米诺环素抑制视网膜小胶质细胞中神经生长因子前体减少

doi:10.3969/j.issn.1673-5374.2013.04.004

摘要/Abstract

摘要：

以前房加压灌注平衡盐溶液建立大鼠急性高眼压模型后，立即腹腔注射90 mg/kg米诺环素连续3d。免疫荧光和Western blot及PCR检测发现，急性高眼压后大鼠视网膜中神经生长因子前体，神经生长因子和p75NTR蛋白及神经生长因子和p75NTR mRNA表达增加，小胶质细胞标志物CD11b和神经生长因子前体、星型胶质细胞标志物胶质纤维酸性蛋白和p75NTR、胶质纤维酸性蛋白和凋亡标志因子Caspase-3双标阳性细胞数量也明显增加，米诺环素治疗后这些蛋白和mRNA表达明显受到抑制。表明米诺环素可抑制急性高眼压后视网膜中小胶质细胞中神经生长因子前体和星型胶质细胞中的p75NTR的表达增加而导致的细胞凋亡增多。

关键词: 神经再生, 生物因子, 神经生长因子前体, P75NTR, 米诺环素, 细胞凋亡, 神经生长因子, 急性高眼压, 视网膜, 图片文章

Abstract:

A rat model of acute ocular hypertension was established by enhancing the perfusion of balanced salt solution in the anterior chamber of the right eye. Minocycline (90 mg/kg) was administered intraperitoneally into rats immediately after the operation for 3 consecutive days. Immunofluorescence, western blot assay and PCR detection revealed that the expression of the precursor form of nerve growth factor, nerve growth factor and the p75 neurotrophin receptor, and the mRNA expression of nerve growth factor and the p75 neurotrophin receptor, increased after acute ocular hypertension. The number of double-labeled CD11B- and precursor form of nerve growth factor-positive cells, glial fibrillary acidic protein- and p75 neurotrophin receptor-positive cells, glial fibrillary acidic protein- and caspase-3-positive cells in the retina markedly increased after acute ocular hypertension. The above-described expression decreased after minocycline treatment. These results suggested that minocycline inhibited the increased expression of the precursor form of nerve growth factor in microglia, the p75 neurotrophin receptor in astroglia, and protected cells from apoptosis.

Key words: neural regeneration, biological factor, precursor form of nerve growth factor, p75 neurotrophin receptor, minocycline, apoptosis, nerve growth factor, acute ocular hypertension, retina, photographs-containing paper, neuroregeneration

. 米诺环素抑制视网膜小胶质细胞中神经生长因子前体减少[J]. 中国神经再生研究(英文版), 2013, 8(4): 320-327.

Xiaochun Yang, Xuanchu Duan. Minocycline inhibits the production of the precursor form of nerve growth factor by retinal microglial cells[J]. Neural Regeneration Research, 2013, 8(4): 320-327.

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引言

材料方法

Design

A randomized, controlled, animal experiment.

Time and setting

The experiment was performed at the Kunming Institute of Zoology, Chinese Academy of Sciences in China from August 2010 to February 2011.

Materials

Healthy, adult, male, clean, Sprague-Dawley rats (n = 160), weighing 250–300 g and aged 3–4 months, were supplied by Kunming Medical College, China, license No. SYXK (Dian) 2005-0003. They were raised in a clean animal room with free illumination, at 20–23°C and humidity at 40–80%. Principles of laboratory animal care were in accordance with the guidelines expounded by the European Community Council Directive (86/609/EEC of 24 November 1986) and the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Methods

Modeling of intraocular pressure

All animals were anesthetized with chloral hydrate (500 mg/kg) and 0.04% (v/v) benoxyl (Santen, Suzhou, China). The pupil was enlarged following treatment with mydrin (Santen, Suzhou, China). In the model group, the intraocular pressure was raised to 14.67 kPa for 60 minutes by enhancing the perfusion of balanced salt solution (Nanjing, Kunming, China) with a 27-gauge needle placed in the anterior chamber of the right eye. The intraocular pressure was achieved by hanging a saline bottle at the height of 146 cm. All procedures were performed under sterile conditions and did not injure any intraocular structures. For the sham-operated controls, the eyes were perfused only, with a bottle hanging at the same height as the operated eye^[36].

Minocycline intervention

In the minocycline group, minocycline (90 mg/kg; Sigma, Santa Clara, CA, USA) was administered intraperitoneally in sterile PBS (20 g/L) into rats immediately after operation for 3 consecutive days. In the PBS group, 0.1 M PBS (4.5 mL/kg) was administered into the other animal models in the same way.

Sample collection

The rats were decapitated following anesthesia with chloral hydrate at 1, 3, 5 and 7 days.

Immunohistochemical detection of p75 neurotrophin receptor, precursor form of nerve growth factor, glial fibrillary acidic protein and CD11B in the rat retina

The eyeballs were excised out and fixed with 4% (w/v) paraformaldehyde (pH 7.4) for 1 hour on ice and then for another half an hour after removal of the cornea, lens and vitreous body. After that, the calyculi ophthalmicuses were placed in 10% (w/v) sucrose julep (pH 7.4) at 4°C to dewater, and then in 20% (w/v) and finally 30% (w/v). Tissues were embedded and cut into transverse frozen slices of 6–10 μm. The frozen slices were incubated with 2% (w/v) normal bovine serum albumin and 0.4% (v/v) Triton X-100 for 1 hour then with rabbit anti- p75 neurotrophin receptor (1:100; Millipore, Billerica, MA, USA) or precursor form of nerve growth factor (1:200; Millipore) polyclonal antibodies, or mouse anti-rat glial fibrillary acidic protein (1:200; Millipore) or CD11B (1:100; Chemicon, Billerica, MA, USA) monoclonal antibodies at 4°C overnight, followed with DyLight^TM 594-conjugated goat anti-rabbit IgG (1:200; ZSGB-Bio, Beijing, China) or DyLight^TM 488-conjugated goat anti-mouse IgG (1:200; ZSGB-Bio, Beijing, China ) for 1 hour at room temperature. For double labeling, two kinds of primary antibodies or secondary antibodies were incubated at the same time. Nuclei were labeled with 4',6-diamidino-2- phenylindole in fluorescent mounting medium (ZSGB-Bio, Beijing, China). In controls, the slices were incubated without primary antibodies. A DX60 fluorescent microscope (Olympus, Jiangsu, China) was used.

Western blot detection of p75 neurotrophin receptor, precursor form of nerve growth factor and nerve growth factor in the rat retina

Fresh retinal tissues were homogenized in radioimmune precipitation assay lysis buffer for 30 minutes on ice, then centrifuged at 25 000 × g for 30 minutes at 4°C. Equal amounts of protein were determined using bicinchoninic acid assay^[37] (Pierce, Pittsburgh, PA, USA). Protein samples (40 μg) were separated on 12% SDS- polyacrylamide gels after being denatured at 95°C for 5 minutes, and then transferred to polyvinylidene difluoride membranes (Roche, Shanghai, China). The membranes were incubated with 5% (w/v) nonfat skim milk in Tris-buffered saline Tween-20 for an hour at room temperature, and then with the primary antibodies rabbit anti-p75 neurotrophin receptor (1:4 000), precursor form of nerve growth factor (1:4 000) polyclonal antibodies, or mouse anti-rat glial fibrillary acidic protein monoclonal antibody (1:2 000), and finally with horseradish peroxidase-labeled goat anti-rabbit monoclonal antibody (1:10 000, KPL, Gaithersburg, MD, USA). Immunoblots were detected by chemiluminescence (SuperSignal, Pierce). The experiment was repeated in triplicate. Rabbit against rat β-actin monoclonal antibody (1:8 000; Millipore) was used as the internal control. The UVP BioDoc-It^TM gel-imaging instrument (UVP Inc., San Gabriel, CA, USA) was used.

Reverse transcription-PCR detection of p75 neurotrophin receptor and nerve growth factor in the rat retina

Total RNA was isolated from the retina using TRIZOL Reagent (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized according to the Platinum Taq DNA polymerase instructions (Invitrogen). Samples were denatured at 94°C for 5 minutes, followed by amplification for 30–35 cycles of 95°C for 30 seconds, 55–60°C for 40 seconds, 72°C for 30 seconds to 1 minute. After that, PCR products were subjected to electrophoresis on a 1.5% agarose gel and visualized using ethidium bromide. The primers were designed using the Oligo6.53 software (SinoGenoMax, Beijing, China).

The primer sequences are as follows:

Negative controls consisted of PCR reactions lacking primers or reverse transcriptase. β-actin was used as the internal control. The experiment was repeated in triplicate. The UVP BioDoc-It^TM gel-imaging instrument (UVP Company) was used.

Apoptosis detection of astroglial cells in the rat retina

The expression of caspase-3 was used to test apoptosis^[38]. Permeated and blocked frozen slices were incubated with primary mouse anti-rat glial fibrillary acidic protein monoclonal antibodies (1:200) and rabbit anti-caspase-3 polyclonal antibodies (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at the same time at 4°C overnight, then incubated with DyLight^TM 594-conjugated goat anti-rabbit IgG (1:200) and DyLight^TM 488-conjugated goat anti-mouse IgG (1:200) at room temperature for an hour. After mounting with DAPI mounting medium, a DX60 fluorescent microscope (Olympus) was used.

讨论

文章快阅

延伸阅读

1 实验设计构思介绍：

一直以来，人们都认为神经营养因子前体蛋白没有生物活性。2001年，Lee和他的同事首次发现，神经营养因子前体蛋白与p75^NTR受体具有很高的结合力，解离常数为1.2×10^-11M（为高亲和力受体），且结合以后诱导细胞死亡。这一研究发现促使人们对神经营养因子前体蛋白和p75^NTR受体的功能进行了大量研究。目前神经生长因子前体与p75^NTR结合后具有增加细胞易损性的作用已经在Alzheimer病、自身免疫性脑膜炎、脊髓损伤等神经系统的退行性病变及体外培养的PC12细胞中得到了证实。已有试验表明p75^NTR在高眼压大鼠视网膜表达增加，但在高眼压大鼠视网膜p75^NTR与神经生长因子前体的变化关系，以及神经生长因子前体的来源目前尚未见报道。

米诺环素在神经退行性疾病中的神经保护作用在以往的研究中已经得到了证实。在小鼠青光眼模型中米诺环素能减少小胶质细胞的激活和促进视网膜神经节细胞轴浆运输和完整性来保护视神经，还能通过增加X连锁凋亡抑制蛋白，survivin和Bcl-2的表达保护星形胶质细胞免于凋亡。但其中的机制目前仍不清楚。因此，我们设计本实验，旨在观察急性高眼压大鼠视网膜星形胶质细胞的损伤与的关系，视网膜神经生长因子前体的可能来源以及米诺环素对青光眼的神经保护作用是否与其影响p75^NTR与神经生长因子前体的表达有关。

2 国内外同类研究水平的介绍：

神经生长因子前体与其凋亡受体p75^NTR结合介导神经凋亡的功能已在多种神经变性疾病中被发现和证实，急性高眼压动物模型中也发现有p75^NTR蛋白的增加，但急性高眼压视网膜神经生长因子前体蛋白和mRNA以及p75^NTR mRNA是否增加目前尚未见报道。

神经生长因子前体可以存在于中枢和外周神经组织，小胶质细胞是脊髓损伤时神经生长因子前体的来源，但急性高眼压视网膜神经生长因子前体的细胞来源目前尚未见报道。

米诺环素的神经营养作用也在多种疾病中得到证实，其中涉及多个机制，如减少小胶质细胞的激活，促进视网膜神经节细胞轴浆运输和完整性，增加X连锁凋亡抑制蛋白, survivin和Bcl-2的表达等。但确切的关键机制目前仍未发现。

3 与其他同类研究的比较：

创新点为：(1)首次研究发现急性高眼压大鼠视网膜星形胶质细胞p75^NTR不仅蛋白表达增加，其mRNA也表达增加。(2)首次发现急性高眼压视网膜神经生长因子前体增加，并且星形胶质细胞的损伤与p75^NTR和神经生长因子前体表达增加有关。(3)首次发现视网膜小胶质细胞是急性高眼压大鼠视网膜神经生长因子前体的重要来源。(4)首次着眼研究米诺环素的神经保护作用与p75^NTR和神经生长因子前体的关系。结果发现视网膜星形胶质细胞表达p75^NTR增加，使小胶质细胞诱导的神经生长因子前体表现出诱导凋亡的作用是星形胶质细胞损伤的重要原因，而米诺环素对青光眼的神经保护作用正是与其抑制p75^NTR和神经生长因子前体的表达有关。

４文章创新性的分析：

文章以“proNGF”和“IOP”在pubmed上检索所有年限的文献报道，仅发现2009年有1篇相关报道。以“proNGF”和“eye”在pubmed上检索2001/2011的文献，结果检索到6篇文献。以“proNGF”和“minocycline”在pubmed上检索，结果检索到2007年有1篇文献。以“proNGF”、“caspase-3 ”和“rat”在pubmed上检索，检到2008年有1篇报道。以“proNGF”、“p75^NTR”和“rat”在pubmed上检索，检到2002/2010共8篇报道。以“proNGF”和“IOP”、“神经营养因子前体”和“米诺环素”分别在CNKI上检索，未检索到文献。在这些国内外文献中，均未见有关急性高眼压大鼠视网膜神经生长因子前体来源的报道，也未见米诺环素在青光眼中的神经保护作用与p75^NTR和神经生长因子前体关系的报道。实验在试验设计和研究上具有创新性，其研究结果在国内外尚未见报道，具有先进性。