神经细胞损伤微环境诱导人脐带间充质干细胞的神经分化

doi:10.3969/j.issn.1673-5374.2012.34.006

摘要/Abstract

摘要：

为研究人脐带间充质干细胞在损伤神经细胞模型诱导下向神经细胞方向分化的情况，取体外分离培养的第5代人脐带间充质干细胞用于实验。采用Transwell共培养体系，将人脐带间充质干细胞分别与正常或受损的PC12细胞/PC12细胞上清/PC12细胞裂解产物共培养。Western blot法和流式细胞仪检测到各培养组中人脐带间充质干细胞均表达乙酰胆碱转移酶和神经细胞特异性标志物微管相关蛋白2，且以与受损PC12细胞共培养的人脐带间充质干细胞中两者表达最强。单独培养的人脐带间充质干细胞不表达乙酰胆碱转移酶和微管相关蛋白2。同时，CCK-8法检测到共培养条件下的人脐带间充质干细胞对受损PC12细胞增殖有促进作用。说明神经组织损伤微环境可有效诱导人脐带间充质干细胞向神经样细胞分化，且诱导分化的人脐带间充质干细胞可促进受损神经细胞的修复。

关键词: 神经再生, 干细胞, 脐带间充质干细胞, 共培养, 诱导, 分化, 神经细胞, 微管相关蛋白2, 受损细胞, Transwell, 再生

Abstract:

This study aimed to investigate the neural differentiation of human umbilical cord mesenchymal stem cells (hUCMSCs) under the induction of injured neural cells. After in vitro isolation and culture, passage 5 hUCMSCs were used for experimentation. hUCMSCs were co-cultured with normal or Aβ_1-40-injured PC12 cells, PC12 cell supernatant or PC12 cell lysate in a Transwell co-culture system. Western blot analysis and flow cytometry results showed that choline acetyltransferase and microtubule-associated protein 2, a specific marker for neural cells, were expressed in hUCMSCs under various culture conditions, and highest expression was observed in the hUCMSCs co-cultured with injured PC12 cells. Choline acetyltransferase and microtubule-associated protein 2 were not expressed in hUCMSCs cultured alone (no treatment). Cell Counting Kit-8 assay results showed that hUCMSCs under co-culture conditions promoted the proliferation of injured PC12 cells. These findings suggest that the microenvironment during neural tissue injury can effectively induce neural cell differentiation of hUCMSCs. These differentiated hUCMSCs likely accelerate the repair of injured neural cells.

Key words: stem cell, umbilical cord mesenchymal stem cell, co-culture, induction, differentiation, neural cell, microtubule-associated protein 2, injured cell, Transwell, neural regeneration, regeneration

. 神经细胞损伤微环境诱导人脐带间充质干细胞的神经分化[J]. 中国神经再生研究(英文版), 2012, 7(34): 2689-2697.

Jin Zhou, Guoping Tian, Jinge Wang, Xiaoguang Luo, Siyang Zhang, Jianping Li, Li Li, Bing Xu, Feng Zhu, Xia Wang, Chunhong Jia, Weijin Zhao, Danyang Zhao, Aihua Xu. Neural cell injury microenvironment induces neural differentiation of human umbilical cord mesenchymal stem cells[J]. Neural Regeneration Research, 2012, 7(34): 2689-2697.

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引言

材料方法

Design

A controlled, observational, in vitro study.

Time and setting

This study was performed at the Experimental Center, China Medical University, between January 2009 and December 2010.

Materials

Umbilical cord tissue from healthy normal full-term delivery babies was provided by the First People’s Hospital of Shenyang, China. According to the Administrative Regulations on Medical Institution issued by the State Council of the People’s Republic of China, mothers or their relatives provided informed consent^[57].

Methods

hUCMSC preparation

Under sterile conditions, umbilical cord tissue was harvested. Following thorough washes with Hanks' balanced salt solution, umbilical vein, umbilical artery and umbilical cord adventitia were removed and chopped into 1 mm × 1 mm × 1 mm pieces. Then, these tissue blocks were transferred into a mixture of collagenase II (1 g/L; Nanjing KeyGen Biotechnology Co., Ltd., Nanjing, China) and DMEM for digestion. Digestion solution containing cells was diluted in 30 volumes of DMEM and centrifuged at 1 500 r/min for 10 minutes, and the supernatant was discarded. Cells were thoroughly resuspended with DMEM/F12 culture medium (Invitrogen, Carlsbad, CA, USA) containing 15% fetal bovine serum, plated into a culture flask at a density of 1 × 10⁶/mL and cultured at 37°C in an incubator containing 5% CO₂. On day 3, after cell adherence, half of the culture medium was refreshed, non-adherent cells were discarded, and culture medium was changed once every 3 days thereafter. When cells reached 90% confluency, they were digested with 0.25% trypsin and 0.02% ethylenediaminetetraacetic acid and passaged at 1:3. Passage 5 cells were used for co-culture experiments.

Analysis of hUCMSC immunophenotype by flow cytometry

Passage 5 hUCMSCs were digested with 0.25% trypsin and 0.02% ethylenediaminetetraacetic acid and then prepared into a single-cell suspension at a density of 1 × 10⁶/mL. After addition of phycoerythrin-labeled mouse anti-human CD34, CD44, CD29, CD45 or CD56 monoclonal antibodies (BD Bioscience Pharmingen Inc., San Diego, CA, USA), the cell suspension was incubated at room temperature for 30 minutes and then centrifuged for 10 minutes at 1 000 r/min. After one PBS wash, the cell suspension was centrifuged at 1 000 r/min for 10 minutes. After cell resuspension with PBS, cell immunophenotype was analyzed using a FACScalibur flow cytometer (Becton Dickinson, San Jose, CA, USA). This experiment was performed in triplicate. The proportion of MAP2-positive cells within the total cell count was calculated using CellQuest software (Becton Dickinson).

PC12 cell culture and passage

PC12 cells (Cell Research Institute, Chinese Academy of Sciences) were used as a substitute for neural cells and were grown in PC12 cell culture medium (containing 10% horse serum and 5% fetal bovine serum 1640). PC12 cells grew as a suspension. After 7–8 passages, cells adhered to the flask and were used for experimentation. PC12 cells were resuspended in PC12 cell culture medium, and a 300 μL aliquot of 1 × 10⁷ cells/mL was transferred into the wells of a 24-well plate^[58-59].

Experimental procedures and grouping

Preparation of injured PC12 cell supernatant: PC12 cells were incubated with 10 μg/mL Aβ_1-40 (Sigma, St. Louis, MO, USA) overnight^[60-63]. After removal of the supernatant, cells were cultured with 1640 culture medium for 24 hours, and then the supernatant was collected as injured PC12 cell-conditioned culture medium. Simultaneously, normal PC12 cell-conditioned culture medium was collected using the same method.

Preparation of injured PC12 cell lysate: Under sterile conditions, PC12 cells were collected during preparation of injured PC12 cell supernatant, pre-cooled with PBS, centrifuged, and washed with sterile PBS three times. After adding a sufficient amount of sterile PBS, cell clumps were dispersed to prepare a single-cell suspension. Cells at a density of 1 × 10⁴ cells/mL were sonicated in an ice bath (350 W; working time: 2 seconds; interval: 10 seconds; 40 times). After decontamination, cells were stored at –80 °C for later use.

Preparation of transwells: 16 wells were selected from the 24-well plate and filled with 400 μL DMEM. hUCMSCs at a density of 1 × 10⁴ cells/well were cultured in the transwells (aperture: 0.4 μm; Corning, Steuben County, NY, USA)^[64].

Preparation of PC12 cells: PC12 cells were digested and plated in the 24-well plate at a density of 1 × 10⁴ cells/well.

hUCMSCs co-cultured with normal or Aβ_1-40-injured PC12 cells, PC12 cell supernatant or PC12 cell lysate: Normal or injured PC12 cells were incubated in the Transwell upper chamber for 24 hours and then transferred into the lower chamber for another 24 hours of culture.

The seven groups used in this study were as follows:

group A: normal PC12 cell supernatant + hUCMSCs; group B: injured PC12 cell supernatant + hUCMSCs; group C: normal PC12 cell lysate + hUCMSCs; group D: injured PC12 cell lysate + hUCMSCs; group E: normal PC12 cells + hUCMSCs; group F: injured PC12 cells + hUCMSCs; group G: hUCMSCs cultured alone.

Western blot analysis

hUCMSCs were washed twice with ice-cold PBS and lysed on ice. The lysing solution was composed of 20 mM Tris-HCl, 1 mM ethylenediaminetetraacetic acid, 50 mM NaCl, 50 mM NaF, 1 mM Na₃VO₄, 1% Triton X-100 and 1 mM phenylmethyl sulfonylfluoride. At 4°C, hUCMSCs were centrifuged at 22 000 × g for 30 minutes. Thesupernatantwas collected for determination of protein concentration using the Coomassie brilliant blue protein assay^[46]. 50 μg of total protein was separated on 12% SDS-PAGE gels and then transferred onto a PVDF membrane. The membrane was blocked with 5% defatted milk at room temperature for 2 hours, incubated with mouse anti-human ChAT and β-actin monoclonal antibodies (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight, and then with goat anti-mouse IgG secondary antibody (1:200; Beijing Zhongshan) at 37°C for 2 hours. Bands were revealed with an enhanced chemiluminescence agent (KeyGen Biotechnology Co., Ltd.). This experiment was performed in triplicate. The absorbance of each protein band was determined using Image J software (National Institutes of Health, Rockville, MD, USA). The absorbance ratio of target protein to β-actin was calculated as the relative expression of target protein.

Expression of neural cell-specific protein MAP2 in hUCMSCs assessed with flow cytometry

hUCMSCs were digested with 0.25% trypsin and 0.02% ethylenediaminetetraacetic acid, and then prepared into a single-cell suspension at a density of 1 × 10⁶/mL. After addition of Cy5-labeled rabbit anti-MAP2 polyclonal antibody (BioLegend, San Diego, CA, USA), hUCMSCs were incubated for 1 hour at room temperature in the dark, centrifuged at 1 000 r/min for 10 minutes, washed three times with PBS, and centrifuged again at 1 000 r/min for 10 minutes. Finally, cells were resuspended, and MAP2 expression in hUCMSCs was detected using a FACScalibur flow cytometer (Becton Dickinson). This experiment was performed in triplicate. The proportion of MAP2-positive cells within the total cell count was calculated using CellQuest software (Becton Dickinson).

Effects of induced hUCMSCs on the proliferation of injured PC12 cells

The proliferation of injured PC12 cells was determined using 24-well plates. The initial cell density was adjusted to 2.5 × 10³cells/well for hUCMSCs and 1 × 10⁵ cells/well for Aβ_1-40-injured PC12 cells. Cell proliferation after 48 hours in culture (5% CO₂, 37 °C) was determined using the Cell Counting Kit-8 assay (Dojindo, Japan). Briefly, 10 μL of Cell Counting Kit-8 reagent was added to each well. After incubation at 37°C for 2 hours, the absorbance of each well was measured at 450 nm with an ELISA reader (Thermo Fisher Scientific, Boston, MA, USA). This experiment was performed in triplicate.

Statistical analysis

All measurement data were statistically processed using SPSS 13.0 software (SPSS, Chicago, IL, USA). One-way analysis of variance was used for comparison between groups. Results were expressed as mean ± SD. A level of P < 0.05 was considered statistically significant.

Author contributions: Jin Zhou and Guoping Tian designed the experiments, analyzed the data and wrote the manuscript. Guoping Tian, Jinge Wang, and Xiaoguang Luo checked and revised the manuscript. Siyang Zhang, Jianping Li, Li Li, Bing Xu, Feng Zhu, Xia Wang, Chunhong Jia, Weijin Zhao, Danyang Zhao, Aihua Xu performed the experiments. All authors approved the final version of the paper.

Conflicts of interest:None declared.

Ethical approval: Animal-related procedures were reviewed and approved by the Animal Care and Use Committee of China Medical University.

Author statements: The manuscript is original, has not been submitted to or 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.

讨论

文章快阅

延伸阅读

实验设计及文章构思：

体外分离培养人脐带来源的间充质干细胞（MSC），取第5代细胞进行实验。观察细胞形态，流式细胞术分析细胞表面标志物的表达。在不同实验组诱导下的hUCMSCs细胞中，采用western blot方法检测ChAT表达水平的变化，流式细胞术分析神经细胞特异性标志物MAP2阳性细胞百分比的变化，MTT方法检测诱导分化后的hUCMSCs对受损细胞增殖的影响。本文拟通过形态学特征和细胞功能两方面验证共培养方法体外诱导hUCMSCs向神经细胞分化的能力。

课题设计中区别于以往相关研究的特点：

研究通过共培养的方法发现与受损细胞共培养的方法可以最大程度的诱导hUCMSCs定向分化，提示神经组织损伤微环境可以有效诱导hUCMSCs定向分化。同时我们又通过研究发现诱导分化的hUCMSCs可以促进受损细胞的增殖，提示受损细胞和hUCMSCs可能存在着相互作用，共同完成组织损伤修复过程。本研究还将ChAT的表达量用于评价诱导分化的hUCMSCs是否具有神经细胞的功能，发现hUCMSCs在各实验组诱导下，ChAT表达增加，而共培养组ChAT的表达水平最高，这表明与受损细胞共培养诱导hUCMSCs向神经细胞分化的效果较好。我们的结果提示hUCMSCs在诱导分化过程中，不仅具有神经细胞形态学特征，并在一定程度上表现出神经细胞的功能。

与国内外同类研究的比较：

	主要观察指标	结论
Gene Therapy. 2011, 18(4): 394-402.	VEGF、NSE、GFAP、nestin、TH	转染VEGF基因的hUCMSCs分化成为多巴胺神经元样细胞，表达NSE、GFAP、nestin、TH，改善PD动物模型的症状并减少多巴胺神经元的丢失。
Stem Cells. 2006, 24(1):115-24.	Shh、FGF8、TH	神经细胞条件培养基、Shh和FGF8共同诱导hUCMSCs向神经细胞分化，表达TH和儿茶酚胺合成酶，分泌多巴胺，缓解大鼠的PD症状。
J Neurosci Res. 2008, 86(9): 1902-15.	Nestin、MAP2、GFAP	与大鼠神经细胞共培养诱导hUCMSCs呈星形胶质细胞样表型，并表达GFAP。
J Neurosci Res. 2008, 86(10):2168-78.	Raf-1、ERK、BDNF、p35、Akt、Bcl2、	BDNF诱导hUCMSCs向神经细胞分化，在分化过程中上调Raf-1和ERK磷酸化以及p35表达，并通过Akt磷酸化和Bcl2表达促进细胞存活。
解剖科学进展. 2012, 18(3):278-281.	VEGF、血管新生	将体外培养的hUCMSCs与VEGF联合植入脑缺血模型大鼠脑内，促进缺血区血管新生和神经功能的恢复。
本文	CD29、CD45、CD34、CD44、CD56、MAP2、ChAT	hUCMSCs在与损伤神经细胞模型共培养后开始表达神经细胞特异性标记MAP2，出现向神经细胞分化的趋势，并表达ChAT，提示可能具有神经细胞的功能

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检索CNKI、PubMed数据库，检索时间不限，检索关键词设定为Umbilical cord mesenchymal stem cells；neuron；differentiation；MAP2；ChAT；分化；神经细胞；基质干细胞；共培养等。检索结果表明，国内外此类神经组织损伤再生修复的研究多集中于利用细胞因子刺激和转染方法观察充质干细胞在形态学和功能上是否有向神经细胞分化的趋势。我们分析认为在脐带间充质干细胞向神经细胞分化过程中，损伤神经细胞的微环境对干细胞分化可能也是有重要作用的，因此在此基础上我们建立了共培养模型，研究在损伤神经细胞诱导下脐带间充质干细胞向神经细胞分化过程中形态和功能的变化，及其对受损细胞增殖的影响，研究它们的相互作用可能更接近于体内情况，将为以后体内应用脐带间充质干细胞治疗神经系统变性疾病提供实验基础。