ABSTRACT
Background Cysteine-rich 61 (Cyr61)/CCN1 has been reported to stimulate retinal neovascularization (RNV) in retinopathy of prematurity (ROP).However, whether CCN1 small interfering RNA (CCN1 siRNA) can inhibit or cure ROP has not been extensively investigated.Objective This study was to investigate the regulation effect of CCN1 specific siRNA expression vector on retinal endothelial cells.Methods Rhesus choroid-retinal vascular endothelial cells (RF/6A) were cultured under the normoxic (normoxia control group) and hypoxic condition (1％ O2,5％ CO2 with 94％ N2) in vitro, and then lipofectamineTM 2000 (LF2000) vector plasmid with or without CCN1 siRNA was transiently transfected in the hypoxic-cultured cells as the CCN1 siRNA transfected group and hypoxic control group, respectively.Reverse transcription PCR was employed to detect the expression of CCN1 siRNA plasmid 24 hours after transfection.The vatality of the cells was assayed by cell counting kit-8 (CCK-8) 0,24,48,72 and 96 hours after cultured.Twenty-four hours after cultured,the apoptosis of the cells was evaluated by flow cytometry, and the expressions of CCN1 and vascular endothelial growth factor (VEGF) proteins were detected by immunofluorescence technique and Western blot assay.Results The expression band of CCN1 siRNA was detected in the cells 24 hours after transfection of CCN1 siRNA.CCK-8 assay showed that RF/6A cells were significantly increased over time, and the proliferating value (absorbancy) of the cells was significantly reduced in the CCN1 siRNA transfected group compared with in the normoxia control group and hypoxic control group (Fgroup =198.45, P＜0.05;Ftime =39.26, P＜ 0.05).The apoptosis rates of the cells were (68.9± 1.1) ％ , (18.9±1.3)％ and (39.6± 1.8)％ in the CCN1 siRNA transfected group, normoxia control group and hypoxic control group,and the apoptosis rates of the CCN1 siRNA transfected group were evidently higher than those of the normoxia control group and hypoxic control group (t =2.93 ,t=2.56 ,both at P＜0.05).CCN1 and VEGF proteins were weakly expressed in the normoxia control group and strongly expressed in the hypoxic control group,however,their expression intensity was evidently weakened in the CCN1 siRNA transfected group.The related expression levels of CCN1 and VEGF proteins in the CCN1 siRNA transfected group were significantly lower than those in the hypoxic control group (both at P＜0.05).Conclusions RNA interference targeting CCN1 can inhibit proliferation and promote apoptosis of RF/6A cells.CCN1 siRNA can arrest RNV probably by downregulating the expression levels of CCN1 and VEGF in the cells.
ABSTRACT
P2 receptors are divided into two subclasses:P2X receptors which are ligand-gated ion channels and P2Y receptors which are G-protein coupled. Several kinds of P2X and P2Y receptor subtypes express in vascular endo-thelial cells and vascular smooth muscle cells. Purinergic signalling plays an important role in vascular diseases such as atherosclerosis, cerebral vessels ageing and blood vessel remodeling. So this signalling pathway may pro-vide a new target to treat vascular diseases.
ABSTRACT
Objective To prepare controlled release microspheres of vascular endothelial growth factor(VEGF)& calcium alginate and observe their effect on proliferation of human umbilical vein endo- thelial cells(HUVEC)in order to provide theoretical basis for controlled release of VEGF facilitating an- giogenesis of tissue engineering bone.Methods VEGF-calcium alginate microspheres were prepared by using the needle extrusion/external gelation method to investigate physicochemical character and in vitro release of VEGF.According to the different ingredients added into the culture medium,the seconda- ry cultured HUVEC were divided into four groups,ie,control group,microsphere group,VEGF group and VEGF-calcium alginate microsphere group,in which the proliferation of the cultured HUVEC was ob- served with cell counting method,MTT method and flow cytometry.Results The calcium alginate mi- crospheres were revealed as spherical shape and evenly distributed,with mean grain diameter of(560?50)?m,carrying capacity of 0.72 ng/mg and the encapsulation efficiency of 54%.Smooth controlled re- lease in VEGF-alginate microspheres lasted for more than 10 days.Proliferation of the cultured HUVEC was accelerated the most in VEGF group at the beginning but in EGF-calcium alginate microsphere group at midanaphase compared with other groups,with statistical difference(P＜0.05).There was no statis- tical difference upon cell counting,cell activity and time point of cell cycle between control group and mi- crosphere group.Conclusion VEGF-sodium alginate microapheres can continue activity of VEGF,re- lease VEGF for over 10 days and promote proliferation cultured HUVEC for a long time.