摘要
目的:构建脾虚痰湿型肺癌肿瘤相关消减cDNA文库,以寻找脾虚痰湿型肺癌证候特异基因。方法:以原发性肺癌脾虚痰湿型的患者肿瘤组织及瘤旁正常组织为材料,分离Poly(A)RNA,反转录合成单链及双链cDNA,经Rsa I酶切,将肿瘤组织cDNA分成两组,分别与两种不同的接头连接,再与正常肺组织cDNA进行两次消减杂交和两次抑制性PCR扩增,将第两次PCR产物与pGEM-T载体连接,转化大肠杆菌DH5α感受态细胞进行文库扩增,构建了消减cDNA文库,同法建立反向消减cDNA文库。对经反向斑点杂交验证,从cDNA文库中随机挑选的200个含差异片段的克隆,碱裂解法提取DNA,从克隆5’端进行单向测序。EST测序序列行生物信息学分析,所有基因聚类用BLASTX(E values<10-5)和BLASTN(E values<10-10)搜索GenBank的非冗余核酸序列数据库及蛋白质库予以注释。结果:经文库扩增,随机挑选克隆进行PCR鉴定,插入片段主要分布在250～1000bp之间,并以反向斑点杂交法进一步验证其阳性克隆,最终获得251个阳性克隆,其中正向消减文库获得165个阳性克隆,反向消减文库获得86个阳性克隆。测序发现了正向消减文库差异基因22个,反向消减文库差异基因6个。结论:成功构建脾虚痰湿型肺癌肿瘤相关消减cDNA文库,发现了那些有明确生物学功能的证候相关基因,其基因表达及分布特征初步反映了肺癌脾虚痰湿型在分子层面的证候特点。
Objective： To construct a normalized cDNA library from lung cancer,to research the related genes of lung cancer with splenic asthenia and phlegm-damp type via functional genomics.Methods： The total RNA was extracted from normal tissue and pathological tissue of cancer with splenic asthenia and phlegm-damp type using TRIZOL respectively.mRNA was reversely transcripted to cDNA using superscript II reverse transcriptase.Second–strand synthesis was performed using DNA polymerase I.the purified cDNA was digested by Rsa I restriction enzyme.After adding adaptor,comparing cDNA of experiment group（as Tester or Driver） with normal control cDNA（as Tester or Driver）.SSH was used to screen differentially expressed genes associated with splenic asthenia and phlegm-damp type.The PCR product was ligated into T-easy plasmid vectors and transformed into E.coli DH 5α.Screened through the blue-white screening system,the positive recombinant clones were emplified by PCR method and identified with dot blot hybridization.The clones derived from different library were grown in LB medium containing ampicilin 100 μg/mL in 96 well plates for 16 h.The plasmids were extracted by alkaline lysis and used for capillary sequencing applying the standard primers,high-quality ESTs were generated.A total of 192 clones produce readable sequences from 5＇ end,yielding 192 ESTs with high quality.These sequences were clustered manually and were searched against the nr database（E values 10-5） and nt database（E values 10-10） for homology comparison using BLASTX and BLASTN.Results： A total of 251 clones were obtained as follows： 165 from the forward subtractive cDNA library and 86 from the reverse subtractive cDNA library.22 differentially expressed genes were found in forword normalized cDNA library,accordingly,6 differentially expressed genes in reword normalized cDNA library.Conclusion：A group of differentially expressed genes with identified function were found.It would be initial characteristic of specific transcripts of lung cancer with splenic asthenia and phlegm-damp type and lays a good foundation for the study of Zheng-hou genomics.
出处
《中华中医药学刊》
CAS
2010年第12期2539-2543,共5页
Chinese Archives of Traditional Chinese Medicine
基金
广东省中医药局科研课题(3060001)
广东省自然科学基金项目(8451040701001281)
关键词
肺癌
脾虚痰湿型
证候基因
抑制性消减杂交
CDNA文库
表达序列标签
转录组
差异表达
Lung cancer
Splenic asthenia and phlegm-damp type
Suppression Subtractive hybridization
cDNA library
Expressed sequences tags
Transcriptome
Differential expression
