Experimental Observation Of Basil Polysaccharide Against VEGF-C/D-VEGFR-2/3Signal Pathway And The Expression Of Bax/Bcl-2of Human Lymphatic Endothelial Cells

OBJECTIVE: TO investigate the effect of basil polysaccharide againstVEGF-C/D-VEGFR-2/3signal pathway of HLECs and the co-culture system bySKOV3-HLECs,and study on the changes of expression level of mRNA and proteinabout Bax/Bcl-2、VEGF-C\D and VEGFR-2/3to provide basis for the clinicalapplication and development of drugs.METHODS:Using analysis methods such as the in vitro tube, enzyme-linked immuno assay,immunocytochemistry,and the quantitative fluorescent RT-PCR assay to make asystematic study on the changes of expression level of mRNA and protein aboutBax/Bcl-2、VEGF-C/D and VEGFR-2/3under different drug concentrations andoxygen concentration.RESULTS:1. Establish of the single culture system and co-culture systemAfter recovery and inoculation, the SKOV3and HLECs are in a good growthcondition, and make generation successfully. After the construction of co-culturesystem, the SKOV3and HLECs are also in a good growth state, and never contactmixed. 2. The effect of basil polysaccharide against the pipe number of HLECsRegardless of the normoxia and hypoxia the basil polysaccharide can reducethe pipe numbers by reducing with increase concentration in vitro, whatever under thecondition of mono-culture and co-culture system.3.The effect of basil polysaccharide against the changes of expression level of mRNAand protein about Bax/Bcl-2、VEGF-C/D and VEGFR-2/3.In the single culture system, basil polysaccharide can raise the mRNA expressionof VEGFR-2/3and Bcl-2under normoxic condition; meanwhile basil polysaccharidecan reduce the mRNA expression of VEGFR-3and raise the mRNA expression ofBax under hypoxic condition.In the co-culture system, we also obtain someinformation form mRNA by RT-PCR: in the normoxia environment the mRNAexpression of VEGFR-3and Bcl-2increases obviously, and the mRNA expression ofBax mRNA inhabits significantly; in the hypoxia condition the expression ofVEGFR-2increases and the expression of Bax mRNA decreases.In the single culture system, the basil polysaccharide can raise the proteinexpression of VEGFR-2/3in the normoxia environment and raise the proteinexpression of VEGFR-3under hypoxic condition. The content level of VEGFR-3didnot change in the co-culture system regardless of normoxia or hypoxia, but the basilpolysaccharide can reduce the expression of VEGFR-3. Meanwhile in the normoxiaenvironment the protein expression of VEGFR-2did not change, and the basilpolysaccharide can inhibit the protein expression of VEGFR-2. And in the hypoxiaenvironment and co-culture system the protein expression of VEGFR-2increases, andthe basil polysaccharide has no effect for it.In the single culture system, basil polysaccharide can increase proteinexpression of the VEGF-C and VEGF-D in normoxic condition, meanwhile in thehyloxic condition basil polysaccharide can reduce it.In the co-culture system, the protein content of VEGF-C and VEGF-D increasedsignificantly in the normoxic and hypoxic environment; under the intervention ofbasil polysaccharide the protein content of VEGF-C can be reduced significantly, and in the hypoxic the protein content of VEGF-D can be also reduced significantly.Further study from this experiment, basil polysaccharide decreases the mRNAexpression of VEGFR-3and Bcl-2in normoxia environment; and the basilpolysaccharide can also decrease the mRNA expression of VEGFR-3and VEGFR-2,and increase the mRNA expression of Bax and Bcl-2; furthermore, there is somedifference among diverse concentrations.CONCLUSION:The basil polysaccharide can regulate the biological activity, through observe thechange of various signal facto; and there is some difference among different oxygenconcentration and drug concentration.