睫状神经营养因子可影响海马区神经祖细胞神经元样细胞的分化

doi:10.3969/j.issn.1673-5374.2013.04.002

摘要/Abstract

摘要：

睫状神经营养因子是直到现在发现的惟一可以促进成体大鼠海马神经祖细胞向胶质和神经元方向分化的神经营养因子，这与自发性分化的结果相似。因此，睫状神经营养因子可能参与了神经干细胞自发性分化过程。为了证实此假说，实验对3月龄成年雄性大鼠海马中分离神经祖细胞进行体外培养，发现其在缺乏成纤维细胞生长因子2和表皮生长因子条件下能自发分化成神经元和神经胶质细胞。Western blot和免疫组化染色结果发现，外源性睫状神经营养因子能够诱导海马神经祖细胞分化成神经元和神经胶质样细胞，第4代海马神经祖细胞表达内源性睫状神经营养因子，睫状神经营养因子抗体能够减缓海马神经祖细胞向神经元和神经胶质细胞的分化。提示内源性睫状神经营养因子介导海马神经祖细胞的自发分化。

关键词: 神经再生, 干细胞, 自发分化, 神经祖细胞, 内源性神经营养因子, 睫状神经营养因子, 基金资助文章, 图片文章

Abstract:

Ciliary neurotrophic factor is the only known neurotrophic factor that can promote differentiation of hippocampal neural progenitor cells to glial cells and neurons in adult rats. This process is similar to spontaneous differentiation. Therefore, ciliary neurotrophic factor may be involved in spontaneous differentiation of neural stem cells. To verify this hypothesis, the present study isolated neural progenitor cells from adult male rats and cultured them in vitro. Results showed that when neural progenitor cells were cultured in the absence of mitogen fibroblast growth factor-2 or epidermal growth factor, they underwent spontaneous differentiation into neurons and glial cells. Western blot and immunocytochemical staining showed that exogenous ciliary neurotrophic factor strongly induced adult hippocampal progenitor cells to differentiate into neurons and glial cells. Moreover, passage 4 adult hippocampal progenitor cells expressed high levels of endogenous ciliary neurotrophic factor, and a neutralizing antibody against ciliary neurotrophic factor prevented the spontaneous neuronal and glial differentiation of adult hippocampal progenitor cells. These results suggest that the spontaneous differentiation of adult hippocampal progenitor cells is mediated partially by endogenous ciliary neurotrophic factor.

Key words: neural regeneration, stem cells, spontaneous differentiation, neural progenitor cells, endogenous neurotrophic factors, ciliary neurotrophic factor, regeneration, grants-supported paper, photographs-containing paper, neuroregeneration

. 睫状神经营养因子可影响海马区神经祖细胞神经元样细胞的分化[J]. 中国神经再生研究(英文版), 2013, 8(4): 301-312.

Jun Ding, Zhili He, Juan Ruan, Ying Liu, Chengxin Gong, Shenggang Sun, Honghui Chen. Influence of endogenous ciliary neurotrophic factor on neural differentiation of adult rat hippocampal progenitors[J]. Neural Regeneration Research, 2013, 8(4): 301-312.

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引言

材料方法

Design

Cell biology contrast observation.

Time and setting

The experiment was performed in New York State Institute for Basic Research, Department of Neurochemistry and the Key Laboratory of Nervous System Disease, Huzhong University of Science and Technology, China in 2011.

Materials

Three-month-old male Wistar rats (specific-pathogen free level) were used for all experiments. Animals were housed in standard cages and allowed free access to food and water. All protocols were conducted in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of China^[63].

Methods

Isolation and culture of progenitor cells from adult rat hippocampus

Adult hippocampal progenitor cells were isolated as described previously^[61]. Briefly, rats were euthanized with a lethal dose of nembutal administered intraperitoneally and the hippocampi were quickly dissected and chopped into 0.5 mm³ pieces in Hibernate A containing 2% B27 supplement and 0.5 mM glutamine at 4°C (Invitrogen/Life Technologies, Grand Island, NY, USA). The tissue pieces were digested with 2 mg/mL papain (Worthington, Freehold, NJ, USA) in Hibernate A/B27 for 30 minutes at 30°C with shaking on a reciprocal shaker at 170 r/min. After washing with warm Hibernate A/B27 once and triturating 15–20 times with a siliconized Pasteur pipette in Hibernate A/B27, the progenitors were enriched on an Optiprep step density gradient and plated onto a 0.01% poly-D-lysine (70– 150 kDa; Sigma, St. Louis, MO, USA) -coated plate (Corning, Corning, NY, USA) in Neurobasal A containing 2% B27 supplement and 0.5 mM glutamine. After culture at 37°C in a CO₂ incubator for 1 hour, the medium was replaced with fresh medium containing 100 IU/mL penicillin, 100 g/mL streptomycin, and 10 ng/mL fibroblast growth factor-2 (Peprotec, Rockhill, NJ, USA). The medium was changed every other day, and the cells were passaged at confluence by trituration with a 1-mL Eppendorf pipette. After the first passage, the fibroblast growth factor-2 concentration was increased to 20 ng/mL. Early-stage cultures from the third to fifth passages and late stage cells after 20 passages (about 6 months culture time) were used for the present study.

Cells that had been cultured in 10-cm-diameter poly-D- lysine-coated dishes (Corning) were used to study the effect of various growth factors. Starting on day 0, 2 × 10⁵ cells/mL were seeded onto the plates, and the fibroblast growth factor-2 concentration was maintained at 20 ng/mL. After 1 day, the cells were treated with ciliary neurotrophic factor (Peprotec) or fibroblast growth factor-2 at different concentrations (0–100 ng/mL for ciliary neurotrophic factor and 0–100 ng/mL for fibroblast growth factor-2) in fresh medium. The medium was changed every other day. The experiments were terminated after 7 days of treatment (14 days for long-term observation), when the fastest growing cultures had reached 95% confluency.

Cytotoxicity assay by lactate dehydrogenase assay

After culture for 5 weeks, a fluorometric assay (CytoTox- One Homogenous Membrane Integrity Assay Kit, Promega) was used for the measurement of lactate dehydrogenase 8 released from damaged or dying cells. Briefly, 100 μL of conditioned media was aliquoted into a black 96-well plate. To each well, 100 μL of the CytoTox- One reagent was added for 10 minutes. Following termination of the enzymatic reaction, resulting fluorescence was measured [560 nm (excitation)/590 nm (emission)] using a Viktor3 ELISA plate reader (Perkin Elmer, Boston, MA, USA). These values were subsequently normalized and expressed as a percentage of total lactate dehydrogenase release (assessed by treating cultures with 1% Triton X-100).

Determination of nestin, tubulin, glial fibrillary acidic protein, 5-bromodeoxyuridine, anti-O4 and IgG expression by immunofluorescence

For immunofluorescence, the cells were cultured on 0.01% poly-D-lysine-precoated Lab-Tek II slides (Nunc; Naperville, IL, USA) for 6–7 days. 5-bromodeoxyuridine (10 ng/mL; Sigma) was added to the cultures for the last 12 hours. Cells were then fixed with 3% paraformaldehyde, permeabilized with 0.5% Triton X-100 in PBS for 5 minutes, blocked with 5% bovine serum albumin in Tris-buffered saline for 10 minutes, and incubated with a mixture of primary antibodies isolated from different species at 37°C overnight, followed by fluorescent secondary antibodies for 2 hours. The following antibodies were used as primary antibodies: mouse anti-rat nestin (1:500; BD Pharmingen San Jose, CA, USA), mouse anti-rat tubulin (Tuj1; 0.5 g/mL; BD Pharmingen), mouse anti-rat glial fibrillary acidic protein (1:2 000; BD Pharmingen); rat anti-5-bromodeoxyuridine (1:1 000; Accurate Chemical & Scientific Co.; Westbury, NY, USA); mouse anti-rat anti-O4 (11 g/mL; Chemicon; Temecula, CA, USA); mouse anti-rat IgG (1:5 000; ICN Pharmaceuticals UP Biochemical; Aurora, OH, USA). The following secondary antibodies were used: Alexa Fluor 488 goat anti-mouse IgG (H+L), Alexa Fluor 355 goat anti-rat IgG (H+L), Alexa Fluor 594 donkey anti-rabbit IgG (H+L), Alexa Fluor 594 donkey anti-mouse IgG (H+L), and Alexa Fluor 488 donkey anti-rat IgG (H+L). All fluorescent secondary antibodies were diluted at 1:1 000 and were purchased from Molecular Probes (Eugene, OR, USA). All antibodies were diluted in blocking solution. Samples were washed three times with Tris-buffered saline and cover-slipped in SlowFade Light Antifade solution (Molecular Probes; Eugene). For 5-bromodeoxyuridine staining, samples were incubated for 30 minutes in 2 M HCl at 37°C and rinsed for 10 minutes in 0.1 M Na₂B₄O₇ (Borax), pH 8.5, and six 15-minute rinses in Tris-buffered saline, pH 7.5, then permeabilized with Triton and incubated with antibodies. In some cases, cells were treated with 50 g/mL 4, 6-diamidino-2-phenylindole (Sigma) for 10 minutes. All images of immunostained cells were taken using a high-resolution digital camera set up for a Zeiss Axiophot equipped for epifluorescence or for a laser scanning confocal microscope (PCM 2000, Nikon, Sendai, Japan). Cells were quantified using a 63 × objective. Eight fields (a total of at least 500 cells) per well of eight-well chamber slides were counted, with at least two wells for each experimental point in each experiment. Cells of all staining intensities were included in the quantification.

Determination of nestin, tubulin, glial fibrillary acidic protein, ciliary neurotrophic factor, and CNPase expression by western blot

Cells were cultured to 90% confluence. For harvesting, the attached cells were rinsed once with cold (4°C) Neurobasal A, immediately followed by lysis buffer (1% SDS, 1%-mercaptoethanol, 1 mM freshly prepared phenylmethylene-sulfonylfluoride, 2 g/mL Aprotinin, 2 g/mL Leupeptin, and 1 g/mL Pepstatin A) for 5 minutes on ice. After the cells were fully lysed, the lysates were harvested using cell scrapers and were boiled for 5 minutes. To break the DNA, the lysates were mechanically triturated seven times using 1 mL insulin syringes (New Medical Tech; Livingston, Scotland) and then aliquoted and stored at –85°C until use. Protein concentrations were determined by the modified Lowry method of Bensadoun and Weinstein^[64]. Protein samples were electrophoresed at least in triplicate on sodium dodecyl sulfate-polyacrylamide gels (8 cm × 6 cm × 0.75 cm or 20 cm × 10 cm × 0.75 cm; 7.5% or 10% acrylamide), transferred to Immobilon membranes (Millipore, Bedford, MA, USA), and probed with primary antibodies. The following primary antibodies were used: mouse anti-rat nestin (1:1 000; Developmental Studies Hybridoma Bank, Iowa City, IA, USA); mouse anti-rat β-tubulin monoclonal antibody and rabbit anti-β-tubulin polyclonal antibody (0.2 ng/mL; Covance, Berkeley, CA, USA); mouse anti-glial fibrillary acidic protein (ascites, SMI 22; 1:5 000; Sternberger); mouse neutralizing anti-ciliary neurotrophic factor (1 g/mL; R&D); mouse anti-CNPase (1:200; Chemicon, Temecula, CA, USA) at 37°C overnight. Generally, 20 g cell lysate protein per lane was applied, unless otherwise indicated. In some cases, bound antibodies were probed with ¹²⁵I-conjugated anti-mouse or anti-rabbit IgG. The radio immunoblots were scanned with a Fuji BAS 1500 Bio Image analyzer (Raytest USA Inc., Wilmington, DE, USA). Images were processed with the Tina software, and the intensity of immunostaining was expressed as pixels per square length.

Statistical analysis

Data were expressed as mean ± SEM. Statistical comparisons were conducted by analysis of variance with post hoc Student-Newman-Keuls test when more than two groups were involved. Intergroup differences were determined using a post hoc Scheffer’s test or paired t-test as appropriate. A value of P < 0.05 was considered statistically significant.

讨论

文章快阅

延伸阅读

1 实验设计构思介绍：

神经祖细胞具有自我更新和多向分化的潜力。在不含有丝分裂原如成纤维细胞生长因子2或表皮生长因子的离体培养条件下，胚胎或成年哺乳动物脑内的神经干细胞会自发性分化为神经元和神经胶质细胞，这就是自发性分化。自发性分化是神经干细胞的一种特性，也是研究定向分化的基础。而有研究报道，外源性睫状神经营养因子引起胚胎神经祖细胞分化为星形胶质细胞，Louis等发现，外源性睫状神经营养因子可以促进少突胶质细胞的存活和成熟，并且在实验中发现外源性睫状神经营养因子可以显着促进成体大鼠海马神经祖细胞向神经元方向分化。因此，睫状神经营养因子是直到现在发现的惟一可以促进成体大鼠海马神经祖细胞向胶质和神经元方向分化的神经营养因子，这与自发性分化的结果相似。因此，睫状神经营养因子可能参与了神经干细胞自发性分化过程。为了证实此假说，设计并实施了这个实验。

2 国内外同类研究水平的介绍：

神经干细胞的发现，大量的研究都集中在了外源性生长因子，细胞因子和生化因素对胚胎和成年神经干细胞发育的影响。以期更好的控制外部因素，得到特定细胞。一部分神经干细胞的增殖和分化机制部分已被研究清楚，很多外源性生长因子已确认是增殖或分化的诱导因素。尽管研究人员已经开始认识到细胞组成和微环境的重要作用，如内源性神经营养因子对神经干细胞发育的影响，但是这方面的研究仍很不足。神经干细胞的自发性分化机制不清楚。

3 与其他同类研究的比较：

自发性分化是神经干细胞的特性，而之前的很多关于神经干细胞定向分化的研究都没有考虑到自发性分化对实验结果的影响，因此，自发性分化成为研究神经干细胞生物学特点及临床应用必须面对的问题。尽管自发性分化的机制不清，但是通过实验可以发现，神经干细胞表达的内源性睫状神经营养因子参与了自发性分化过程。