A Experimental Research Of The Expression Of Shh And Gli-1 In Oral Squamous Cell Carcinoma And The Correlation Between Microvessel Density And Clinical Pathology

Objective: The present study aimed to investigate the significance of Hedgehog(Hh)signaling pathway in association with clinicopathology parameters and its effect on angiogenesis in oral squamous cell carcinoma(OSCC).Methods:(1)With the consent of 80 patients,postoperative cancer tissue specimens that pathologically proved OSCC were collected.The expression of Shh,Gli-1 and CD34 were done on primary tumors in OSCC samples from 80 patients through immunohistochemical analysis.The arithmetic of Microvesseldensity(MVD)depends on the expression of CD34.Then their clinicopathological features and relationship between Shh,Gli-1 and MVD were analyzed individually.(2)SCC4,SCC9,SCC25 and Cal27 cell lines were cultured in vitro,and the Western Blot was used to examine the expression of Shh in OSCC cell lines.MVs were derived by differential centrifugation from OSCC cell lines that with higher expression of Shh protein.Then transmission electron microscopy was used to observe the morphology of MVs.In endothelial cells(ECs)culture medium MVs were co-cultured with human umbilical vein endothelial cells(HUVEC),and the interaction mode between them was observed in virtue of fluorescence microscope.Results :(1)The result of Immunohistochemistry showed: a.The expression of Shh,Gli-1 protein and MVD were significantly correlated with lymph node metastasis and tumor clinical stage in OSCC,and had no relationship with tumor size,age and sex.b.Spearman correlation analysis showed that the expression of Shh protein was significantly correlated with Gli-1(P=0.008).c.Spearman correlation analysis and linear regression analysis showed that the expression of Shh protein was positively correlated with MVD.(r = 0.696,P <0.01).(2)The result of Western Blot showed:a.Shh protein was expressed in all four kinds of OSCC cell lines,which was relatively high in Cal27 cell line.b.The content of Shh protein in MVs derived from Cal27 cell line was significantly higher than that in Cal27 cells.(3)MVs in Cal27 cells were successfully isolated by low temperature differential centrifugation,and the stable morphology of MVs was observed under transmission electron microscope.(4)With the help of fluorescence electron microscopy,we observed a perfect fusion of HUVEC co-cultured with MVs and green fluorescent material aggregated in the cytoplasm of ECs.Conclusion: 1.The expression of Shh,Gli-1 and change of microvesseldensity in oral squamous cell carcinoma were significantly correlated with lymph node metastasis and TNM stage in clinical pathological features,and Shh was significantly positively correlated with MVD,therefore the abnormal expression of Shh and Gli-1 may be involved in the occurrence,invasion and metastasis of oral squamous cell carcinoma.2.MVs derived from Cal27 cell line,with high expression of Shh protein,were fused with human umbilical vein endothelial cells in vitro,and the data suggested that OSCC derived Shh carried by MVs may facilitate the tumor growth and modulate the preparation of a vascular network in primary tumor and/or premetastatic niche.