| Objectives:(1)To explore the effects of tumour related cytokins in targeting lymphatic metastasis by detecting the expression of them in different microenvironments(lymph node and bone marrow)at different stages of oral squamous cell carcinoma(OSCC).(2)To explore the influence of tumour cells on the microenvironment and lymphatic metastasis by detecting the expression of related cytokines before and after co-culture of oral cancer cells and microenvironment.Methods:(1)4-nitroquinoline-1-oxide(4NQO)was used to build an oral squamous cell carcinoma and lymph node metastasis model of Balb/c mice.The microenvironments(both lymph nodes and bone marrow)of normal mice(normal group,blank control group),mice used 4NQO for 8 weeks(8 weeks group,negative control group),mice with dysplasia(dysplasia group),mice with OSCC(OSCC group)and mice with lymphatic metastasis(metastasis group)were collected.(2)Immunohistochemical staining was used to detect the expression of transforming growth factor-?1(TGF-?1)?transforming growth factor-?2(TGF-?2),Interleukin-10(IL-10)and vascular endothelial growth factor-A(VEGF-A)in submandibular lymph nodes and bone marrow of mice at different stages to understand their role in lymphatic metastasis of oral cancer.(3)Liquid chip method was used to dectect the expressions of cytokines granulocyte-macrophage colony-stimulating factor(GM-CSF),interferon-gamma(IFN-?),IL-10,Interleukin-12(IL-12p40),Interleukin-6(IL-6),CXC chemokines2(CXCL2),tumour necrosis factor-?(TNF-?)and VEGF-A in the microenvironment of different organs at different stages of OSCC to understand their role in lymphatic metastasis of oral cancer.(4)Transwell chamber was used to establish different microenvironments and SAS cell co-culture model at different stages.At 0h,24 h,48h and 72 h of co-culture,the supernatant of the microenvironment was collected and the expression of each cytokine was detected by liquid chip to investigate the influence of tumour cells on cytokines in the microenvironment.Results:(1)Immunohistochemical staining showed that the results of cytokines in bone marrow smears could not be statistically treated.The expression levels of IL-10 and VEGF-A in the lymph nodes of normal group were higher than those of 8 weeks group(P<0.005).The expression levels of IL-10 and VEGF-A in the lymph nodes of 8 weeks group showed no significant difference from those of dysplasia group(P>0.05),while gradually increased in the lymph nodes of OSCC group and metastasis group(P<0.005).There was no statistically significant difference in the expression of TGF-?1 in the lymph nodes of normal group and 8 weeks group(P>0.05),but the expression level of TGF-?1 increased gradually in the stages of dysplasia,OSCC and metastasis group(P<0.005).The expression level of TGF-?2 in the submandibular lymph nodes of normal,8 weeks and dysplasia group was gradually increased(P<0.005).There was no statistically significant difference between the expression level of TGF-?2 in the lymph nodes of OSCC and dysplasia group(P>0.05).The expression level of TGF-?2 in metastasis group was significantly higher than that in lymph nodes of OSCC group(P<0.005).(2)The expression of IFN-??IL-10?IL-12p40?IL-6?CXCL2?TNF-? in the lymph node was higher than that in bone marrow in normal,8 weeks,dysplasia,OSCC and metastasis group(P < 0.05),while the expression of VEGF-A within the lymph node was lower than that in bone marrow(P < 0.05).There was no significant difference in the expression of CXCL2 between lymph nodes and bone marrow in dysplasia and OSCC group(P>0.05).(3)In lymph nodes,the expression of IFN-? in normal,dysplasia,OSCC and metastasis group was lower than that in 8 weeks group(P<0.05).The expression of IFN-? in dysplasia group was higher than that in normal group(P<0.05).The expressions of IL-10 in normal and metastasis group were higher than that in 8 weeks and dysplasia group(P<0.05).The expression of IL12p40 in 8 weeks and metastasis group was higher than that in normal group(P<0.05).The expression of IL12p40 in 8 weeks group was higher than that in OSCC group(P<0.05)while IL12p40 in metastasis group was higher than that in dysplasia and OSCC group(P<0.05).The expression of IL-6 in the normal group was higher than that in 8 weeks and dysplasia group(P<0.05).The expression of IL-6 in 8 weeks group was lower than that in dysplasia,OSCC and metastasis group(P<0.05).IL-6 in dysplasia group was lower than OSCC and metastasis group(P<0.05).The expression of CXCL2 in the normal and OSCC group was higher than that in 8 weeks group(P<0.05).The expression of TNF-? was higher in normal and 8 weeks group than OSCC group(P<0.05).The expression of TNF-? was higher in metastasis group than dysplasia and OSCC group(P<0.05).The expression of VEGF-A in normal group was higher than that in dysplasia and OSCC group(P<0.05).The expression of VEGF-A in dysplasia group was higher than that in metastasis group(P<0.05).There was no significant difference of expression of these cytokines in lymph node between other groups(P>0.05).In bone marrow of each stage of OSCC,the expressions of IFN-? in normal,8 weeks and metastasis group were higher than that in dysplasia and OSCC group(P<0.05).IFN-? in dysplasia was higher than that in OSCC group(P<0.05).The expression of IL12p40 in normal group was higher than that in dysplasia,OSCC and metastasis group(P<0.05).IL12p40 in 8 weeks group was higher than that in dysplasia and OSCC group(P<0.05).IL12p40 in metastasis and dysplasia group were higher than OSCC group(P<0.05).The expression of IL-6 in 8 weeks group was lower than that in dysplasia and OSCC group(P<0.05).The expression of CXCL2 in 8 weeks group was higher than that in dysplasia and metastasis group(P<0.05).CXCL2 in OSCC group was higher than that in metastasis group(P<0.05).The expression of TNF-? in normal group was higher than that in dysplasia and metastasis group(P<0.05).TNF-? in 8 weeks and metastasis group were higher than that in dysplasia group(P<0.05).The expression of VEGF-A in the normal group was higher than that in dysplasia,OSCC and metastasis group(P<0.05).VEGF-A in 8 weeks group was higher than that in dysplasia,OSCC and metastasis group(P<0.05).While the expression of these cytokines in bone marrow in other groups showed no statistical significance(P>0.05).(4)SAS had no significant effect on the expression levels of GM-CSF,IFN-?,IL-12p40,and CXCL2 in bone marrow at different stages,while the expression levels of GM-CSF,IFN-?,IL-10,TNF-? and VEGF-A in lymph nodes of normal group,8 weeks group and dysplasia group significantly increased with co-culture time.Conclusions:(1)The expression differences of IFN-?,IL-10,IL-12p40,IL-6,CXCL2,TNF-?,VEGF-A,TGF-?1 and TGF-?2 in submandibular lymph nodes at different stages in the occurrence and development of OSCC until metastasis suggest that these cytokines may be involved in the formation process of submandibular lymph node metastatic carcinoma of OSCC.(2)These results showed that cytokines IFN-?,IL-10,IL-12p40,IL-6,TNF-?in lymph nodes were higher than those in bone marrow at different stages of OSCC and lymphatic metastasis,and the expressions of CXCL2 in lymph nodes of normal,OSCC and metastasis group were higher than those in bone marrow.These differences in expression may be an important reason for the targeting of lymphatic metastasis in OSCC.(3)Co-culture of tumour cells with microenvironment of bone marrow and lymph nodes at different stages showed that compared with bone marrow,tumour cells had a greater influence on the expression of cytokines in lymph nodes.This suggests that the targeting of lymphatic metastasis of OSCC may be the result of the combined action of tumour cells and cytokines. |
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