The Inhibitory Effect Of Culture Supernatant Of Human GRC-1 Cells Transfected With PF4 CDNA On The VEGF Expression And The Growth Of ECV304 Cells

Primay renal cell carcinoma which account for about 2%-3% of all malignant solid tumours are less occurred.But in the field of urology, they are constantly occurred.In our country the renal cell carcinoma hold the second place inferior to the carcinoma of bladder in the urologic malignant tumours.And they are the most commonly occurred maglignant solid tumours in adult kidneys.But the operation treatment is regard as the only effective method.Additonal chemistical therapy and radiational therapy have not any effects on the renal cell carcinoma.The tolerance to the cytotoxicitic drugs may be the major course. But with the importance of antiogenesis increasing in the tumours' growth , incursion and metastasis , anti-antiogenesis have been raised as a new managment which come to be widely acknowledged.Platelet factor-4 (PF4) is a chemokine with high affinity for sulfated glycosaminoglycans like heparin and heparan sulfate. It is a 7.8kD proteoglycan .It is synthesised by megakaryocyte and storaged in a granules and excreted by activated blood plateles.Some experiments shown that human platelet factor4 inhibits tumor growth through anangiogenesis-dependent mechanism which can weaken the effecs ofVEGF,bFGF, MMP and inhibite the gene p21 down-regulating.AIM: To construct the eukaryotic expression vector pcDNA3-PF4-SS, and todetect the effect of the culture supernatant of transfected GRC-1 cells on theVEGF expression in transfected GRC-1 cells and the growth of ECV304cells.To investigate the inhibitive effects of hPF4 on GRC-1 cells andprovide some important basic documents to prevent and cure the kidneyneoplasms.Methods: 1. The construction and identification of the eukaryotic expression vector pcDNA3-PF4-SS. With the endonucleas Bgl II ,BamR I and T4DNA ligase hPF4 cDNA was connected to the recombinant eukaryotic expression vector pcDNA3-CD34-SS,which was identified with Bgl II /BamH I digestion. The eukaryotic expression vector pcDNA3-PF4-SS was constructed successfully.2. The transfection and identification of GRC-1 cells . The pcDNA3-PF4-SS was tranfected stably into GRC-1 cells with lipofectamine mediation.which was growing in log phase.With 400-1200mg/L drug dense the cells positive to G418 were chosen.When the drug dense reach 1000ug/ml,the controlled cells were completely dead. The transfected GRC-1 cells were cultured.With RT-PCRthe transfected GRC-1 cells were identified .3. The VEGF expression in transfected GRC-1 cells was detected by immunohistochemical staining.With VEGF polyclonal antibody the GRC-1-PF4 and GRC-1 cells were detected by immunohistochemical staining.and then to compare the each expression levels.4. The effect of the culture supernatant of transfected GRC-1 cells on ECV304 cells. Divided the ECV304 cells into three groups which weregrowing in log phase,and added the culture supernatant of GRC-1-PF4 cells, GRC-1cells and RPMI1640 culture media into each one . Detect the growth effects by MTT colorimetry and cells counting. The results was analysized by SPLM statistical software.Results: 1. The eukaryotic expression vector pcDNA3-PF4-SS was constructed and identified with Bgl II /BamH I digestion.A 250bp specific fragment was amplified with electrophoresis.which was the same size as the PF4 reported in literatures.This certified that PF4-cDNA had been linked into pcDNA3-CD34-S and pcDNA3-PF4-SS had been constructed successfully.2. A 250bp specific fragment was amplified by RT-PCR in the transfected GRC-1 cells but nothing was found in GRC-1cells.This certified that PF4-cDNA had been transfected into the GRC-1 cells .called GRC-1-hPF4 cells3. The VEGF expression in transfected GRC-1 cells was detected by immunohistochemical staining.The immunocytohistochemical staining showed that the pale-brown positive reactant could be seen in the cytoplasm and cytomembrane of GRC-1 cells transfected by pcDNA3-PF4-SS, and the colour is lighter than the ones not tran...