腫瘤缺氧區域對於化學治療與放射治療都具有一定程度的抵抗性；無論是藥物的傳遞與效果不佳，或是放射線造成的傷害無法被固定，使腫瘤細胞無法完全清除。因此提高藥物對於缺氧區的傳遞是目前治療上迫切需解決的問題。本研究將介白素三號(Interleukin-3, IL3)基因插入帶有六重複Hypoxia response element(HRE)序列的載體，再利用微脂體轉染入小鼠攝護腺癌腫瘤細胞(TRAMP-C1)。使用藥物Deferoxamine或缺氧無菌培養箱(1%O2、5%CO2、94%N2)之培養條件，證明TRAMP-HRE-IL3釋放IL-3之能力有明顯的提升。利用pre-IR照射模組，比較TRAMP、TRAMP-IL3、TRAMP-HRE-IL3於動物體內生長及探討腫瘤微環境的改變。結果顯示受IL-3影響，腫瘤微環境中巨噬細胞有更多聚集的現象，而TRAMP-HRE-IL3由於在缺氧區有局部性高濃度的IL-3表現，因此相對於其於兩細胞株之Control之缺氧區約有1.7倍多之巨噬細胞浸潤，而細胞移動實驗更加確認巨噬細胞移動增加之能力與IL-3有直接相關。此外，由於IL-3所導致的脾臟腫大之副作用也因IL-3釋放受HRE調控而大幅減少。因此我們改善了原本利用IL-3於腫瘤基因治療之副作用，並建立了可在缺氧區提升巨噬細胞聚集程度之腫瘤微環境，欲配合以單核球-巨噬細胞作為藥物載體之系統，與放射線治療做為結合，期望能得到更佳的腫瘤治療效果。

Tumor hypoxic regions are more resistant to chemo- and radiation- therapy due to the ineficiency of drug delivery or unfixable DNA damages resulted from low concentration of oxygen. Therefore, therapeutic strategy improvements are desperately needed to treat these tumor microenvironments.
Murine prostate cancer, TRAMP-C1, was used as a research model. A vector of six-tandem repeats of bidirectional enhancers, hypoxia response element (HRE), inserted with murine interleukin-3 (mIL-3) as a working gene at one arm was used to create a new cell line named TRAMP-C1-HRE-IL3, in order to regulate the secretion of IL-3 within hypoxic regions for monitoring changes in the tumor micrenvironments and macrophage aggregation.ELISA and RT-PCR results showed that the expression of IL-3 was induced after Deferoxamine (DFX) treatment or hypoxia-mimic environment incubation (1% O2、5% CO2、94% N2).
The tumor growth and microenvironment changes in three different cancer cell lines, TRAMP-C1, TRAMP-C1-IL3 and TRAMP-C1-HRE-IL3, on C57BL/6J mice were compared. The data showed that TRAMP-C1- HRE-IL3 tumors can enhance the migration of macrophages toward hypoxia regions to as high as 1.7 folds comparing with the other two types of control tumors, and have the similar aggregation patterns in the pre-IR tumor model.
Further experiments proved that this migration enhancement is directly related to IL-3 secretion from cancer cells. Additionally, spleen enlargement, a side effect from original IL-3 gene therapy, is greatly reduced by using TRAMP-C1-HRE-IL3.
To sum up, the side effects of using IL-3 were lowered by regulating its secretion with HRE, and at the same time, a macrophage migration and aggregation favorable environment was created within the tumor hypoxic regions. In combination with monocyte-macrophage drug delivery system and radiation-therapy, it is hoped that it will resulted as a better anti-cancer strategy.

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