氧糖剥夺培养脑片缺氧诱导因子1α及少突胶质细胞系基因1的表达

doi:10.3969/j.issn.1673-5374.2013.04.005

摘要/Abstract

摘要：

少突胶质细胞系基因1在少突胶质细胞表达中启动了修复受损神经元髓鞘外衣的过程，对髓鞘修复至关重要。转录因子缺氧诱导因子1α在早产儿缺氧缺血性脑损伤发生过程中发挥重要作用，是否对少突胶质细胞系基因1具有直接调控作用尚不明确。实验对SD大鼠全脑脑片进行静态培养，氧糖剥夺处理后，分别转染缺氧诱导因子1α或少突胶质细胞系基因1。免疫组织化学染色结果显示，转染前氧糖剥夺大鼠脑组织缺氧诱导因子1α和少突胶质细胞系基因1的表达均上调，缺氧诱导因子1α更明显。转染缺氧诱导因子1α后8 h，氧糖剥夺大鼠脑组织少突胶质细胞系基因1的表达上调，转染1 d时少突胶质细胞系基因1的表达达到高峰；转染少突胶质细胞系基因1后，氧糖剥夺大鼠脑组织缺氧诱导因子1α表达无显著变化。结果证实，缺氧诱导因子1α对缺氧脑损伤组织少突胶质细胞系基因1有调控作用，推测此调节作用有可能是缺氧诱导因子1α实现神经修复作用的途径之一。

关键词: 神经再生, 脑损伤, 生物因子, 缺氧诱导因子1α, 少突胶质细胞系基因1, 氧糖剥夺, 脑片培养, 免疫组织化学, 少突胶质细胞, 髓鞘修复, 早产, 大鼠, 基金资助文章, 图片文章

Abstract:

Oligodendrocyte lineage gene-1 expressed in oligodendrocytes may trigger the repair of neuronal myelin impairment, and play a crucial role in myelin repair. Hypoxia-inducible factor 1α, a transcription factor, is of great significance in premature infants with hypoxic-ischemic brain damage. There is little evidence of direct regulatory effects of hypoxia-inducible factor 1α on oligodendrocyte lineage gene-1. In this study, brain slices of Sprague-Dawley rats were cultured and subjected to oxygen-glucose deprivation. Then, slices were transfected with hypoxia-inducible factor 1α or oligodendrocyte lineage gene-1. The expression levels of hypoxia-inducible factor 1α and oligodendrocyte lineage gene-1 were significantly up-regulated in rat brains prior to transfection, as detected by immunohistochemical staining. Eight hours after transfection of slices with hypoxia-inducible factor 1α, oligodendrocyte lineage gene-1 expression was upregulated, and reached a peak 24 hours after transfection. Oligodendrocyte lineage gene-1 transfection induced no significant differences in hypoxia-inducible factor 1α levels in rat brain tissues with oxygen-glucose deprivation. These experimental findings indicate that hypoxia-inducible factor 1α can regulate oligodendrocyte lineage gene-1 expression in hypoxic brain tissue, thus repairing the neural impairment.

Key words: neural regeneration, brain injury, biological factors, hypoxia-inducible factor 1α, oligodendrocyte lineage gene-1, oxygen-glucose deprivation, brain slice culture, immunohistochemistry, oligodendrocyte, myelin repair, premature delivery, rat, grants-supported paper, photographs-containing paper, neuroregeneration

. 氧糖剥夺培养脑片缺氧诱导因子1α及少突胶质细胞系基因1的表达[J]. 中国神经再生研究(英文版), 2013, 8(4): 328-337.

Hong Cui, Weijuan Han, Lijun Yang, Yanzhong Chang. Expression of hypoxia-inducible factor 1 alpha and oligodendrocyte lineage gene-1 in cultured brain slices after oxygen-glucose deprivation[J]. Neural Regeneration Research, 2013, 8(4): 328-337.

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引言

材料方法

Design

A randomized, controlled, animal experiment.

Time and setting

Experiments were performed in the Department of Pediatrics, Beijing Friendship Hospital Affiliated to Capital Medical University from May 2009 to May 2010.

Materials

Twelve 3-day-old Sprague-Dawley specific pathogen-free rats, irrespective of gender, weighing 8– 10 g, were provided by Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., China. The license number is SCXK (Beijing) 2007-0001. Experimental disposals were in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of China^[47].

Methods

Preparation of brain slices

According to the methods of Adamchik et al ^[48-49] with some modifications, brain slices were prepared. In brief, Sprague-Dawley rats were disinfected with 75% alcohol and killed. The brains were quickly placed in precooled anatomical buffer for 15 minutes. The buffer contained 64% Dulbecco’s modified Eagle’s medium, 32% Hank’s balanced salt solution (Gibco, New York, NY, USA), 6.5 g/L D-glucose, 2.98 mg/L hydroxyethyl piperazine ethanesulfonic acid (Sigma, St. Louis, MO, USA), 100 U/mL penicillin, 100 µg/mL streptomycin, and 2.5 µg/mL amphotericin (Gibco). The meninges were removed under an anatomical microscope (Leica, Wetzlar, Germany). Brain tissues were cut into slices using a microtome (TED PELLA, Redding, CA, USA), at 450 μM thickness. Brain slices were cultured in 1 mL of complete culture medium (containing 50% Dulbecco’s modified Eagle’s medium, 25% Hank’s balanced salt solution, 25% equine serum (Gibco), 2.98 mg/L hydroxyethyl piperazine ethanesulfonic acid, 100 U/mL penicillin, 100 µg/mL streptomycin, 2.5 µg/mL amphotericin, and 6.5 g/L D-glucose) in 6-well culture plates (Costar, Vernon, CA, USA). Then, cultured slices were transferred to Millicell culture plates (Gibco) and incubated at 37°C in an incubator (Heraeus, Wetzlar, Germany) in 95% O₂ + 5% CO₂ and saturated humidity. Half of the culture medium was changed twice per week.

Establishment of oxygen-glucose deprived models

Brain slices cultured for 36 hours were respectively placed in 92% nitrogen + 8% oxygen, glucose-free culture medium, in a 37°C incubator for the oxygen- glucose deprivation group, hypoxia-inducible factor 1α transfection group and oligodendrocyte lineage gene-1 transfection group. Oxygen and glucose supply was restored 60 minutes later^[50]. Hematoxylin-eosin staining was applied to observe nuclear pyknosis, karyorrhexis, and fusion.

Target gene transfection

Brain slices were transfected with 2 × 10⁷ IU Ad5- oligodendrocyte lineage gene-1 and Ad5-hypoxia- inducible factor 1α virus (Vector Gene Technology Co., Ltd., Beijing, China). In brief, 2 × 10⁷ IU virus was diluted to 400 μL complete culture medium and transfected for 4 hours. Then, virus was discarded and fresh normal medium was added to Millicell culture plates^[51]. The blank control group and oxygen-glucose deprivation group received no treatment.

Morphology of brain slices

Cultured brain slices were observed twice a week under an inverted microscope (Olympus, Tokyo, Japan), to detect brain slice viability, growth and structure.

Frozen sections of brain slices

The cultured brain slices were fixed with 10% formaldehyde and incubated with 30% sucrose overnight. Each brain slice was then cut into 2–3 frozen sections at 20 µm thickness.

Hematoxylin-eosin staining for brain slices and histological changes under light microscope

After 4 hours of transfection, brain slices were stained with 10% Harris hematoxylin for 3 minutes and differentiated with 0.5% hydrochloric acid alcohol. Blue dye was developed with saturated lithium carbonate water. Slices were rinsed with distilled water for several seconds and stained with 0.5% eosin for 1 minute. Afterwards, slices were dehydrated in graded ethanol, cleared in xylene, and mounted with gum. Cell morphology and nuclei were observed under an optical microscope (Olympus).

Electron microscope observation

Rat cerebral cortex, hippocampus and lateral ventricle tissue were fixed with 2.5% glutaraldehyde for 2 hours, rinsed with PBS three times, and fixed with 1% osmic acid for additional 2 hours, and finally dehydrated in gradient alcohol, for epoxy propane/resin replacement. Slices were embedded in pure resin, trimmed, and cut into semithin sections. Sections were stained with azure-methylene, and positioned under light microscope. Then semithin sections were further sliced into ultrathin sections and stained with uranyl acetate- lead citrate, finally observed under an H-7650 electron microscope.

Expression of hypoxia-inducible factor 1α and oligodendrocyte lineage gene-1 in rat brain tissues as detected by immunohistochemical staining

Immunohistochemical analysis was performed at 4 hours, 8 hours, 1 day, and 3 days after transfection. Frozen sections were soaked in distilled water for 10 minutes and treated with 3% H₂O₂ for 10 minutes. Then, sections were rinsed with distilled water twice for 5 minutes each; citrate buffer was added and sections were microwaved for 1–2 minutes, to make the solution boiling. Afterwards, sections were soaked in cold water to cool them to room temperature, and rinsed with PBS twice for 5 minutes. Sections were blocked with 5% bovine serum albumin at room temperature for 20 minutes, and incubated with rabbit anti-oligodendrocyte lineage gene-1 polyclonal antibody (1:3 000; Chemicon, CA, USA) or rabbit anti-hypoxia-inducible factor 1α polyclonal antibody (1:100; Boster, Wuhan, China) at 4°C overnight. Sections were then rinsed with PBS three times for 5 minutes each time, and incubated with goat anti-rabbit IgG (Boster) at 37°C for 20 minutes. After three 5-minute PBS washes, sections were incubated with SABC at 37°C for 20 minutes, and again rinsed with PBS four times, for 5 minutes each time. Sections were developed with 3,3’-diaminobenzidine, to 50 seconds, and counterstained with hematoxylin for 40 seconds, followed by a series of procedures: dehydration, transparency and mounting. Five to ten visual fields (1 000 × magnification) randomly selected from each frozen section were used for image analysis. Immunohistochemical images were analyzed using Image Pro Plus 6 software (Media, Cybernetics, Baltimore, MD, USA), and the average absorbance value was measured.