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2 RT-PCR

3 % ras HER2/neu P53 P16 CTA CTA MAGE BAGE GAGE SSX NY-ESO-1 HOM-Tes-14/SCP-1 MAGE-1 3 NY-ESO-1 suppression subtractive hybridization, SSH cdna SSH GenBank 2

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5 [ ] [ ] 1 suppression subtractive hybridization SSH RT-PCR RNA cdna BiostarH RT-PCR 35 MAGE-1-3 SSX NY-ESO-1mRNA CTA 3 RT-PCR DNA 4 NY-ESO-1 35 [ ] 1 SSH EST GenBank C20/AF C34/AY RT-PCR AY (C12/PPP1CB C18/caluminin C38/S100A2 C40/HSNOV1 C41/OCIA) AF (C2/HSP90 C27/Ferrintin C33/gp96)

6 DNA 3 35 MAGE-1-3 SSX NY-ESO % 12/ % 20/ % 6/ % 6/35 20% 7/ % 9/ % 13/35 7 CTA 7 26/35 (74.3%) 23/35 (65.7%) RT-PCR CTA CTA p> NY-ESO-1 NY-ESO-1 mrna NY-ESO-1 [ ] 1 SSH 6 / CTA CTA CTA CTA - 5

7 ABSTRACT Identification and Preliminary Analysis of Tumor Associated Genes in Lung Cancer Candidate of M.D Hui Zhao Supervisor: Prof. Jiyan Xie Vice Supervisor: Prof. Guoliang Zhang Department of Thoracic Surgery, Peking University People s Hospital Objective: By way of analysis genes differentially expressed in human lung cancer and normal lung tissues, to identify tumor associated genes in lung cancer, and to analyse their roles in the development of lung cancer. Methods: (1) Through screening a subtracted cdna library of human lung cancer constructed by using suppression subtracted hybridization (SSH) method in our previous research works, clones representing mrnas that are truly differentially expressed in lung cancer but not in its adjacent non-cancerous tissues were selected out to be identified by semi-quantitative RT-PCR in 30 patients of no-small cell lung cancer (NSCLC). (2) The total RNA were extracted from the mass of no-small cell lung cancer (NSCLC) and the adjacent normal lung tissues in 10 patients. The cdnas were labeled by different fluorescence after the total RNA were probed by reverse transcriptase. The two different probes hybridized with BiostarH-1 1 microarray and the signals were analysed by computer. (3) Reverse-transcription polymerase chain reaction (RT-PCR) was used to investigate the expression of the MAGE-1,-3, SSX-1,-2,-4,-5, and NY-ESO-1 genes in 35 lung cancer samples and corresponding non-tumorous lung tissue. 3 samples selected randomly from each CTA PCR positive results were sequenced. (4) Immunohistochemistry was used to investigate the expression of NY-ESO-1 antigen at the protein level in above 35 lung cancer samples and corresponding non-tumorous lung tissue. Results: (1) 10 differentially expressed gene cdna fragments of lung cancer were obtained by SSH. Among them six were already known genes; two sequences were already identified but their functions were still unknown (hypothetical protein); 6

8 two were novel (GenBank accession number were AF and AY respectively). The results from semi-quantitative RT-PCR showed that the transcription expression level of PPP1CB, caluminin, S100A2, HSNOV1, OCIA and AY was down-regulated in some cases of NSCLC, while the transcription of HSP90, Ferrintin, gp96 and AF was up-regulated in some of NSCLC. (2) The data showed that there were a lot of different expressed genes between NSCLC and normal lung tissues. Compared with the normal lung tissues, 113 genes were up-regulated and 112 genes were down-regulated in tissues of NSCLC. The profile of these differentially expressed genes mainly focus on immunologic genes, metabolized genes, protein translation and synthesis, signal transduction genes, oncogenes and tumor suppressor genes, apoptosis related genes, etc. While others like cellular period protein, cytoskeleton and movement protein, receptor genes, DNA damage, repair and recombination, ect. have a little relevancy with this profile. (3) of 35 tumor samples, the MAGE-1,-3, SSX-1,-2,-4,-5, and NY-ESO-1 mrna expression were detected in 34.3% 12/35, 57.1% 20/35, 17.1% 6/35, 17.1% 6/35, 20% 7/35, 25.7% 9/35, 37.1% 13/35, respectively. At least one of these genes was expressed in 26/35 (74.3%). Two or more of these genes were expressed in 23/35 (65.7%). None non-tumours lung tissue was positive for these genes. The DNA sequence confirms that the RT-PCR products were truly CTA cdna. Moreover, there were no significant correlation between CTAs expression with clinicopathological parameters. (4) No nomal lung tissues showed NY-ESO-1 reactivity, and its expression in testis was restricted to germ cell particularly spermatogonia. Co-typing of NY-ESO-1 antigen and mrna expression in a panel of lung cancers showed a good correlation. There is great variability in NY-ESO-1 expression in individual tumors, ranging from an infrequent homogeneous pattern of staining to highly heterogeneous antigen expression. Conclusion: (1) Six already known genes identified by SSH technique have been already implicated in the pathogenesis of lung carcinogenesis, or they are involved in immunological defense mechanism in human body. Two hypothetical 7

9 proteins probably also play an important role in lung cancer pathogenesis. The function of two novel genes in lung carcinogenesis are under-researching. (2) cdna microarray is not only a powerful technique to study the gene expression profile, but also offers a useful way to understand the pathogenesis of lung cancer. (3) The cancer-testis antigens are expressed in a hige percentage of lung cancer, all of these genes are negative in non-tumorous lung tissue. These cancer-testis antigen gene products are potential targets for antigen-special immunotherapy of lung cancer. The coexpression pattern of these antigens provides a foundation for developing a polyvalent lung cancer vaccine. Key Words: lung cancer, gene, cancer-testis antigen, cdna Microarray, Reverse-transcription polymerase chain reaction (RT-PCR), immunohistochemistry 8

10 5 15% (1) 20 (2) 1 / SCLC myc C-myc N-myc L-myc bcl-2 NSCLC ras K-ras N-ras H-ras HER-2/neu (3,4,5) D1 NSCLC 9

11 1.1 myc myc C-myc N-myc L-myc DNA myc myc SCLC NSCLC myc myc myc myc (7) myc SCLC NSCLC (8) myc 1.2 ras ras K-ras N-ras H-ras N-ras 1p13 K-ras 12p12.1 H-ras 11p ras 4 21KD P21 G P21 GTP GDP ras GTP K-ras K-ras 12 K-ras 12 GGT K-ras 12 CGT GTT ras NSCLC 15-20% 20-30% K-ras K-ras 30% K-ras 12 G-T K-ras K-ras 9 10 ras 10

12 ras (11-14) ras ras ras ras ras ras (15) I II (16) 1.3 HER-2/neu EGF 7p12~13 EGFR HER-1 17q21 HER-2/neu c-erbb-2 17q11.2~12 EGFR HER-2/neu RNA HER-2/neu 1/3 NSCLC HER-2/neu 5 P185 HER-2/neu 17 HER-2/neu HER-2/neu HER-2/neu HER-2/neu Herceptin 18 Herceptin II Herceptin 11

13 1.4 bcl-2 bcl-2 18q21.3 p53 bcl-2 p53 bcl-2 19 bcl-2 bcl-2 98% D1 Cyclins CDK G0/G1 S D1 Cyclin D1 D1 NSCLC D1 CDK p53 p53 80% p53 p53 p KD p53 DNA p21 p53 Rb 12

14 S DNA p53 DNA p53 p53 80% p53 23 p p53 p % 41.2% 21 57% 53.6% 70.6% p53 p53 (24) 1996 p53 10 p % p % p %p53 p53 (25) p53 p Murakami 70 PCR-DGGE(PCR-denaturing gradient electrophoresis) p53 41% 29/70 p53 17 p53 37 P= p53 cox p53 Kaplan-meier p53 (26) p53 p53 p53 Roth 1993 p53 p53 (27) 13

15 p53 p53 (28) 1996 p (29) p53 p53 I II p53 p53 (30) 2.2 Rb Rb 13q14.2 Rb Rb 105KD G0/G1 S G0/G1 S SCLC Rb SCLC NSCLC Rb NSCLC Rb Rb G0/G1 S (31,32) D1 P16 NSCLC Rb D1 CDK 4 6 Rb p16 CDK4 6 Rb Rb 2.3 p16 p16 9 9q21 CDK 4 6 p16 14

16 2 CD2-1 MTS-1 p16 p16 p53 p16 CDK4 G1 S p53 p21 p16 p Minna 3p 9p p16 9p21 p53 ras p16 NSCLC p16 9%-11% p16 NSCLC 50% 2001 Jin 106 I~II p % 5 45% p % p=0.006 p16 (33) p16 p53 1/4 p16 -CDK4 p16 NSCLC MD Anderson p16 (34) NSCLC Kawabe p16 p53 p16 p16 (35) 2.4 FHIT 1982 Wang Pen 3 100% 3p 50% 80 3p 3p 3p14~ Ohta 3p14.2 fragile histidine 15

864 现代药物与临床 Drugs & Clinic 第 31 卷第 6 期 2016 年 6 月 of apoptosis related factors, decrease the incidence of adverse reactions, which is of great

864 现代药物与临床 Drugs & Clinic 第 31 卷第 6 期 2016 年 6 月 of apoptosis related factors, decrease the incidence of adverse reactions, which is of great 现代药物与临床 Drugs & Clinic 第 31 卷第 6 期 2016 年 6 月 863 曲妥珠单抗联合多西紫杉醇治疗 Her-2 阳性乳腺癌的临床研究 * 周永安, 刘训碧黄石市中心医院 ( 普爱院区 ) 乳腺肿瘤外科, 湖北黄石 435000 摘要 : 目的探析注射用曲