The Research Of The Effect Of Protein Tyrosine Kinase Inhibitor Genistein On The In Vitro Behavior Of Renal Cell Carcinoma Cell Line GRC-1 And Its Relative Mechanism.

Renal cell carcinoma is a common urinary malignant carcinoma.According to epidermiological investigation,the oriental incidence is obliviously lower than occidental.A possible reason for this phenomenon is probably the dietary difference:the eastern people usually takes more soybean.We investeigate the effect of genistein-a soybean extracted tyrosine kinase inhibitor,on the in vitro behavior of renal cell carcinoma cell line GRC-1 to make it clear that whether protein tyrosine kinase inhibitor can be employed in the therapy of kidney neoplasm and in inhibiting tumor invasion.And relative mechanism is also discussed.OBJECTIVE:It's its invasive ability that makes tumor so harmful.And when necessary auxiliary surgical measures are taken,tumor can be cured if its invasive ability is curbed.Tyrosine kinase receptor can transfer exocytic signal to intracellular membrane receptor.Many membrane receptors transfer cellular signals via mitogen activated protein kinases(MAPK) pathway.The phosphorylation of growth factor receptor may induce the synthesis of many kinds of protein compounds(Shc/Grb2,Sos et al), catalysis the dissection of GTP,activate the GTP-binding protein Ras,whose activation may induce the activation of serine threonine protein kinase Rafl.And the activation of Rafl mayfurther induce the phosphorylation of a sery of reactive proteins who are involved in the activation and phosphorylation of MAPK.And invasion-related genes are thus activated.In this experiement,we expose the renal cell granular carcinoma cell GRC-1 to genistein,a PTK inhibitor.And with the help of electron microscope,flow cytometry,laser confocal microscope,high performance papillaryelecrophoresis,immunohistochemistry,etc,we investigated the variation of biological behavior of renal cell carcinoma under the exposure of protein tyrosine kinase,and further investigated the mechanism of invasive ability decrease.Thus give academic support for employing protein kinase inhibitor to kidney neoplasm chemotherapy. METHOD:1. We use MTT method to observe the effect of genistein on the growth and proliferation ability of renal cell carcinoma cells,and caculated the IC50.2. Using flow cytometry,we investigate the variation of cell cycle and the relation between variation and dosage.3. We investigated the variation of invasive ability with the help of transwell model.4. Observing the infrastructure change of the tumor cells with electron microscope.5. Learn the morphology change of the tumor cells and the arrange pattern of the intracellular vimentin with cell skeleton staining,laser confocal microscope,immunochemohistology to analysis the cell skeleton change after intercepting of the PTK signaling pathway andthe relationship between invasive ability and cell skeleton.6. With high performance papillary electrophoresis,we observed the effect of genistein on the tumor cell's secretion of MMP.7. We carried out cellular adhensive experiment to obsen'e the effect of genistein on the cellular adhension.And further investigated the relationship between invasive ability and adhensive ability.RESULTS:1. MTTGenistein can obviously inhibit tumor proliferation at low concentration and within short time.Its ICso is merely 13 u mo 1/1(3.5 V- g/ml),and is far lower than 10 u g/ml,the in vivo superior drug limit.2. Flow cytometry:Under the exposure of genistein,the mitosis behavior of the GRC-1 cells is axed.The proliferation of tumor cells is intercepted at G1/S,G2/M phases.3. Transwell:The invasive ability of tumor cells is sharply lowered.4. Electron microscope:After exposure of genistein,many apoptosis cells appeared. Lysosome allayed drastically, endoplasmic reticulum is dilated, mitochondrion swelled,and intracellular capsules appeared.Cell surface microvillus and cell junction are greatly reduced.And the remain cell juctions are severely deformed.Cell nucleus is swelled,and the nucleolus is dil...