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研究生:張秋足
研究生(外文):Chiou-Tuz Chang
論文名稱:人類早期單核球細胞株HL-CZ自體分泌介白質4及其及其與細胞自體融合關係之探討
論文名稱(外文):Studies on the secretion of interleukin - 4 and the relation of autofusion in the human promoncyte cell line, HL-CZ
指導教授:呂健惠
指導教授(外文):Chien-Hui Lieu
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學生物技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
中文關鍵詞:介白質4細胞融合早期單核球細胞株
外文關鍵詞:interleukin - 4cell fusionHL-CZ
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HL-CZ 屬於人類早期單核球細胞,細胞大小不一,和 C 肝病人血清一起培養後發現呈現2-5倍大的細胞會增多。之前學長從流式細胞儀觀察到 HL-CZ 有大小兩群細胞,以細胞分離技術將細胞分成此兩群後分別培養,經一段培養時間細胞又會回復其原本比例,推測細胞中存有不均等分裂與細胞自體融合等現象存在。爲證實細胞融合的存在以及何種因素所致故進行本實驗。首先,將 HL-CZ 分別處理兩種活體細胞追蹤染劑:25 μM CMTMR 的紅色螢光和10 μM CMFDA 的綠色螢光,之後等比例混合一起培養,經24小時後以流式細胞儀分析,觀察到 HL-CZ 細胞具有自體融合的現象,融合比例在10-18% 之間。將 HL-CZ 以細胞分離技術分離細胞的大小,經由 Liu’s stain 發現細胞在越低轉速(細胞越大)細胞核的個數也越多,印證 HL-CZ 具有細胞自體融合的現象。 本實驗以 RT-PCR 測得 HL-CZ 會合成 IL-4及IL-4 receptor 的 mRNA,並且經由 ELISA 得知其可表現 IL-4 蛋白質。在培養不同天,IL-4 receptor mRNA 表現量相差不大;而且 IL-4 蛋白質表現量相當,沒有明顯的改變。 將 CCE 分離後的細胞進行 in-situ RT-PCR,之後再以 Image Quant 做相對定量,結果顯示越大顆的細胞其細胞內合成的 IL-4 mRNA 越多,且由 ELISA 得知其分泌到培養基的 IL-4 蛋白質也有增多的趨勢。進一步在 HL-CZ 培養基中添加 neutralized anti-IL4 antibody,48小時後利用流式細胞儀分析,發現細胞融合的百分比有下降並且下降程度與劑量成正相關,證實 IL-4 在 HL-CZ 細胞融合中扮演重要的角色。本實驗證明人類早期單核球細胞株HL-CZ會自體分泌介白質4及分泌的介白質4之現象與細胞融合有關。
HL-CZ is a cell line of promocyte. When they were co-cultured with hepatitis C patient serum, they formed large cells with size 2-5 larger than others. Our previous study indicated that individually cultured the different size cells obtained from the counter-flow centrifuge elutriation (CCE), the percentage of the large cells and small cells was always kept constant. In this experiment, we treated HL-CZ individually with two different living cell tracker probes: 25 μM CMTMR -- the red fluorescent dye or 10 μM CMFDA -- the green fluorescent dye. After 24 hours of co-culturing the two population cells, we found the two dyes could be detected in the same cells. It indicated that auto-fusion phenomenon was exited. The fusion percentage was 10-18%. When we used CCE to separate the different sizes of HL-CZ, we found that the cells at lower speed fractions (the large ones) had more nuclei than cells at high speed fraction. This data verified there was auto-fusion phenomenon in HL-CZ. We also found HL-CZ produced IL-4 mRNA、IL-4 receptor mRNA and IL-4 protein which can be detected by RT-PCR or ELISA. The amount of IL-4 receptor mRNA and IL-4 protein were expressed almost uniform at different subculture days. After the cells were separate by CCE, the cells were subjected to in-situ RT-PCR and determined the relative amounts of the IL-4 mRNA in different elute fractions by the Image Quant software. The results indicate the larger sizes of the cells, the more expression of IL-4 mRNA and the IL-4 protein. Addition of the neutralized anti-IL4 antibody into the culture medium in 48 hours, the cell fusion percentages were decreased and showed a dose-dependent manner. The data suggests IL-4 can induce auto-fusion in HL-CZ. Take together, our study revealed that HL-CZ can secrete interleukin-4 and that may lead to auto-fusion of cells.
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