Human Nerve Schwann Cells Destroyed By Salivary Adenoid Cystic Carcinoma Cells With Or Without Inhibited By RNAi

Objective: Schwann cell is a kind of glial cell wrapping peripheral nerves. The cell memrane of Schwann cells forms the myelin sheath of medullae nerve fibers. So, Schwann cells are the chief cell components of peripheral nerves.Salivary adenoid cystic carcinoma (SACC) is one of the commonest malignancies in salivary gland, which tends to invade nerves at early stage and lead to paresthesia, numbnesss and pain. Previous research revealed that the neural invasion by tumor cells was only regarded as space occupation, and the nerve itself did not suffer from damage. However, it is not clear if the tumor cells invade and damage nerve directly by their neurotropic growth.The neoplastic myoepithelial cells (NMCs) in SACC with the potential of bilateral differentiation secrete proteoglycans (PGs). PGs are macromolecules composed of core protein and GAG chains. As the major components of extracellular matrix, PG play an important role in the processes such as growth, adhesion, migration, and differentiation of tumor cells. The role of PGs in neural invasion and damage by SACC still needs further research. The initial step of PG biosynthesis is that xylosyltransferase (XT) catalyzes transfer in the cells. XT-I known as the chain-initiating enzyme is involved in rate-limiting step in the biosynthesis of GAG-containing PG. It is essential for PG biosynthesis.The technology of RNA interference (RNAi) post-transcriptional gene silence (PTGS) caused by the introduction of a homologous and complementary double-stranded RNA (dsRNA), result in degradation of mRNA .In this study, human Schwann cells of peripheral nerve were co-cultured with SACC-83 cells, and the invasion and damage of human Schwann cells by SACC-83 cells was observed before and after inhibition of PG biosynthesis of SACC-83 cells by RNAi, in order to discuss and prove if SACC-83 cells may damage human Schwann cells directly, and if PG plays a role in the process of nerve damage by SACC-83 cells.Method:1 The method of tissue culture was used for primary culture of human Schwann cells in DMEM:F12 containing 20% Fetal Boving Serum(FBS). Human Schwann cells were identified by immunocytochemical stain against S-100 monoclone antibody. And SACC-83 cells (RPMI 1640 contained 15% FBS) were identified by immunocytochemical stain against CK and Myosin monoclone antibody.2 The XT-â… gene expression of SACC-83 was silenced by transfection of constructed XT-â… gene expression vectors shRNA-WJ4 (inhibited group) and shRNA-HK (negative control group) into SACC-83 cells by LipofectameneTM 2000. Group of SACC-83 cells (untransfection group) was not transfected any vector.3 The expression of the mRNA level of XT-â… gene in 3 group of SACC-83 was detected by the Real-time PCR technology.4 The culture medium was collected in 48h after transfection. The contents of proteoglycans of each group were tested by Blyscan Assay Kit.5 Three groups including SACC-83-WJ4, SACC-83-HK, and SACC-83 cells respectively co-cultured with human Schwann cells in 24-well cell culture plate for 24 h, and were observed by the inverted microscope and scanning electron microscope (SEM). SACC-83 cells and human Schwann cells were distinguished by immonocytochemical stain, so as to count the human Schwann cells invaded and calculate the rate of invasion. Statistical analysis showed that all the data were analyzed by the software SPSS13.0, analyzed for statistical significance by using one way ANOVA. P<0.05 was considered statistically significant.Result:1 The identification of human Schwann cells of primary generation by immunocytochemical stain against S-100 showed positive reaction in the cytoplasms.2 The SACC-83 cells transfected with shRNA-WJ4 and shRNA-HK by Lipofectin expressed green fluorescent protein (GFP) stably. The transfection efficiency was 51.44% and 58.70% respectively.3 After 48h transfection of shRNA-WJ4, the expression of XT-I was inhibited 49% by Real-Time PCR.4 The content of PGs was down-regulated 41.32% in 48h after transfection of shRNA-WJ4.5 Tumor cells of SACC-83 group tended to gather and attack human Schwann cells together; as a result of the cytoplasms disintegration and nucleus pyknosis of human Schwann cells was found. In group of SACC-83-WJ4, tumor cells did not invade and damage human Schwann cells.Conclusion1 SACC-83 cells invaded and destroyed peripheral nerves directly.2 Proteoglycans secreted by SACC-83 cells played an important role in the process of nerves damage.