| Objectives Specific esterase staining and immunohistochemical methods were used to observe the expression changes of tumor infiltrating neutrophils(TINs)in cervical cancer,and the effects on the expression of CD8~+T lymphocytes and some tumor promoters.The relationship between the expression of TINs infiltration,the expression of CD66b~+TINs and the clinicopathological parameters of CSCC patients in cervical cancer tissues of CSCC group were analyzed.To understand the role of TINs expression in the development of cervical cancer,provide experimental evidence for the prevention and treatment of cervical cancer.Methods A total of 159 patients who underwent hysterectomy or cervical biopsy at the Tangshan Workers'Hospital affiliated to North China University of Science and Technology from September 2014 to September 2017 were selected.The specimens were collected in strict accordance with the inclusion and exclusion criteria collected from cervical tissue specimens.Among them,36 patients underwent total hysterectomy for benign uterine diseases(control group,normal cervical tissue specimens);47 patients with cervical intraepithelial neoplasia(CIN)grade I(CINI)(CINI group,specimens of cervical tissue);34 patients with CINII/III grade(CINII/III group,specimens of cervical tissue were retained);42 patients with squamous cell carcinoma of the cervix(CSCC)(CSCC group,specimens of cervical cancer tissue).The experimental research was approved by the Ethics Committee of Tangshan Workers'Hospital.All specimens were obtained with the informed consent of the patient,and Specimen tissues were grouped by two pathologists according to the 2009 FIGO standard.Pathological types and grades were confirmed according to WHO standard.Record the patient's basic information(see Table 2)and perform statistical analysis on the information.Specific esterase staining was used to detect the infiltration of TINs in different pathological cervical tissues.Immunohistochemistry was used to detect the expression of CD66b~+TINs(CD66b infiltrating TINs surface-specific molecular marker)and immune cell CD8~+T cells in each tissue sample.According to the average percentage of CD66b~+TINs cells,the CSCC component was the CD66b~+TINs high expression group and the CD66b~+TINs low expression group.RT-qPCR was used to detect the mRNA expression of some tumor promoters:tissue degradation enzymes(MMP-9,MMP-3,MMP-1),vascular endothelial growth factor(VEGF),cytokines(IL-11,IL-8,IL-18)and chemokines(CCL2,CCL5,CXCL10)in the CD66b~+TINs high expression group and CD66b~+TINs low expression group in the CSCC group.The relationship between the expression of TINs infiltration,the expression of CD66b~+TINs and the clinicopathological parameters of CSCC patients in cervical cancer tissues of CSCC group were analyzed.Results 1 Statistical analysis of the subject's profile:There were no significant differences in the general conditions(age,menarche age,menstrual cycle,menstrual days,number of pregnancies,abortion times)between the control group,CINI group,CINII/III group and CSCC group(all P>0.05),indicating that the experimental samples are comparable.2 Specific esterase staining showed that the positive response of neutrophils infiltrating the tumor microenvironment was reddish-brown granules located in the cytoplasm.The results showed that the percentage of positive staining cells in the control group,CINI group,CINII/III group and CSCC group was 14.36%±2.09%?20.57%±3.86%?33.61%±5.94%?56.71%±8.13%,respectively.The difference was statistically significant(?~2=140.89,P<0.01).The expression of TINs in CINI group was significantly higher than that in control group(P<0.05).The expression of CINII/III group was significantly higher than that of CINI group and control group(all P<0.05).The expression of CSCC group was significantly higher than that of CINII/III group,CINI group and control group,the difference was statistically significant(all P<0.05).3Immunohistochemical staining results:The percentage of cells infiltrated by CD66b~+TINs in the control group,CINI group,CINII/III group and CSCC group increased sequentially,8.07%±1.63%?10.97%±2.98%?15.71%±3.47%?24.83%±4.29%,respectively.The difference was statistically significant(?~2=124.24,all P<0.01).The percentage of CD8~+T cells in the cervical lesions of each group increased first and then decreased,which were 7.41%±1.35%?10.13%±3.22%?6.36%±2.51%?3.07%±1.09%,respectively(?~2=103.75,all P<0.01).The infiltration expression of CD66b~+TINs and CD8~+T cells in CINII/III group(r=-0.139,P=0.030)and CSCC group(r=-0.275,P=0.034)was negatively correlated.4 According to the degree of infiltration of CD66b~+TINs in cervical cancer tissues,it was divided into high expression group and low expression group.Total RNA was extracted from cervical tissues of both groups and detected by RT-qPCR.Among them,the mRNA relative expression levels of tumor promoting factor tissue degrading enzymes(MMP-9,MMP-3,MMP-1),vascular endothelial growth factor(VEGF),cytokines(IL-11,IL-8,IL-18)and chemokines(CCL2,CCL5,CXCL10)were shown as follows:Compared with the low expression group of CD66b~+TINs,the cervical tissues with high expression were up-regulated:MMP-9 was up 2.756 times,MMP-3 was up 1.252 times,MMP-1 was up 1.475 times,VEGF was up 1.310 times,IL-11 was up 2.215 times,IL-8 was up 1.654 times,IL-18 was up 3.082 times,and CCL2 was up 1.627.Times,CCL5 is up 2.173 times,CXCL10 is up 1.462 times,all P<0.05.5 The correlation analysis between the percentage of TINs infiltration,the expression of CD66b~+TINs and the clinicopathological parameters of CSCC patients showed:Percentage of TINs infiltration and expression of CD66b~+TINs in CSCC group were not related to age,tumor size,tumor differentiation and clinical stage(all P>0.05).But all related to lymph node metastasis(P<0.05).Conclusions Increased TINs infiltration during cervical carcinogenesis;The increase of TINs infiltration inhibits the expression of immune cell CD8~+T cells in the cervical microenvironment.At the same time promote the expression of a variety of tumor-associated promoting factors,directly or indirectly promote the development of cervical cancer,TINs may become a new target for the treatment of cervical cancer.In the identification experiment of TINs,the specific esterase staining method has the characteristics of rapid,sensitive and high specificity,which can replace the immunohistochemistry method.Figure 5;Table 8;Reference 220... |
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