| Sialidase is a kind of glycosidase,which can hydrolyzing sialic acid from non-deoxidized end glycogen and glycolipid。Human sialidases are divided three kinds, among which human membrane-associated membrane (hmSD) is more important . HmSD is unique in specifically hydrolyzing gangliosides, thought to participate in cell differentiation and transmembrane signaling, thereby playing crucial roles in the regulation of cell surface functions. Many recent studies indicate that high expression of hmSD in cancer cells leads to protection against programmed cell death, probably modulation of gangliosides. Therefore further investigation may provide a possible sialidase target for diagnosis and therapy of prostate cancer. Objective: To construct a expression plasmid carrying human membrane sialidase gene and in the pc-3 cell .and investigate the relativity of hmSD and cell apoptosis, so we can lay a foundation for study of the connection hmSD and caricinoma more.Methods:1. A complementary DNA (cDNA) clone for human membrane sialidase was obtained from testicle organization by RT-PCR, then subcloned into pECFP-C1 vector, which express green fluorescent protein(GFP)., to form the recombinant pEGFP-C1-hmSD plasmid. The plasmid was transfected into pc-s cells by the liposome mediated process. The cells expressing GFP were obtained and identified with fluorescent micorgraphy and detecting the activity of the sialidase.2. NaBT was added to the culture media of three kinds of cells for the same time and then the following assays were proceeded: ①Detect the inhibitory rate of cell growth by MTT assa; ②etect apoptosis and the changes of cell cycle by flow cytometry.Results: 1. The specific band ,the molecular size of 1300bp,could be seen in the garose gel eletrophorosis of PCR amplified human membrane sialidase2. The cDNA was approved to tally with the one on genebank absolutely after analysis 3. Digested with BglII and EcoRI, both the bands of about 1300bp were released from pMD18-T-hmSD and pEGFP-C1-hmSD recombinant plasmids 4. Using G418 for two weeks, we acquired PC-3 cells transfected with pEGFP-C1-hmSD and pEGFP-C1,both of which were observed green fluorescent, plasma membrane expression of GFP in the former.5. detecting the activity of the sialidase. of three kinds of cells, that of pECF-C1-hmSD is significantly up-regulated (p<0.05)。6. Cell growth was significantly inhibited after the addition of NaBT, while the rate of inhibitory of pECFP-C1-hmSD was lower significantly than the others7.The apoptosis rates of PC-3,pEGPF-C1,pEGFP-C1-hmSD were different by FCM, the latter is low remarkablyDiscuss: 1. Selecting appropriate organization and reagent is crucial since the amount of hmSD is few and increasing the reaction cycles in PCR would also be advisable. We adopt gold tag (Roche) taq, the first-class Taq, and hot-start PCR technique to insure to acquire the specific target gene.2. we selected pEGFP-C1 vector expressing green fluorescent protein as the eukaryon vector due to the lack of antibody of hmSD, GFP and hmSD gene are behind the same promoter, so once PC-3 cell transfected pEGFP-C1-hmSD recombination plasmid show green fluorescence and the activity of sialidase detected was proved high. ,we could say we had acquired the cell strain which could express hmSD stably.3.PC-3 cells transfected pEGFP-C1-hmSD and pEGFP-C1 plasmid were observed green fluorescence and plasma membrane expression of GFP in the former. The succedent mensurate can show we obtained the pc-3 cells transfected pEGFP-C1-hmSD expression plasmid successfully. the pEGFP-C1-hmSD plasmid in the pc-3 cells will be the potentially used in the investigation of tumors.4. The results of MTT and FCM demonstrated the remarkable lower apoptosis rates of pEGFP-C1-hmSD than the others. It suggests that hmSD may play crucial roles in apoptosis regulation of prostate cancer cells. According to recent reports, apoptosis is suppressed in hmSD over expressing cells with increase in Bcl-2 expression. hmSD inhibi... |
