Effects Of Ginseng On Molecules Mechanism In Lung Cancer Cells Based On Tumor Associated Macrophage

Chinese medicine is the characteristic method of treatment of malignant tumor,that the superiorities of improve patients' life quality,prolong survival advantage and ease side effects,the curative effect and security are widely acknowledg.In traditional chinesemedicine,Yi qi fu zheng is the main principle at tumor treatment,ginseng as a represent medicine wildly used in clinical.In recent years,the study found that ginseng has effect of regulate immunocyte and improve the immune function.Tumor growth is based on Tumor Microenvironment,that constantly provides a energy source for the de velopment of tumor.Tumor associated macrophages is the main immunocyte,involve ment the process of growth,invasion and metastasis of tumor,the key to band the deve lopment of tumor and Tumor immuno suppression.Therefore,by defining the adjust ent of ginseng to TAMs immunocompetence,to study its mechanism of tumor therapy1.Literature reviewBy means of reviewing relevant literatures,the current status of studied on TME knowledges of TCM,application in tumor immune microenvironment of Yi qi fu zheng and adjustment of ginseng to TME,research the relationship of TCM and TME.Summarizing the relationship betweenTME and tumor development,TME and TAMs,the phenotype of TAMs,TAMs improve the tumor groth,which lays the foundtion for providing practical research programs and determing the study of examined indicator and the starting point on this thesis.2.Build and identification the model of TAMs2.1 Induction of THP-1 cells from three different conditions to build and optimization TAMs model.The three different conditions;PMA+5%FBS,PMA+IL-13andIL-4+5%FBS,PMA+IL-13andIL-4.The microscope observation method demonstrated that the suspension of THP-1 cells become adherent TAMs cells after induction,serum-freeTA Ms cells had poor state,part of death.Q-PCR analysis showed that CD68 were expres sed highly in three conditions,IL-12 expressed lowly in serum containing,IL-10 exp-ressed highly in containing of interleukin.The results indicated that the process of in-ducing must provide the needed nutrients-FBS,interleukin cound increase the expres sion of M2 phenotype,and selection model for induced conditions was PMA+IL-13,IL-4+5%FBS.2.2 Validation of the TAMs model.Q-PCR results showed that the markers of M1 phenotype significantly decreased(P<0.01),the markers of M2 phenotype obviously increased.ELISA showed that the TAMs model highly secreted of IL-12,lowly secreted IL-10.FCM tested the TAMs had high expression of CD206.All the results indicated that the model success.3.The effect of ginseng and its main components on phenotype of TAMs cells3.1 Ginseng quality control and water extract of ginseng preparation.Emploed HPLC test the contents of ginsenoside Rg1?Re?Rb1,The result indicated that the contents of ginsenoside Rg1?Re?Rb1 requirements of the pharmacopoeia,the main material composition in the water extraction liquid had be soluted.3.2The effect of WEG on phenotype of TAMs cells.The result Q-PCR showed that WEG cound increased the mRNA expression of IL-12?IL-10?iNOS,decreased the mRNA expression of CD206 and remained statistically significant.ELISA showed that WEG cound increased the secretion of IL-12 in supernatant,suppression IL-10 Western blot assay demonstrated that WEG cound significantly increased the protein expression of iNOS and decreased the protein expression of CD206.The result indica ted that WEG cound adjust the markers of TAMs,changed its phenotype.3.3 The effect of main components on phenotype of TAMs cells.The result Q-PCR showed that GPS and GTS cound increased the mRNA expression of IL-12?IL-10,GTS cound decreased the mRNA expression of CD206 and GPS cound increased the mRNA expression of iNOS.ELISA showed that both GPS and GTS cound increased the secretion of IL-12 and IL-10 in supernatant.Western blot assay demonstrated that GPS cound significantly increased the protein expression of iNOS and GTS cound decreased the protein expression of CD206.The result indicated that main components of ginseng could adjust the markers of TAMs,but the markers were different.4.Effect of ginseng on lung cancer A549 in co-culture system with TAMs and A5494.1 Build the model of co-culture system and effect ofthis system on lung cancer A549 cell proliferation and migration.The microscope observation and MTT method demon strated that co-culture system cound promoted the A549 cells proliferation.The Real-time Cell Analysis(RTCA)tested that this system cound significantly promoted the cell migration of lung cancer.Indicated that simulated tumor immune microenvir onment with TAMs and A549,which promoted the biological behavior of tumor cells.4.2 The effect of ginseng and its main components on lung cancer A549 cell proliferation in different conditions.The result of MTT showed that WEG and GPS cound promote the A549 cells proliferation,GTS inhibited yet;however,in co-culture system of transwell WEG and GTS cound inhibit the A549 cells proliferation,GPS promoted yet.Indicated that WEG may has the inhibition of A549 cells by regulated TAMs immunocompetence.4.3 The effect of ginseng and its main components on on lung cancer A549 cell migration in co-culture system.The Real-time Cell Analysis(RTCA)tested that WEG and GPS significantly inhibit the cell migration of lung cancer.Immunofluorescence High content screening(HCS)analysised that WEG and GPS cound significantly decrease the number of cell microfilament skeleton,cytoskeleton area,No effect of GTS on lung cancer A549 cell migration.The results indicated that WEG snd GPS adjust TME inhibiting migration ability of lung cancer A549 cells,but,GTS may has not adjustment for tumor metastasis.