Expression Of Esophageal Cancer Four Syndromes Associated Autoantigens And Its Regulation Of Removing Heat And Phlegm Formula

Objective:To study the distribution of esophageal cancer(EC)associated autoantigens CK13,CK16,CaD and ACTG2 expression in EC four syndromes,then further explore the regulatory effects of removing heat-phlegm formula and its two separated prescription(removing heat prescription and removing phlegm prescription)on the proliferation of EC cell and EC associated autoantigens expression,so as to provide basis for the molecular pathogenesis of EC syndromes and clinical medication.Methods:1.Comparative analysis between EC autoantigens-antibodies and patients’ self-serum on EC tissue by using western blot method.2.Immunohistochemical method was used to analyze the expression of CK13,CK16,CaD and ACTG2 in EC four syndromes of normal tissue,para-carcinoma tissue and carcinoma tissue.3.MTT method was used to detect the effects of removing heat and phlegm formula and its two separated prescription(removing heat prescription and removing phlegm prescription)on the proliferation of EC109 cell,EC9706 cell and TE-1 cell.4.Flow cytometry method was used to analyze the effects of removing heat and phlegm formula and its two separated prescription(removing heat prescription and removing phlegm prescription)on the cell cycle of EC109 cell,EC9706 cell and TE-1cell.5.Western blot method was used to analyze the effects of removing heatand phlegm formula and its two separated prescription(removing heat prescription and removing phlegm prescription)on CK13,CK16,CaD and ACTG2 protein expression of EC109 cell,EC9706 cell and TE-1 cell.Results:1.Onset age of different syndrome EC patients was different,onset age of EC patients with phlegm and qi stagnation syndrome was 60.6±8.1,blood stasis syndrome was 61.5±11.6,body fluid insufficiency and heat agglomeration syndrome was 65.6±8.1,qi and yang insufficiency syndrome was 68.4±7.3.2.Expression of Autoantigens CK13,CaD,ACTG2 and CK16 were detected in EC tissue of phlegm and qi stagnation syndrome,blood stasis syndrome,body fluid insufficiency and heat agglomeration,and qi and yang insufficiency syndrome respectively,autobodies of autoantigens could be used to detect expression of EC tissue autoantigens.3.Positive expression rates and positive expression level of CaD in EC patient carcinoma tissue were higher than its normal tissue and para-carcinoma tissue,and expression of para-carcinoma tissue was higher than normal tissue.Positive expression rates of CaD in normal tissue,para-carcinoma tissue and cancer tissue of EC patients was 50.9%,62.2%,72.8%,and its level was 0.0085±0.0048,0.0107±0.0056,0.0177±0.0103;Positive expression level of CK16 and ACTG2 in EC carcinoma tissue was higher than normal tissue and para-carcinoma tissue,and expression of para-tissue was higher than normal tissue,positive expression level of CK16 in normal tissue,para-carcinoma tissue and cancer tissue of EC patients was0.0076±0.0033,0.0158±0.0065,0.0356±0.0165,positive expression level of ACTG2 in normal tissue,para-carcinoma tissue and cancer tissue of EC patients was0.0091±0.0039,0.0136±0.0043,0.0214±0.0110;Positive expression level of CK13 in EC carcinoma tissue was lower than normal tissue and para-carcinoma tissue,and expression of para-tissue was lower than normal tissue,positive expression level of CK13 in normal tissue,para-carcinoma tissue and cancer tissue of EC patients was0.2053±0.0311,0.1633±0.0280,0.0412±0.0239.4.Expression of CK13,CaD,ACTG2 and CK16 in EC patients four syndromes carcinoma tissue was different: Positive expression rates and positive expression level of CaD in blood stasis syndrome was 100%(6/6)and 0.0364±0.0051 respectively,which was higher than phlegm and qi stagnation syndrome(29/34,0.0168±0.0088),body fluid insufficiency and heat agglomeration syndrome(5/8,0.0159±0.0045)and qi and yang insufficiency syndrome(5/7,0.0166±0.0089);Positive expression level of ACTG2 in body fluid insufficiency and heat agglomeration syndrome was0.0346±0.0100,which was higher than phlegm and Qi stagnation syndrome0.0201±0.0106,blood stasis syndrome 0.0244±0.0043 and Qi and Yang deficiency syndrome 0.0235±0.0077;Positive expression level of CK16 in Qi and Yangdeficiency syndrome was 0.0595±0.0131,which was higher than phlegm and Qi stagnation syndrome 0.0325±0.0167,blood stasis syndrome 0.0227±0.0154 and body fluid insufficiency and heat agglomeration syndrome 0.0393±0.0068;Positive expression level of CK13 in Qi and Yang deficiency syndrome was 0.0174±0.0058,which was lower than phlegm and Qi stagnation syndrome 0.0452±0.0292,blood stasis syndrome 0.0385±0.0209 and body fluid insufficiency and heat agglomeration syndrome 0.0330±0.0251;Expersion of CK13,CK16,CaD,ACTG2 had no significance in different syndromes of EC normal tissue and para-carcinoma tissue.5.Removing heat and phlegm formula and its two separated prescription(removing heat prescription and removing phlegm prescription)all had inhibitory effects on the proliferation of EC9706,EC109 and TE-1 cells,and the removing heat and phlegm foumula had the strongest inhibitory effect,then the removing heat group,the removing phlegm group.6.Removing heat and phlegm formula and its two separated prescription(removing heat prescription and removing phlegm prescription)all had effects on the cell cycle of EC9706,EC109 and TE-1 cells: Compared with the blank control group,cells of S phase in the removing heat group,the removing phlegm group and the whole formula group of EC109 and TE-1 cells decreased,and the removing phlegm group changed the most;EC9706 cells of removing heat group in S phase increased significantly,G2 phase cells decreased significantly,removing phlegm group G1 phase cells decreased,G2 phase cells increased significantly,the whole formula group cells in G1 phase increased significantly,while its cells in G2 phase decreased significantly.7.Removing heat and phlegm formula and its two separated prescription(removing heat prescription and removing phlegm prescription)could varying degrees regulate CK13,CK16,CaD,ACTG2 expression in EC9706,EC109 and TE-1 cells.Compared with the blank control group,the expression of CK13 in all drug group increased,while the CK16,CaD and ACTG2 decreased.Conclusion:1.There were expression of CK13,CK16,CaD and ACTG2 in EC patients carcinoma tissue,of which,expression of CK16,CaD and ACTG2 increased,while expression of CK13 decreased.2.Expression of CK13,CK16,CaD and ACTG2 all changed in the pathological process of EC different synddromes,of which,expression of CK13 in phlegm and qi stagnation syndrome has great change,expression of CaD in blood stasis syndromehas great change,expression of ACTG2 in body fluid insufficiency and heat agglomeration has great change,and expression of CK16 in qi and yang insufficiency syndrome has great change.3.Removing heat and phlegm formula and its two separated prescription(removing heat prescription and removing phlegm prescription)all have inhibitory effects on the proliferation of EC9706,EC109 and TE-1 cells,and present dose-dependent and time-dependent relationship.4.Removing heat and phlegm formula and its two separated prescription(removing heat prescription and removing phlegm prescription)all have effects on the cell cycle of EC9706,EC109 and TE-1 cells,of which,the drug groups’ effects of EC109 and TE-1 cells were mainly in S phase,and that of EC9706 was mainly in S phase and G2 phase.5.The inhibitory effects of removing heat and phlegm formula and its two separated prescription(removing heat prescription and removing phlegm prescription)on the proliferation of EC109,EC9706 and TE-1 cells have relationship with the expression level of CK13,CK16,CaD,ACTG2,which provides molecular basis for the treatment of EC with removing heat-phlegm formula.6.EC pathogenesis of phlegm stagnation,blood stasis,fluid insufficiency and qi deficiency have relationship with change of autoantigens CK13,CK16,CaD,ACTG2 expression.