Study On Prevention Effects Of Immunoconjugate Of Monoclonal AnticollageⅣ-Genistein On Capsule Opacification

After-cataract, or posterior lens capsule opacification, is the most important factor on the effect of vision recovery after cataract surgery. PCO can be treated by YAG laser posterior capusulotomy, which carries a small risk of sight threatening complications such as macular edema or retinal detachment or increased intraocular pressure. So drug to prevent after-cataract become the emphasis of research at present.After cataract surgery, residual lens epithelial cells (LECs) proliferate in the defective lens capsule and migrate onto the posterior capsule to make posterior capsule opacify. Growth factors play an important roll on proliferation of LECs, while protein tyrosine kinases family is center link of growth factors in signal-transducing functions.Genistein, as a strong inhibitor of protein tyrosine kinases, can inhibit proliferation of LECs by regulating cell cycle, cellular transformation and inducing apoptotisis.In our previous studies, genistein can inhibit rabbit LECs proliferation in vitro, the effects of genistein on human LECs have not been reported.Since the early 1980s, the research of targeted drug conjugates had made huge progress using carrier of mAb. Target drug therapy can improve treatment effect by carring drug selectively to target. This method can improve drug's effect, increasing effectiveness, and lessening side effects.Cell can surivive, prolifere and migrate, only when it can adhere extracellular matrix.Posterior capsular membrane play an critical role in adhesion and migration of LECs in vivo, and it is the specifically interior condition to growth for LECs. So we assume that a greater concentration of drug can be allowed with increasing effectiveness and lessening side effects, if the drug distribute well-proportional on posterior capsule membrane.Typeâ…£collagen and laminin are characteristic components of posterior capsule membrane.Targeting genistein to posterior capsule membrane was considered by covalently linking genistein to monoclonal anticollagenâ…£,which can specific bind with collagenâ…£on posterior capsule membrane.We presume genistein might be distributed evenly to posterior capsule bag by this way, and inhibite proliferation of LECs on posterior capsule effectively.PurposeTo set up appropriate method to generate immunoconjugate which genistein could be coupled to anti-collagen monoclonal antibody. The activity of antibody and drug could maintain well in the immunoconjugate; The immunoconjugate of monoclonal anticollageâ…£-genistein can effectively inhibit LEC cells from proliferation. This study could provide the scientific basis for target therapy to prevent PCO in vivo.MethodsThree sections were involved in this studySection 1 Antiproliferation Effects of Genistein on Cultured Human Lens Epithelial Cell In Vivo1. Frozen LECs-B₃ cell lines were defrozed and cultured in DMEM medium supplemented with 10% fetal bovine serum, in 5%CO₂ saturated atmosphere.2. LEC-B₃ were incubated with 0-75mg/L genistein for 12-72 hours, and the survival rate of LEC-B₃ was determined by tetrazolium assay(MTT).3. Flow cytometric analysis was employed to study the influence of different concentrations of genistein on cell cycles and cell apoptosis of LEC-B₃. 4.Western blot was used to determine the expression of P21, P53 and CyclinD1 in LEC-B3 which were incubated with 0-75mg/L genistein for 48 hours.5.To testify the expression of P21's mRNA and P53' mRNA and CyclinD1's mRNA in LEC-B₃ which were incubated with 0-75mg/L genistein for 48 hours by RT-PCR.Section 2 Generation and characterization of immunoconjugate of genistein to anti-collagenâ…£monoclonal antibody1. Genistein was conjugated to anti-collagenâ…£monoclonal antibody by the active ester intermediate.2. The immunoconjugate was purified by sephadex G25 column chromatography. The molar ratio of genistein and monoclonal antibody in the conjugate was measured by Bradford protein assay and UV absorption spectra assay.3. The immunological activity of the antibody in conjugate was determined by immunohistochemistry method.Section 3 Antiproliferation Effects of Immunoconjugate of Monoclonal Anticollageâ…£-genistein on Human Lens Epithelial Cells In Vivo1,The morphological changes of LEC-B3 cells treated by immunoconjugate for 48 hours were observed by inverted microscopy.2,Apoptosis death of LEC-B3 which was treated by immunoconjugate for 48h was observed by fluorescence microscopy after Acridine orange staining.3,LEC-B3 were incubated with immunoconjugate for 12-48 hours, and the survival rate of LEC-B₃ was determined by tetrazolium assay(MTT).Results1.LEC-B₃'s growth curve was mapped, 3-5days after incubation are logarithmic growth stage. 2.Genistein ranging from 6.25～75mg/L inhibited hLBCs proliferation and had dose-dependent and time-dependent effect. 75mg/L of genistein exhibited antiproliferative effect at 12 hours(P＜0.05), and the antiproliferative effects of 12.5, 25, 50 and 75mg/L of genistein showed statistically significant difference compared with the control group at 24, 48 hours(P=0.03).All concentrations of genistein had obvious antiproliferative effects at 72 hours.3.The flow cytometric analysis suggested that cell cycle was blocked at G₁-S stage.Apoptosis of LEC-B₃ was found after the treatment with 12.5mg/L genistein for 48 hours and was more obvious after being treated with 50mg/L genistein for 48 hours. The rate of apoptosis was 12.72%, 51.83%, 59.3% respectively in 12.5, 25, 50mg/L genistein groups at 72 hours.4.The expression of P21 mRNA and protein was gradually increased with the increasing dose of genistein, while the expression of cyclinD₁ mRNA and protein was gradually decreased, there was no significant difference on the expression of P53 mRNA and protein between the control and treated group.5. Genistein could be coupled to anti-collagen monoclonal antibody by the active ester intermediate. Molar ratio of genistein to antibody in immunoconjugate is 439, and it has a good character for binding lens capsule.6.Apoptotic bodies could be found under fluorescence microscopy after LEC cells treated with immunoconjugate for 48h with AO staining.7.Immunoconjugate effectively inhibited LEC cells growth in a time-dependent manner in MTT assay, and the rate of inhibition was 28.5%, 50.3%, 76.9% respectively at 12,24,48 hours.The antiproliferation effect of immunoconjugate was more sensitive compared with unconjugated genistein and had statistically significant.Conclusion1.Genistein can inhibite proliferation of HLEC-B₃ cell through both cell-cycle arrest and apoptosis induction.2.Genistein can be successfully conjugated with anti-collagenâ…£monoclonal antibody by the active ester intermediate. The conjugates maintein a good character for binding lens capsule.3.The immunoconjugate of monoclonal anticollagenâ…£-genistein conjungated can effectively inhibit LEC cells from proliferation in vivo.