增加多巴胺合成的骨髓间充质干细胞

doi:10.3969/j.issn.1673-5374.2012.34.002

摘要/Abstract

摘要：

以往研究表明将酪氨酸羟化酶或Neurturin基因修饰的细胞分别移植到帕金森病大鼠脑内，均能明显改善帕金森病大鼠的行为、提高纹状体内多巴胺含量。实验将酪氨酸羟化酶和Neurturin基因修饰的骨髓间充质干细胞移植到帕金森病模型大鼠的损伤侧纹状体内。移植后大鼠损伤侧纹状体中酪氨酸羟化酶、Neurturin和多巴胺及其代谢产物3,4-二羟基苯乙酸表达增加，且纹状体多巴胺能神经突触后膜上D2受体密度下降，大鼠运动功能出现明显改善。表明酪氨酸羟化酶-Neurturin基因修饰骨髓间充质干细胞的移植不但能够增加多巴胺的合成，并且能明显改善帕金森病大鼠的行为能力。

关键词: 帕金森病, 酪氨酸羟化酶, Neurturin, 骨髓间充质干细胞, 移植, 多巴胺, 基因治疗, 神经退行性疾病, 再生, 神经再生

Abstract:

Previous studies showed that tyrosine hydroxylase or neurturin gene-modified cells transplanted into rats with Parkinson’s disease significantly improved behavior and increased striatal dopamine content. In the present study, we transplanted tyrosine hydroxylase and neurturin gene-modified bone marrow-derived mesenchymal stem cells into the damaged striatum of Parkinson’s disease model rats. Several weeks after cell transplantation, in addition to an improvement of motor function, tyrosine hydroxylase and neurturin proteins were up-regulated in the injured striatum, and importantly, levels of dopamine and its metabolite 3,4-dihydroxyphenylacetic acid increased significantly. Furthermore, the density of the D2 dopamine receptor in the postsynaptic membranes of dopaminergic neurons was decreased. These results indicate that transplantation of tyrosine hydroxylase and neurturin gene-modified bone marrow-derived mesenchymal stem cells increases dopamine synthesis and significantly improves the behavior of rats with Parkinson’s disease.

Key words: Parkinson’s disease, tyrosine hydroxylase, neurturin, bone marrow-derived mesenchymal stem cells, transplantation, dopamine, gene therapy, neurodegenerative disease, regeneration, neural

. 增加多巴胺合成的骨髓间充质干细胞[J]. 中国神经再生研究(英文版), 2012, 7(34): 2653-2662.

Yue Huang, Cheng Chang, Jiewen Zhang, Xiaoqun Gao. Bone marrow-derived mesenchymal stem cells increase dopamine synthesis in the injured striatum[J]. Neural Regeneration Research, 2012, 7(34): 2653-2662.

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引言

材料方法

Design

A randomized, controlled, animal study.

Time and setting

The study was performed at the Henan Provincial People’s Hospital, China from May 2010 to June 2012.

Materials

One hundred and ninety male Sprague-Dawley rats, 2–3 months old, 180–200 g, were provided by the Experimental Animal Center of Henan Province (animal license: No.4104035). Experimental procedures were performed in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of China^[32].

Methods

In vitro culture and identification of rat BMSCs

A total of 20 rats were ether-anesthetized and decapitated. Bone marrow was obtained from rats under sterile conditions. BMSCs were separated in vitro by density gradient centrifugation for primary culture at 37°C in air containing 5% CO₂^[33]. Sub-cultured BMSCs were monitored using the MTT method^[34] to guarantee appropriate growth. Passage 3 BMSCs were analyzed by flow cytometry and immunohistochemistry for the expression of CD29 and CD34, and prepared for transformation with the pIRES-TH-NTN vector in vitro ^[35-36].

Construction and expression of pIRES-TH-NTN vector in vitro

The TH and NTN gene sequences were cloned from pMD18T-TH and pcDNA-NTN (TaKaRa Bio, Dalian, China), respectively. The constructed pIRES-TH-NTN vectors were obtained from the Molecular lab in Zhengzhou University. Liposome 2000 (Invitrogen, Chicago, IL, USA) was used to transfect BMSC lines with pIRES-TH-NTN, and the transfected cells were selected under G418 (Inalco, Paris, France). The co-expression of TH and NTN mRNA in TH-NTN-BMSCs was assessed with real-time PCR^[37]. Primers used for amplification of the 284-bp TH were 5’-TCT GGA ACG GTA CTG TGG CT-3’ and 3’-CAA TGT CCT GGG AGA ACT GG-5’. Primers used for amplification of the 332-bp NTN were 5’-CCC TGC TGT CTG TCT GGA TGT G-3’ and 3’-ACG GTT TCG TCC GAC GTG TAG-5’. NTN and TH protein expression in cell lysates was measured by enzyme-linked immunosorbent assay^[38] at 24, 36, 48 and 72 hours to evaluate transfection efficiency. After G418 selection, stably transfected TH-NTN-BMSCs were prepared for transplantation.

Establishment of PD models

A PD model was established by two-point injection of 10 μL 6-OHDA (Sigma, St. Louis, MO, USA) into the right striatum^[39]. After 2 weeks, the surviving PD model rats were peritoneally injected with apomorphine (0.5 mg/kg; Sigma) to test their behavior, and successful PD model rats were confirmed^[28]. Rats in the control group were injected with 10 μL sodium chloride (0.9%) into the right striatum.

Cell transplantation for PD

Two weeks after 6-OHDA injection, successfully modeled rats in the BMSC and TH-NTN-BMSC groups were transplanted with 5 μL of BMSC or TH-NTN-BMSC cell suspension (1 × 10³ cells/μL) into the right striatum. BMSCs and TH-NTN-BMSCs were labeled with 5-bromodeoxyuridine (10 μM; Sigma) 3 days before transplantation^[40].

Behavioral assessment after cell transplantation

After BMSC or TH-NTN-BMSC transplantation, model rats in the PD, BMSC and TH-NTN-BMSC groups were peritoneally injected with apomorphine (0.5 mg/kg, Sigma) four times, and subsequently monitored for behavior at 2, 4, 6 and 8 weeks after transplantation. The number of rotations to the uninjured side (left) was recorded for 30 minutes after apomorphine injection.

Immunohistochemistry for TH and NTN expression in the corpus striatum

At 8 weeks after cell transplantation, rats in all groups were deeply anesthetized with chloral hydrate and cardially perfused with 4% paraformaldehyde. Formalin-fixed paraffin-embedded right corpus striatum tissue (1 mm³) was cut into 4-µm-thick sections, deparaffinized in three changes of xylene, and rehydrated through a graded alcohol:water series. Antigen retrieval was performed in 10 mM of sodium citrate buffer at pH 6 in an automated pressure cooker. The slides were then rinsed in running water for 5 minutes. Endogenous peroxidase was blocked with 3% hydrogen peroxide in water for 15 minutes at room temperature. The slides were then rinsed in water and immersed in PBS for 15 minutes at room temperature, followed by blocking in PBS with 5% goat serum (Vector Laboratories, Hamilton, Canada) for 1 hour. Sections were then incubated with mouse anti-rat TH monoclonal antibody (1:500; sc-47708, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or rabbit anti-rat NTN monoclonal antibody (1:400; ab49203, Abcam, London, UK) diluted in blocking solution and incubated overnight in a humidified chamber at 4°C. After washing three times with PBS, the slides were incubated with biotinylated goat anti-mouse or goat anti-rabbit IgG at adilution of 1:500 for 30 minutes at room temperature (Zymed, San Diego, CA, USA). Diaminobenzidine was used as the chromogen substrate, and hematoxylin was used as the nuclear counterstain. The primary antibody was replaced with PBS or normal rabbit serum (1:100) as a negative control. Slides were imaged by light microscopy (Philips, Amsterdam, Netherlands) and labeled TH and NTN intensity scores were quantified by software Image J (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA).

Western blot analysis of TH and NTN protein expression in the right striatum

Pre-cleared right striatum brain tissue lysates were boiled at 100°C for 5 minutes in SDS-loading buffer. Equal amounts of protein per sample were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% non-fat dry milk in PBST for 2 hours at room temperature and reacted with the primary antibodies (diluted to 0.5 µg/mL in PBST/5% milk; same antibodies as for immunohistochemistry) at 4°C overnight, followed by incubation with HRP-conjugated secondary antibodies (same antibodies as for immunohistochemistry; 1:2 000) for 45 minutes at 37°C Immunoreactive bands were visualized using enhanced chemiluminescence (GE Healthcare, London, UK). Bands were analyzed using Typhoon 9410 and ImageQuant TL v2008.01 (GE Healthcare).

High performance liquid electrochemical detection of DA and DOPAC content in striatum

Brain samples from the left or right hemispheres containing striatum and substantia nigra were hydrolyzed in a high performance liquid electrochemical measurement system^[41]. DA and DOPAC in the left and right corpus striatum were detected and recorded by the high performance liquid electrochemical measurement system.

Ultrastructure of the striatum of model rats observed by post-embedding immunogold electron microscopy

The procedure for post-embedding immunogold electron microscopy was adapted from Bergersen and colleagues^[42]. Small rectangular pieces from the corpus striatum region were typically 0.5 mm × 0.5 mm × 1 mm. In the control group, the samples were picked from the left corpus striatum (non-operated and without 6-OHDA injection) in PD model rats. In the PD, BMSC and TH-NTN-BMSC groups, the samples were taken from the right striatum (6-OHDA injection and cell transplantation side). All samples were obtained at 8 weeks after cell transplantation. Ultrathin sections (5 μm) were incubated overnight with primary antibodies against the dopamine D1 receptor (Abcam-ab20066, London, UK) or dopamine D2 receptor (Abcam- ab21218, UK) diluted in TBST containing 2% HAS. The concentrations of the dopamine receptor antibodies were 5 µg/mL. Bound antibodies were visualized by incubating for 2 hours with goat anti-rabbit immunoglobulin secondary conjugated with 10-nm (diameter) colloidal gold (1:20; British Biocell International, London, UK). Sections were observed in a Philips CM20 electron microscope. Images were taken randomly at a primary magnification of × 43 000. Ten individual synapses with clearly defined postsynaptic densities were imaged from each animal. The lengths (nm) of the postsynaptic membranes were marked, and the densities of gold particles were calculated as previously described^[42]. For density calculations, gold particles situated within 25 nm (i.e., approximately the same distance as the lateral resolution of the immunogold method)^[42] on either side of the midline of the membranes were included in the analysis, and the area containing gold particles was assumed to be a rectangle with a 50-nm width.

讨论

文章快阅

延伸阅读

1 研究背景

帕金森病是常见的神经系统退行性疾病，其基本的临床症状包括静止性震颤、僵硬、运动迟缓和姿势异常等。其主要病理变化是黑质多巴胺能神经元的变性，结果导致纹状体内多巴胺及其代谢产物3,4-二羟基苯乙酸含量明显减少。目前治疗帕金森病的主要方法有药物治疗、手术治疗、细胞移植和基因治疗。干细胞生物学技术的发展，提供了采用基因修饰的骨髓间充质干细胞治疗神经系统病变的可能。骨髓间充质干细胞移植由于是自体移植，可以避免排异反应及伦理学问题。并且由于易于体外培养和扩增，成为一种理想的基因载体。

酪氨酸羟化酶是多巴胺合成过程中的限速酶，是神经系统内多巴胺能神经元的蛋白标志。研究发现把外源性的酪氨酸羟化酶基因导入帕金森病模型大鼠纹状体内，可增加脑内多巴胺的水平，改善阿扑吗啡诱导的旋转症状，对帕金森病有治疗作用。Neurturin在结构和功能上与胶质细胞源性神经营养因子有关，二者同属于胶质细胞源性神经营养因子亚家族，对多巴胺能神经元具有特异性的营养、支持、保护和损伤修复作用。研究证实Neurturin在体内外都能促进胚胎中脑神经元的存活，阻止神经退行性病变的发生。总之，Neurturin对于帕金森病的治疗作用值得探索。

以往研究表明将酪氨酸羟化酶或Neurturin基因修饰的细胞分别移植到帕金森病大鼠脑内，均能明显改善帕金森病大鼠的行为、提高纹状体内多巴胺含量。如果将2种基因联合移植入纹状体内，是否会产生比单独移植更好的疗效呢？实验将进行多基因修饰骨髓间充质干细胞的移植研究。

2 外审专家意见及作者答复

问题：酪氨酸羟化酶阳性细胞包括多巴胺能神经细胞和去甲肾上腺素能神经细胞，只有酪氨酸羟化酶和芳香族L氨基酸脱羧酶阳性，而多巴胺β羟化酶阴性的细胞才是多巴胺能神经细胞，因此建议在图3结果中加入L氨基酸脱羧酶和多巴胺β羟化酶的检测结果。

回复：实验中检测酪氨酸羟化酶阳性细胞的目的不是为了区分多巴胺能或去甲肾上腺素能神经细胞，而是考虑到酪氨酸羟化酶为多巴胺形成过程中的限速酶。检测酪氨酸羟化酶表达量的变化，是为解释多巴胺及其代谢产物发生变化的可能机制。