| Summary: | 博士 === 國立臺灣大學 === 醫學工程學研究所 === 102 === Currently, the mainstay for the treatment of salivary gland carcinoma is radiation and chemotherapy. However, all these treatments are associated with severe short- and long-term side effects. One of the major side effect is that may cause dysfunction of the salivary glands and lead in the desiccant oral environment. Therefore, a potential solution for patients to retrieve their lost salivary gland functions, tissue engineering of an auto-secretory device is profoundly needed. Previous studies demonstrated that we had set up a stable protocol for primary cultivation of human salivary gland cells from surgery specimens. However, the largest challenge in implementing this solution is the primary culture of human salivary gland cells, because the cells are highly differentiated and difficult to expand in vitro. In this study, we combine the gene regulation and biomaterials to address whether the combination have a well effect on the salivary gland cells. In addition, creating a DNA vaccine for salivary gland carcinoma to prevent the tumor regrowth after surgical operation is also a topic in this study.
In the first work, we successfully established the tumor cell line, which carrying the oncogene-LMP1, to mimic the salivary gland tumor cells. The established tumor cell line exhibited the significantly higher proliferation rate and higher migration capability. In this work, we have demonstrated that pcDNA3.1/CRT/LMP1 DNA vaccinia was effective in preventing but less effective for treating LMP1-associated tumors in the tumor animal model. Because dendritic cells (DCs) can be engineered with various kinds of tumor-associated antigens to elicit robust immune response against cancer cells, DC vaccine can be combined with DNA vaccine to enhance the immunotherapy for salivary gland carcinoma.
The induction of monocyte differentiation into dendritic cells (DCs) is the essential protocol for the DC-mediated cancer immunotherapy. In the second work, monocytes isolated from mouse bone marrow were cultured on chitosan substrate to evaluate the effect of the chitosan culture system on the induction and tumor protection of DCs. Compared to tissue culture polystyrene (TCPS), the chitosan culture system could enhance monocyte aggregation and detachment with increased MTT reduction activity and expression of DC marker CD11c and LPS co-receptor CD14. Moreover, compared to TCPS, chitosan could enhance lipopolysaccharides (LPS)-stimulated DCs to secrete higher amount of IL-12. More importantly, vaccination of tumor lysate-pulsed DCs harvested from chitosan could increase cytotoxic T-lymphocyte (CTL) activity and showed significantly enhanced anti-tumor effect than those from TCPS. Therefore, the current study demonstrated that a protocol to culture DCs on a less-adherent chitosan substrate followed by treatment with tumor lysate has the potential in future DC-based vaccine application.
The final work is to combine the gene regulation and biomaterial to improve the salivary acinar cell regeneration. Traditionally, biomaterials can be physically or chemically modified for improving cell attachment, proliferation and function. Conversely, this work tried to modify human parotid gland acinar cells (ACs), not biomaterials, with enhanced expression of N-acetylglucosamine on the cell surface by delivering N-acetylglucosaminyl transferase V (GnT-V) gene into the cells. We hypothesized that upregulation of N-acetylglucosamine expression on the AC surface could enhance the AC-chitosan interaction due to chitosan consisting of the similar structure, D-glucosamine and N-acetylglucosamine. Compared to primary ACs, the GnT-V-transfected ACs could greatly enhance N-acetylglucosamine expression and was more effective in attachment onto chitosan surface. Moreover, upregulation of GnT-V expression could function in the proliferation of ACs on chitosan and the replicative capacity of human ACs was maintained after prolonged culture. More importantly, over-expression of GnT-V facilitated the secretion of α-amylase, the most abundant enzyme in human saliva, in ACs cultivated on chitosan, while primary cells could not exhibit such behavior.
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