Establishment of the targeted cell line for anticancer therapy

• Main Authors: Ju-Chan Tai, 戴儒君
• Other Authors: Wen-Jen Yu
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/84858148943168634993

碩士 === 弘光科技大學 === 生物科技研究所 === 100 === Somatostatin receptors (SSTRs) are known as growth inhibitory receptors and reported to regulate various exocrine and endocrine homeostasis. Five SSTR subtypes (SSTR1–5) were documented and classified as the family of G-protein coupled receptors which possess seven transmembrane domains. The expression profiles of endogenous SSTRs are mainly in brain, pancreas, stomach and kidney tissues which differ from those in tumor tissues. Somatostatin and its analogue were found to inhibit the neuroendocrine gastroenteropancreatic tumor growth. Therefore, it is possible to develop the strategy for targeting therapy via SSTRs pathways. However, absence or few SSTRs expression were found in the cancer cell lines which derived from tumor tissues with aboundant SSTRs expresion. It is not appropriate to use the commencial tumor cell lines for the evaluation of targeting therapy via SSTRs pathway. In this study, we co-constructed the plasmid with SSTR cDNA, EGFP and luciferase reporter genes, and puromycin resistant genes (SLPE2) for the establishment of SSTR stable cell lines. The objective of this study was to generate the stable cell lines which over-express SSTR and suitable for the platforms for targeting therapy in vitro or in vivo. The SLPE2 plasmid was confirmed by restriction map and transfected into various cell lines. Cells carried the SLPE2 plasmid were enriched by antibiotics selection and approved by reporter gene and SSTR gene expression using RT-PCR and Western blotting. The affinity to targeting drug and cytotoxicity of selected SSTR all lines were further examined by fluorescence activated cell sorting (FACS) and MTT, respectively. In addition, the animal tumor model was generated by injecting 1x106 SSTR cells into the right hind legs of nude mice. Three stable SSTR cell lines (T47D, AR42J, MCF7) with normal growth rate and cell morphology were successfully established for the subsequent experiments. The results of cell and animal experiments indicated that the targeting drug (SST-Lipo-Dox) caused an adverse effects on the SSTR tumor cell growth and reduced the side effects of tumor therapy in invo. In the presents study, we successfully generated three stable SSTRs cell lines (T47D, AR42J, MCF7) with dual reporter genes (EGFP, luciferase) that can be used as in vitro and in invo platforms for the targeted anti-cancer drug screening through the SSTR pathway.