemokine receptors in epithelial cell layers of oral lichen planus.[J] Oral Pathol Med. 2006,35(3):167-74
24 Mattila R, Alanen K, Syrj?nen S Immunohistochemical study on topoisomerase IIalpha, Ki-67 and cytokeratin-19 in oral lichen planus lesions[J ]. Arch Dermatol Res. 2007 ,298(8):381-8
25 Tao XA, Li CY, Xia J, Yang X ,et al.Differential gene expression profiles of whole lesions from patients with oral lichen planus[J ]. J Oral Pathol Med. 2009 ,38(5):427-33
26 Fan Y, Zhan Z, Liu J.Detection of differentially expressed genes in oral lichen planus[J ].Hua Xi Kou Qiang Yi Xue Za Zhi. 2007 ,25(4):378-82.
27 Jeffery I B, Higgins D G, Culhane A C. Comparison and evaluation of methods for generating differentially expressed gene lists from a) microarray data[J ]. BMC Bioinformatics, 2006, 26(7): 359
28 Wang S J, Tsai C A, et al. Selection of differentially expressed genes in microarray data analysis[J ]. Pharmacogenomics J, 2007,7(3):212-20.
29 Cui X, Churchill G A. Statistical tests for differential expression in cDNA microarray experiments[J ]. Genome Biol, 2003, 4(4): 210
30 Pavlidis P, Li Q, Noble W S. The effect of replication on gene expression microarray experiments[J ]. Bioinformatics, 2003,19 (13):1620-1627
31 Zhang M, Yao C, Guo Z, et al. Apparently low reproducibility of true differential expression discoveries in microarray studies[J ]. Bioinformatics, 2008, 24(18):2057-2063
32 Allison D B, Cui X, Page G P, et al. Microarray data analysis: from disarray to consolidation and consensus[J ]. Nat Rev Genet, 2006, 7(1): 55-65
33 Guo L, Lobenhofer E K, Wang C, et al. Rat toxicogenomic study reveals analytical consistency across microarray platforms[J ]. Nat a) Biotechnol, 2006, 24(9):1162-1169
34 Shi L, Reid L H, Jones W D, et al. The MicroArray Quality Control(MAQC) project shows inter- and intraplatform reproducibility of gene expression measurements[J ]. Nat Biotechnol, 2006, 24(9):1151-1161
35 Yang D, Li Y, Xiao H, et al. Gaining confidence in biological interpretation of the microarray data: the functional consistence of the significant GO categories[J ]. Bioinformatics, 2008, 24(2): 265-271
36 Breitling R, Armengaud P, Amtmann A, et al. Rank products: a simple, yet powerful, new method to detect differentially regulatedgenes in replicated microarray experiments[J ]. FEBS Lett, 2004, 573(1-3): 83-92
37姚晨,张敏,邹金凤,等.可识别多种癌症的基因功能模块[J ].中国科学C辑:生命科学2009 ,39(11): 1092 -1096
2詹启敏.分子肿瘤学[M].北京:人民卫生出版社,2005,225-226,265-266
3 Bascones C,conzalez-Moles MA,Esparza G,et al.Apopotsis and cell cycle arrest in oral lichen planus Hypothesis on their possible influence on its malignant transformation[J].Arch oral Biol,2005,50(10):873-81
4程克斌,王乐民,部恒骏,等.肺栓塞一深静脉血栓形成相关基因群表达谱变化的研究.中华医学杂志,2007, 87(34):2420-2422
5 TOM P MONIE,ANDREW J PERRIN,JAMES R BIRTLEY,et al.Structural insights into the transcriptional and translational roles of Ebp[J].Eur Mole Biol Orga(EMBO) 2007,26:3936–3944
6 XUEWU ZHANG,KERRY A,PICKIN,RON BOSE,et al.Inhibition of the EGF receptor by binding of MIG6 to an activating kinase domain interface[J] . NATURE, 2007,11;450(29): 741-745
7 Lee HJ,Song JY,Kim JW,et al.Association study of polymorphisms in synaptic vesicle-associated genes SYN2 and CPLX2 with schizophrenia[J].Behav Brain Funct.2005,31(1):15
8林元珠,高顺强,徐世正,主编.现代儿童皮肤病学[M],北京:学苑出版社,2008,463
9 XU J,LU S,TAO J,et al.CD72 polymorphism associated with child-onset of idiopathic thrombocytopenic purpura in Chinese patients[J].J Clin Immunol.2008,3;28(3):214-9
10 WANG J,TABA Y,PANG J,et al. GIT1 mediates VEGF-induced podosome formation in endiothelial cells:critical role for PLC gamma Arterioscler[J]. Thromb Vasc Biol.2009.9;29(2):202-8
11 SATO H,SUZUKI-INOUE K,INOUE O,et al.Regualation of adaptor protein GIT1 in platelets,leading to the interaction between GIT1 andintegrin alpha(11b)beta3[j].Biochem Biophys Res Commun.2008,368(1):157-61
12 STOCKTON R,REUTERSHAN J,SCOTT D,et al.Induction of vascular permeability:beta PIX and GIT1 scaffold the activation of extracellular signal-regulated kinase by PAK[J].Mol Biol Cell.2007,18(6):2346-55
13 JONE NP,KATAN M.Role of phospholipase C gamma in cell spreading requires association with a beta_Pix/GIT1-containing complex,leading to activation of Cdc42 and Rac[J]Mol cell Biol 2007,27(16):5790-805
14 HONG SJ,CHOI HJ,HONG S,et al.Transcription factor GATA-3 regulates the transcriptional activity of dopamine Mol Immunol, 2007 ,44(12):3100-3111
15 20 Stehlik C, Hayashi H, Pio F, Godzik A, et al. CARD6 is a modulator of NF-kappa B activation by Nod beta-hydroxylase by interacting with Sp1 and AP4[J].neurochem Res.2008,33(9):1821-31
16 LASKY-SU J,NEALE BM,FRANKE B,et al. Genome-wide association scan of equantitative traits for attention deficit hyperactivity disorder indentifies novel associations and confirms candidate gene associations[J].Am J Med Genet B Neuropsychiatr Genet.2008, 147B(8): 1345-54
17 TOGSVERD M,WERGE TM,TANKO LB,et al.Cognitive performance in elderly women:significance of the 19bp insertion/deletion polymorphism in the 5' flank of the dopamine beta-hydroxylase gene, educational level,body fat measures,serum triglyceride,alcohol consumption and age[J]. J Geriatr Psychiatry.2007,22(9):883-9
18 Magalh?es A, Marcos NT, Carvalho AS, et al. Helicobacter pylori cag pathogenicity island-positive strains induce syndecan-4 expression in gastric epithelial cells.FEMS Immunol Med Microbiol, 2009 ,56(3): 223-232.
19 Weidinger S, Klopp N, Rummler L, et al.Association of NOD1 polymorphisms with atopic eczema and related phenotypes.J Allergy Clin Immunol, 2005 ,116(1):177-184.
20 Uehara A, Fujimoto Y, Fukase K, et al.Various human epithelial cells express functional Toll-like receptors, NOD1 and NOD2 to produce anti-microbial peptides, but not proinflammatory cytokines.1- and Cardiak-mediated pathways.J Biol Chem,2003 ,278(34):31941-31949
21 Chuang JY, Yang WH, Chen HT, et al. CCL5/CCR5 axis promotes the motility of human oral cancer cells. J Cell Physiol, 2009 ,220(2):418-426.
22 ALESSANDRA MARINI, MD,_MARTIN WAGENMANN, MD,et al. Squamous Cell Cancer and Human Papillomavirus Infection in Oral Lichen Planus[M]: the American Society for Dermatologic Surgery, Blackwell Publishing 2007,33(6):756–760
23程克斌,王乐民,部恒骏,等.肺栓塞一深静脉血栓形成相关基因群表达谱变化的研究[J].中华医学杂志.2007,87(34):2420-2422
1 Campbell AM , Fckdahl TT , Fowlks E , et al. Colabative programs : genome consortium for axctive teaching ( GCAT) [J ] . Science ,2006 ,311 :1103-1104
2詹启敏.主编.分子肿瘤学[M].北京:人民卫生出版社,2005:619
3周小玉,许进明,华岚,等.微柱凝胶法鉴定ABO血型正反定型不一致原因分析[J ] .临床输血与检验, 2007 , 9(4) :367
4 Zhou L, Lim QE, Wan G,et al. Normalization with genes encoding ribosomal proteins but not GAPDH provides an accurate quantification of gene expressions in neuronal differentiation of PC12 cellsal. [J ]BMC Genomics. 2010 ,29(11):75 [Epub ahead of print]
5胡玉崇,唐慧君.应用基因芯片筛查宫颈癌的相关基因.[J ]内蒙古医学杂志,2008,40(1)43-83
6宋健.胰腺癌甲基化异化基因的检测、芯片筛查和诊断研究第二军医大博士论文2008
7詹启敏.主编.分子肿瘤学[M].北京:人民卫生出版社,2005:225-226,265-266
8李秉琦.主编.口腔黏膜病学[M].第二版北京:人民卫生出版社.2003,83-85
9 Petruzzi M, De BenedittisM. Oral lichen planus: a preliminary clini-cal study on treatment with tazarotene. Oral Dis ,2002,8:291-295
10张陆壮.中国医科大学硕士学位论文2003年5月
11 Ran Ping,Yong Jun-guang,Shi Yong-de,et al.Analysis and significance of whole blood apparent viscosity,casson viscosity and yield stree in hemorheology.Chinese Journal of Clinical Rehabilitation. 2005, 15(9): 192-193
12何巍,季旭东,李龙江等.口腔鳞状细胞癌组织中Bax和CDK4的表达. [J ]郑州大学学报(医学版) 2003,39(6):1017-1019
13周刚.武汉大学博士学位论文2004年4月
14姚希,沈佳丽,殷操等.口腔扁平苔藓Caspase-3,TNF-α和TNFRΙ的表达及与细胞凋亡的关系. [J ]第四军医大学学报,2006,27(7):580-583
15张延琳,王嘉陵,丁蕾等.扁平苔藓患者血小板聚集和血液流变学研究. [J ]口腔临床医学杂志,2005,21(8):502-503
16刘青,周威,吴织芬等.口腔扁平苔藓中cyclinD1和E2F-1的表达及意义. [J ]临床口腔医学杂志2005,21(2):82-83
17章卫平,陈宇,耿宁等.口腔扁平苔藓中基质金属蛋白酶及其组织抑制剂的表达. [J ]中华口腔医学杂志,2006,41(7):420-421
18尚云峰,柳志文,付本燕等.口腔黏膜扁平苔藓患者口腔黏膜组织中cyclin D_1,ki-67及凋亡的表达研究. [J ]口腔医学,2005,25(1):19-21
19耿玉兰,李增宁,康琼英等.口腔扁平苔藓患者唾液bFGF检测及意义. [J ]实用口腔医学杂志,2005,18(3):355-356
20付洁,陈伟,孙正等.口腔扁平苔藓组织中表皮生长因子及其受体表达变化的研究[J ].中华口腔医学杂志,2005,40(6):455-458
21邸萍,高岩. CD44在口腔扁平苔藓中的表达及意义. [J ]现代口腔医学杂志, 2003,17(5):410-412
22许彦枝,尚丽乔,刘铁军.藓化饮对口腔黏膜癌前病变的逆转作用. [J ]肿瘤防治研究,2008,35(12):874-877
23 Ichimura M, Hiratsuka K, Ogura N,et al. Expression profile of chemokines and chemokine receptors in epithelial cell layers of oral lichen planus.[J] Oral Pathol Med. 2006,35(3):167-74
24 Mattila R, Alanen K, Syrj?nen S Immunohistochemical study on topoisomerase IIalpha, Ki-67 and cytokeratin-19 in oral lichen planus lesions[J ]. Arch Dermatol Res. 2007 ,298(8):381-8
25 Tao XA, Li CY, Xia J, Yang X ,et al.Differential gene expression profiles of whole lesions from patients with oral lichen planus[J ]. J Oral Pathol Med. 2009 ,38(5):427-33
26 Fan Y, Zhan Z, Liu J.Detection of differentially expressed genes in oral lichen planus[J ].Hua Xi Kou Qiang Yi Xue Za Zhi. 2007 ,25(4):378-82.
27 Jeffery I B, Higgins D G, Culhane A C. Comparison and evaluation of methods for generating differentially expressed gene lists from a) microarray data[J ]. BMC Bioinformatics, 2006, 26(7): 359
28 Wang S J, Tsai C A, et al. Selection of differentially expressed genes in microarray data analysis[J ]. Pharmacogenomics J, 2007,7(3):212-20.
29 Cui X, Churchill G A. Statistical tests for differential expression in cDNA microarray experiments[J ]. Genome Biol, 2003, 4(4): 210
30 Pavlidis P, Li Q, Noble W S. The effect of replication on gene expression microarray experiments[J ]. Bioinformatics, 2003,19 (13):1620-1627
31 Zhang M, Yao C, Guo Z, et al. Apparently low reproducibility of true differential expression discoveries in microarray studies[J ]. Bioinformatics, 2008, 24(18):2057-2063
32 Allison D B, Cui X, Page G P, et al. Microarray data analysis: from disarray to consolidation and consensus[J ]. Nat Rev Genet, 2006, 7(1): 55-65
33 Guo L, Lobenhofer E K, Wang C, et al. Rat toxicogenomic study reveals analytical consistency across microarray platforms[J ]. Nat a) Biotechnol, 2006, 24(9):1162-1169
34 Shi L, Reid L H, Jones W D, et al. The MicroArray Quality Control(MAQC) project shows inter- and intraplatform reproducibility of gene expression measurements[J ]. Nat Biotechnol, 2006, 24(9):1151-1161
35 Yang D, Li Y, Xiao H, et al. Gaining confidence in biological interpretation of the microarray data: the functional consistence of the significant GO categories[J ]. Bioinformatics, 2008, 24(2): 265-271
36 Breitling R, Armengaud P, Amtmann A, et al. Rank products: a simple, yet powerful, new method to detect differentially regulatedgenes in replicated microarray experiments[J ]. FEBS Lett, 2004, 573(1-3): 83-92
37姚晨,张敏,邹金凤,等.可识别多种癌症的基因功能模块[J ].中国科学C辑:生命科学2009 ,39(11): 1092 -1096