| Objective:Immune tolerance in tumor microenvironment is closely related to tumor occurrence and development,but its specific mechanism is still unknown,chemokines and their receptors also play an important role in tumor progression.Whether there is a correlation between chemokine receptor and immune tolerance in oral squamous cell carcinoma needs further study.This subject takes oral squamous cell carcinoma tissue as the research object,detects the expression of chemokine receptors CCR9,PD-L1/PD-1,p-Akt and the infiltration of CD8+T cells and CD4+Foxp3+T cells,and explores the formation factors of immune tolerance in oral squamous cell carcinoma from the perspective of chemokine receptors.Methods:(1)Clinical specimen collection:HE tissues of oral and maxillofacial surgery department of the affiliated stomatological hospital of Kunming medical university from January 2011 to June 2018 and the tissues diagnosed as oral squamous cell carcinoma by postoperative pathology and the corresponding adjacent tissues were collected.(2)Immunohistochemical method:The expression of PD-1,PD-L1,CCR9,p-Akt protein and CD8+T cell infiltration in tumor were detected by immunohistochemical method.Interpretation of immunohistochemical results:the staining intensity and the number of positive cells were comprehensively analyzed,and the results were determined by semi-quantitative integration method.Five high power fields were randomly selected from each slice to judge the results.The specific criteria were:(1)positive cell number:≤5%was counted as 0;6%-25%is counted as 1 point;26%-50%is counted as 2 points;51%-75%are scored as 3 points;>75%is 4 points;(2)Positive intensity:pale yellow is scored as 1 point;Yellow marks 2 points;Brown is scored 3 points;Judging the positive grade by the product of positive cell number and positive intensity:0 is negative(-);1-4 is weak positive(+);5-8 is positive(++);9-12 is strongly positive(++).(3)Immunofluorescence double labeling method:Immunofluorescence double labeling method was used to detect the infiltration of CD4+Foxp3+T cells in tumor.Interpretation of fluorescent double-standard results of immune tissues:comprehensive analysis was carried out by fluorescence staining intensity and positive cell number,and the results were determined by semi-quantitative integration method.Five high power fields were randomly selected from each slice to judge the results.The specific criteria were:(1)positive cell number:≤5%was counted as 0;6%-25%is counted as 1 point;26%-50%is counted as 2 points;51%-75%are scored as 3 points;>75%is 4 points;(2)Positive intensity:weak is scored as 1 point;medium marks 2 points;strong is scored 3 points;judging the positive grade by the product of positive cell number and positive intensity:0 is negative(-);1-4 is weak positive(+);5-8 is positive(++);9-12 is strongly positive(++).(4)Data processing:Results SPSS 25.0 statistical software was used for statistical analysis.The counting data are expressed by the rate and composition ratio,the grade data are expressed by nonparametric Wilxon rank sum test,the inter-group difference analysis is performed by nonparametric Kruskal-Wallis test(i.e.H test),the correlation analysis is performed by Spearman grade correlation analysis,and the difference is statistically significant with P<0.05.Results:(1)Among 81 tissue specimens,53 were male and 28 were female.The patients were 19-67 years old,with an average age of 53.2 years old.48 cases were highly differentiated,22 cases were moderately differentiated and 11 cases were poorly differentiated.Cervical lymph node metastasis occurred in 20 cases.(2)Immunohistochemical staining showed that CCR9,PD-1,PD-L1,and p-Akt were mainly expressed in OSCC tissues,and the expression in OSCC tissues was significantly stronger than that in adjacent tissues,with statistically significant difference(P<0.05).CCR9,PD-L1 and p-Akt are mainly expressed in tumor cells,and PD-L1 is also expressed in stroma,but mainly expressed in tumor cells.PD-1 is expressed in tumor cells and interstitial cells,but mainly in interstitial cells.CD8+T cell infiltration exists in OSCC tissue,and the infiltration is mainly positive.According to Spearmnan grade correlation analysis,CCR9,PD-L1,and p-Akt are differentially expressed in OSCC tissues with different degrees of differentiation,and are correlated with the degree of differentiation(P<0.05).The infiltration of CD8+T cells in OSCC tissue is related to their differentiation degree(P<0.05).The expression of CCR9,PD-L1,p-Akt in OSCC tissue is correlated with cervical lymph node metastasis(P<0.05).(3)The results of immunofluorescence double-label method showed that CD4+Foxp3+T cells infiltrated in OSCC tissue,and the infiltration was mainly positive,and the infiltration was significantly higher than that of the adjacent tissues,with statistical significance(P<0.05).According to Spearmnan grade correlation analysis,the infiltration of CD4+Foxp3+T cells in OSCC tissues with different differentiation degrees is different,and is correlated with the differentiation degree(P<0.05).The infiltration of CD4+Foxp3+T cells in OSCC tissue is correlated with cervical lymph node metastasis(P<0.05)(4)Further analysis of immunohistochemical and immunofluorescence double-labeled results by SPSS 25.0 shows that the expression of CCR9 is positively correlated with PD-L1,p-Akt expression and CD4+Foxp3+T cell infiltration(P<0.05).The expression of PD-L1 is positively correlated with the expression of PD-1,p-Akt and the infiltration of CD4+Foxp3+T cells(P<0.05).There was a positive correlation between PD-1 expression and CD8+T cell infiltration(P<0.05).p-Akt expression was positively correlated with CD4+Foxp3+T cell infiltration(P<0.05).Conclusion:The expression of CCR9 and PD-L1 in oral squamous cell carcinoma is correlated with cervical lymph node metastasis of oral squamous cell carcinoma,which may be one of the important factors for cervical lymph node metastasis of oral squamous cell carcinoma.The expression of CCR9 in oral squamous cell carcinoma is related to the infiltration of CD4+Foxp3+T cells,which may be one of the reasons for the formation of immunosuppression in oral squamous cell carcinoma. |
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