淋巴管生长因子VEGF-C与口腔癌淋巴道转移关系研究

The lymphatic metastasis is a common complication of squamous cell carcinoma (SCC) rising at oral and maxillofacial region and usually indicates a poor prognosis. In a long period, the treatment of lymphatic metastases has been inefficient due to the poor understanding of the mechanism during tumor cells spreading through the lymphatic system.The vascular endothelial growth factor C (VEGF-C), a member of VEGF/PlGF growth factor family, is a ligand for lymphatic endothelium special receptor, VEGF receptor (VEGFR)-3 (or Flt4). The binding of VEGF-C to VEGFR-3 will active the autophosphoryla tion of the receptor and stimulate the proliferation of lymphatic endothelial cells, which is important in the lymphangiogenesis during the lymphatic metastasis of malignances. The present research was designed to explore the relationship between VEGF-C and the jugular lymphatic metastases of oral carcinoma. The expressions of VEGF-C and VEGFR-3 in normal oral tissue, inflamating tissue, leukoplakia, oral carcinoma and lymphatic metastases were detected by immunohistochemistry and RT-PCR. Blood and lymphatic vessel differential stain was determined by enzyme-histochemical technique. Automated image analysis quantification was applied to determine the immunoreactivities of different lesions and the morphological quantitative changes. Then , using PCR amplification of reverse- transcribed mRNA, VEGF-C cDNA was cloned from human tongue squamous cell carcinoma. After sequenced, the fragment was subcloned into plasmid vector pBKCMV and induced its expression in Ecoli. Then the fragment was used to construct a recombined plasmid (pcDNA3.1(+)-VEGF-C), which was cloned into a lingual SCC cell line (Tca8113) by lipid-mediated transfection. The expression of VEGF-C was detected by Western blotting and immunohistochemical staining using rabbit anti-human VEGF-C polyclone antibody, which recognizes the C-terminal sequence of VEGF-C precursor. The proliferating ratio of transfected cells was assayed by MTT. The cell linage stably expressing VEGF-C was cultured and proliferated in vitro. The hetero-transplanted tumor mold was established using BALB/C nude mice by subcutaneous injection of tumor cells. The tumorigenic and metastatic property of the transfected cell was investigated. The RT-PCR and immunostaining positive rate of VEGF-C was significantly higher in squamous carcinoma tissue than in normal tissue and benign lesions(P<0.05). Good correlations of VEGF-C expression in squamous carcinomas were noted with pathological grades(P<0.05), lymph node metastasis(P<0.01), but not with clinical grades(P>0.05). The number of VEGFR-3 staining lymphatics significantly higher in VEGF-C-positive squamous carcinomas, lymph node metastasis and spuamous carcinomas than in VEGF-C-negative (P<0.01), non-metastasis(P<0.01) and normal mucosa, leukoplakia(P<0.05), respectively. But its relation to the pathological grades and T grades was not significantly statistical difference（P>0.05）. The lumen area (Area) (p<0.05), diameter (Diameter) (P<0.05), the number of total (TNa) (P<0.01) and opening (ONa) (p<0.05) lymphatics in unit area of squamous cell carcinoma were bigger than those in normal tissues. The Area(P<0.05), Diamter(P<0.05), TNa (P<0.01)in metastasis group and VEGF-C-positive group were significantly bigger than those in non-metastasis group and VEGF-C-negative group, respectively. The lymphatic TNa (P<0.01)in VEGF-C-positive group was bigger than that of VEGF-C-negative group. Comparing with those in normal tissues group and non-metastasis group, the blood (P<0.01) and lymphatic TNa(P<0.01) were significant increasing in squmaous carcinoma group and metastasis group, respectively. The lymphatic TNa increased gradually with the increasing of clinical grades(p<0.01) and pathological grades(p<0.05). Comparing with normal control group, the lymphatic ONa of peri-inflammation in different acute periods significantly increased（P<0.05）, but the TNa was not statistic difference（P>0.05）. The lymphatic TNa （P<0.05）and ONa（P<0.05） of chronic inflammation

淋巴管内皮长生因子（VEGF-C）与淋巴管的生长、增殖有关，通过对其在人口腔良、恶性肿瘤、炎症、创伤，以及鼠高低淋巴道转移肿瘤模型的病变组织、淋巴转移灶、血液中和蛋白表达的检测，与正常组织比较，确定VEGF-C表达与癌周淋巴管的数量变化、淋巴道转转移相理的地位和在诊断、治疗中应用的可能性。