藉由篩選ENU突變鼠以尋找參與在干擾素訊息傳遞中新穎且重要的分子

Abstract

干擾素是具有免疫調節，抗細胞增生，以及抗病毒感染能力的細胞素。根據許多研究指出，雖然干擾素主要是活化JAK/STAT訊息傳遞路徑，仍有一些輔佐的調控機制參與在干擾素訊息傳遞中。為了進一步研究參與在干擾素訊息傳遞中詳細的調控機制並發現從未被界定的機轉，我們採用遺傳學的方法，藉由篩選ENU突變鼠來尋找對干擾素有異常反應的小鼠。 為了找出對干擾素反應異常的小鼠，我們發展了一套兩步驟的篩選策略。首先，利用流式細胞儀來觀察可被干擾素直接調控的組織相容性複合體 (MHC)的表現量，當找到異常鼠後，再利用及時定量聚合酵素鏈鎖反應觀察其他可被干擾素誘導的基因群表現量來確認此異常的表徵。藉由這個篩選策略，我們從134個家族，總共約2800隻ENU突變鼠中篩選出三個對干擾素反應異常的家族。他們分別是對α干擾素反應很低的117i，以及對α干擾素和γ干擾素反應都很低的243g和248k。使用反轉錄定量聚合酵素鏈鎖反應(RT-QPCR)，我們發現在117i家族中藉由ISRE啟動的基因，例如OAS和PKR，都無法被α干擾素所誘導。然而對γ干擾素的反應卻是正常。將突變鼠與正常鼠進行交配後，我們證明了α干擾素的反應不良症是隱性遺傳的表徵。因為，當一隻異常鼠和正常鼠交配後，我們並沒有其子代中找到有異常α干擾素反應的老鼠。然而，我們確實可以在正常的F1(117i x wild type)相互交配的子代中找到異常鼠。 突變鼠造血細胞的發育表現正常。在突變鼠中，無論在胸腺，骨髓，脾臟或周邊血液中，T細胞，B細胞，單核球以及顆粒球細胞群的分布皆與正常鼠類似。另外，由高劑量LPS所引發毒性休克的敏感度在突變鼠與正常鼠之間是相類似的。為了尋找造成α干擾素低反應的可能機制，我們利用西方墨點法觀察STAT1, STAT2 以及 STAT3的表現。有趣的是，我們發現STAT2無論在突變鼠的脾臟細胞或者在周邊血液細胞中都無法被偵測到。這個結果暗示造成突變鼠對α干擾素反應過低但對γ干擾素反應正常的原因，很可能是由於失去STAT2蛋白質。然而，由於STAT2 mRNA 在突變鼠細胞中仍可被α干擾素誘發，縱使被誘發的mRNA量相較於正常鼠來的低，這個結果仍顯示失去STAT2蛋白質的表現很可能是在轉錄後或者轉譯的層級發生問題。基因定位以及STAT2基因定序應可幫助我們精確地找出真正造成干擾素反應異常的因素。

Interferons (IFNs) are cytokines that have immunmodulatory, anti-proliferation, and antivirus ability. Although IFNs activate JAK-STAT pathway, several studies suggest that there may be some alternative regulatory mechanisms of IFN signaling. To further study the fine regulatory mechanisms and to discover those undefined IFN signaling machineries, we have taken a genetic approach to screen ENU-mutagenized mice that displayed altered IFN responses. We have developed a two-step screening strategy, first by flow cytometry to monitor surface markers like MHC molecules that are tightly regulated by IFNs and then by real-time QPCR for different sets of IFN-inducible genes to screen mice for abnormal responses. We have screened some 2800 mice from 134 pedigrees and find that three pedigrees shown impaired IFNs responses. They are 117i, which shows hypo-responsiveness to IFNα, 243g and 248k, which have hypo-responsiveness to both IFNγ and IFNα. Using RT-QPCR, we have shown that 117i pedigree fails to induce ISRE-containing genes such as OAS and PKR in response to IFNα. However, the response to IFNγ is normal. By crossing the mutant mice to wild type mice, we have demonstrated that the hypo-responsiveness to IFNα in 117i mice is inheritable and is a recessive trait because no altered IFNα response is found in the offspring of a cross between wild type and deviant mice. However, we do observe mice with hypo-responsiveness in the offspring from a cross between two F1 (117i x wild type) mice that do not display the phenotype. The development of hematopoietic cells appears to be normal in the mutant mice as T cell, B cell, monocyte and granulocyte populations in bone marrow, thymus, spleen and PBL of mutant mice are comparable to that of wild type (WT) mice. In addition, the susceptibility of high dose LPS-induced toxic shock in the mutant mice is also similar to that of WT mice. In searching for possible mechanisms accounting for the impaired response to IFNα, we perform Western blotting using antibodies to STAT1, STAT2, and STAT3. Interestingly, we found that STAT2 protein is not detected in either PBL or splenocytes of the mutant mice, suggesting that the loss of STAT2 in mutant mice may result in the altered response to IFNα but not IFNγ. However, mRNA of STAT2 is still present and inducible, though at lower level, in mutant cells in response to IFNα, suggesting that the absence of STAT2 may lie in a post-transcriptional or translational mechanism. Genetic mapping and sequencing of STAT2 genome should be able to allow us to pinpoint the gene that causes the altered IFN response.