The Study On Effects Of Rh-endostatin And Docetaxel On HUVECs

Partâ… The study on effects of rh-endostatin and docetaxel in phenotypes of HUVECs in different activating levelsObjective:1 To simulate vascular endothelial cell activation,proliferation status in vitro.2 To study the effects of rh-endostatin and docetaxel in phenotypes of HUVECs in different activating levels and further reveal the mechanisms of rh-endostatin.3 To study the effects of rh-endostatin and docetaxel in proliferation,apoptosis and cell cycle of HUVECs and further explore the relevant mechanisms.4 To assess the combination effects of rh-endostatin and docetaxel on HUVECs.If synergistic effect was observed,we further investigate the relationship between the effect and the optimum time,sequence or dose of the combination regimens,and explore the possible mechanisms in order to instruct the clinic.Methods:1 To detect the markers CD146 and CD105 on the surface of confluent HUVECs, stimulated HUVECs with TNF-αby flow cytometry;2 The effects of rh-endostatin,docetaxel and rh-endostatin+docetaxel on the confluent HUVECs,stimulated HUVECs with TNF-αwere indicated by the variation of the expressions of CD 146 and CD 105 by flow cytometry;3 The effects of different administrations on stimulated HUVECs with VEGF were revealed by the variation of the expressions of CD146,CD105 and CD62E by flow cytometry;4 The effects of rh-endostatin and docetaxel on HUVEC proliferation were studied by means of MTT;5 The cell apoptosis and cell cycle were determined by flow cytometry;6 The data were analyzed by One-way ANOVO.The relationship between HUVEC cell surface markers and drug effects was analyzed by SPSS13.0.Results:1 The percentage of cell markers CD146 and CD105 after treatments with different drugs:(1) CD146(1) Confluent HUVECs after treatments with drugs by 48h:control,different concentrations of single rh-endostatin and rh-endostatin 300μg/ml+docetaxel, CD146 expression differences were statistically significant(all P＜0.05).(2) Confluent HUVECsâ‘ docetaxel between 24h and 48h or 72hâ‘¡rh-endostatin 100μg/ml+docetaxel between 24h and 72hâ‘¢rh-endostatin 300μg/ml +docetaxel between 24h and 48h,CD146 expression differences were statistically significant (all P＜0.05).(3) Stimulated HUVECs with TNF-αafter treatments with drugs by 48h:â‘ control and rh-endostatin 300μg/mlâ‘¡rh-endostatin 200μg/ml and rh-endostatin 300μg/ml +docetaxelâ‘¢rh-endostatin 300μg/ml and rh-endostatin300μg/ml+docetaxel, CD146 expression differences were statistically significant(all P＜0.05).(2)CD105(1) Confluent HUVECs after treatments with drugs by 48hâ‘ control and docetaxelâ‘¡control and rh-endostatin 300μg/ml+docetaxelâ‘¢rh-endostatin 100μg/ml and rh-endostatin 300μg/ml+docetaxelâ‘£rh-endostatin 200μg/ml and rh-endostatin 300μg/ml+docetaxel⑤rh-endostatin 100μg/ml+docetaxel and rh-endostatin 300μg/ml+docetaxel,CD105 expression differences were statistically significant (all P＜0.05).(2) Stimulated HUVECs with TNF-αafter treatments with drugs by 24h:â‘ control and docetaxelâ‘¡control and each combined treatment group,CD105 expression differences were statistically significant(all P＜0.05).(3) Stimulated HUVECs with TNF-α:docetaxel,combined treatment groups between 24h and 48h or 72h,CD105 expression differences were statistically significant(all P＜0.05).(3) Confluent HUVECs after treatments with drugs by 48h:CD146 and CD105 expressions showed a linear trend,the correlation coefficient was 0.908 (P=0.002).(4) Stimulated HUVECs with TNF-αafter treatments with drugs by 48h: CD146 and CD105 expression showed non-linear trend,the correlation coefficient was -0.226(P=0.590).2 The percentage of cell markers CD146,CD105 and CD62E on stimulated HUVECs with VEGF after treatments with different administrations.(1) CD146(1) CD146 expression differences were statistically significant(all P＜0.05) between control or VEGF groups and simultaneous,docetaxel followed by rh-endostatin(sequence 1) groups.(2) CD146 expression differences were statistically significant(all P＜0.05) between rh-endostatin group and docetaxel,simultaneous,sequence 1 groups.(3) CD146 expression differences were statistically significant(all P＜0.05) between sequence 2 group and simultaneous,sequence 1 groups.(2) CD105There were no statistical significances among all groups.(all P＞0.05)(3) CD62E(1) CD62E expression differences were statistically significant(all P＜0.05) between control group and docetaxel,simultaneous,sequence 1 groups.(2) CD62E expression differences were statistically significant(all P＜0.05) between rh-endostatin group and docetaxel,simultaneous,sequence 1 groups.(3) CD62E expression differences were statistically significant(all P＜0.05) between sequence 1 group and VEGF,sequence 2 groups.3 The effects of the two single drugs on inhibition of HUVEC proliferation:The inhibition rate of rh-endostatin increased significantly after 48h and kept stable in a relatively high level after 96h～120h;Docetaxel had marked inhibition rate after 48h.4 The effects of different administrations on inhibition of HUVEC proliferation: The joint had a more obvious role than monotherapy and simultaneous administration had the highest inhibition rate(77.4%).5 Apoptosis study demonstrated that docetaxel followed by rh-endostatin had the maximum apoptosis rate(34.7%).Cell cycle study exhibited that compared with control,cell proportion slightly increased at G₀/G₁ phase after single rh-endostatin;increased at G₂/M phase after single docetaxel;increased at G₀/G₁ phase after docetaxel followed by rh-endostatin;increased at G₂/M phase after simultaneous administration;increased at G₀/G₁ and reduced at G₂/M phase after rh-endostatin followed by docetaxel.Conclusions:1 CD146,CD105 expressions on confluent HUVECs could be reduced by certain concentrations of Rh-endostain and/or docetaxel.2 Rh-endostatin could promote cell adhesion and might have a weak role in inhibiting cell activation within a certain period of time when endothelial cells were in the state of excessive activation.With the time extended,cells occurred apoptosis or necrosis by rh-endostatin.Docetaxel could directly inhibit cell activation,proliferation,adhesion,and induce cell apoptosis or necrosis.3 Rh-endostatin with certain concentrations combined with docetaxel were stronger than single drugs on effects of inhibiting cell activation,proliferation,adhesion, induction of apoptosis or necrosis.Simultaneous and docetaxel followed by rh-endostatin groups had more obvious function.Rh-endostatin followed by docetaxel group inferiored to single docetaxel.Therefore,Simultaneous and docetaxel followed by rh-endostatin styles were the first choices in the combined administrations.The synergistic effect could be associated with cycle distribution and apoptosis,etc. Partâ…¡The study on effects of rh-endostatin and docetaxel on functions of HUVECsObjective:1 To show the effects of rh-endostatin and docetaxel on the expressions of EMMPRIN and MMP-2 of HUVECs.2 To observe the effects of rh-endostatin and docetaxel on HUVEC tube formation.3 To assess the combination effects of rh-endostatin and docetaxel on HUVECs.If synergistic effect was observed,we further investigate the optimum time, sequence or dose of the combination regimens and the possible mechanisms in order to instruct the clinic.Methods:1 The expressions of EMMPRIN and MMP-2 were analyzed by immunohistochemistry and Western blot;2 The effects of drugs on aCEC tube formation were observed and photographed by inverted contrast-phase microscope;3 The data were analyzed by One-way ANOVO.The relationship between the functions of HUVECs and drugs were analyzed by SPSS13.0.Results:1 Immunohistochemistry showed that compared with control,cell morphology and density changed and the expressions of both EMMPRIN and MMP-2 reduced after treatments.2 Western blot showed that both rh-endostatin and docetaxel could diminish the expressions of both EMMPRIN and MMP-2.3 Tube formation study indicated that rh-endostatin could promote the formation of HUVEC tube-like structure within a certain period of time.With the time extended,the ability of tube formation was cut down.Docetaxel could directly inhibit the formation of tube-like structure.Rh-endostatin combined with docetaxel produced a synergistic inhibitory effect,and docetaxel followed by rh-endostatin and simultaneous treatments had more obvious inhibition.Conclusions:1 Both rh-endostatin and docetaxel with certain concentrations could diminish the expressions of EMMPRIN and MMP-2.2 Docetaxel could directly inhibit cell activation,proliferation,adhesion,the formation of tube-like structure,induce cell apoptosis or necrosis,diminish vascular basement membrane degradation and reduce vascular permeability because of its cytotoxicity.3 Rh-endostatin could promote endothelial cell adhesion,collection,promote cell differentiation and maturation,and promote the formation and differentiation of tube-like structure;and it could diminish vascular basement membrane degradation by MMPs,therefore,vascular permeability,IFP and vascular pressure could be reduced and vasculars could be rearranged within a certain period of time.It means that rh-endostatin had a role in vascular normalization within a certain period of time.With the time extended,cells occurred apoptosis or necrosis,and the ability of HUVEC tube formation was weaken.Therefore,the effect of rh-endostatin's vascular normalization might be short-term.4 Simultaneous and docetaxel followed by rh-endostatin administrations exhibited better inhibitory effect on HUVEC tube formation.The effect mechanisms could be associated with docetaxel's stronger cytotoxicity,higher capacity of destroying cell structure than rh-endostatin;on the other hand,expressions of adhesion molecules could be reduced after simultaneous and docetaxel followed by rh-endostatin administrations.Rh-endostatin followed by docetaxel was weaker than the above combined administrations,which might be due to the expressions of CD146 and CD62E were increased and the two adhesion molecules could contribute to the formation of tube-like structure;docetaxel could inhibit cell activity,proliferation,adhesion,etc,so the number of tube-like structures was diminished and less than rh-endostatin's.