Research On Influence Of TRPV1-targeted Drug On Microvascularization In A 3D-culture System

Part 1 Construction of three-dimensional microvascular networkObjective:The process of constructing a microvascular network,a new blood vessel aggregation.It is essential for constructing engineering structures in many diseases and surgical procedures.Vascularization in vitro can maintain cellular activity,induce tissue differentiation,and promote the transplantation of implantation.As a seed cell-endothelial cell(EC),a core role in the formation of neovascularization and existing blood vessels.Therefore,this study applies cell co-culture(HUVECs and HSFs)technology to generate microvascular networks in 3D fibrin buildings,explore environmental factors affecting blood vessels,optimize the construction of microvascular network conditions;Under the most appropriate conditions of the above experiment,HUVECs is co-cultivated,and the observation HUVECs and ADSCs can also successfully form a pipe network structure;HUVECs is the first choice for regenerative vascular seed cells,but its source is limited.Therefore,this study also applies SD rats adult cell-abdominal artery ECs,adding the most appropriate conditions of the experiment to explore the pipe network,observing whether the aortic ECs and HSFs in the SD rats can also successfully form a microvascular network,verify construct purpose of microvascular network conditions.In order to achieve the laid foundation of the preparation method of the rapid internal endothelial and improved transplantation,optimization,and simplify the preparation method of tissue engineering angiogenesis.Methods:(1)HUVECs observed morphology under different concentrations fetal bovine serum(2%,5%,10%),HUVECs and HSFs were co-cultured under different cell density(0.75×10~6/m L,1×10~6/m L,1.5×10~6/m L,3×10~6/m L),different concentrations of fibrinogen(1 mg/m L,5 mg/m L,10 mg/m L,25μL fibrinogen solution)and thrombin(0.5 mg/m L,1 mg/m L,10 mg/m L,25μL thrombin solution),and different concentrations of cross-linking agent genipin(0.05 mg/m L,0.5 mg/m L,1 mg/m L,3mg/m L,6 mg/m L),to observe the mixed culture of HUVECs and HSFs in fibrin.Observe the netting effect of 1,3,5 and 7 days,and monitor the process of blood vessel formation in vitro by immunofluorescence and confocal imaging technology;With the most suitable condition for forming microvascularization,HUVECs and ADSCs were treated with ADSCs at a proportion of 5:1,and the 3,5,and 7 days of networked effect,the focusing technology monitors the processes of the outer microvascular formation.(2)From the healthy SD rats,the culture of abdominal main artery ECs is separated,and the third-generation cell having a good growth condition is selected,and the cell growth morphology is observed;the abdominal artery ECs verifies the tube capacity by Matrigel matrix gel,and determines nitric oxide.Secure and immunofluorescence staining observe the expression of the associated antigens;inquiry under the basis of HSFs,the most suitable condition for forming microvascularization,SD rats abdominal aorta ECs and HSFs were co-cultured in 5:1ratio.3,5 and 7 days of surfacing effect,the process of monitoring the formation of the outer microvascular formation under fluorescence microscope.Results:(1)When the cell ratio of HUVECs and HSFs is 5:1,FBS concentration 5%,cell density 3×10~6cells/m L,fibrinogen concentration 5 mg/m L,thrombin concentration 0.5 mg/m L,crosslinking agent genipin concentration at 0.05 mg/m L,microvascularization can be better formed.The longest stable maintenance of the micro-blood network is 7 days;Under 3D fiber protein constructs,HUVECs and ADSCs also showed a better microvascular network on day 3 and stabilized for 7days.(2)Success SD rats abdominal aorta ECs.The abdominal artery ECs is formed into a fibrous shape due to the low cell density,and the cellular density increases,the cell morphology is arranged in a paved pebble-like;The identification results show that the abdominal aorta ECs has vascular ability and can secrete nitric oxide and high expression ECs markers(VIII factor,CD31,VWF);SD rats in the fibrino matrix ECs and HSFs can also a preferred microvascular network can show a better microvascular network on day 3,and maintain 7 days.Conclusion:(1)Constructing a microvascular network is affected by FBS concentration,cell density,and fibrin,while the concentration of thrombin on cell morphology and constituent microvascular network is not large,but will affect the coagulation time of fibrino matrix,crosslinking agent genipini concentration directly affects the stability and hardness of fibrin matrix;HUVECs and ADSCs can also generate microvascular networks in 3D fibrinogen under 3D fibrinogen under normal conditions.(2)In the formation of microvascularization,the rat adult cell(abdominal aorta ECs)can also generate a microvascular network in 3D fibrinogenic buildings,and verify that the 3D fibrinogen construct conditions have certain common applications.Part 2 Effect of TRPV1 target drug on three-dimensional microvascular networkObjective:Transient receptor potential vanilloid 1(TRPV1)is a multimodal cation channel,an important participant in ECs migration,proliferation and microangiogenesis.Previous studies have found that capsaicin(Cap)and rutaecarpine(Rut)are both TRPV1 receptor agonists,and capsazepine(CAPZ)is a TRPV1 receptor antagonist.They can affect the generation of a single ECs phylogenesis in an in vitro 2D environment.Therefore,this study was HUVECs with different cells combined with different cells,and the effect of the TRPV1 receptor agonists Cap,Rut and TRPV1receptor antagonists CAPZ were observed whether there was still an effect on the microvascular network.At the same time,different concentrations of CAP,Rut,CAPZ(1μM,3μM,10μM)were also affected by the microvascular network in the 3D environment.Methods:HUVECs and ADSCs,at the same time,HUVECs and Human skin fibroblast cells(HSFs)were placed on the basement membrane matrigel and mixed and co-cultured.To explore the effect of adding different concentrations of Cap,Rut,and CAPZ(1μM,3μM,10μM)on the formation of microvascular networks under the condition of co-cultivation of two kinds of cells.At the same time,different concentrations of CAP,Rut,CAPZ(1μM,3μM,10μM)were also affected by the microvascular network in the 3D environment.Results:(1)In a 2D environment:when HUVECs is combined with ADSCs,the TRPV1receptor agonist Cap increases with the concentration,which can significantly increase the number of microvascular network intersections,and the difference has statistically significant(P<0.05),but angiogenesis there is no significant effect on the length of the branch,no significant difference(P>0.05);With the increase in concentration,the TRPV1 receptor agonist Rut did not have a significant effect on the effect of the microvascular formation.Differences have statistically significant(P<0.05);HUVECs is combined with HSFs,the TRPV1 receptor agonist Cap increases with the concentration increases,and the length of the microvascular network intersection and the length of the branch are statistically significant.P<0.05);TRPV1receptor agonist Rut promotes the effect of intersection at low concentration,and the difference has statistically significant(P<0.05),but the length of the vascular branch has no significant effect,no significant difference(P>0.05)The TRPV1 receptor antagonist CAPZ,at a high concentration,significantly inhibited microvascular mesh,and the difference has statistically significant(P<0.05).(2)In a 3D fiber protein environment:HUVECs and ADSCs mixed co-cultured in fibrino matrix,at a concentration of 1μM,3μM,10μM,and Cap promotes the number of intersections of microvascular network,and the difference has statistically significant(P<0.05However,Cap has no significant effect on the length of the vascular branch,and there is no significant difference(P>0.05);Rut promotes the effect of microvascular measuring network,and the difference has statistical significance(P<0.05),CAPZ significantly inhibits microvascular network,difference It has statistical significance(P<0.05).Conclusion:(1)Cap,Rut,CAPZ has different degrees of impact on the microvascular network formed by cells in a two-dimensional environment in vitro:TRPV1 receptor agonist Cap and Rut at 1μM,3μM,10μM,for different The microvascular network of cells has different degrees of affected,but the TRPV1 receptor antagonist CAPZ has significantly inhibited the construction of microvascular networks as concentrations.(2)In a more real biological 3D environment,the TRPV1 receptor agonist Cap and Rut can promote microvascular mesh,while TRPV1 receptor antagonist CAPZ significantly inhibits microvascular web.