| Objective:To investigate the mechanism that AG490intervene JAK2/STAT3signaling pathway in retinoblastoma cell line,in order to explore the effect of AG490on the HXO-RB44cell lines in vivo anti-proliferative and cell cycle,in addition,to observe the effect of AG490on the STAT3,p-STAT3,VEGF protein expression.To explore the effect that JAK2/STAT3to regulate the transcriptional activation of VEGF in retinoblastoma, estimate the value of the target therapy in RB.Method:(1)We cultured the human retinoblastoma HXO-RB44cell line.(2)Essay grouping and measure:experimental group:A、B、C、D、E、 F、E group treated with different concentrations (6.25μM,12.5μM,25μM,50μM,100μM),the control group treated with of RPMI-1640. To measured by follow method:1)Cell proliferation in response to AG490were measured by MTS.2)Cell cycle distribution and apoptosis rate were analyses were measured by flow cytometry.3)To detect different protein’s (Stat3, p-Stat3, VEGF) expression by western bolt.statistical analysis was used to check the significant differences among them.Results:HXO-RB44cell line treated with AG490after48hours(1)Cell viability were measured by MTS assay.The cell group followed concentration gradient (6.25μM,12.5μM,25μM,50μM,100μM,200μM) increased with proliferation rate reduce,The inhibition ratio of six experimental increased:7.18±8.78%,20.02±2.21%,31.92±5.30%,58.61±4.27%,62.17±6.05%,72.67±1.51%, compared with the control group,6.25uM have no significant difference(P>0.05),the other results have significant difference(P>0.05), it have significant difference.(P<0.05);(2) Flow cytometry results demonstrated:Select the experimental from MTS assay:(6.25μM、12.5μM,25μM,50μM,100μM),the group cycle which followed concentration gradient increased with apoptosis rate increase:8.94±2.21%,12.86±2.12%,13.59±1.22%,26.51±2.01%,35,37±1.95%, compared with the control group(the apoptosis:4.10±2.35%),have significant difference (P<0.05).50uM and100uM cell lines compared to the other concentration gradient group was shown to increase in the proportion of cells in the Gl-phase while decrease in the proportion of cells in the S-phase have significant difference (P<0.05).(3) Western blot results demonstrate:The gray level of STAT3are:2.11±0.23,1.77±0.18,1.27±0.18,1.15±0.19,0.77±0.11,demonstrate that STAT3expression reduced while the followed concentration gradient increase.compared with the control group(the average of the gray level:3.84±0.21),have significant difference(P<0.05). The gray level of p-STAT3are:1.12±0.12,1.11±0.11,0.82±0.09,0.38±0.04,0.35±0.04,demonstrate that p-STAT3expression reduced while the followed concentration gradient increase.compared with the control group(the average of the gray level:1.52±0.14),have significant difference(P<0.05).The gray level of VEGF are:2.56±0.11,2.51±0.14,1.96±0.18,1.81±0.12,1.31±0.06,demonstrate that VEGF expression reduced while the followed concentration gradient increase.compared with the control group(the average of the gray level:2.61±0.15),6.25μM、12.5±M concentrations group have no significant difference (P>0.05),the other results have significant difference (P<0.05).Conclusion1Janus Kinase inhibitor AG490inhibited the growth and proliferation of the retinoblastoma cell lines by concentration and time dependent.2.Janus Kinase inhibitor AG490down-regulates the expression of VEGF by blocked JAK2/STAT3signaling pathway in order to inhibited the proliferation of RB.4.These results provides evidence that AG490,a specific inhibitor of JAK2/STAT3signaling pathway can be thought of as a potential pharmacological target for the therapeutic management of RB and pathological angiogenesis. |
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