Cellular responses to baculovirus transduction in human mesenchymal stem cells and induced pluripotent stem cells

桿狀病毒在人類間葉幹細胞和老鼠多功能幹細胞之免疫途徑探討

陳冠宇(Guan-Yu Chen)

指導教授 ： 胡育誠

國立清華大學/工學院/化學工程學系/博士(2011年)

https://doi.org/10.6843/NTHU.2011.00633

摘要

桿狀病毒已成為新興的基因治療載體，間葉幹細胞(MSCs)則是非常有潛力被應用於組織再生。我們已成功應用桿狀病毒於人類間葉幹細胞(hMSCs)的基因改質，並在裸鼠內刺激硬骨新生，顯示桿狀病毒/基因治療結合間葉幹細胞於再生醫學的潛力。為釐清應用桿狀病毒於hMSCs改質的安全性，以在未來推向臨床應用，本研究先探討病毒轉導如何影響hMSC的基因表現，是否誘發了免疫反應，並更進一步評估TLR3與相關訊號傳遞路徑在此扮演的角色。我們首先發現桿狀病毒轉導hMSCs後不會對細胞特性造成太大影響，但卻引發不同細胞激素(IL-6和IL-8)的分泌；後續也進一步確認桿狀病毒轉導與細胞激素反應具有關聯性。接下來我們也成功發現桿狀病毒轉導hMSCs後引發了TLR3訊息傳遞路徑上的基因表現，並以流式細胞儀與共軛焦顯微鏡分析TLR3分子確實被桿狀病毒活化。進一步我們也分析TLR3路徑下游的訊息傳遞分子(TRIF、IRF3和NF-κB)也被桿狀病毒活化，確認TLR3訊息傳遞途徑確實被啟動。最後我們也用反向證明的方式，利用siRNA抑制TLR3，再分析抑制TLR3後，桿狀病毒轉導的確會影響細胞激素表現與細胞遷移。在我們的研究中也探討了誘導性多功能幹細胞(iPSCs)，iPSCs具備與胚胎幹細胞相似的細胞特性，未來被期待能夠取代胚胎幹細胞並應用於基因治療，我們發現病毒在iPSCs中不會造成細胞增生、凋亡以及分化的影響，並且也不會引發太強烈的免疫反應，這是因為iPSCs中TLRs、MDA5、DAI和NF-κB表現量不明顯，而RIG-I、AIM2和IRF3在病毒轉導後不會被活化所造成，這些免疫現象與病毒在轉導hMSCs後有很大的差異性。本研究是第一個探討病毒進入hMSC內藉由TLR3訊息傳遞途徑引發細胞反應的研究，也是第一個探討病毒進入iPSCs後對細胞的影響及免疫相關途徑的研究，對於幹細胞在再生醫學/基因治療的應用有重大影響。

關鍵字

桿狀病毒 ； 人類間葉幹細胞 ； 多功能系幹細胞 ； 免疫反映 ； 訊息途徑

並列摘要

Human mesenchymal stem cells (hMSCs) and mouse induced pluripotent stem cells (iPSCs) can be genetically modified with viral vectors and hold promise as a cell source for regenerative medicine, yet how hMSCs and iPSCs respond to viral vector transduction remains poorly understood, leaving the safety concerns unaddressed. Hereby we explored the responses of hMSCs and iPSCs against an emerging DNA viral vector, baculovirus (BV). First, we uncovered that BV transduction perturbed the transcription of 816 genes associated with 5 signaling pathways in hMSCs. Surprisingly, toll-like receptor-3 (TLR3), a receptor that generally recognizes double-stranded RNA, was apparently upregulated by BV transduction as confirmed by microarray, PCR array, flow cytometry and confocal microscopy. However, we can not find any TLRs activation after BV transduction in iPSCs. Cytokine array data showed that BV transduction triggered robust secretion of IL-6 and IL-8, but not of other inflammatory cytokines and IFN-b in hMSCs. BV transduction activated the signaling molecules (e.g. TRIF, NF-κB and IRF-3) downstream of TLR3, while silencing the TLR3 gene with small interfering RNA considerably abolished the cytokine expression and promoted cell migration. Surprisingly, our data indicated that BV tranduction of iPSCs only elicited mild immunse responses. These data demonstrate, for the first time, that a DNA viral vector can activate the TLR3 pathway in hMSCs but not in iPSCs, and lead to a cytokine expression profile distinct from that in immune cells. These findings underscore the importance of evaluating whether TLR3 signaling cascade plays roles in the immune response provoked by other DNA vectors (e.g. adenovirus). Nonetheless, BV transduction barely disturbed the surface marker expression and only induced transient and mild cytokine response, thereby easing the safety concerns of using BV for hMSCs and iPSCs engineering.

參考文獻

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