細胞激素免疫療法的發展，讓癌症在治療上多了一項非常有力的工具，因為免疫反應可以被強化進而對抗腫瘤細胞。如介白素三號（IL-3）在植入腫瘤細胞後，可以減緩腫瘤在體內的生長，並且產生特定的抵抗力。目前在臨床的治療上，經由搭配化學療法，可用於復發的乳癌、小細胞肺癌等，並促使病人的顆粒球及血小版再生速度加快。雖然在實驗動物身上及臨床試驗都可以看到不錯的療效，但介白素三號在體內高濃度的循環，仍會引發一些副作用，最明顯的是可以看到動物身上的脾臟都發生病變而變的異常腫大。因此我的研究目的主要在於，阻斷介白素三號蛋白質在製造後會送出細胞外的途徑，使其合成後能夠停留在細胞內，希望這樣能夠改善副作用的發生而且能同時達到治療效果。
利用基因重組技術，將介白素三號的信號序列截短或完全刪除後與質體相接，構築成pIRESpuro-IL3、pIRESpuro-IL3(64)、pIRESpuro-IL3(66)、pIRESpuro-IL3(168)及pIRESpuro-IL3(169)等質體DNA，送入Nfsa及Fsa細胞中表現。利用PT-PCR的方法觀察這些轉染細胞中的mRNA，發現送入的質體DNA都有被表現出來，而在測試IL-3是否製造的實驗中，得知分泌型的IL-3的確都在細胞的上清液，而非分泌型IL-3不會被送出來反而會留在細胞內，且經由細胞內染色可知停留的位置是在細胞質的區域裡。

而在探討IL-3基因的植入是否會引起功能性的變異時，發現製造分泌型IL-3的細胞變的對TNF-α很敏感，而製造非分泌型IL-3的細胞與原始細胞相同，則是非常不敏感，甚至低濃度的TNF-α會有促使細胞增生的情形。而評估對於放射線的反應時，也發現非分泌型IL-3基因的植入並不會改變細胞對放射線的敏感度。最後經由注射可製造非分泌型IL-3的腫瘤細胞在老鼠體內，觀察是否有副作用產生，發現非分泌型IL-3並不會如分泌型IL-3般使脾臟腫大，因此副作用發生的情況可以被改善。

此份研究顯示藉由改造IL-3的信號序列，可以建立非分泌型介白素三號之模式，未來在應用上可以讓非分泌型介白素三號大量在腫瘤細胞中製造，再藉由放射治療照射釋放出來，不僅達到腫瘤局部位置治療的效果，還可以遏止副作用的發生，並減輕有害副作用的程度。

Abstract
It has been shown that the cytokine immunotherapy, which can enhance immune response to fight tumor, is a powerful tool for cancer therapy. Interleukin 3 (IL-3)-transduced into tumor, for example, can suppress tumor growth and generate the specific immunity against parental tumor. Although animal experiments and clinic trials show promise results, high concentration of IL3 circulating in body causes side effects. The most significant appearance resulted from high concentration of IL-3 in the blood is splenomegaly. The aim of this study is to examine whether the interrupting of the pathway that leads to IL-3 secretion could reduce the side effect during IL3 immunotherapy.

Using gene recombination techniques, we construct pIRESpuro-IL3, pIRESpuro-IL3(64), pIRESpuro-IL3(66), pIRESpuro-IL3(168) and pIRESpuro-IL3(169) plasmids, which have different regions of signal sequence truncated. These plasmids were then transfected into NFsa and Fsa cells. Following puromycin selection, the mRNA expression of each construct was detected by RT-PCR. ELISA assay demonstrates that the deletion of signal sequence (clones from the transfection of pIRESpuro-IL3(64), pIRESpuro-IL3(66), pIRESpuro-IL3(168) and pIRESpuro-IL3(169) plasmids) prevents the secretion of IL-3 into medium. Immunocytochemistry further confirms that non-secreted form of IL-3 was indeed produced in cytosol region.

To investigate whether the transfection of various forms of IL-3 constructs can induce the functional alteration, we performed TNF-alpha responsive assay and intrinsic radiosensitivity assay. The TNF-alpha responsive data show that the cells transfected with secretory form of IL-3 become more sensitive to TNF-alpha killing than those transfected with non-secreted form of IL-3. Furthermore, cells transfected with non-secreted form, like parental tumor, can proliferate under low concentration of TNF-alpha. The result from intrinsic radiosensitivity assay shows that the transfection of non-secreted form of IL-3 do not alter radiosensitivity. In animal experiments, mice bearing tumors transfected with non-secreted form of IL-3 do not develop splenomegaly. This suggests that the side effects reported from previous IL-3 gene immunotherapy can be reduced by this approach.

In conclusion, this study suggests that it is possible to establish the model of non-secreted form of IL-3 by changing IL-3 signal sequence although its biological function needs further investigation. The potential of non-secreted form of IL-3 model in cancer therapy is in combining with radiotherapy. The aim of this approach does not only intend to enhance tumor curing rate, but it can also reduce the side effect.

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