ABSTRACT
Objective: To evaluate the effect of clusterin with and without leader sequence on overexpression preventing apoptosis in human prostate LNCaP cells. Methods:The plasmid pIRES2 EGFP was used to generate the clusterin expression constructs with full length or without the leader sequence (designated as pIRES2 EGFP/cluac, pIRES2 EGFP/clubc, respectively). Western blot analysis was employed to compare clusterin expression levels in the lysis and supernatant fluid of clusterin transfected LNCaP cells in vitro . The distribution of different functional domains of clusterin in cells was detected with Immunocytochemical staining. The clusterin's protective role of Na 2SeO 3 induced apoptosis in LNCaP cells was examined by flow cytometry (FCM) and fluorescence microscope. Results: Clusterin expression was detected in the lysis and supernatant fluid of pIRES2 EGFP/cluac transfected LNCaP cells, while clusterin was found only in lysis liquid of pIRES2 EGFP/clubc transfected LNCaP cells, but not found in their supernatant fluid. The distribution of cluserin in the plasm of pIRES2 EGFP/cluac transfected cells was aggregative, and on the other hand, clusterin distributed dispersedly in pIRES2 EGFP/clubc transfected cells. Its anti apoptotic property in LNCaP cells was proved by FCM and fluorescence microscope.Conclusion: It is apparent that clusterin plays an important role in preventing apoptosis in prostate cancer, and the presence of the leader sequence is necessary for clusterin's anti apoptotic function.
ABSTRACT
Objective To construct a suppression subtractive hybridization (SSH) library of human renal cell carcinoma. Methods Poly A + RNA was isolated from human renal cell carcinoma (RCC) tissues and matched normal kidney tissues, respectively. Then double strand cDNA were synthesized and restricted by Rsa I.RCC cDNA were divided into two groups and ligated with either adaptor 1 or adaptor 2.After RCC cDNA hybridized with normal kidney cDNA twice and underwent nested PCR,the PCR products were cloned into pT Adv vector and transformed E.coli TOP10F′.Some positive clones randomly picked up were digested and sequenced. Results The SSH library contained 414 positive clones. Random analysis of 265 clones with enzyme restriction showed that 246 clones contained cDNA fragments which were distributed mainly between 300～900 bp. Among 40 arbitrary clones derived from the above 246 clones, No.170 clone is a previously unknown sequence and the other 39 clones were derived from 35 known genes. Conclusions The quality of the SSH library of human RCC is reliable and its construction would lay the foundation for further screening differentially expressed genes in RCC.
ABSTRACT
Objective To investigate the effect and s ig nificance of GYLZ-RCC18 (Genebank accession number:BE825133) on RCC cell line GRC-1. Methods To detect the expression of GYLZ-RCC18 by means of RT-PCR in both the renal cell carcinoma cell line G RC-1 and the normal kidney cell line HK-2. 20bp GYLZ-RCC18 antisense oligon u clotide packed with liposome was transfected into GRC-1 cell.The change in gro wth speed,proliferation activity,apoptosis,mortality and morphology of GRC-1 w ere observed. Results The expression of GYLZ-RCC18 in R CC was much higher than in the normal kidney.After transfection of GYLZ-RCC18 a ntisense oligonuclotide,the mortality of GRC-1 increased significantly.At the s ame time the proliferation activity and growth speed were retarded remarkably.Th e antisense oligonuclotide induced apoptosis of GRC-1 throughout the observati on time. Conclusions GYLZ-RCC18,a RCC relative novel ge ne,overexpression would stimulate the growth and proliferation activity of RCC.A poptosis and mortality of the RCC cell were also retarded.So,transfection of ant isense oligonuclotide could inhibit the generation and development of RCC provid ing a new approach to the research of RCC.