恒河猴脂肪间充质干细胞向多巴胺能神经元的分化

doi:10.3969/j.issn.1673-5374.2012.34.001

摘要/Abstract

摘要：

实验用携带能够启动神经元细胞的发育程序促进胚胎干细胞向多巴胺能神经元分化的Lmx1a基因的重组腺病毒感染恒河猴脂肪间充质干细胞，同时与胚鼠神经干细胞进行非接触性共培养，并应用音猥因子和成纤维细胞生长因子8诱导细胞分化。免疫荧光染色见细胞表达神经元特异性标志物神经元特异性烯醇化酶和β-微管蛋白Ⅲ；RT-PCR结果显示经诱导的恒河猴脂肪间充质干细胞除表达特异性烯醇化酶和β-微管蛋白Ⅲ mRNA外，还表达多巴胺能神经元标志物酪氨酸羟化酶、Nefm、GFRα2、Nurr1；且表达多巴胺能神经元标志物的细胞明显多于未经Lmx1a基因诱导的恒河猴脂肪间充质干细胞。结果表明，Lmx1a的调控和与神经干细胞联合共培养的诱导策略能够显著提高恒河猴脂肪间充质干细胞向多巴胺能神经元分化的效率。

关键词: 恒河猴, 脂肪间充质干细胞, 分化, LMX1a, 多巴胺能神经元, 共培养, 神经干细胞, 帕金森, 神经退行性疾病, 神经再生

Abstract:

LIM homeobox transcription factor 1a (Lmx1a) has the capacity to initiate the development program of neuronal cells and promote the differentiation of embryonic stem cells into dopaminergic neurons. In this study, rhesus adipose stem cells were infected with recombinant adenovirus carrying the Lmx1a gene and co-cultured with embryonic rat neural stem cells. Cell differentiation was induced using sonic hedgehog and fibroblast growth factor-8. Immunofluorescence staining showed that cells were positive for neuron-specific enolase and β-tubulin III. Reverse transcription-PCR results demonstrated that rhesus adipose stem cells were not only positive for neuron-specific enolase and β-tubulin III, but also positive for the dopaminergic neuron marker, tyrosine hydroxylase, neurofilament, glial cell line-derived neurotrophic factor family receptor α2 and nuclear receptor related factor 1. The number of Lmx1a gene-infected cells expressing the dopaminergic neuron marker was substantially greater than the number of cells not infected with Lmx1α gene. These results suggest that Lmx1a-mediated regulation combined with the strategy of co-culture with neural stem cells can robustly promote the differentiation of rhesus adipose stem cells into dopaminergic neurons.

Key words: rhesus, adipose stem cells, differentiation, LIM homeobox transcription factor 1a, dopaminergic neurons, co-culture, neural stem cells, Parkinson’s disease, neurodegenerative disease, neural regeneration

. 恒河猴脂肪间充质干细胞向多巴胺能神经元的分化[J]. 中国神经再生研究(英文版), 2012, 7(34): 2645-2652.

Yan Zhou, Maosheng Sun, Hongjun Li, Min Yan, Tianhong Xie. Differentiation of rhesus adipose stem cells into dopaminergic neurons[J]. Neural Regeneration Research, 2012, 7(34): 2645-2652.

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引言

材料方法

Design

A neural induction study combining genetic engineering and cellular biology.

Time and setting

The experiment was performed in the Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College in China between 2010 and 2012.

Materials

A 6-year-old male rhesus monkey, weighing 6 kg, provided by the Institute of Medical Biology, Chinese Academy of Medical Science & Peking Union Medical College in China, was used for the isolation of rhesus adipose stem cells. Embryonic day 14.5 Sprague-Dawley rats purchased from Kunming Medical College were used for the isolation of rat ventral mesencephalic neural stem cells. Every effort was made to minimize the number of animals used and their suffering. All experimental procedures were conducted in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of China^[33].

Methods

Isolation and characterization of rhesus adipose stem cells

Rhesus adipose stem cells were isolated from 3 to 5 g of adipose tissue obtained from rhesus omentum under aseptic conditions. Rhesus adipose stem cells were cultured as previously described^[34]. To induce adipogenic differentiation, rhesus adipose stem cells at passage 3 were plated in Corning 6-well plates (1 × 10⁵ cells per well) containing Dulbecco’s modified Eagle’s medium (high glucose), 10% fetal bovine serum, 1 μM dexamethasone, 10 μg/mL insulin, 200 μM indomethacin (Sigma, St. Louis, MO, USA) and 0.5 mM 3-isobutyl-1- methylxanthine (Sigma). The medium was discarded every 3 days. After 2 weeks of adipogenic stimulation, cells were stained with oil red O to detect lipid vacuoles^[24]. To induce osteogenic differentiation, rhesus adipose stem cells were plated (1 × 10⁵cells per well) in Corning 6-well plates containing Dulbecco’s modified Eagle’s medium (high glucose), 10% fetal bovine serum, 10 nM dexamethasone, 10 mM β-glycerophosphate disodium salt hydrate and 150 μM L-ascorbic acid-2- phosphate (Sigma). Medium was changed every 3 days. After 3 weeks of osteogenic stimulation, cells were stained with alizarin red^[24]. Alkaline phosphatase activity was assayed using an alkaline phosphatase activity staining kit (Genmed Scientifics, Inc., Washington DC, USA)^[21].

Isolation and characterization of neural stem cells

Under aseptic conditions, neural stem cells were harvested from the mesencephalic tissue of Sprague-Dawley rat embryos at embryonic day 14.5 as described previously^[35]. The cells were grown in suspension in Dulbecco’s modified Eagle’s medium/F12 medium with B27 supplement (Gibco, Carlsbad, CA, USA) and 20 ng/mL human basic fibroblast growth factor (Millipore, Billerica, MA, USA). Cells were allowed to proliferate and generate neurospheres, and were passaged every 3 days to maintain appropriate density. Seven days later, adherent cells were purified by adding arabinoside cytosine (Sigma). The medium was changed to neural basal medium (Gibco) containing 2% B27. Neural stem cells were identified by labeling for nestin (rabbit anti-rat; Millipore) and neurons derived from neural stem cells were detected by labeling for tyrosine hydroxylase (rabbit anti-rat; R&D Systems, Inc., Minneapolis, MN, USA) using immunofluorescence staining.

Treatment with Lmx1a and co-culture with neural stem cells

We constructed an E1/E3-deleted, replication-deficient recombinant human adenovirus serotype 5 containing Lmx1a gene using the AdEasy^TM Adenoviral Vector System (Stratagene, Stevens Creek Blvd Santa Clara, CA, USA). To differentiate rhesus adipose stem cells into dopaminergic cells in vitro, rhesus adipose stem cells were trypsinized and then plated at a final density of 1 × 10⁵ cells/cm² on poly-D-lysine-coated culture wells with coverslips. Rhesus adipose stem cells were infected with Ad-Lmx1a at a multiplicity of infection of 6 in 1 mL of culture medium containing 5% fetal bovine serum for 6 hours at 37°C, followed by fresh medium replacement containing 10% fetal bovine serum.

For co-culture of rhesus adipose stem cells and neural stem cells, 2.5 × 10⁵ fetal neural stem cells derived from Sprague-Dawley rat embryos at embryonic day 14.5 were seeded onto poly-D-lysine-coated hanging cell culture inserts with a 0.4 µm poly(ethylene terephthalate) membrane (Millipore, Billerica, MA, USA) that is sufficient to allow for protein transfer. One day later, the membrane insert was immersed in a well containing Ad-Lmx1a infected rhesus adipose stem cells. The medium was replaced with 50 ng/mL sonic hedgehog (R&D Systems, Inc.) plus 50 ng/mL fibroblast growth factor-8 (R&D Systems, Inc.) medium (Figure 5).

Reverse transcription-PCR analysis

To determine whether co-culture with neural stem cells combined with Lmx1a transfection could induce neuron- like cells from rhesus adipose stem cells, reverse transcription-PCR was performed using rhesus-specific primers for neuron-specific genes and β-actin. Total RNA was isolated from induced rhesus adipose stem cells using an RNA extraction kit (Omega, Germany) and cDNA was synthesized using M-MULV reverse transcriptase (Fermentas International Inc., Burlington, Canada). DNA was amplified with rhesus-specific primers (Table 1) according to the following conditions: 95°C for 5 minutes; 35 cycles of 95°C for 30 seconds, 55°C for 30 seconds and 72°C for 30 seconds; and 72°C for 10 minutes. The samples were stored at 4°C.

Immunofluorescence staining

To examine the expression of Lmx1a and neuron-specific genes in rhesus adipose stem cells after infection and induction, cells were fixed with 2% paraformaldehyde containing 0.2% Triton X-100 for 20 minutes at 4°C, then fixed with pre-cooled 98% methanol for 10 minutes at 4°C. After washing with ice-cold PBS, samples were blocked with 2% bovine serum albumin (Sigma) in PBS for 1 hour at 37°C. The slides containing cells were incubated with antibodies, such as Lmx1a (0.1 μg/mL; Santa Cruz Biotechnology, Santa Cruz, CA, USA), β-tubulin III (0.1 µg/mL, Millipore), neuron-specific enolase (0.1 μg/mL; Thermo Fisher Scientific, Barrington, IL, USA) and tyrosine hydroxylase (0.1 μg/mL, Millipore) for 1.5 hours at 37°C. After incubation, cells were washed with PBS and incubated with goat anti-rabbit IgG-DyLight™ 549 (0.5 μg/mL, Rockland Immunochemicals Inc., Gilbertsville, PA, USA) or mouse anti-rabbit IgG-FITC (0.5 μg/mL; Chemicon, Santa Cruz, CA, USA) for 1 hour at 37°C. The slides were then washed three times with PBS and covered with a cover slip, followed by staining with 4, 6-diamidino-2- phenylindole dihydrochloride (Sigma). The negative controls were performed in parallel. Samples were analyzed using a fluorescence microscope (Nikon, Tokyo, Japan).

Statistical analysis

SPSS 15.0 (SPSS, Chicago, IL, USA) was used for statistical analysis. Data were expressed as mean ± SD. A two-tailed Student’s t-test was used to determine the statistical significance. A value of P < 0.05 was considered statistically significant.

Acknowledgments

We are grateful to Mr. Huaisheng Shi from Central Laboratory, Institute of Medical Biology, Chinese Academy of Medical Science & Peking Union Medical College for assistance with image capture during the experiments.

讨论

文章快阅

延伸阅读

1所投文章国内外研究状况的微观分析

干细胞治疗神经退行性疾病研究的重点在于是否能够找到可替代缺损细胞的细胞源，诱导分化是其中的一个重要研究途径。目前干细胞的诱导分化研究主要集中于在体外通过化学试剂或信号分子如RA，bFGF和BME等刺激干细胞分化，但都存在很多问题。基因敲除实验已经确认一些转录因子如LIM homeobox transcription factor（包括Lmx1a和Lmx1b），Msx1，Nurr1，Pitx3，En等在神经元细胞的分化和成熟过程中的重要作用。研究发现Lmx1a在中枢神经系统胚胎发育过程中对中脑多巴胺神经元的形成过程起到了重要调控作用。

研究者向小鼠的胚胎干细胞中转入Lmx1a基因，在SHH的诱导环境下诱导出多巴胺能神经元细胞。过表达Lmx1a基因可以导致胚胎中脑前体细胞表达3倍的酪氨酸羟化酶（TH）。因此，SHH/Lmx1a/Msx1信号通路在早期DA神经元分化成熟过程中起到非常重要的调控作用。

在本研究中，我们通过腺病毒载体传递Lmx1a，对恒河猴脂肪间充质干细胞（rASCs）进行基因修饰，强制表达Lmx1a，确定干细胞特定分化方向，在可溶性分子SHH和FGF-8引导作用下和神经干细胞协同促分化作用下进一步将rASCs向多巴胺能神经元细胞定向诱导。该研究在国内外还未见报道。

2 .所投文章创新性的分析

用清华同方、中国知网全文数据库检索近三年所载相关文章，查询结果大致如下：

研究方法	研究结果
共培养研究	人脐带间充质干细胞（hUC-MSCs）与胰岛细胞共培养可诱导hUC-MSCs分化为可释放胰岛素的胰岛样细胞。
	hMSCs在共培养的微环境中,可以分化成内皮细胞表型
	分离培养的骨髓间充质干细胞与心肌细胞共培养，Notch信号系统对骨髓间充质干细胞分化为心肌细胞起正向调控作用。
	成骨细胞与BMSC共同培养应用于BMSC的成骨诱导,从诱导效率和诱导后成骨细胞的分化活性方面,都能证实该共同培养方法的有效性。
	转染isl基因的BMSCs不仅可以稳定表达Isli蛋白,而且在微环境共培养条件下,可以提高BMSCs向心肌细胞分化的能力。
共培养诱导神经元分化	MSCs可以促进NSCs分化为神经元,而且在NSCs诱导下部分MSCs可以转化为神经元和星形细胞。
	MSCs与雪旺细胞共培养可以分化MSCs为神经元样细胞。
	hMSCs促进体外培养的NSCs向少突胶质细胞分化,可能与hMSCs分泌可溶性因子相关。

（1） 理论假设创新

SHH/Lmx1a信号通路调控脂肪间充质干细胞（ASCs）协同神经干细胞分泌促神经分化因子可提高ASCs分化为多巴胺能神经元细胞的能力。

（2） 技术方法创新

传统的细胞移植治疗要么只是经过简单的化学诱导剂诱导，分化方向不特异且诱导成功的神经元比例较低，要么是直接移植干细胞于脑内，容易形成畸胎瘤且针对帕金森病没有特异性治疗作用。

本研究首次通过Lmx1a基因转染恒河猴脂肪间充质干细胞并与神经干细胞非接触性共培养来诱导脂肪间充质干细胞分化为多巴胺能神经元样细胞。当经过Lmx1a基因的修饰后，对rASCs分化方向进行调控，最佳时机诱导后，将细胞移植进入帕金森氏病猴模型，治疗更为特异。

（3） 结果创新

通过以上方法诱导后的细胞能够表达神经元特异性标志，与单独和神经干细胞共培养诱导方法相比，能够显著提高分化效率。

（4）其他创新

既往研究表明,理想的替代细胞资源是进行细胞移植治疗的关键。与骨髓间充质干细胞和脐带间充质干细胞相比，rASCs原料丰富，取材方便而且在体外培养操作简单，可以为帕金森病治疗及将来可能的临床实验研究提供充足的细胞源；rASCs利于自体移植，在将来用于临床应用时，免疫原性低，不需经过严格配对使用，这一点在治疗神经系统疾病方面显得尤为重要。

所有种类的动物模型中，在非人灵长类动物模型中所观察到的帕金森氏病疾病特征与在人类所观察到的临床症状最为相似，更接近人类，是更为理想的验证分化细胞的生理功能的动物模型。取自恒河猴自身的脂肪间充质干细胞可以用于自体移植，为将来可能的帕金森氏病的细胞移植临床治疗提供实验依据。

3. 所投文章区别于他人他篇的特点

通过PubMed检索，Barzilay等通过慢病毒携带Lmx1a基因进入骨髓间充质干细胞（MSC），可以将MSC诱导为表达Pitx3，En，TH的神经元细胞，并且Lmx1a基因的过表达并不影响MSC本身的基因表达，如RUNX2。该研究与本研究较为相近，但存在以下不同：

（1）技术方法的不同

本研究主要通过腺病毒将Lmx1a基因携带进入恒河猴脂肪间充质干细胞中，并与神经干细胞共培养，将内源性和外源性诱导因素进行很好的结合。

（2）其他方面的不同

所选用的间充质干细胞不同，本研究选用的脂肪间充质干细胞尤其是恒河猴的脂肪间充质干细胞的研究很少，利于后期动物实验的进行，且获得的资料珍贵。

4 专家意见与答疑

专家意见：本研究结果还需进一步明确.如Fig 5.研究者用NSE,β-tubulin III等标记神经元,但未有参考标记等研究,那么作者又是如何得到NSE阳性细胞和β-tubulin III阳性细胞数;并且结果未在同一视野.

作者答疑：在Fig.5中，NSE,β-tubulin III荧光染色结果为阳性结果较多的视野取的照片，实验过程中同时做DAPI染色，从而计算阳性细胞比例。NSE,β-tubulin III阳性细胞数计算是重复实验三次以上算出的平均值，图片未在结果中显示。由于条件有限，未能做免疫共聚焦，在未来的研究工作中我们会更好地完善实验，增加结果的说服力。