| BackgroudRetinoblastoma(RB)is a common intraocular malignant tumor in infants and young children,accounting for 3% of all children's malignant tumors.It has a family genetic tendency and can be monocular,binocular,or simultaneous.Its clinical manifestations are complex and can be expressed as mydriasis,white pupil disease,strabismus.Retinoblastoma can cause changes in the fundus,and in severe cases it can move away and be life-threatening.Treatment methods for retinoblastoma include: local treatment(radiotherapy,intravitreal injection therapy and ocular artery intervention therapy),chemotherapy and surgical treatment.Although early diagnosis and comprehensive treatment methods makes it possible for developed countries to completely cure retinoblastoma cells,they still can cause serious damage to vision,However,the cure rate in developing countries is extremely low.The chemotherapy drugs used commonly have the disadvantages of single target and large toxic side effects.The most commonly used gene drugs are small interfering RNA(siRNA).However,they are easily degraded and not easily absorbed by cells,resulting in poor treatment of tumors.Therefore,new treatment strategies are urgently needed.In recent years,polymer micelles have been widely used as a new type of nanocarriers in the treatment of tumors.They can be used to load gene drugs and chemotherapy drugs to increase the enrichment of gene drugs and chemotherapy drugs at the tumor site and achieve the effect of tumor targeted therapy,thereby reducing its toxic and side effects on normal tissues.PurposeTo explore the role of polymer nanomicelles in combined delivery of chemotherapy drugs and genetic drugs to retinoblastoma cells,to observe the synergistic inhibitory effect of drugs on retinoblastoma cells,and to study the mechanism of drugs on tumor angiogenesis.MethodsIn previous studies,we have synthesized a new cationic triblock copolymer,poly(ethylene glycol)-poly(?-caprolactone)-g-polyethyleneimine(PEG-PCL-g-PEI,abbreviation For PCI),prepare polymers loaded with celastrol and siRNA,Nano-ZSE potential and particle size analyzer to detect changes in potential and particle size.Agarose electrophoresis gel test to detect the polymer's ability to complex nucleic acids.Cell uptake experiments Detection of efficient bioavailability of polymers.CCK8 method to detect the proliferation of human Y79 cells and human EA.hy926 cells.The cell scratch test and invasion test to detect the migration and invasion ability of EA.hy926 cells.Western blot detection of HIF-1? and HIF-1?/VEGF/VEGFR signaling pathway by Western blot.ResultsIn this study,the use of polymer nanomicelles combined with celastrol and HIF-1? siRNA to regulate HIF-1?/VEGF inhibited human retinoblastoma cells to synergistically inhibit tumor cells.Previous studies have confirmed that the prepared positive ion nanomicelles are spherical,uniform in size,with an average particle size of 24 nm,and a surface potential of + 8.5 m V.Celastrol released early and then slowly released.With the lowering of p H,it releases faster,which indicates that cationic nanomicelles can be used to carry celastrol for experimental study.The agarose electrophoresis gel test shows that the nanoparticles can effectively adsorb negatively charged small interfering RNA.At the same time,as the ratio of nitrogen and phosphorus increases,the polymer micelles can completely complex the small interfering RNA.The cell uptake test shows that the celastrol-loaded polymer micelles can efficiently enter tumor cells(Y79 cells)and release celastrol from the cytoplasm to achieve anti-tumor effect.Cell proliferation experiments show that the polymer micelles containing celastrol can effectively reduce the viability of tumor cells and inhibit the proliferation of tumor cells,while the blank micelles had no effect on the proliferation of tumor cells,indicating that the polymer micelles were not toxic to the cells.In the co-culture system of human Y79 cells and human EA.hy926 cells,the results show that after pretreating Y79 cells with celastrol,Y79 cells can inhibit the proliferation of vascular endothelial cells.We tested the combined transmission of celastrol and HIF-1? siRNA by Western blot acting on tumor cells,and observed that the expression of HIF-1? and VEGFA are reduced,especially the synergistic effect of the both can significantly reduce the expression of HIF-1?.In the co-culture of Y79 cells and EA.hy926 cells system,migration and invasion test studies have shown that tumor cells pretreated by celastrol can inhibit the migration and invasion of vascular endothelial cells,and western blot results show that tumor cells that were pretreated by celastrol can downregulate the expression of vascular endothelial cells receptors(VEGFR1 and VEGFR2)and celastrol can down-regulate the VEGFR1 and VEGFR2 receptor expression.Conclusions1.The polymer micelle carrier can transmit celastrol and HIF-1? siRNA to tumor cells,and can effectively inhibit the expression of HIF-1? and VEGFA in Y79 cells.2.We found that celastrol not only directly inhibited tumor cell proliferation,but also inhibited the proliferation,migration and invasion of vascular endothelial cells by regulating tumor cells.3.Celastrol can not only directly down-regulate the expression of vascular endothelial cell receptors,but also down-regulate the expression of vascular endothelial cell receptors by acting on tumor cells.4.In summary,the inhibitory effect of celastrol on tumor angiogenesis may be through the HIF-1?/VEGF/VEGFR channel.5.This study provides a basis for the combined treatment of retinoblastoma with chemotherapy drugs and gene drugs. |
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