Studies Of The Effect Of Hepatocyte Growth Factor On Human Lens Epithelial Cells

IntroductionPosterior capsule opacification (PCO) is the most important factor on the effect of vision recovery. There isn't any excellent way to prevent beforehand now. Thus, it's very important to study the mechanism to prevent posterior capsule opacification.The main factor of PCO is the lens epithelial cells' proliferation, migration and transformation. It's found that many kinds of cell factors, blood - aqueous barrier's destruction, mediators of inflammation releasing and complement activation play important roles. But there is few study on hepatocyte growth factor's effect on PCO.Hepatocyte growth factor (HGF) is a kind of polypetide growth factor. c -Met is a kind of oncogene. The transmembrane protein coded with c - Met is the acceptor of HGF. HGF's density in epithelium is extremely high. Binding with c - Met, HGF can activate tyrosine protein kinas (TPK) , and accommodate the cellular's fission, proliferation, differentiation and migration. So we assume that HGF and c - Met can be found in the human lens epithelial cells. Cataract surgery , blood - aqueous barrier's destruction and inflammation can activate the HGF's expression by the PI3K and MAPK signal conduction to inhibit apoptosis and promote proliferation. The result is that the human lens epithelial cells migrate towards posterior capsule.PurposeThe aim of the present study is to detect the expression of HGF and c -Met, to study HGF's effect on promoting migration and anti - apoptosis in hu-man lens epithelial cells and to investigate the PI3K and MAPK signal conduction inhibiting apoptosis and promoting proliferation.MethodsThere were three sections in this studySection 1 Detection of HGF and c - Met expression1. Recovery culture of frozen stored cells, the third - generation cell is used for the lab study. Cell culture human's lens epithelial cells (LECs) were cultured in DMEM medium supplemented with 10% fetal bovine serum, in 5% CO2 saturated atmosphere.2. To testify the expression of HGF's mRNA and c - Met's mRNA by RT -PCR;Using Trizol reagent of total mRNA was extracted from LECs. The first chain of cDNA was reverse transcription, and PCR products of HGF and c - Met were amplified. After agarose gel electrophoresis, images were made and PCR products were scanned by KODAK image analysis system.3. Western blot was used to determine the expression of HGF and c - Met;Cells sediment was resolved, dissociated, centrifugalized and extracted the supernatant. The sample was electrophoreted by SDS - PAGE. Then damp — dry transfer was done. The first antibody and the second antibody were added in, room temperature incubating and washing the membrane, ECL for coloration.Section 2 Test of effect of HGF1. DNA ladder was used to evaluated the apoptosis of lens epithelial cells. Group 1 is ActD(50ng/ml). Group 2 is ActD(50ng/ml) + HGF( lOOng/ml).2. Western blot test evaluate the expression of caspase - 3. Group 1 is ActD (50ng/ml) and group 2 is ActD (50ng/ml) + HGF (lOOng/ml).3. Promoting proliferation was evaluated by MTT. Cells were cultured in 96 -well plate without FCS overnight. Cells were treated by different density ofHGF;the negative control was treated in DMEM only. Then 20ul MTT were added in for 2 h. Discarding the DMEM, cells were treated by lOOul DMSO forreading the absorbance at 570nm.4. Western blot test evaluated the expression of Bel -2.Section 3 Study of HGF signal conduction on promoting proliferation and inhibiting apoptosis1. PI3K/AKT in the signal conduction of inhibiting apoptosis was evaluated. (1) DNA ladder was used to evaluate the apoptosis of lens epithelial cells by LY294002, the inhibitor of PI3K. (2) AKT.p - AKT' s protein expression were evaluated by Western blot.2. ERK in the signal conduction of promoting proliferation was evaluated. (1) MTT was used to analyse the effect of proliferation by HGF and U0126, the inhibitor of ERK. (2) Bel - 2, ERK and p - ERK' s expression were evaluated by Western blot.Results1. The mRNA and protein of HGF and c - Met can be detected in human lens epithelial cells.2. The proliferation increased when the density of HGF increased. When the density was 50ng/ml the proliferation was most significant in 24h (P < 0. 05).3. HGF can inhibit the apoptosis induced by ActD in human lens epithelial cells. There wasn' t any DNA ladder in the group ActD + HGF, and the expression of caspase - 3 was lower than the other group. The Bel - 2 was higher than the other group especially in lh.4. The inhibitor of ERK, U0126 can inhibit the promotion of proliferation by HGF in 24h by the density of 50ng/ml ( P < 0.05). p - ERK' s expression was lower than the other group significantly, never were the ERK.5. The inhibitor of PI3K, LY294002 can inhibit the anti - apoptosis by HGF. p - AKT and Bel - 2' s expression were decreased, never was the AKTfc.Conclusions1. HGF and c - Met expressed in human lens epithelial cells.2. HGF promoted proliferation and resisted apoptosis.3. ERK signal path took part in the promoting proliferation, PI3K signal path took part in the resisting apoptosis.