Separation And Identification Of Nuclear Matrix Proteins Expressed Differentially Between Human Esophageal Immortalized And Carcinomatous Cell Lines

Esophageal carcinoma(EC) is one of the most common malignant tumors in China, and its etiology and pathogenesis remain to be determined. Comparing with the nomal esophageal epithelial tissue, nuclear matrix proteins(NMPs) which differentially expressed in EC had been detected. This hints that some NMPs may take part in the generation and development of EC. However, which NMPs expressed differentially in EC remains unknown.. Base on this, the objective of this study was to separate and identify the differentially expressed NMPs between SHEE and SHEEC by 2-DE, MALDI-TOF- MS, RT-PCR, cDNA clone and sequencing analysis, which may contribute to further study the pathogenesis of esophageal carcinoma.Main procedures and methods:1 The NMPs were extracted from SHEE and SHEEC cell lines. The quality of NMPs was monitored by western blotting analysis. NMPs of SHEE and SHEEC were analyzed by 2-DE, silver staining and PDQuest6.2 image analysis software. Three spots in which differentially expressed NMPs was more obvious were selected and analyzed with MALDI- TOF-MS.2 STRBP8, one of the differentially expressed NMPs in EC, was further verified by RT-PCR, cDNA clone and sequence analysis: Total RNA was obtained from SHEE and SHEEC cell lines. The STRBP8 cDNA obtained by RT-PCR was linked to pGEM-T easy vector, and then the pGEM-T easy-STRBP8 was introduced into TOP10F' E. coli competence cell. The positive clones were obtained by screening, and then the plasmids of positive clones were sequenced and analyzed with BLAST.3 The structure and function of STRBP8 was preliminarily predict by bioinformatics.Main results:1 Western blotting analysis revealed that DNA topoisomerase II and PCNA were detected in NMPs and a majority of Histone were lost in NMPs of SHEE and SHEEC. three EC associated NMPs including cytoskeletal tropomyosin, FKBP6 and STRBP8 were preliminarily identified by 2-DE and MALDI-TOF-MS respectively.2 STRBP8 cDNA was amplified successfully by RT-PCR: Positive clones were obtained after the pGEM-T easy-STRBP8 was introduced into TOP10F' E. colicompetence cell. The result of sequence analysis was revealed that the sequence of STRBP8 cDNA was identical entirely to the published sequence of Genebank database.3 The structure and function of STRBP8 was preliminarily predict by bioinformatics. several glycosylation site and phosphorylation site were found in STRBP8, mostly random coil and partially a-helix and p-sheet were appeared in the secondary structure of STRBP8.Conclusion:1 2-DE-silver staining procedure was established and partial procedure was Optimized in our laboratory. A mature way for 2-DE image analysis was established.2 The differentially expressed NMPs extracted from SHEE and SHEEC were studied using 2-DE and MALDI-TOF-MS, and three differentially expressed NMPs were preliminarily identified. It hints that these proteins were connected with EC.3 STRBP8 mRNA was only detected in SHEEC by RT-PCR, cDNA clone and sequence analysis.4 the structure and function of STRBP8 was preliminarily predict by bioinformatics which made foundation for studying the function of STRBP8 in EC.