帕金森病纹状体提取液诱导骨髓间充质干细胞向神经元样细胞分化

doi:10.3969/j.issn.1673-5374.2012.34.004

摘要/Abstract

摘要：

实验以6-羟基多巴胺注射于右侧前脑建立帕金森病模型大鼠，分别以10%，60%毁损侧和未毁损侧纹状体诱导液培养从股骨和胫骨中分离的骨髓间充质干细胞，结果显示帕金森病大鼠毁损侧纹状体提取液诱导后，骨髓间充质干细胞向双极或多极化形态发展；胶质纤维酸性蛋白、巢蛋白和神经特异性烯醇化酶阳性细胞比例增加；且随帕金森病大鼠毁损侧纹状体提取液浓度的增加，神经特异性烯醇化酶阳性细胞比例增加。而未毁损侧纹状体诱导液只能增加胶质纤维酸性蛋白阳性细胞比例。提示帕金森病大鼠毁损侧纹状体提取液能够诱导骨髓间充质干细胞向神经元样细胞分化。

关键词: 骨髓间充质干细胞, 帕金森病, 纹状体提取液, 诱导分化, 神经细胞, 胶质纤维酸性蛋白, 巢蛋白, 神经特异性烯醇化酶, 神经干细胞, 再生, 神经再生

Abstract:

A rat model of Parkinson’s disease was established by 6-hydroxydopamine injection into the medial forebrain bundle. Bone marrow-derived mesenchymal stem cells (BMSCs) were isolated from the femur and tibia, and were co-cultured with 10% and 60% lesioned or intact striatal extracts. The results showed that when exposed to lesioned striatal extracts, BMSCs developed bipolar or multi-polar morphologies, and there was an increase in the percentage of cells that expressed glial fibrillary acidic protein (GFAP), nestin and neuron-specific enolase (NSE). Moreover, the percentage of NSE-positive cells increased with increasing concentrations of lesioned striatal extracts. However, intact striatal extracts only increased the percentage of GFAP-positive cells. The findings suggest that striatal extracts from Parkinson’s disease rats induce BMSCs to differentiate into neuronal-like cells in vitro.

Key words: bone marrow-derived mesenchymal stem cell, Parkinson’s disease, striatal extract, induced differentiation, nerve cell, glial fibrillary acidic protein, nestin, neuron-specific enolase, neural stem cell, regeneration, neural regeneration

. 帕金森病纹状体提取液诱导骨髓间充质干细胞向神经元样细胞分化[J]. 中国神经再生研究(英文版), 2012, 7(34): 2673-2680.

Xiaoling Qin, Wang Han, Zhigang Yu. Neuronal-like differentiation of bone marrow-derived mesenchymal stem cells induced by striatal extracts from a rat model of Parkinson’s disease[J]. Neural Regeneration Research, 2012, 7(34): 2673-2680.

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引言

材料方法

Design

An in vitro cytology study.

Time and setting

The experiment was performed at the Central Laboratory of the Second Affiliated Hospital, Soochow University, China from March 2006 to May 2007.

Materials

Ten healthy Sprague-Dawley rats of a clean grade, female or male, aged 2–3 weeks and weighing approximately 60–90 g, were used in this study. Another 40 male rats, aged 2–3 months and weighing 220–240 g, were used to establish Parkinson’s disease models. All rats were provided by the Animal Center of the Medical College, Soochow University (License No. SYXK (Su) 2007-0035), housed in a temperature-controlled environment with free access to food and water in a 12-hour light/12-hour dark cycle, and handled in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of China^[37].

Methods

Unilateral 6-hydroxydopamine injection for establishment of Parkinson’s disease models

Rats were anesthetized with 3.6% chloral hydrate (1 mL/100 g, supplemented as necessary) by intraperitoneal injection, and then fixed in a stereotaxic frame (KOPF Company, Hamburg, Germany). A 10 μL Hamilton syringe was lowered into the right medial fore-brain bundle at two coordinates (from the bregma: right 2.5 mm; posterior 1.8 mm; depth 7.5 mm, and right 2.5 mm; posterior 1.8 mm; depth 8.0 mm; according to The Stereotaxic Atlas of Rat Brain^[38]). Four microliters of 6-hydroxydopamine (Sigma, St. Louis, MO, USA) were infused at 0.5 μL/min into each point. The microsyringe remained in place for 5 minutes prior to slow withdrawal^[39].

Apomorphine-induced rotational behavior for assessment of models

Apomorphine-induced rotational behavior was observed to determine whether the creation of the 6-hydroxydopamine-induced lesion had been successful^[39]. At 4 weeks post-lesion, rats were placed in a quiet environment. Counting of rotations began 5 minutes after subcutaneous injections of apomorphine (0.5 mg/kg diluted in 0.9% saline), and rotational asymmetry was monitored over 30 minutes. Rats with at least 210 rotational turns in 30 minutes were regarded as successfully modeled and selected for subsequent use.

Preparation of striatal extracts

We prepared intact striatal extracts (on the left side) and lesioned striatal extracts (on the right side). Under deep anesthesia, the selected model rats were sacrificed by rapid decapitation after the rotational behavior test. Intact and lesioned striata were dissected on dry ice. Striata were weighed and homogenized with specific volumes of PBS in a proportion of 50 mL PBS (0.1 mM) per 100 mg wet weight. Tissue homogenates were centrifuged at 10 000 × g at 4 °C for 1 hour. After filter sterilization, the resulting supernatants were stored at –70 °C until use.

After rapid warming, intact and lesioned striatal extracts were respectively mixed with serum-free culture medium (50% L-DMEM and 50% F12) to prepare 10% and 60% I-SM and L-SM, according to the concentration ratio of the volumes of striatal extracts and total solution.

Isolation and culture of BMSCs

Rats were sacrificed and bone marrow was harvested under sterile conditions by aspiration from femurs and tibias in a rinse solution consisting of 10% fetal bovine serum (Sijiqing Biological Engineering Material Co., Ltd., Hangzhou, China), 45% L-DMEM (Gibco, Carlsbad, CA, USA), 45% F12 (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin). After standing for 2–3 minutes, the supernatant containing the cells was recovered and centrifuged at 800 r/min for 5 minutes. The supernatant was discarded, and complete culture medium consisting of 15% fetal bovine serum, 42.5% L-DMEM, 42.5% F12, 100 U/mL penicillin, and 100 μg/mL streptomycin was added. Cells were resuspended and seeded in 75 mL culture flasks at a density of 1 × 10⁶ cells/mL at 37°C in a humidified atmosphere with 5% CO₂. Culture medium was changed every 3–4 days.

Induced differentiation of BMSCs using striatal extracts

Second passage BMSCs at 80% confluence were seeded at a density of 2 × 10⁵/mL on coverslips in six-well plates. Culture medium was changed after 3 days. In experimental groups, BMSCs were cultured with 10% I-SM (10% I-SM group), 10% L-SM (10% L-SM group), 60% I-SM (60% I-SM group), or 60% L-SM (60% L-SM group). In control groups, cells were cultured in medium consisting of 15% fetal bovine serum, 42.5% L-DMEM, and 42.5% F12, or serum-free medium (50% L-DMEM and 50% F12). Cells and their morphological changes were observed under an inverted phase contrast microscope (Olympus, Tokyo, Japan) at 48 hours.

Immunohistochemistry for detection of nestin, GFAP, NSE and tyrosine hydroxylase

BMSCs were washed with PBS, blocked with serum in 0.3% Triton X-100/PBS, and incubated for 1 hour at room temperature with mouse anti-rat nestin (1:200; Chemicon, Santa Cruz, CA, USA), mouse anti-rat GFAP (1:600; DAKO, Glostrup,Denmark), rabbit anti-rat NSE (1:600; NeoMarkers, Fremont, CA, USA) and mouse anti-rat tyrosine hydroxylase (1:200; Chemicon) monoclonal antibodies. After three rinses with PBS, cells were incubated with biotinylated goat anti-mouse/rat IgG (1:200; Chemicon) at 37°C for 15 minutes. The cells were incubated with avidin biotinylated horseradish peroxidase for another 15 minutes, and then diaminobenzidine for 2 minutes followed by rinsing under tap water to terminate the reaction. For imaging, 10 non-overlapped fields of view were randomly selected from each sample under a light microscope (Olympus) and photos were taken using a medicine image analysis system (Motic Med 6.0, Motic, Xiamen, China). Brown or deep-brown cells were considered positive cells, and the number of deep-blue cell nuclei was regarded as the total cell number. Percentages of nestin-, GFAP-, NSE- and tyrosine hydroxylase-positive cells = nestin-, GFAP-, NSE-, and tyrosine hydroxylase-positive cells/total cell number × 100%, respectively.

Statistical analysis

Data were expressed as mean ± SD, and compared between two groups by one-way analysis of variance. All analyses were performed using SPSS 11.5 software (SPSS, Chicago, IL, USA). P-values less than 0.05 were considered statistically significant.

Author contributions: Xiaoling Qin conceived and designed the study, analyzed the data, performed the statistical analyses and wrote the manuscript. Zhigang Yu participated in its coordination and helped to edit the manuscript. Wang Han helped to analyze the data, perform the statistical analyses and write the manuscript. All authors approved the final manuscript.

Conflicts of interest: None declared.

Ethical approval: The study was approved by the Animal Ethics Committee of the Second Affiliated Hospital of Soochow University, China.

Author statements: The manuscript is original, has not been submitted to and is not under consideration by another publication, has not been previously published in any language or any form, including electronic, and contains no disclosure of confidential information or authorship/patent application disputations.