雷帕霉素通路的激活参与脊髓损伤修复

doi:10.3969/j.issn.1673-5374.2013.02.001

摘要/Abstract

摘要：

雷帕霉素靶蛋白通路在调节神经元生长，增殖和分化方面有重要作用。为了更好的认识雷帕霉素靶蛋白通路在脊髓损伤形成过程中的作用，实验应用改良的重物自由落体打击法建立脊髓损伤大鼠模型，腹腔注射雷帕霉素靶蛋白通路激活剂ATP和雷帕霉素靶蛋白激酶抑制剂雷帕霉素干预7d。损伤后1，2，3，4周，应用BBB评分评估大鼠运动功能，应用免疫组化、免疫印迹和实时定量聚合酶链反应检测脊髓神经干细胞标志物nestin, 神经元标志物神经元特异核蛋白, 神经元特异性烯醇化酶, 轴突标志物神经丝蛋白200, 星形胶质细胞标志物胶质纤维酸性蛋白和雷帕霉素靶蛋白通路丝氨酸/苏氨酸蛋白激酶、雷帕霉素靶蛋白、信号转录和转录激活因子3的表达。证实ATP介导的丝氨酸/苏氨酸蛋白激酶/雷帕霉素靶蛋白/信号转录和转录激活因子3通过诱导内源性神经干细胞增多，诱导神经发生和轴突生长，抑制过度反应性星形胶质化，改善脊髓损伤大鼠运动功能。

关键词: 神经再生, 脊髓损伤, 丝氨酸/苏氨酸蛋白激酶, 哺乳动物, 雷帕霉素靶蛋白, 信号转导及转录激活因子3, ATP, 信号通路, 功能恢复, 雷帕霉素, 图片文章

Abstract:

The mammalian target of rapamycin (mTOR) pathway plays an important role in neuronal growth, proliferation and differentiation. To better understand the role of mTOR pathway involved in the induction of spinal cord injury, rat models of spinal cord injury were established by modified Allen's stall method and interfered for 7 days by intraperitoneal administration of mTOR activator adenosine triphosphate and mTOR kinase inhibitor rapamycin. At 1–4 weeks after spinal cord injury induction, the Basso, Beattie and Bresnahan locomotor rating scale was used to evaluate rat locomotor function, and immunohistochemical staining and western blot analysis were used to detect the expression of nestin (neural stem cell marker), neuronal nuclei (neuronal marker), neuron specific enolase, neurofilament protein 200 (axonal marker), glial fibrillary acidic protein (astrocyte marker), Akt, mTOR and signal transduction and activator of transcription 3 (STAT3). Results showed that adenosine triphosphate-mediated Akt/mTOR/STAT3 pathway increased endogenous neural stem cells, induced neurogenesis and axonal growth, inhibited excessive astrogliosis and improved the locomotor function of rats with spinal cord injury.

Key words: neural regeneration, spinal cord injury, serine/threonine-specific protein kinase, mammalian target of rapamycin pathway, signal transduction and activator of transcription 3, adenosine triphosphate, signal pathway, rapamycin, photographs-containing paper, neuroregeneration

. 雷帕霉素通路的激活参与脊髓损伤修复[J]. 中国神经再生研究(英文版), 2013, 8(2): 101-110.

Zhengang Sun, Lingyun Hu, Yimin Wen, Keming Chen, Zhenjuan Sun, Haiyuan Yue, Chao Zhang. Adenosine triphosphate promotes locomotor recovery after spinal cord injury by activating mammalian target of rapamycin pathway in rats[J]. Neural Regeneration Research, 2013, 8(2): 101-110.

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引言

材料方法

Design

A randomized, controlled, animal experiment.

Time and setting

This study was performed at the Department of Orthopedics, Central Research Institute of Lanzhou General Hospital, Lanzhou Military Command of Chinese PLA, from October 2008 to December 2009.

Materials

Totally 128 adult specific pathogen-free female Sprague-Dawley rats, weighing 200–250 g, were provided by Laboratory Animal Center of Gansu College of Traditional Chinese medicine (license No. SCXK (Lu) 20030010) and housed in facilities maintained at 22 ± 2°C under a 12-hour light/dark cycle. Before surgery, all animals were acclimatized for 5–7 days and allowed free access to food and water. All animal experiments were performed in strict accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of China^[30].

Methods

Spinal cord injury model preparation

Rats were anesthetized with sodium pentobarbital (30 mg/kg, i.p.) and placed in a prone position on a heating pad to maintain a constant body temperature. Under aseptic conditions, a longitudinal incision (about 5 cm) was made at the midline of the back exposing the paravertebral muscles. These muscles were dissected to expose T₇_-₁₁ vertebrae. The dura mater of the spinal cord was exposed via a three-level T₈_-₁₀ laminectomy, and covered with a metal gasket consistent with the length and curvature of spinal cord T₈_-₁₀. Spinal cord injury was induced by epidural weight drop using a modified Allen's stall with damage energy of 50 g-cm force (4 g × 12.5 cm). The wound was closed and disinfected. A successful animal model of spinal cord injury should meet the following criteria: at the moment of the ball impacting the dura mater of spinal cord, animals showed body jitter, hindlimb retraction, tail spastic swing, and hindlimb flaccid paralysis. The sham-operated group animals underwent a T₈_-₁₀ laminectomy without weight- drop injury.

Animal treatment and sample preparations

In the ATP group, following spinal cord injury induction, rats were daily administered ATP (40 mg/kg, i.p.; Qilu Pharmaceutical Corp, Shandong, China) for 7 successive days. In the control group, following spinal cord injury induction, rats were daily administered sterile saline (40 mg/kg, i.p.) for 7 successive days. In the block/interruption group, following spinal cord injury induction, rats were daily administered ATP (40 mg/kg, i.p.; Qilu Pharmaceutical Corp) and rapamycin (3 mg/kg, i.p.; Sigma, St. Louis, MO, USA)^[31] for 7 days. In the sham-operated group, rats were subjected to laminectomy but without spinal cord injury induction and then received physiological saline administration (40 mg/kg, i.p.) for 7 successive days (Table 1).

All rats received gentamicin solution administration daily starting 30 minutes post-trauma followed by repeated injections (b.i.d., 2 mg/kg) for 7 successive days. After spinal cord injury induction, rats were housed for a survival period of 1–4 weeks. During this time period, all rats was subjected to treadmill training for 30 minutes per day at 8:00 p.m. and the bladder of each rat was manually emptied twice a day until the rats regained normal bladder function. Rats were anesthetized with an overdose of sodium pentobarbital (60 mg/kg) at 1–4 weeks post-trauma in each group, respectively (n = 8) to harvest 1.5 cm length spinal cord samples centered at the epicentre site. Rostral samples were removed by median cross-section, fixed overnight with 4% paraformaldehyde solution prepared in PBS at 4°C for 24 hours and further processed for histological examination. Caudal samples were divided into two copies from the mid-sagittal plane and kept in liquid nitrogen for protein measurements.

Behavioral analysis

Locomotor activity was evaluated using the BBB locomotor rating scale^[32]. Functional tests were performed before surgery and each week during the 4-week survival period. Considering the significant difference between day and night rat activities, all rats were scored in an open field at 8:00 p.m. for 4 minutes. Two independent examiners blinded to experimental design were asked to evaluate the locomotor function of each animal.

Spinal cord immunohistochemistry

Samples were fixed and embedded in paraffin. Serial para-sagittal sections (5 μm) were made from spinal cord centered on the injury epicenter and mounted on poly-L- lysine-coated glass slides. Sections were dewaxed in xylene, rehydrated in gradient ethanol, and washed in 0.1 M PBS (pH 7.5). Endogenous peroxidase activity was quenched with 3% H₂O₂ at room temperature for 10 minutes, sections were washed three times in distilled water, and then heated in 10 mM citrate buffer (pH 6.0) for antigen retrieval. After cooling, sections were washed three times in PBS and then blocked in 5% bovine serum albumin (Boster, Wuhan, China) for 1 hour at room temperature. Incubation with the indicated primary antibodies (Table 2) was performed overnight at 4°C. inhibitor cocktail (Sigma) and phosphatase inhibitor cocktail (Sigma) for 30 minutes at 4°C. After three washes in PBS for 5 minutes each, the sections were reacted with biotin-conjugated goat anti-rabbit/ mouse IgG (Boster) at room temperature for 30 minutes, and then incubated with horseradish peroxidase- conjugated streptavidin (Boster) for 30 minutes at room temperature. Following washes in PBS, sections were incubated with 0.05% diaminobenzidine (Boster), counterstained with hematoxylin, sealed by DPX mountant, covered, and then visualized using an Olympus light microscope (DP71, Olympus, Japan) at 100 × and 400 × magnification. Negative control sections were treated with the identical procedure except that the primary antibodies were omitted. Cell counts were determined using unbiased sampling: three sampling frames of 350 μm × 350 μm were selected per section at random within the traced region.

Western blot analysis

Samples were quickly removed from liquid nitrogen, homogenized by adding lysate buffers (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1mM ethylenediamine tetraacetic acid; 1% NP-40; 0.25% Na-deoxycholate;1 mM phenylmethyl sulfonylfluoride; 0.1% sodium dodecyl sulfate) and supplemented with protease inhibitor cocktail (Sigma) and phosphatase inhibitor cocktail (Sigma) for 30 minutes at 4°C. After homogenates were centrifuged at 12 000 r/min for 15 minutes at 4°C, the supernatants were collected and total protein contents were determined using commercial background- corrected absorbance assay kits (BIOS, Beijing, China). The samples (8 μg protein) were added to 1/2 sample buffer (2% sodium dodecyl sulfate, 10% glycerol, 0.1% bromophenol blue, 2% 2-mercaptoethanol, and 50 mM Tris-HCl) and subjected to 5–17% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Table 2). After electrophoresis, the proteins were transferred onto polyvinylidene difluoride membranes (0.45 μm) (Millipore Corp, Billerica, MA, USA), and blocked in 2% nonfat dry milk buffer for 2 hours at room temperature. After one wash with PBS + 0.05% Tween-20 (PBS-T), the membranes were incubated overnight at 4°C with primary antibodies (Table 2) diluted in antibody dilution (PBS-T + 2% nonfat dry milk + 1% bovine serum albumin). After three washes with PBS-T, the membranes were incubated for 2 hours with horseradish peroxidase-conjugated secondary antibody (sheep anti-rabbit/mouse, 1:5 000) in the dilution described above. Following extensive washes in PBS-T, the signals were detected with an immobilon western chemiluminescent horseradish peroxidase substrate (Millipore Corp, Billerica, MA, USA) and exposed on X-ray film (Kodak, China). For semiquantitative analysis of the expression level of Akt, P-Akt, mTOR, P-mTOR,

STAT3, P-STAT3, NeuN, NSE, nestin, NF200, and GFAP, the integrated absorbance of each band was measured by Image-Pro PLUS (Meyer Instruments, Houston, TX, USA). The integrated absorbance was normalized to that of β-actin (Zhongshan, Beijing, China) and expressed as arbitrary units. Each sample was assayed in triplicate and averaged.

Statistical analysis

All data were presented as mean ± SD. The differences were analyzed by one-way analysis of variance and repeated-measures analysis of variance. Results were considered significantly different at P < 0.05. All analyses were performed using SPSS 16.0 statistical software (SPSS, Chicago, IL, USA).

讨论

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专家问题及作者答疑：
问题1：截瘫大鼠的术后护理，尤其是定期排尿等，是造模成功的关键。术后大鼠存活率如何？请说明。
回复：研究脊髓损伤修复的最基本条件是建立理想的标准化的SCI模型。具备理想模型的条件是：与临床相似性；动物实验的可操作性和可重复性；脊髓损伤模型的可调控性。国内外有关脊髓损伤造模的研究较多，传统的方法有脊髓撞击伤法、脊髓挤压伤法、脊髓缺血损伤法、脊髓横断法、脊髓放射性损伤、脊髓化学损伤等方法。脊髓撞击伤模型的病理过程与人类脊髓急性损伤非常相近，可引起早期脊髓组织出血、水肿和坏死。脊髓撞击伤模型是模拟人类急性脊髓损伤中最常见类型之一，其中改良Allen重物打击法最常用。改良的Allen重物打击法可以保持硬脊膜的完整，防止外源性成分的浸入，还可以防止脊髓和脑脊液的外漏、并可以精确控制损伤的程度。但是这种造模方式也存在不足之处，如手术技术熟练程度，暴露的脊髓未严格固定导致每次撞击部位不一致等。

在实验中，采用自制的Allen重物打击器，应用不同的下落高度，造成脊髓损伤程度不同的模型。由此可见改良的Allen重物打击法所致脊髓损伤程度一致性较高，可以满足本实验的要求。
改良Allen重物打击法制造脊髓损伤造模是非常适用的。实验采用4 g钢球重物下落损伤大鼠脊髓T8-10部位。当高度为8 cm时，第2天大鼠后肢运动功能即有明显恢复，21天后后肢运动功能基本恢复正常，只能用于对轻度脊髓损伤后病理改变的研究，不能够用于对治疗干预的研究。重物下落高度为15和20 cm，手术后大鼠死亡较多，对实验结果影响较大，并且损伤严重，恢复差。重物下落高度为12.5 cm时，术后1天BBB评分为0.80±0.23，21天后为7.97±0.93，可有较大的恢复潜力，适合治疗干预的研究。

实验采用的实验大鼠均为雌性，因为雌性大鼠尿道短，有利于人工排尿，有利于大鼠的存活率。我们做信号通路的128只SD大鼠：实验组死亡2只、对照组1只、阻断组2只，假手术组未死亡，死亡的大鼠再补充同样的SD大鼠。总的大鼠成活率为96.1%。

问题2：脊髓损伤修复的可能通路较多，请着重阐明选择Akt/mTOR/STAT3研究的缘由。
回复：影响脊髓损伤修复的通路还有很多（如ROCK和JAK 、ERK/MAPK信号通路等）。哺乳类动物雷帕霉素靶蛋白（The mammalian target of rapamycin，mTOR）是一类脯氨酸调控的丝氨酸-苏氨酸（Ser/Thr）蛋白激酶，是结构复杂的大分子蛋白。研究表明人与鼠的mTOR序列一致性达98% ，而与非哺乳动物TOR1、TOR2同源性仅为43%、45%，有助于通过大鼠试验研究人类脊髓损伤修复。

丝氨酸/苏氨酸蛋白激酶（protein kinase B，Akt）通过激活或抑制底物在调解细胞的存活、增殖和分化中起重要的作用。此外，最近研究表明在哺乳动物细胞中发现的三种Akt的亚型 (Akt1, Akt2 和 Akt3)是神经轴突生长和神经元分化的重要调解器。mTOR是丝氨酸/苏氨酸激酶的磷脂激酶相关家族的成员之一。通过生长因子、神经营养素、细胞因子等激活信号，活化的mTOR在调整细胞生长、增殖、分化中起关键作用，活化的mTOR能被雷帕霉素阻断（一种专门抑制mTOR激酶的抑制因子）。在神经元分化中mTOR 是Akt的直接效应物，可被活化的Akt磷酸化。信号转录和转导激活因子3 (signal transducers and activator of transcription 3，STAT3)可以被许多细胞因子和生长因子激活，并很容易被mTOR激酶磷酸化。最近，证据表明在中枢神经系统中活化的STAT3在控制神经元生长、增殖、分化和神经轴突生长起重要的作用。

mTOR是一个重要的信号转导分子，外界刺激激活mTOR后，引起通路下游的信号分子的表达与活性增加，影响细胞生长周期进程，促进细胞生长、增殖、分化。许多细胞因子如LIF、BMP2和CNTF等能激活mTOR，引起下游STAT3信号分子磷酸化，促进神经干细胞分化为神经胶质细胞。有研究发现Nogo-66通过激活mTOR/STAT3信号转导通路促进神经干细胞诱导分化为星型胶质细胞，加入雷帕霉素抑制该通路后，神经干细胞分化为胶质细胞明显减少。我们从干预Akt/mTOR/STAT3信号转导通路治疗脊髓损伤提供了新思路。所以我们研究Akt/mTOR/STAT3通路与脊髓损伤的关系。

问题3：实验只采用了mTOR抑制剂雷帕霉素干预，并未采用 Akt/mTOR/STAT3通路中Akt和 STAT3的抑制剂进行干预，因而Akt/mTOR/STAT3通路与ATP相关性缺乏直接证据，推测的成分居多，因而结论略显主观。
回复：设计的实验是观察mTOR通路对脊髓损伤的影响，作者查阅了大量的文献，发现雷帕霉素为mTOR信号通路的抑制剂，所以我们采用了 mTOR通路抑制剂雷帕霉素干预，观察mTOR信号的改变，同时观察 mTOR通路上游因子Akt和mTOR通路下游的因子 STAT3和 p70S6 激酶的变化（实验观察是 STAT3）。最初设计mTOR上游因子 Akt，随着雷帕霉素干预是不会改变的，但是实际是雷帕霉素阻断后，上游因子 Akt磷酸化后也随着降低。我们查阅文献发现 mTOR是PI3激酶信号通路的因子，是Akt的下游因子，活化的Akt可以直接或间接的磷酸化mTOR。在哺乳动物细胞中，存在2种mTOR复合物，即mTORC1和mTORC2。mTORC1对大环内酯类抗生素雷帕霉素抑制作用敏感，在激活的mTOR信号通路，Akt起着非同寻常的作用，因为它可以作用于mTORC1上游区和mTORC2下游区。mTORC2可以在Ser473位点磷酸化Akt，Ser473位点磷酸化Akt 再磷酸化 Thr308位点的Akt，然后导致Akt的活化。所以，虽然mTORC2对雷帕霉素的长期治疗不敏感，但仍能被雷帕霉素间接抑制，导致抑制Akt。这和我们的实验结果符合的。同时雷帕霉素阻断后，mTOR通路下游的因子 STAT3和 p70S6激酶都随之降低

最初选择ATP作为治疗脊髓损伤的药物，是因为 ATP有作为神经递质调节神经系统的功能，还有营养脊髓神经元、促进轴突生长的作用。Holton等首次发现神经末梢可以释放ATP，表明ATP可作为神经递质行使其功能。Dalzi等发现ATP通过脊髓神经元和雪旺细胞表面P2Y受体亚型介导神经细胞的电生理变化和细胞增殖，保护受损的神经元。Andres等研究发现在中枢神经系统，细胞外ATP通过肌酸激酶/磷酸肌酸系统促进神经元前体细胞分化。众所周知ATP介导的信号在中枢神经系统中有广泛的作用，包过作为神经递质，调整细胞生长、增殖、分化。神经系统损伤后，ATP作为一个关键的因素，启动蛋白激酶级联信号，通过激活PI3激酶/Akt信号通路，保护神经作用。ATP、激素、生长因子激活mTOR后，能促进蛋白质的合成，促进细胞生长和增殖。此外，在细胞生长、增殖和分化条件下，已经确立ATP与mTOR 信号系统的关系，ATP可以作为调整有丝分裂原激活mTOR 信号系统的底物。此外，在细胞增殖、修复和重塑中，细胞外的ATP可以诱导Akt活化并刺激mTOR磷酸化。

问题4：BBB是分析Locomotor activity的金标准吗？
回复：BBB评分法是评估大鼠后肢运动功能恢复情况的有力证明方法。术后进行大鼠后肢运动功能的评价，同一组个体之间的BBB评分最大差距在3分内，所有实验动物在麻醉清醒后立即进行BBB评分，双下肢功能均为完全性瘫痪，可能是由于脊髓遭受强烈创伤后发生脊髓休克，脊髓功能暂时处于生理停滞状态，这种情况到第2天已基本恢复，大鼠双下肢功能BBB评分应从术后1天后开始。

采用BBB评分法评估大鼠后肢运动功能恢复情况优点：
(1)BBB评分法是专门用于评价实验大鼠脊髓损伤后双后肢运动功能恢复情况。
(2)BBB评分法可见进行性排列，体现了脊髓损伤恢复的全过程。
(3)BBB评分法记分不是以前累计求和的方法，而是每一个分数都有一个独立的评分标准。该方法不需特殊的设备，易于推广。但其评分标准较复杂，要求观察人员进行一定的培训，以尽量避免主观因素的影响。

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