Expression Of Skp2 And Relationship With P27^kip1 Protein In HLEs

ObjectiveCataract is the world's first a blinding eye disease. In the global 40 million blind people, Cataract blindness due to about 46%. The primary means to current treatment of cataract is extraction of the lens opacification by surgery. But the occurrence of posterior capsular opacification (hereinafter PCO) after surgery, that is, after cataract, will cut down the elevated vision again after operations, impact of surgical results. The incidence rate of PCO is 30-50% in adult and 100% in children. Therefore, the control of PCO has been subject to the attention of the people. Currently, histopathology has been confirmed that reliquus epithelium of lens hyperplasia before capsule and ambitus, migrate to posterior capsular and metaplasia is the cause of PCO. To prevent the occurrence of PCO, we must prevent the proliferation of reliquus epithelium of lens.The proliferation of reliquus epithelium of lens also follow the cell cycle. The pathway of ubiquitin-proteasomes is the important quality control in protein, participating several cell physiological process, as the regulate of cell cycle, cellular proliferation and cell differentiation, as well as signal conduction et cetera. From the G1 phase to enter S phase, Skp2 is the essential factor to cell and the essential F-box family member to duplication of DNA duplication, mainly through the ubiquitin-proteasomes degradation pathway degradatiohn a wide range of target protein, closely related to the cell proliferation that regulate by cell cycle. At present, there is no reports about expression of Skp2 in HLEs and the effect on cell proliferation at home and abroad. Also there is no reports about research of relationship with p27^kip1 protein in HLEs. It's of actual significance to do research on expression of Skp2 and relationship with p27^kip1 protein in HLEs, which can stop the proliferation of lens epithelial cells from the perspective of the regulation of cell cycle and prevent happening of PCO.Methods1 Cell climbing slideInoculating the epithelium of lens in six shadow mask by 1×10⁶ cells per hole. Puting coverslips in the bottom of the hole and making cells grown adhere the slide. Adding each hole 2.5 ml by BFGF containing different concentrations of the 10%FBS culture medium. Control groups are joined with the same volume PBS of the 10%FBS culture medium. They are trained 48 hours in Sterile constant temperature box by 37℃, 5%CO₂,saturated humidity.2 immunocytochemistryCell climbing slide should be removed carefully, washed by PBS, fixed by ice acetone(4℃). We detect the expression of Skp2 in the epithelium of lens by immunocytochemistry.3.image collectUtilizing the HPIAS1000 high definition color pathoanalytical system, enlarging 400 times coincidence and random selecting 10 fields of vision no overlap from cell climbing slide, portraying the positive area of the cell which be measured, automatic measured and procure Integrated OD Average by computer.4. statistics analyzeAdopting one way analysis of variance and Spearman's correlation from ranks, all data are manipulated in SPSS 10.0 software, size of testα= 0.05. Results1.Detecting the expression of Skp2 in the epithelium of lens by immunocytochemistry.Skp2, p27^kip1, PCNA can be detected in epithelium of lens on cell climbing slide. Skp2 and PCNA are nuclear brown coloring, p27^kip1 is nuclear and cytoplasm brown coloring.2. image analysis resultDiscovery by I.O.D comparison:There was a significant difference between albumen express of Skp2, p27^kip1, PCNA cultured with bFGF containing different concentrations culture medium and blank control group.3 dependablity analytic resultDuring the process of epithelium of lens proliferation, the expression of Skp2 has a positive correlation with PCNA, (r=0.706, P<0.05) , has a negative correlation with p27^kip1(r=-0.679, P<0.05) . The expression of p27^kip1 has a positive correlation with PCNA (r=-0.668,P<0.05) .Conclusions1.Skp2 can be detected in epithelium of lens.2. Expression of Skp2 is strengthen with increasing concentration of bFGF.3. During the process of epithelium of lens proliferation,the expression of Skp2 has a positive correlation with PCNA,and has a negative correlation with p27^kip1.