| PrefaceCataract is one of the most common eye diseases that causes visual disorder and blindness. At present, extracapsular cataract exraction ( ECCE) combined with the implantation of intraocular lenses is the most effective method to treat cataract . However, lens epithelial cells remained after the operation may invade the capsular membrane by migration and proliferation, which can reach the surface of intraocular lens and the posterior capsular, causing posterior capsular opacification (PCO) ,and effecting visual function. PCO incidence rate has declined greatly by various method, but it is still a intractable problem. Lens epithelial cells remained after the surgery proliferate as other cells according to cell proliferatory rule, which is effected by many factors. If the molecular mechanism of the lens epithelial cell proliferation can be clearified and its proliferation can be inhibited at molecular level, PCO prevention may be greatly improved.PCO is started by the inviromental stimulating signals. Many studies have shown that cellular factors are very important in the PCO formation. Fibroblast growth factor (FGF) is one of the most widely distributing cellular growth factors. Recent studies showed that basic fibroblast growth factor (bFGF) increased in aqueous humor soon after the cataract surgery. As one of the most important causes of PCO, bFGF is a starting factor in several signaling pathways, promoting lens epithelial cell proliferation and differentiation. Three subtypes of FGF receptors are all expressed in lens of many kinds of living things, but onlythe expression of FGF-R1 is constant . FGF-R1 may play a role in PCO formation. Phosphatidylinositol 3 kinase(PI-3k) / protein kinase B (PKB) is one of the most important signal transduction pathways that control cellular skeleton and apoptosis. We have attached great importance to the role of PI-3k / PKB in the regulation of cell proliferation . PKB, the most important part in PI-3k / PKB signal system, is single chain structured,and when Ser473 in the regulated area changes its structure after phosphoration, this change release its inhibition on catalytic area, thus the enzyme shows its activity. Phosphorated ser473 may combined with Ser473-R, which reflect PKB activity.Phophatase and tensin homology deleted on chromosome 10 (PTEN) gene was discovered in 1997. Phosphatase of PTEN is homogenous with double specific protein tyrosine phophatase (FTP). The Phosphatase of PTEN is ,so far, the first discovered inhibiting gene with phosphatase. It can dephosphate tyro-sine , serine or threonine. Iipid phosphatase of PTEN can inhibite the activity of PI-3k, the activity of PKB and cell proliferation, playing the role of negative feed back to PI-3k / PKB signal system.At present, PKB, PTEN are mostly studied in tumor. Eventhough there are some studies about the effects of PKB, bFGF, FGF-R1 on incubted lens epithelial cells and lens crystallines in vitro, there is no study on the proliferation of lens epithelial cells after the surgery in animal models. There is no study about the PTENmRNA on the proliferation as well. Our study use proliferation cell nuclear antigen ( PCN A) as proliferation control to test the expression of PKB, PTEN, bFGF and FGF-R1 at different time after the surgery in rabbits. The results give us experimental data about the molecular mechanisms of the proliferation of lens epithelial cells and these data may make it possible to regulate cell proliferation.Material and MethodFirst part: Effect of PKB on the proliferation of rabbit epithelial cells after aspiration of lens cortex1. 42 white healthy rabbits ( averege weight 1. 5 kg) were put into twogroups, test group (36) and control group (6) Randomly. In control group, 6 rabbits without any treatment were killed and the lens were extirpate for test. In test group ,36 rabbits anesthetized were operated lens cortex absorption by the same surgeon.. Every 6 rabbits were killed and prepared the lenses for test in lday, 3day, 1 week,2 week, 1 month and 2month after operations. In every group 6 rabbits, 6 lenses from 3rabbits ( 42 lenses in all) were fixed by 4% formaldehydum polymerisatum and embedded by paraffin wax. . Equatorial portion were obtained within 2mm of equator from the 6 lenses of other 3 rabbits in each group,and were frozen in -70° C.2. The expressions of PCNA , PKB and Ser473-R in equator lens epithelial cells in different periods were detected by immunohistochemistry method ( SABC) and PKBmRNA were detected by RT-PCR. In the immunohistochemistry results, the average absorption optical density (A value) of PCNA , PKB and Ser473-R in unit area were analyzed by Metamorph software. RT-PCR results of PKBmRNA were analyzed by Kodak ID image software and the relative mRNA quantity were obtained. The linear correlation between PKB, PKBmRNA, Ser473-R and PCNA were tested.Second part; Effect of bFGF, FGF-RlmRNA on the proliferation of rabbit lens epithelial cellsThe animal medol and spicemens were as same as first part. The expressions of bFGF mRNA and FGF-RlmRNA of lens epithelial cells in equator were detected by RT-PCR method. The results were analyzed by Kodak ID image software and the relative mRNA quantity were obtained. The linear correlation between bFGF N FGF-RlmRNA and PCNA were tested.Third part; Effect of PTENmRNA on the proliferation of rabbit lens epithelial cells after aspiration of lens cortexThe animal medol and spicemens were as same as first part. The expressions of PTENmRNA of lens epithelial cells in equator were detected by hybridization in situ and RT-PCR method. The average absorption optical density (A value) of the results of hybridization in situ in unit area were analyzed by Metamorph software. RT-PCR results were analyzed by Kodak ID image software and the relative mRNA quantity were obtained. The linear correlation between PTEN-mRNA and PCNA were tested.Statistical analysis: All t test and linear correlation test were analized with SPSS10.0 Statistical softwareResultsFirst part: Effect of PKB on the proliferation of rabbit epithelial cells after aspiration of lens cortex1. The expression of PCNA in the nucleus of normal rabbit LECs was positive , and the nuleus of positive PCNA cells was stained brown-yellow. The absorption optical density (A value) of normal rabbit LECs was 0.46 ±0.04. The expression was strongest on the postoperative 1 day, the A value was 0. 86 ±0.02, P<0.01 in comparision with preoperative ones, and the expression maintained high level during postoperative 1 ~ 7 day. After 2 weeks, the expression was gradually decreased, and until 1 ~ 2 months, it restored to its preoperative level.2. The results of immunohistochemistry showed: The expression of PKB in the cytoplasm of normal rabbit LECs was weak positive, and the cytoplasm of positive PKB cells was stained brown - yellow. The absorption optical density (A value) of normal rabbit LECs was 0. 33 ±0.02. The expression was slightly stronger on the first day after operation , the " A" value was 0.37 ±0.02, and the expression maintained slightly high level during postoperative 3 days ~ 2weeks, but P > 0. 05 in comparision with preoperative one. After 2 weeks, the expression was gradually decreased, and until 1 ~ 2 months, it restored to its preoperative level. The correlation coefficient between PKB and PCNA was: r = 0. 906992. The results of RT-PCR: The relative quantities of PKBmRNA changed insignificantly in every postoperative periods, P >0.05 in comparision with preoperative one. The correlation coefficient between PKBmRNA and PCNA was: r= -0.36727.3. The expression of Ser473-R in the cytoplasm of normal rabbit LECs was weak positive. The absorption optical density of normal rabbit LECs was 0.15 ± 0. 01. The expressions was strongest on the postoperative lday, the A value was 0. 54 ±0.03, P <0. 01 in comparision with preoperative one, and the expressionmaintained high level during postoperative 1 ~3days. After 1 week, the expression was gradually decreased, and until 1 ~ 2 months, it restored to its preoperative level. There was positive correlation between Ser 4 7 3 - R and PCNA ( r = 0.972957).Second part; Effect of bFGF, FGF-RlmRNA on the proliferation of rabbit lens epithelial cells1. The expressions of bFGFmRNA changed insignificantly in every postoperative periods. The correlation coefficient between bFGFmRNA and PCNA was: r= -0.51697.2. The expression of FGF-RlmRNA was strongest on the postoperative 3th day, and the relative quantity was 130. 35 ±5.41, P <0.01 in comparision with preoperative one,which was 86. 15 ±6.71, and it was gradually decreased after the postoperative 1 week. Until 1 ~ 2 months postoperatively, the expression of FGF-RlmRNA restored to its preoperative level. There was positive correlation between FGF-RlmRNA and PCNA (r =0.923522).Third part: Effect of PTENmRNA on the proliferation of rabbit lens epithelial cells after aspiration of lens cortex1. The results of hybridization in situ showed: The expression of PTENmRNA in the cytoplasm of normal rabbit lens epithelial cells was positive. The expressions in the equatorial region on the postoperative 1 ~ 3 days was weaker , the A value was 0. 20 ±0. 02, and 0. 19 ±0. 02 respectively, than preoperative ones (P <0. 01). The expressions of PTEN was gradually increased after the postoperative 1 week. Until 1 ~ 2 months postoperatively, the expressions of PTEN restored to its preoperative level. There was negative correlation between PTENmRNA and PCNA (r= -0.979).2. The results of RT-PCR; The expression of PTENmRNA in the normal rabbit LECs was positive, and the relative quantity was 155.70 ±5.90, but the expression on the postoperative 1 day was weakly positive,it was 101.33 ±5. 09, P < 0. 01 in comparision with preoperative ones. The expression was gradually increased after the 1 week. Until 1 ~2 months,it restored to its preoperative level . There was negative correlation between PTENmRNA and PCNA ( r =-0.94863).
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