中文摘要
細胞激素(cytokine)在造血作用與免疫反映的調節上扮演了一個重要的角色而且還能影響淋巴細胞的生長。介白素10 (interleukin 10或IL-10) 是一種親多組織的間白素(pleiotropic cytokine) 且其可以作用在不同的細胞種類上，它原被定義為抑制免疫力的細胞激素，但是它也扮演了一個免疫誘發的角色。最近被發現的介白素10家族的成員包括IL-19、IL-20、IL-22、MDA-7 (IL-24)與AK155 (IL-26)等。
人類IL-22是由180個胺基酸組成，其序列與老鼠IL-22相比有78%相同，與人類IL-10相較有26%相同。IL-22是由IL-9刺激老鼠T淋巴球(T lymphocyte)被發現的；在體內與體外實驗中，對老鼠施打IL-22後，發現老鼠會產生急性時期反應物(acute phase reactants)，因此我們猜測IL-22在免疫系統中，扮演的重要的角色。IL-22的訊息傳導過程需要受器綜合體(receptor complex)，其包含同為IL-10的受體成員之一的IL-10R2c與細胞激素第二家族新成員之一的IL-22R1。最近被發現的IL-22 binding protein (IL-22BP)又稱CRF2-10可以用來調節IL-22的生物功能，在體外實驗中發現，CRF2-10可以與IL-22結合並阻止IL-22與其細胞膜上的專一受器結合，進而達到抑制因IL-22所引發的細胞訊息傳導之作用。
在我們實驗室中，利用人類IL-22BP的基因當作probe 而找到老鼠的IL-22BP基因，hIL-22BP與mIL-22BP 的胺基酸序列有66.9%相同. mIL-22BP有230胺基酸，比hIL-22BP少一個.
為了進一步研究IL-22的生物功能和IL-22與其結合蛋白CRF2-10間之交互影響，我們純化重組IL-22與人類的CRF2-10蛋白質來進行功能分析，實驗證實IL-22可以促使人類或老鼠的肝臟細胞中的STAT3磷酸化，我們亦證明CRF2-10可以抑制STAT3的活化在有IL-22的刺激下。為了要去探索IL-22未知的生物功能，我們將IL-22分別去刺激人類的T細胞、B細胞與單核球後，發現B細胞會明顯的產生reactive oxygen species (ROS)，所以我們推測IL-22與細胞的apoptosis過程有關。
將recombinant hIL-22 打入兔子的皮下組織中，經過多次免疫後，已取得IL-22的polyclonal antibodies (Ab)，經由western blot證實，所得之polyclonal Ab不但可以認識human IL-22亦可認識mouse IL-22。這個IL-22的polyclonal Ab對於未來在in vivo 的實驗，將非常有用。
近期文獻中，發現有另二種human IL-22的binding protein (CRF2-10S與CRF2-10L)，我們已將此二種蛋白質送入E. coli中大量表現並純化出來，初期的實驗結果顯示，當IL-22誘發人類肝臟細胞HepG2的STAT3磷酸化時，在沒有post translational modification修飾的CRF2-10仍具有抑制 STAT3磷酸化的效用，而CRF2-10S與CRF2-10L卻無此抑制功能；但在大鼠的肝臟細胞H4IIE中，CRF2-10S有些許的抑制STAT3磷酸化能力而CRF2-10L並沒有此抑制功能。
體內自然存在的三種alternatively splice binding proteins，若可專一性的與IL-22結合並進而調控其在體內的免疫作用，在未來有機會運用到醫療上，來治療IL-22所引發的免疫反應。

Cytokines play important roles in the regulation of hematopoiesis and immune responses and can influence lymphocyte development. Interleukin 10 (IL-10) is a pleiotropic cytokine that acts on many different cell types. It was originally identified as an immunosuppressive cytokine, but it also plays an immunostimulatory role. Several members of the IL-10 family, including IL-19, IL-20, IL-22 [IL-10-related T-cell-derived inducible factor (IL-TIF)], IL-24 [melanoma differentiation-associated antigen 7 (MDA-7)], and IL-26 ( AK155), have only been discovered recently.
IL-22 was originally identified as a cytokine induced by IL-9 in murine T lymphocytes. The production of acute phase reactants stimulated by IL-22 in vitro and in vivo suggests its role in inflammatory response. IL-22 signals through a receptor complex that contains a functional component of the IL-10 signaling molecule, IL-10R2c, and a new member of the class II cytokine receptor family, IL-22R1. Recently discovered IL-22 binding protein(IL-22BP; renamed as CRF2-10) appears to be potent regulators of IL-22 activities. Interference with the binding of IL-22 to their membrane receptors and, thus, inhibition of cytokine signaling has been demonstrated in vitro.
In our laboratory, we has isolated an mIL-22BP genomic clone by homology hybridization using human cDNA clone as a probe. The amino acid sequences of hIL-22BP and mIL-22BP are 66.9% identical. The mIL-22BP gene encodes a protein of 230 amino acids, one amino acid less than hIL-22BP.
In order to investigate the IL-22 biological function and the interaction of IL-22 with its binding protein in the future, we have purified the recombinant IL-22 and hCRF2-10 for functional assay. We demonstrated that hCRF2-10 can neutralize STAT3 activation in two hepatoma cell lines (Hep G2 and H4IIE) stimulated with either human or mouse IL-22. In order to further explore novel biological functions of IL-22, we treated T cells, B cells, and monocytes with IL-22 and analyzed the result of stimulation. We found that human B cells produced reactive oxygen species (ROS) when it was stimulated with hIL-22.
To get the polyclonal antibody against hIL-22 recombinant protein, we immunized two rabbits with hIL-22 several times. Western blot was performed to prove that the polyclonal antibody can recognize both hIL-22 and mIL-22.
Two alternatively splice variants, hCRF2-10L and hCRF2-10S have also been discovered. We have purified the recombinant proteins of these two splice variants from E. coli, and investigate their functions. The hCRF2-10S seemed to partially inhibit STAT3 activation induced by both hIL-22 and mIL-22 in rat H4IIE cells, but not in human HepG2 cells.
Future more comprehensive study of molecular mechanism of these three binding protein (hCRF2-10, hCRF2-10S, and hCRF2-10L) interacting with IL-22 will provide us some insights of their potential uses in clinics.

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