瘦素對間葉幹細胞與 3T3-L1 脂肪前驅細胞 分化之影響

The effects of leptin on the differentiation of mesenchymal stem cells and 3T3-L1 cells

韓雅如(Ya-Ju Han)

指導教授 ： 江伯倫

國立臺灣大學/醫學院/口腔生物科學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201602830

摘要

小鼠骨髓間葉幹細胞具有免疫調節與多向分化潛能，可分化為易誘發炎症的脂肪細胞。3T3-L1 脂肪前驅細胞為目前常用於脂肪細胞研究的模型。瘦素為促進發炎的脂肪細胞激素，能抑制脂肪分化以及促進 T 淋巴細胞增生反應。本研究將瘦素加入小鼠骨髓間葉幹細胞與 3T3-L1 脂肪前驅細胞，探討瘦素對於兩者分化能力的影響。首先，將所培養之小鼠骨髓間葉幹細胞進行表面抗原、分化能力與免疫抑制能力的鑑定。以流式細胞技術鑑定間葉幹細胞表面抗原，實驗結果指出有表現CD29、CD73、CD105 和 Sca1，不表現 CD44、CD34、CD11b、B220 與 CD90.2。將間葉幹細胞分別培養於脂肪分化培養液、硬骨分化培養液以及軟骨分化培養液，並以 Oil Red O、Alizarin Red S 和 Alcian Blue 染色，確認具有分化能力。將小鼠骨髓間葉幹細胞與經由抗體 CD3ε/28 刺激後的 T 淋巴細胞混合培養，實驗結果發現間葉幹細胞可有效抑制 T 淋巴細胞的增生。接著，將小鼠骨髓間葉幹細胞和3T3-L1 脂肪前驅細胞施予瘦素，進行酵素免疫分析實驗，測定 IL-1RA 與 IL-6 的表現量，並測定兩者施予瘦素後的脂肪分化能力。本研究中所施予瘦素的濃度均無細胞毒性。酵素免疫分析實驗結果指出，IL-6 沒有在範圍內、沒有表現，僅有加入1000ng/mL 的瘦素至間葉幹細胞的組別，其 IL-1RA 分泌量有顯著下降趨勢，其餘組別沒有顯著影響。而分化能力實驗結果指出，施予 100ng/mL、1000ng/mL的瘦素、培養 14 天的間葉幹細胞；以及施予 10-8M、10-6M 的瘦素、培養 8 天的3T3-L1 脂肪前驅細胞，兩者上機測定 Oil red O 於 510nm 的吸光值與未加入瘦素的對照組吸光值相比，其吸光值下降，脂肪分化能力下降。本研究推論，瘦素有較大趨勢影響細胞走向發炎反應，但不走向脂肪分化。與瘦素互為拮抗的脂肪細胞激素為脂聯素，兩者對於在肥胖相關疾病當中，交互作用尚未非常明瞭，未來可再探討是否會進一步影響間葉幹細胞的免疫調節。本研究將瘦素加入間葉幹細胞與3T3-L1 脂肪前驅細胞，探討對於分化的影響，對於未來間葉幹細胞脂肪分化機制與分化過程中的發炎走向，可做為臨床應用之參考。

關鍵字

間葉幹細胞 ； 3T3-L1 脂肪前驅細胞 ； 脂肪細胞 ； 脂肪細胞激素 ； 瘦素 ； 脂肪分化 ； 免疫調節 ； 脂聯素

並列摘要

Mouse mesenchymal stem cells (MSCs), which could differentiate into adipocytes, have the characterization of immune regulation and multi-lineage differentiation potential. 3T3-L1 cells is used in biological research on adipose tissue. Leptin is an adipokine, secreted by adipocytes, which could induce inflammation, inhibit adipogenesis and promote the T cell proliferation. In this research, we treated MSCs and 3T3-L1 cells with leptin to investigate the effect of differentiation ability. At first, we confirm the surface markers, differentiation ability, and immune regulation ability of the MSCs. The flow data shows the positive surface markers are CD29, CD73, CD105 and Sca1 while the negative surface markers are CD44, CD34, CD11b, B220 and CD90.2. MSCs cultured in adipogenic medium, osteogenic medium, and chondrogenic medium, were stained with the Oil Red O, Alizarin Red S, and Alcian Blue, respectively. Data shows the MSCs have the differentiation ability. Through the stimulation of the antibody CD3ε/28 and co-culturing the MSCs with the T cells, the mesenchymal stem cells could effectively inhibit the proliferation of the T cells. Then, treated mouse mesenchymal stem cells and 3T3-L1 cells with leptin, assayed the level of IL-1RA and IL-6 by ELISA, and assayed the adipogenic ability. In this research, the concentration of leptin is non-cytotoxic. The ELISA data shows IL-6 is not detected. IL-1RA is significant decreased while the MSCs treated with 1000ng/mL leptin, however, other groups have no effects. Compared to the control group, the OD value for Oil red O at 510nm of the MSCs (treated with 100ng/mL, 1000ng/mL for 14 days) and the 3T3-L1 cells (treated with 10-8M、10-6M for 8 day), are decreased and the adipogenic ability are also decreased. This research suggested that the leptin have a tendency to induce an inflammatory response, but not to the adipogenesis. In this study, treating mesenchymal stem cells and 3T3-L1 cells with leptin to investigate the effect of the differentiation, could be used as the clinical application of the mechanism and inflammation during differentiation of mesenchymal stem cells in the future.

並列關鍵字

mesenchymal stem cells ； 3T3-L1 cells ； adipocytes ； adipokines ； leptin ； adipogenesis ； immune regulation ； adiponectin

參考文獻

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