維生素對小鼠骨髓間質幹細胞的分化及免疫調節特性之影響

Effects of vitamins on the differentiation and immune regulatory properties of mouse bone marrow mesenchymal stem cells

王資婷(Zih-Ting Wang)

指導教授 ： 林璧鳳

國立臺灣大學/生命科學院/生化科技學系/碩士(2017年)

https://doi.org/10.6342/NTU201703440

摘要

間質幹細胞 (Mesenchymal stem cells, MSC) 具有多元的分化能力，且能調節免疫反應，協助組織修復，目前已被應用於細胞療法。在特定誘發條件下，MSC會分化為脂肪、軟骨及成骨細胞。有文獻發現當MSC分化為脂肪細胞後，免疫調節能力會減弱，顯示MSC的分化與免疫調節能力有關。維生素A與D也被發現會影響MSC的分化，是否也影響MSC的免疫調節能力尚未可知。因此，本研究目的欲探討維生素對MSC分化及免疫調節能力的影響。實驗方法為從BALB/c小鼠骨髓分離出MSC，分析表面抗原不具CD11b、CD34、CD90.2、CD45R (B220)，且呈現CD29、CD44、CD73、Sca1、CD105等，並分別用不同分化劑培養基誘發生成脂肪細胞、軟骨細胞或成骨細胞，以鑑定所得MSC之分化能力。同時也驗證可抑制T細胞的增生，是具免疫調控能力的MSC。確認每批所培養細胞具MSC特性後，分別將MSC培養於含不同濃度維生素A、維生素D或葉酸的培養基，於第3、7、14天，分析維生素對MSC分化為脂肪細胞的影響，同時也檢測發炎相關基因的表現量；以及與分離自脾臟的CD4+ T細胞共培養後，測定T細胞增生能力，分析維生素對MSC免疫調節能力的影響。實驗結果顯示，培養7天時，維生素A會抑制MSC表現PPARγ、C/EBPα、Ap2、Glut4等基因，且有助於MSC的增殖；維生素D在0.1 nM濃度下則會抑制MSC表現C/EBPα及Ap2，但不利於MSC的增殖；葉酸也有助於MSC的增殖，僅在100 μM 濃度下會抑制C/EBPα的基因，在20 µM 濃度下皆不影響MSC上述基因的表現。在免疫調節能力方面，此3種維生素皆不改變MSC抑制T細胞增生的能力，維生素A及葉酸培養7天時會抑制Tnfα、Tgfb的表現量，且維生素A培養14天時會促進Il-6的表現。綜合以上，在含維生素A微環境MSC會增殖，也較不易趨向分化為脂肪細胞；在含維生素D微環境中雖較不利於MSC增殖，但也會抑制脂肪相關基因的表現量；在含葉酸微環境中MSC也可增殖，且處於較未分化狀態。此添加維生素對MSC增殖與調控分化過程的研究成果，可做為開發MSC體外量產技術之參考。

關鍵字

間質幹細胞 ； 維生素A ； 維生素D ； 葉酸 ； 脂肪細胞分化

並列摘要

Mesenchymal stem cells (MSCs) are multipotent stem cells which have regenerative and immunoregulatory properties. These features make MSCs being applied in cellular therapy an attractive approach. MSCs can differentiate into osteocytes, chondrocytes and adipocytes under certain culture condition. Recent studies have demonstrated that MSC might lose immunoregulatory properties during the adipogenic differentiation, suggesting its relationships with immunity. Vitamin A and D have been known to regulate the differentiation of MSCs, and might have effects on the immunoregulatory properties of MSCs. Hence, the aim of this study is to investigate the effects of vitamins on differentiation and immunoregulatory properties of MSCs. First, MSCs were isolated from 4-week-old BALB/c bone marrow, characterized by specific surface markers, differentiation tendency to adipocytes, chondrocytes and osteocytes under appropriate condition, and the immunoregulatory properties to supress T cells proliferation. After confirming all the above properties of MSCs, MSCs were cultrured in medium containing different concentrations of all trans-retinoic acid, 1α,25-(OH)2D3 and folic acid. Differentiation and immunoregulatory properties were determined on days 3, 7 and 14. These results showed that after a 7-day culture, vitamin A reduced the gene exprsesion of PPARγ, C/EBPα, Ap2, Glut4, and promoted the proliferation of MSCs. Vitamin D under 0.1 nM reduced the gene exprsesion of C/EBPα, Ap2, but also inhibited the proliferation of MSCs. Folic acid promoted the proliferation of MSCs and under 100 μM could reduce the expression of C/EBPα. On the other hand, all of these vitamins did not alter the inhibitory effect of MSC subsets on CD4+ T cell proliferation. Vitamin A and folic acid reduced the gene expression of Tnfα, Tgfb after a 7-day culture. Besides, Vitamin A enhanced the expression of Il-6 after a 14-day culture. In conclusion, Vitamin A could promote the proliferation of MSCs and inhibited the adipogenesis of MSCs. Vitamin D could inhibit the expression of adipocyte-related gene, but also inhibit the proliferation of MSCs. Folic acid could also promote the proliferation of MSCs, and maintain MSCs in an undifferentiated state. This research suggested that addition of vitamins at certain concentration might alter MSC proliferation and differentiation, which provides information for the expansion of MSCs.

並列關鍵字

Mesenchymal stem cells ； vitamin A ； vitamin D ； folic acid ； adipocyte differentiation

參考文獻

Li, D., Wang, P., Li, Y., Xie, Z., Wang, L., Su, H., Deng, W., Wu, Y. and Shen, H. (2015). All-trans retinoic acid improves the effects of bone marrow-derived mesenchymal stem cells on the treatment of Ankylosing Spondylitis: an in vitro study. Stem Cells Int 2015: 484528.

Abbas, M. A. (2017). Physiological functions of Vitamin D in adipose tissue. J Steroid Biochem Mol Biol 165(Pt B): 369-381.

Abe, I., Shirato, K., Hashizume, Y., Mitsuhashi, R., Kobayashi, A., Shiono, C., Sato, S., Tachiyashiki, K. and Imaizumi, K. (2013). Folate-deficiency induced cell-specific changes in the distribution of lymphocytes and granulocytes in rats. Environ Health Prev Med 18(1): 78-84.

Aggarwal, S. and Pittenger, M. F. (2005). Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood 105(4): 1815-1822.

Ahima, R. S. (2008). Revisiting leptin's role in obesity and weight loss. J Clin Invest 118(7): 2380-2383.