Differentiation of mesencephalic neural stem cells from rat embryos induced by interleukin-1 beta in vitro

Abstract

AIM：Effective proliferation in vitro of neural stem cells and a large amount of dopaminergic neuron differentiation are important in the neural stem cell transplantation for Parkinson disease. This article explores the culture methods for the differentiation of mesencephalic neural stem cells from embryonic mice into dopaminergic neurons by interleukin-1β in vitro.

METHODS：Experiments were conducted in the Laboratory of Biology, Experimental Center, Medical College, Wuhan University from April to December 2005. ①The pregnant 12-day rat embryos were provided by the Experimental Animal Center of Medical College of Wuhan University. Certificate number of animal was Kunzi 2005. Animal intervention met the animal ethical standard. ②Meninges of fetal rats were isolated and midbrain was obtained, digested with trypsin, centrifuged and passaged by the mechanical method. Neurosphere cultured for 5-7 days was collected and incubated at (20-30)/cm2. Cells in the interleukin-1β group were incubated in DMEM/F12 medium containing 200 ng/L interleukin-1β and fetal bovine serum of 0.1 volume fraction. Cells in the blank control group were incubated in the DMEM/F12 medium containing fetal bovine serum of 0.1 volume fraction. ③Mononuclear cells differentiated from 80% cells at days 10-12 were determined by the immunocytochemical technique. Positive cell rate of tyroxine hydroxylase was detected by the flow cytometry.

RESULTS: ①Immunocytochemistry: Cells in neurospheres expressed nidogen antigen and differentiated into neuro-specific enolase and glial fibrillary acidic protein. Tyroxine hydroxylase was found in the interleukin-1β group, showing large cell body, round and ellipse. Tyroxine hydroxylase was seen in cytoplasm and cluster-shape processes. Tyroxine hydroxylase in the blank control group was different from that in the interleukin-1β group in amount and appearance. ②Positive cell rate of tyroxine hydroxylase was higher in the interleukin-1β group than in the blank control group (P < 0.01).

CONCLUSION: Interleukin-1β can promote the differentiation of mesencephalic neural stem cells into dopaminergic neuron with enough amount and mature appearance and function.

Ding JG, Ding WJ, Li G.Differentiation of mesencephalic neural stem cells from rat embryos induced by interleukin-1 beta in vitro.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(3):406-410(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-3/3k-406(ps).pdf]

摘要
目的：在神经干细胞移植治疗帕金森病过程中，有效的神经干细胞体外增殖与多巴胺能神经元的大量定向诱导分化尤为关键。以白细胞介素1β为诱导剂，观察鼠胚胎中脑神经干细胞向多巴胺能神经元的分化。
方法：实验于2005-04/12在武汉大学医学院实验中心结构生物学实验室完成。①动物：清洁级孕12 d昆明鼠胚胎，由武汉大学医学院实验动物中心提供，动物质量合格证号为昆字2005，实验过程中对动物的处置符合动物伦理学标准。②实验方法：分离胎鼠脑膜，取中脑组织，胰蛋白酶消化，离心过滤后机械法传代。取传代培养5~7 d的神经球，按（20~30）个/cm2接种，白细胞介素1β组加入含200 ng/L白细胞介素1β、体积分数为0.1胎牛血清的DMEM/F12培养基进行诱导分化，空白对照组单纯加入体积分数为0.1胎牛血清的DMEM/F12培养基予以自然分化。③实验评估：诱导10~12 d，待80%细胞从神经球迁移出来并分化为单细胞时行免疫细胞化学鉴定，流式细胞术检测酪氨酸羟化酶阳性细胞率。
结果：①免疫细胞化学鉴定：神经球细胞表达巢蛋白抗原，能分化为神经元特异性烯醇化酶、胶原纤维酸性蛋白阳性细胞。白细胞介素1β组可见酪氨酸羟化酶阳性细胞，胞体较大，圆形和椭圆形居多，酪氨酸羟化酶位于胞质及突起中，胞核无表达，突起呈酪氨酸羟化酶阳性神经元典型的串珠样形态。空白对照组酪氨酸羟化酶阳性神经元在数量、细胞成熟形态上均未达到白细胞介素1β组水平。②中脑神经干细胞向多巴胺能神经元分化的阳性率：白细胞介素1β组酪氨酸羟化酶阳性细胞率明显高于空白对照组（P < 0.01）。
结论：白细胞介素1β可明显促进中脑神经干细胞分化为数量足够、形态及功能成熟的多巴胺能神经元。
关键词：中脑神经干细胞；多巴胺能神经元；白细胞介素1β；酪氨酸羟化酶

丁继固，丁文杰，李光.白细胞介素１β体外诱导鼠胚胎中脑神经干细胞的分化[J].中国组织工程研究与临床康复，2008，12(3):406-410 [www.zglckf.com/zglckf/ejournal/upfiles/08-3/3k-406(ps).pdf]