脐带间充质干细胞向神经元样细胞诱导后仍保持免疫调节和抗氧化能力

doi:10.3969/j.issn.1673-5374.2012.34.003

中国神经再生研究(英文版) ›› 2012, Vol. 7 ›› Issue (34): 2663-2672.doi: 10.3969/j.issn.1673-5374.2012.34.003

• 原著:神经损伤修复保护与再生 • 上一篇下一篇

脐带间充质干细胞向神经元样细胞诱导后仍保持免疫调节和抗氧化能力

收稿日期:2012-09-29 修回日期:2012-11-16 出版日期:2012-12-05 发布日期:2012-11-16
作者简介:李建军★，硕士，副主任医师，麻醉科，济南250012，山东大学齐鲁医院，山东省，China250012
基金资助:
山东省自然科学基金项目2011GSF11801; 山东省创新基金项目2012ZD023; 国家重大基础研究发展项目2012CB966504.

Umbilical cord-derived mesenchymal stem cells retain immunomodulatory and anti-oxidative activities after neural induction

Jianjun Li¹, Dong Li², Xiuli Ju², Qing Shi², Dakun Wang², Fengcai Wei³

Department of Anesthesiology, Qilu Hospital, Shandong University, Jinan 250012, Shandong Province, China

Received:2012-09-29 Revised:2012-11-16 Online:2012-12-05 Published:2012-11-16
Contact: Dong Li, M.D., Associate professor, Cryomedicine Laboratory, Qilu Hospital, Shandong University, Jinan 250012, Shandong Province, China lidong73@sdu.edu.cn
About author:Jianjun Li★, Master, Associate chief physician, Department of Anesthesiology, Qilu Hospital, Shandong University, Jinan 250012, Shandong Province, China
Supported by:
This work was supported by grants from the Shandong Province Science and Technology Program, Grant No. 2011GSF11801; and the Innovation Fund Project of Shandong University, Grant No. 2012ZD023; the Major State Basic Research Development Program, Grant No. 2012CB966504.

摘要/Abstract

摘要：

间充质干细胞若在诱导分化后仍保持免疫调节和抗氧化能力将有助提高细胞替代疗法的治疗效果。实验中从人脐带分离间充质干细胞，经碱性成纤维细胞生长因子、神经生长因子、表皮生长因子、脑源性神经营养因子和forskolin诱导后，分化呈圆形，出现较长突起，表达神经元标记物Tuj1，神经丝蛋白200、微管相关蛋白2和神经元特异性烯醇化酶的神经元样细胞。神经诱导后Nestin表达显著降低，但诱导前后免疫调节和抗氧化相关基因的表达无明显变化。间充质干细胞和诱导细胞的混合淋巴细胞的培养结果也没有显著差异，但诱导的神经元样细胞中人类白细胞抗原G的表达显著下降。提示以生长因子方法诱导脐带间充质干细胞向未成熟神经元样细胞分化后仍能够保持细胞免疫调节特性和抗氧化能力。

关键词: 脐带, 间充质干细胞, 免疫调节, 氧化应激, 抗氧化, 细胞分化, 神经诱导, 神经生长因子, 混合淋巴细胞培养, 干细胞, 再生, 神经再生

Abstract:

The immunomodulatory and anti-oxidative activities of differentiated mesenchymal stem cells contribute to their therapeutic efficacy in cell-replacement therapy. Mesenchymal stem cells were isolated from human umbilical cord and induced to differentiate with basic fibroblast growth factor, nerve growth factor, epidermal growth factor, brain-derived neurotrophic factor and forskolin. The mesenchymal stem cells became rounded with long processes and expressed the neural markers, Tuj1, neurofilament 200, microtubule-associated protein-2 and neuron-specific enolase. Nestin expression was significantly reduced after neural induction. The expression of immunoregulatory and anti-oxidative genes was largely unchanged prior to and after neural induction in mesenchymal stem cells. There was no significant difference in the effects of control and induced mesenchymal stem cells on lymphocyte proliferation in co-culture experiments. However, the expression of human leukocyte antigen-G decreased significantly in induced neuron-like cells. These results suggest that growth factor-based methods enable the differentiation of mesenchymal stem cell toward immature neuronal-like cells, which retain their immunomodulatory and anti-oxidative activities.

Key words: umbilical cord, mesenchymal stem cell, immunomodulation, oxidative stress, neural induction, neural regeneration

. 脐带间充质干细胞向神经元样细胞诱导后仍保持免疫调节和抗氧化能力[J]. 中国神经再生研究(英文版), 2012, 7(34): 2663-2672.

Jianjun Li, Dong Li, Xiuli Ju, Qing Shi, Dakun Wang, Fengcai Wei. Umbilical cord-derived mesenchymal stem cells retain immunomodulatory and anti-oxidative activities after neural induction[J]. Neural Regeneration Research, 2012, 7(34): 2663-2672.

参考文献

[1] Williams AR, Hare JM. Mesenchymal stem cells: biology, pathophysiology, translational findings, and therapeutic implications for cardiac disease. Circ Res. 2011;109(8): 923-940.

[2] Musumeci G, Lo Furno D, Loreto C, et al. Mesenchymal stem cells from adipose tissue which have been differentiated into chondrocytes in three-dimensional culture express lubricin. Exp Biol Med (Maywood). 2011; 236(11):1333-1341.

[3] Matsushita K, Morello F, Wu Y, et al. Mesenchymal stem cells differentiate into renin-producing juxtaglomerular (JG)-like cells under the control of liver X receptor-alpha. J Biol Chem. 2010;285(16):11974-11982.

[4] Johnson TV, Bull ND, Hunt DP, et al. Neuroprotective effects of intravitreal mesenchymal stem cell transplantation in experimental glaucoma. Invest Ophthalmol Vis Sci. 2010;51(4):2051-2059.

[5] Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284(5411):143-147.

[6] Sanchez-Ramos JR. Neural cells derived from adult bone marrow and umbilical cord blood. J Neurosci Res. 2002; 69(6):880-893.

[7] Sanchez-Ramos J, Song S, Cardozo-Pelaez F, et al. Adult bone marrow stromal cells differentiate into neural cells in vitro. Exp Neurol. 2000;164(2):247-256.

[8] Woodbury D, Schwarz EJ, Prockop DJ, et al. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res. 2000;61(4):364-370.

[9] Chen J, Li Y, Wang L, et al. Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats. Stroke. 2001;32(4): 1005-1011.

[10] Kurozumi K, Nakamura K, Tamiya T, et al. Mesenchymal stem cells that produce neurotrophic factors reduce ischemic damage in the rat middle cerebral artery occlusion model. Mol Ther. 2005;11(1):96-104.

[11] Mahmood A, Lu D, Wang L, et al. Treatment of traumatic brain injury in female rats with intravenous administration of bone marrow stromal cells. Neurosurgery. 2001;49(5): 1196-1204.

[12] Alexanian AR, Fehlings MG, Zhang Z, et al. Transplanted neurally modified bone marrow-derived mesenchymal stem cells promote tissue protection and locomotor recovery in spinal cord injured rats. Neurorehabil Neural Repair. 2011;25(9):873-880.

[13] Nauta AJ, Fibbe WE. Immunomodulatory properties of mesenchymal stromal cells. Blood. 2007;110(10): 3499-3506

[14] Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood. 2005;105(4):1815-1822.

[15] Atoui R, Shum-Tim D, Chiu RC. Myocardial regenerative therapy: immunologic basis for the potential "universal donor cells". Ann Thorac Surg. 2008;86(1):327-334.

[16] Chen YT, Sun CK, Lin YC, et al. Adipose-derived mesenchymal stem cell protects kidneys against ischemia-reperfusion injury through suppressing oxidative stress and inflammatory reaction. J Transl Med. 2011;9:51.

[17] Sun DC, Li DH, Ji HC, et al. In vitro culture and characterization of alveolar bone osteoblasts isolated from type 2 diabetics. Braz J Med Biol Res. 2012;45(6): 502-509.

[18] Park HE, Kim D, Koh HS, et al. Real-time monitoring of neural differentiation of human mesenchymal stem cells by electric cell-substrate impedance sensing. J Biomed Biotechnol. 2011;2011:485173.

[19] Kubulus D, Roesken F, Amon M, et al. Mechanism of the delay phenomenon: tissue protection is mediated by heme oxygenase-1. Am J Physiol Heart Circ Physiol. 2004;287(5):H2332-2340.

[20] Brazelton TR, Rossi FM, Keshet GI, et al. From marrow to brain: expression of neuronal phenotypes in adult mice. Science. 2000;290(5497):1775-1779.

[21] Nakano K, Migita M, Mochizuki H, et al. Differentiation of transplanted bone marrow cells in the adult mouse brain. Transplantation. 2001;71(12):1735-1740.

[22] Kohyama J, Abe H, Shimazaki T, et al. Brain from bone: efficient "meta-differentiation" of marrow stroma-derived mature osteoblasts to neurons with Noggin or a demethylating agent. Differentiation. 2001;68(4-5): 235-244.

[23] Alexanian AR, Maiman DJ, Kurpad SN, et al. In vitro and in vivo characterization of neurally modified mesenchymal stem cells induced by epigenetic modifiers and neural stem cell environment. Stem Cells Dev. 2008;17(6):1123-1130.

[24] Muñoz-Elías G, Woodbury D, Black IB. Marrow stromal cells, mitosis, and neuronal differentiation: stem cell and precursor functions. Stem Cells. 2003;21(4):437-448.

[25] Woodbury D, Reynolds K, Black IB. Adult bone marrow stromal stem cells express germline, ectodermal, endodermal, and mesodermal genes prior to neurogenesis. J Neurosci Res. 2002;69(6):908-917.

[26] Barnabé GF, Schwindt TT, Calcagnotto ME, et al. Chemically-induced RAT mesenchymal stem cells adopt molecular properties of neuronal-like cells but do not have basic neuronal functional properties. PLoS One. 2009; 4(4):e5222.

[27] Peng J, Wang Y, Zhang L, et al. Human umbilical cord Wharton's jelly-derived mesenchymal stem cells differentiate into a Schwann-cell phenotype and promote neurite outgrowth in vitro. Brain Res Bull. 2011;84(3): 235-243.

[28] Ladak A, Olson J, Tredget EE, et al. Differentiation of mesenchymal stem cells to support peripheral nerve regeneration in a rat model. Exp Neurol. 2011;228(2): 242-252.

[29] Khoo ML, Tao H, Meedeniya AC, et al. Transplantation of neuronal-primed human bone marrow mesenchymal stem cells in hemiparkinsonian rodents. PLoS One. 2011;6(5): e19025.

[30] Pacary E, Legros H, Valable S, et al. Synergistic effects of CoCl(2) and ROCK inhibition on mesenchymal stem cell differentiation into neuron-like cells. J Cell Sci. 2006; 119(Pt 13):2667-2678.

[31] Padovan CS, Jahn K, Birnbaum T, et al. Expression of neuronal markers in differentiated marrow stromal cells and CD133+ stem-like cells. Cell Transplant. 2003;12(8): 839-848.

[32] Tondreau T, Lagneaux L, Dejeneffe M, et al. Bone marrow-derived mesenchymal stem cells already express specific neural proteins before any differentiation. Differentiation. 2004;72(7):319-326.

[33] Salim A, Nacamuli RP, Morgan EF, et al. Transient changes in oxygen tension inhibit osteogenic differentiation and Runx2 expression in osteoblasts. J Biol Chem. 2004;279(38):40007-40016.

[34] Dehmelt L, Smart FM, Ozer RS, et al. The role of microtubule-associated protein 2c in the reorganization of microtubules and lamellipodia during neurite initiation. J Neurosci. 2003;23(29):9479-9490.

[35] Bunnell BA, Betancourt AM, Sullivan DE. New concepts on the immune modulation mediated by mesenchymal stem cells. Stem Cell Res Ther. 2010;1(5):34.

[36] Zhang Q, Shi S, Liu Y, et al. Mesenchymal stem cells derived from human gingiva are capable of immunomodulatory functions and ameliorate inflammation-related tissue destruction in experimental colitis. J Immunol. 2009;183(12):7787-7798.

[37] Spaggiari GM, Capobianco A, Abdelrazik H, et al. Mesenchymal stem cells inhibit natural killer-cell proliferation, cytotoxicity, and cytokine production: role of indoleamine 2,3-dioxygenase and prostaglandin E2. Blood. 2008;111(3):1327-1333.

[38] DelaRosa O, Lombardo E, Beraza A, et al. Requirement of IFN-gamma-mediated indoleamine 2,3-dioxygenase expression in the modulation of lymphocyte proliferation by human adipose-derived stem cells. Tissue Eng Part A. 2009;15(10):2795-2806.

[39] Wang M, Yang Y, Yang D, et al. The immunomodulatory activity of human umbilical cord blood-derived mesenchymal stem cells in vitro. Immunology. 2009; 126(2):220-232.

[40] Ding Y, Xu D, Feng G, et al. Mesenchymal stem cells prevent the rejection of fully allogenic islet grafts by the immunosuppressive activity of matrix metalloproteinase-2 and -9. Diabetes. 2009;58(8):1797-1806.

[41] Chabannes D, Hill M, Merieau E, et al. A role for heme oxygenase-1 in the immunosuppressive effect of adult rat and human mesenchymal stem cells. Blood. 2007;110(10): 3691-3694.

[42] Schäfer S, Calas AG, Vergouts M, et al. Immunomodulatory influence of bone marrow-derived mesenchymal stem cells on neuroinflammation in astrocyte cultures. J Neuroimmunol. 2012;249(1-2):40-48.

[43] Hunt JS, Petroff MG, McIntire RH, et al. HLA-G and immune tolerance in pregnancy. FASEB J. 2005;19(7): 681-693.

[44] Lu LL, Liu YJ, Yang SG, et al. Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesis-supportive function and other potentials. Haematologica. 2006;91(8):1017-1026.

[45] Lee OK, Kuo TK, Chen WM, et al. Isolation of multipotent mesenchymal stem cells from umbilical cord blood. Blood. 2004;103(5):1669-1675.

[46] Elkabetz Y, Panagiotakos G, Al Shamy G, et al. Human ES cell-derived neural rosettes reveal a functionally distinct early neural stem cell stage. Genes Dev. 2008;22(2): 152-165.

[47] Barber RD, Jaworsky DE, Yau KW, et al. Isolation and in vitro differentiation of conditionally immortalized murine olfactory receptor neurons. J Neurosci. 2000;20(10): 3695-3704.

[48] Ohori Y, Yamamoto S, Nagao M, et al. Growth factor treatment and genetic manipulation stimulate neurogenesis and oligodendrogenesis by endogenous neural progenitors in the injured adult spinal cord. J Neurosci. 2006;26(46):11948-11960.

[49] Kuçi S, Kuçi Z, Kreyenberg H, et al. CD271 antigen defines a subset of multipotent stromal cells with immunosuppressive and lymphohematopoietic engraftment-promoting properties. Haematologica. 2010;95(4):651-659.

引言

材料方法

Design

A comparative observation at the cellular and molecular levels.

Time and setting

The experiments were performed at the Cryomedicine Laboratory of Qilu Hospital, Shandong University, China from October 2011 to May 2012.

Methods
Isolation and culture of MSCs

Umbilical cords (n = 10; clinically normal pregnancies) were obtained from Qilu Hospital of Shandong University after normal full-term deliveries. Informed consent was obtained from the mothers. Tissue collection for research was approved by the Ethics Committee of Qilu Hospital. Umbilical cords were excised and washed in 0.1 M PBS (pH 7.4) to remove excess blood. The cords were dissected and blood vessels removed. The remaining tissue was cut into small pieces (1–2 cm²) and placed in plates containing low-glucose Dulbecco’s modified Eagle’s medium (Gibco-BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco-BRL), 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco-BRL). Cultures were maintained at 37°C in a humidified atmosphere with 5% CO₂. The medium was changed every 3–4 days. After 7 to 12 days in culture, adherent cells were observed growing out from the individual tissue explants. The adherent fibroblast-like cells became confluent after 2–3 weeks in culture. They were then trypsinized using 0.25% trypsin (Gibco-BRL) and passaged at 1 × 10⁴cells/cm² in the medium described above. After three or more passages, the cells were analyzed^[44].

Cell surface antigen phenotyping by flow cytometry

Passages 15–17 cells were collected and treated with 0.25% trypsin. The cells were individually stained with fluorescein isothiocyanate or phycoerythrin-conjugated anti-marker monoclonal antibodies in 100 µL PBS for 15 minutes at room temperature or for 30 minutes at 4°C, as recommended by the manufacturer. The antibodies used were specific for the human antigens, CD29, CD34, CD44, CD45, CD73, CD90, CD105 and CD106 (10 µL for 1 × 10⁶ cells; SeroTec, Raleigh, NC, USA). Cells were analyzed on a flow cytometry system (Cytometer 1.0, Cytomics^TMFC500, Beckman Coulter, Brea, CA, USA). Positive cells were counted and the signals for the corresponding immunoglobulin isotypes were compared^[45].

Determination of the differentiation capacities of the cultured cells

To investigate the differentiation potential of the fibroblast-like cells, passage 4 cells were cultured under conditions appropriate for inducing their differentiation into each lineage. The cell density was 2 × 10⁴ cells per cm², and the medium was replaced every 3–4 days for the differentiation assays. The osteogenic differentiation medium consisted of low-glucose Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 0.1 µM dexamethasone, 50 mM β-glycerol phosphate and 0.2 mM ascorbic acid (Sigma, St. Louis, MO, USA). The adipogenic differentiation medium consisted of high-glucose Dulbecco’s modified Eagle’s medium supplemented with 0.25 mM 3-isobutyl-1-methylxanthine, 0.1 µM dexamethasone, 0.1 mM indomethacin (Sigma), 6.25 μg/mL insulin (Peprotech EC Ltd., London, UK) and 10% fetal bovine serum (Gibco-BRL). Cells kept in the regular growth medium served as controls. For evaluation of mineralized matrix formation, cells were fixed with 4% formaldehyde and stained with 1% alizarin red S (Sigma) solution in water for 10 minutes. Positive staining was indicated by brown staining of the cells. For alkaline phosphatase staining, cells were incubated with 300– 400 μL (enough to coat the well) nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate chromogen solution (Jiancheng Bioengineering, Nanjing, China) for 8–10 minutes at room temperature. Positive staining was indicated by distinct purple staining of the cell surface. For oil red O staining, cells were fixed with 4% formaldehyde, stained with oil red O(Sigma) for 10 minutes, and then counterstained with Mayer hematoxylin (Sigma) for 1 minute. The formation of neutral lipid vacuoles was visualized by red staining^[45].

Neuronal differentiation of MSCs

To initiate neural differentiation, subconfluent MSCs at passages 4–7 were treated with neural induction medium consisting of Dulbecco’s modified Eagle’s medium containing 5% fetal bovine serum, 2% B27 (Invitrogen, Carlsbad, CA, USA), 5 μM forskolin (Sigma), 125 µM 3-isobutyl-1-methylxanthine (Sigma), 10 µM β-mercaptoethanol and the following cytokines: 10 ng/mL basic fibroblast growth factor, 50 ng/mL nerve growth factor, 10 ng/mL epidermal growth factor, 10 ng/mL brain-derived neurotrophic factor, and 10 ng/mL fibroblast growth factor-2 (all from Peprotec, Rocky Hill, NJ, USA). The cells were incubated for 7 days before fixation or harvesting^[46-48].

Immunocytochemical analysis of the expression of neuron-specific markers after neural induction

Immunocytochemical staining was performed in cells grown on glass coverslips. After washing in PBS, cells were fixed with 4% paraformaldehyde (pH 7.4) for 10 minutes and rinsed again with PBS. The cells were permeabilized with 0.1% Tween-PBS for 5 minutes and treated with 3% bovine serum albumin/0.1% Tween/PBS for 30 minutes. Thereafter, they were incubated overnight at 4°C with a primary monoclonal antibody (mouse anti-human nestin, Abcam, 1:250; rabbit anti-human Tuj1, Abcam, 1:200; mouse anti-human neurofilament 200, Sigma, 1:50; rabbit anti-human microtubule-associated protein 2, Sigma, 1:250; rabbit anti-human neuron-specific enolase, Abcam, 1:500) in 1% bovine serum albumin/PBS. Cells were then incubated with goat anti-mouse or mouse anti-rabbit secondary antibodies (1:2 000; Zhongshan Golden Bridge, Beijing, China) at room temperature for 1.5 hours. The 3,3’-diaminobenzidine peroxidase substrate system (Sigma) was used. Images were captured with an Olympus MagnaFire S99800 digital camera mounted on an IX71 Olympus inverted microscope (Olympus, Tokyo, Japan).

Peripheral blood mononuclear cell proliferation assay after neural induction

Neurally induced MSCs and the untreated control MSCs must adapt to co-culture medium (RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin) by graded reduction of the proportion of Dulbecco’s modified Eagle’s medium. Subsequently, MSCs were plated in triplicate in 96-well plates in 100 μL of co-culture medium at 100% confluence. Allogeneic peripheral blood mononuclear cells were isolated from peripheral blood by Ficoll/Hypaque gradient centrifugation, resuspended at 4 × 10⁵/mL, and added to wells (4 × 10⁴ cells/well in 100 μL of medium) containing or lacking MSCs in the presence of 10 μg/mL phytohemagglutinin (Sigma). After 48 hours, 100 μL of cells from each well were transferred to new 96-well plates containing 10 μL of Cell Counting Kit-8 reagent (Dojindo, Kumamoto, Japan). The absorbance at 450 nm was measured with a model 450 microplate reader (Bio- Rad Labs, Richmond, CA, USA). All experiments were performed in triplicate and were repeated at least twice^[49].

Real-time quantitative reverse transcription-PCR analysis of immunomodulatory gene mRNA expression after neural induction

Total RNA was purified from each sample of UC-MSCs using Trizol (Invitrogen) as per the manufacturer’s protocol. Measures were taken to guarantee that equal amounts of RNA from each subject were used. The expression levels of immunomodulatory genes were analyzed by quantitative reverse transcription-PCR. Total RNA was reverse transcribed to cDNA with the Omniscript cDNA Synthesis Kit (Qiagen, Hamburg, Germany) according to the manufacturer’s instructions. Briefly, approximately 0.2 µg of total RNA was reverse transcribed in a 20 µL reaction mixture containing the following components: 1 × RT buffer, deoxynucleotide triphosphate mix (5 mM each), RNase inhibitor (10 U/µL RNase Out, Invitrogen), and 4 U Omniscript RT. The samples were incubated at 37°C for 60 minutes. The resulting cDNA was stored at –80°C prior to real-timePCR.

All reagents and primers were obtained from Bioasi Co., Ltd., Shanghai, China. Beta-actin was validated as an internal control. The expression of each gene relative to β-actin was determined using the 2^-△CT method, where ^△CT = (CT_{target gene}– CT_β-actin). Real-time PCR conditions were as follows: 95°C for 4 minutes, 94°C for 15 seconds, and 60°C for 1 minute, for a total of 40 cycles. Quantitative RT-PCR was performed using an ABI 7500 PCR system (Applied Biosystems, Foster City, CA, USA) and the SYBR green I dye (Toyobo, Osaka, Japan). Successful amplification was defined by the presence of a single dissociation peak on the thermal melting curve. Data were analyzed with Sequence Detection Software 1.4 (Applied Biosystems). Results are given as the fold change relative to untreated control group cDNA, while the control group values are set at fold change = 1. Reported data are representative of at least three independent experiments. The primers used are listed below.

Western blot analysis of heme-oxygenase-1 expression in MSCs after neural induction

For heme-oxygenase-1 western blot analysis, after separation by 7.5% SDS-PAGE, proteins (70 μg) were transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked for 1 hour in 5% non-fat milk in Tris-buffered saline containing 0.05% Tween 20 and incubated with the mouse anti-heme-oxygenase-1 antibody (7.25 μg/mL; Abcam, Cambridge, UK) and mouse anti-GAPDH antibody (1:1 000; Abcam) overnight at 4°C. After washing in Tris-buffered saline containing 0.05% Tween 20, membranes were incubated for 1 hour at room temperature with peroxidase-labeled goat anti-mouse secondary antibodies (1:2 000; Zhongshan Golden Bridge). The immunoreactive bands were visualized using a SuperEnhanced chemiluminescence detection kit (Applygen Technologies Inc., Beijing, China) and subsequent exposure of the blots to Biomax L film (Kodak). Image analysis software, GIS 1D Ver 4.0 (Tanon Science & Technology Co., Ltd., Shanghai, China), was used to determine band intensities. GAPDH was used as the internal control.

讨论

文章快阅

延伸阅读

1中文摘要

脐带间充质干细胞是神经组织工程修复理想的细胞来源，它易于获得，没有伦理学障碍。大量报道证明其可被诱导为神经样细胞，可在神经系统中应用以替代受损的功能细胞。脐带间充质干细胞同样还具有免疫调节和抗氧化应激功能。但向神经方向诱导过间充质干细胞是否仍然具有这两个特性仍未知。我们本研究目的在于研究向神经诱导了的间充质干细胞是否还具有免疫调节功能和抗氧化应激功能。我们首先从脐带中分离间充质干细胞，然后用碱性成纤维细胞生长因子、神经生长因子、表皮生长因子、脑源性神经营养因子和forskolin对间充质干细胞进行神经诱导，诱导7天后，间充质干细胞获得了成纤维样形态，表达Tuj1, 神经丝蛋白200、微管相关蛋白2和神经元特异性烯醇化酶等神经标志，Nestin表达下调，但在淋巴细胞混合培养中仍然能抑制淋巴细胞的增殖，免疫抑制和抗氧化相关基因表达也未显著改变，仅有人类白细胞抗原G表达下调，说明生长因子诱导的神经样间充质干细胞让然具有免疫调控能力和抗氧化应激能力，但免疫特权特点有所下降。

2中文内容介绍

间充质干细胞发挥治疗作用，无非是功能替代、因子分泌、免疫抑制和抗氧化应激。生长因子诱导分化，可以促进间充质干细胞的神经分化，使其在体内更好的发挥功能替代作用，如果同时还保留未分化前的免疫调节和抗氧化应激功能，无疑更有利于间充质干细胞在细胞治疗中发挥作用。文章通过定量PCR和混合淋巴反应，研究了神经诱导前后间充质干细胞的免疫调节和抗氧化应激功能，证明了生长因子为基础的神经诱导方案对其这2个功能影响不大。

3实验设计及文章构思特点

①基于生长因子的，温和的神经诱导方案，并检测神经标志物的表达。

②定量PCR检测免疫调节和抗氧化应激功能相关的关键基因表达。

③混合淋巴反应检测间充质干细胞的免疫抑制功能。

4与国内外同类研究的比较

①已有部分文献研究了造骨、脂肪分化对间充质干细胞免疫调控能力的影响，但没讨论对抗氧化应激能力的影响

②未见研究神经分化对间充质干细胞免疫调控和抗氧化应激能力的影响相关文献报道

③神经诱导方案是已经比较成熟的体系，检测的神经标志物也是常见的。

脐带间充质干细胞向神经元样细胞诱导后仍保持免疫调节和抗氧化能力

Umbilical cord-derived mesenchymal stem cells retain immunomodulatory and anti-oxidative activities after neural induction

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