Study On The Function And Mechanism Of CDK1 In Retinal Neovascularization

ObjectiveTo explore the role and mechanism of cyclin-dependent kinases 1(CDK1)in retinal neovascular diseases such as retinopathy of prematurity,diabetic retinopathy and so on.To find new targets for the prevention and treatment of diseases related to retinal neovascularization.MethodsForty-eight healthy C57 BL / 6J mice were selected and randomly divided into a control group and an OIR model group,each with 24 mice.The model group was constructed using a hyperoxia-induced method to construct an OIR mouse model.The retina of mice was stained with GS-IB4 fluorescence staining technique,and the retinal neovascularization was observed to verify whether the OIR mouse model was successfully constructed.To construct a mouse model of diabetic retinopathy.Thirty-six 6-to 8-week-old C57 BL / 6J mice were selected,with a body weight of 18-22 g,and randomly divide into a control group and experimental group,each with 18 mice.The mice in the experimental group were continuously injected with streptozotocin(STZ)at a low dose.This method was used to construct a mouse model of type I diabetes,and it was fed for 5 months.The retinal tissue sections were stained with HE staining and Evans blue perfusion angiography.The retinal vascular occlusion,leakage,and retinal neovascularization were observed and identified whether the mouse model was successfully constructed.CDK1 expression was observed in mouse retinas by immunohistochemical staining and immunofluorescence.CDK1 was detected in mice retinas by Western Blot and real-time fluorescent reverse transcription polymerase chain reaction(RT-PCR).CDK1 small interfering RNA was constructed(control group si NC,experimental group as si CDK1-1,si CDK1-2),small interfering RNA was transfected into human umbilical vein endothelial cells,and CDK1 expression in HUVECs was detected by Western Blot and RT-PCR To verify the effect of transfection.The effects of si CDK1 on the function of HUVECs were tested using the method of Ed U cell proliferation test,Transwell cell migration test and Matrigel matrix gel-like formation.Flow cytometry was used to detect the effect of si CDK1 on the cell cycle of HUVECs,and Western Blot and RT-PCR were used to detect the expression of cell cycle-related proteins.Flow cytometry was used to detect the effect of si CDK1 on the apoptosis of HUVECs cells.Using Western Blot and RT-PCR to detect the expression level of apoptosis-related proteins and explore the mechanism of CDK1 on HUVECs.Results1.The OIR mouse model was successfully constructed.Compared with the normal mouse retina,GS-IB4 fluorescence staining showed that the retina of the OIR mouse showed high-fluorescence neovascularization areas,the neovascularization was tortuous and disordered,and the blood vessels were unclearly layered.2.The mouse diabetic retinopathy model was successfully constructed.Compared with the control group,the blood glucose of the mice in the experimental group was significantly higher than 16.5mmol / L,and the difference was statistically significant(p <0.05).After 5 months of feeding,the mice in the experimental group found that the whole retinal layer became thinner.The inner nuclear layer and outer nuclear layer cells are disorderly arranged,the blood vessels are tortuous,vascular occlusion and vascular leakage are seen,and a small amount of retinal neovascularization can be seen.3.CDK1 expression was increased in retinal neovascularization in mice.Immunofluorescence showed that CDK1 was highly expressed in the retina of OIR mice.Western blot and RT-PCR results showed that the expression level of CDK1 gene and protein was significantly increased,and the difference was statistically significant,p <0.001.4.CDK1 is highly expressed in the retina of diabetic retinopathy mice.Immunohistochemical staining showed that CDK1 was highly expressed in the retina of diabetic retinopathy mice.Western blot and RT-PCR results showed that CDK1 gene and protein expression levels were significantly increased,the difference was statistically significant,p <0.01.5.si CDK1 inhibits HUVECs proliferation,migration and tube-like formation.Western blot and RT-PCR results showed that compared with the control group,HUVECs in the si CDK1 transfected group had lower levels of CDK1 gene and protein expression(si CDK1-1,p <0.01;si CDK1-2,p <0.01).Ed U experiment results showed that HUVECs cell proliferation in the si CDK1 intervention group decreased significantly(si CDK1-1,p <0.01;si CDK1-2,p <0.01);Transwell experiments showed that HUVECs cell migration in the si CDK1 intervention group significantly decreased(si CDK1-1,p <0.01;si CDK1-2,p <0.01);Matrigel-like tube formation experiments showed that the tubular structure of HUVECs cells in the si CDK1 intervention group was significantly reduced(si CDK1-1,p <0.01;si CDK1-2,p <0.01).6.Low expression of CDK1 inhibits retinal neovascularization by inducing G2 / M cell cycle arrest and inducing apoptosis.Flow cytometry cell cycle results showed that compared with the negative control group HUVECs,the cells in the si CDK1 intervention group had an increase of about 10% in G2 / M phase cells.Flow cytometry apoptosis results showed that compared with the negative control group HUVECs.The early and late apoptotic cells in the intervention group increased by about 9% compared with the control group.Western Blot and RT-PCR results showed that compared with the control group,HUVECs in the si CDK1 group showed lower levels of cell cycle-related molecules such as cyclin B,cyclin D,and CDK2(si CDK1-1,p <0.01;si CDK1-2,p <0.01);Increased expression levels of apoptosis-related molecules such as PARP1,p21 and p53(si CDK1-1,p <0.01;si CDK1-2,p <0.01).ConclusionCDK1 is highly expressed in mice retinal neovascularization.CDK1 disruption can inhibit vascular endothelial cell proliferation,cell migration,and cell-like formation by inducing cell cycle arrest and inducing apoptosis,thereby inhibiting retinal neovascularization.