https://scholars.lib.ntu.edu.tw/handle/123456789/160399
標題: | 藉由建立B淋巴球體外培養系統以探討STAT3對其早期發育過程所扮演的角色 Establishment of In vitro Culture System to Investigate the Role of STAT3 in Early B-cell Development |
作者: | 莊淑樺 Chuang, Shu-Hua |
關鍵字: | 淋巴球;體外培養系統;CLP;STAT3 | 公開日期: | 2007 | 摘要: | 在骨髓幹細胞分化成B細胞的過程中,轉錄因子扮演一個相當重要的角色。 我們發現STAT3此轉錄因子缺乏時,會使B淋巴球的發育產生缺陷而使成熟B細胞的數量變少。在STAT3基因剔除鼠(STAT3 knockout mice) 的骨髓中, pro-B、pre-B以及未成熟B細胞的百分比及數目均有顯著下降,然而pro-B 的前趨細胞pre-pro B的數目則有增加的現象。另外,在IL-7的刺激下,我們發現缺乏STAT3的pro-B及pre-B的增殖現象比控制組來得低,其中原因之一是因為這種異常鼠對IL-7有反應的細胞數目降低,而不是因為這些細胞表面的IL-7接受器(IL-7 receptor)數目的減少或訊息傳遞有缺陷。這些結果暗示STAT3 在早期B細胞分化中扮演相當重要的角色。 在此,我們利用體外培養的方式,進一步去探討STAT3在早期B 細胞分化角色的機制。首先,我們將LSKs或CLPs從骨髓中純化出來以後.將此細胞和伽瑪-射線照射後的基質細OP9共同培養,並同時加入B細胞生長所需的激素如:IL-7、SCF和Flt3L。我們發現LSKs細胞無法有效的分化成B細胞,但卻可以分化出大量的顆粒性白血球和巨噬細胞,相反CLPs無論在正常老鼠或缺乏STAT3的老鼠都可以有效的分化出B細胞。有趣的是,從STAT3基因剔除鼠的CLPs分化出的B細胞數目相對的比正常老鼠少很多,另外我們也利用反轉錄定量聚合酵素鏈鎖反應的方式去觀察其B細胞發育所需之基因如: E2A、EBF、pax5、RAG1、RAG2、Igα、Igβ、VperB 、λ5等的表現,然而異常老鼠的表現量和正常老鼠並無顯著差異。 我們利用limiting dilution 實驗,探討STAT3是否影響前趨細胞分化成B細胞的機率,發現在缺乏STAT3的前趨細胞分化成B細胞的機率較低。若把IL-7拿掉,則使缺乏STAT3之前趨細胞分化成B細胞的能力大為下降;若不添加Flt3L的狀況下,則其分化能力沒有顯著差異。這些結果暗示CLPs在沒有STAT3後對IL-7引起B細胞之分化的影響有提高,而對Flt3L所導致B細胞分化的能力消失了。 綜合以上結論,我們進一步去證實之前在in vivo的研究結果,亦即STAT3 對B細胞前趨細胞的形成是必須的。 Transcription factors are critical for instructing the development of B lymphocytes from multipotential progenitor cells in the bone marrow. Previously, we have shown that STAT3KO mice displayed impaired B lymphopoiesis. Reduced pre-B, pro-B immature and mature B cells but accumulated pre-pro B stage of BM cells was found in mice lacking STAT3. The pro-B and pre-B population lacking STAT3 were hyporesponsive to IL-7 because of a decreased number of IL-7-responsive cells rather than decreased expression or signaling of IL-7Rα. These data indicated that STAT3 played a critical role in early B cell development. Here, we set up an in vitro culture system, to elucidate the mechanisms by which STAT3 regulates early B cell development. LSKs or common lymphoid progenitors (CLPs) were first isolated and co-cultured with γ-irradiated OP9 in the presence IL-7, SCF and Flt3L. We found that LSKs of both control and STAT3KO mice did not sufficiently support B cell development. Instead, myeloid cells such as granulocytes and macrophages were preferentially developed in these culture conditions. In contrast, CLPs had higher potential to develop into B cell lineages in both mice. Interestingly, the absence of STAT3 greatly reduced the numbers of B cell developed from CLPs despite that relative percentage was similar between control and STAT3KO mice. Moreover, we found the expression of genes that were critical for B cell development such as E2A, EBF, pax5, Igα, Igβ,VperB, λ5, RAG1 and RAG2 was comparable in control and STAT3KO cells, suggesting that STAT3 might affect on cell proliferation rather than on cell differentiation. In limiting dilution assay, we investigated the frequency of B progenitor cells in the presence or absence of STAT3. We found that a decreased frequency of progenitor cells was observed in STA3KO mice. Moreover, left out exogenous IL-7 but not Flt3L substantially reduced the developmental potential of STAT3KO CLPs, suggesting that IL-7 dependent B lymphopoiesis was increased and that Flt3L-mediated B lymphoipoesis was impaired in the absence of STAT3. Taken together, these results were agreed with our previous in vivo studies, that formation progenitor cell was dependent on STAT3. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/63336 | 其他識別: | en-US |
顯示於: | 免疫學研究所 |
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ntu-96-R94449001-1.pdf | 23.31 kB | Adobe PDF | 檢視/開啟 |
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