The Influences Of The Solvents Of KH902 Affect Human Lens Epithelial Cells

Purpose: KH902 is a new developed genetic engineering drug. It is injected into the vitreous cavity to inhibit the neovascularization. This trial is to investigate the influnces of the 6 different solvents of KH902 affect the in vitro cultured human lens epithelial cells line SRA01/04 (HLEC SRA01/04) and provide laboratory evidence for the solvents selection. Methods: There are 6 groups of solvents according to the results of previous trial: group A.10mM histidine+ 9%trehalose+ 0.05%Tween-20; group B.10mM succinate+ 9%trehalose+ 0.05%Tween-20; group C.50mM PB+ 0.05%Tween-20; group D.10mM succinate+ 9%trehalose+ 0.05%Tween-20+ 6%Mannitol; group E.10mM succinate+ 9%trehalose+ 0.05%Tween-20+ 0.6%Mannitol; group F.PBS. These solvents are added into the cultured HLEC. Measurements: 1.Use the inverted biological phase contrast microscope to observe and record the HLEC growth situation. 2. Measure the expression of Bcl-2 and Bax by immunohistochemical method, and take the mammary cancer cells as the positive control; 3. The methyl thiazolyl tetrazolium (MTT) colorimetric method is used to measure the cells' activity, taking normal cultured HLEC as control group; 4. Measure the expression of Apo2.7 on the apoptosis HLEC by flow cytometric analysis (FCM) and take the H₂O₂ inducing apoptosis HLEC as the positive control. Result: 1. The outcome of HLEC growth situation: cells' deaths in group D are obvious. 2. The outcomes of immunohistochemical method: after 5 days influenced by these solvents, the sequence of expression of Bax: group D>group A,group B,group F>group C,group E; the sequence of expression of Bcl-2: group C,group E>group A,group B,group D>group F. 3. The outcomes of MTT: during 1 week culturing period, comparing with the control group the activity of the cells influenced by group D are lower(P<0.05). Comparing among the solvents groups, the activity of the cells influenced by group D are the lowest. The activities of the HLEC of all the groups descend from 5th to 7th day. 4. The outcomes of flow cytometric analysis: after 5 days influenced by these solvents the expression of Apo2.7 is positive control group>group D>group F>group A> group B>group E >group C (P<0.05). Group D has the highest apoptosis rate while group C has the lowest. There is no significant statistics between group A and B.Conclusions: among the 6 groups of solvents, group C (50mM PBS+ 0.05%Tween-20) has the weakest suppression and inducing-apoptosis effects on the in vitro cultured HLEC, while group D ( 10mM succinate+9%trehalose+ 0.05%Tween-20+ 6%Mannitol) has the strongest. So group C is better than others to be the components of KH902, while group D shouldn't be chosen. The antibody Apo2.7 could be used to investigate the early-apoptosis HLEC. The more the apoptosis cells there are, the more the Apo2.7 will be expressed.