| Objective:The high-trend of lymph node metastasis and potential risk of distant metastasis of nasopharyngeal carcinoma(NPC),impact on the patient's prognosis seriously.In this study,we would be the first to clear the relationship between the expression of lymphangiogenesis factors Ang-1 /Ang-2(angiopoietin,Ang) and lymph node metastasis in NPC,in order to explain their role in tumor-lymphangiogenesis.And then,in vitro,we would use PDT to intervene with NPC cells including CNE-2 cells (tumorigenicity,low metastasis) and 5-8Fcells(high tumorigenicity,high metastasis),to observe the expression of Ang-1,Ang-2.In addition,the other possible mechanisms of anti-lymph node metastasis of PDT will be involved.This study would lay the foundation for experiments in vivo.Methods:1) To marked the lymphatic microvessel by using D2-40 monoclonal antibody,and the related researchs between lymphatic microvessel density(LMVD) and clinicopathological parameters were studied.2) To detect the expression levels of Ang-1,Ang-2 via immunohistochemical technology,and the related researchs were conducted with clinicopathological parameters and LMVD,respectively.3) To grope the half lethal doses of PDT to NPC cells including CNE-2 and 5-8F by using fluorescence detection techniques and CCK-8 colorimetric assay,with blank group,photosensitizer group and laser group as control.4) Using the plate clone assay to observe the change of morphology and proliferation of NPC cells survived from PDT,and to establish their own stability passage cell lines for following experiments.5) Using established cell lines of NPC cells survived from PDT to study their changes of proliferation,adhesion(CCK-8 colorimetric assay) and migration(wound scratch assay),compared to the NPC cells having not been treaded with PDT.6) Using immunocytochemistry and image analysis software IPP6.0 to deal with semi-quantitative of expression levels of Ang-1 and Ang-2.Results:1) D2-40 was successfully used to marked lymphatic microvessel in NPC;LMVD of NPC with lymph node metastasis was higher than that withdout lymph node metastasis(P＜0.01);there was not significant difference between gender,age and clinical stage.2) Positive expression rate of Ang-1 and Ang-2 was 46.15%and 63.5%,respectively;Incidence of lymph node metastasis when Ang-1 or Ang-2 positive was higher than the incidence when Ang-1 or Ang-2 negative(P＜0.05),with gender,age,T stage and other factors had no significant correlation,the highest incidence of lymph node metastasis was found when Ang-1 and Ang-2 positive,followed by Ang-2 positive, but there was no significant difference when Ang-1 and Ang-2 both negative.3) Analysis of contrasting Ang-1 and Ang-2 expression to LMVD showed that when expression of both positive(+/+),LMVD was significantly higher than(+/-),(-/+) and(-/-)groups.4) We succeeded in finding the half lethal dose(LD50) of CNE-2 and 5-8F cells,the former was 0.25mmol/L-2h-1.4J/cm2,the latter was 0.50mol/L-2h-1.21J/cm2.5) Plate clone assay was successfully conducted,observing changes in cell morphology,NPC cells survived from PDT were mostly polygonal.6) It was found that the proliferation of a certain declined,no change in adhesion,migration had declined markedly in NPC cells survived from PDT.7) Expression level of Ang-1,Ang-2 decreased significantly in NPC cells survived from PDT.Conclusions:1) Tumoral lymphatic microvessel might be an important factor in lymph node metastasis in NPC.2) The expressions of Ang-1,Ang-2 in NPC were closely related to lymph node metastasis,and may be involved in formation of lymphatic vessels that promoting lymphatic metastasis;as a result,their cound be used as the risk factors of predicting lymph node metastasis and the candidates of therapeutic target of anti- lymphatic metastasis in the future.3) Varying degrees of decline or decrease in proliferation,migration and the expression of Ang-1,Ang-2 in NPC cells survived from PDT,might be other important mechanisms of inhibitiing tumor metastasis of PDT.
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