Vol.14 No.3 September 2009

DOI: 10.2478/s11658-009-0004-6 Volume 14 (2009) pp 363-376
Title RNA INTERFERENCE AGAINST Biot2, A NOVEL MOUSE TESTIS - SPECIFIC GENE, INHIBITS THE GROWTH OF TUMOR CELLS
Authors Chun-Ting Wang1, +, Peng Zhang2, +, Yong-Sheng Wang1, Xu-Zhi Ruan1, Zhi-Yong Li1, Feng Peng1,3*, Han-Shuo Yang1* and Yu-Quan Wei1
Abstract Biot2 is a novel murine testis-specific gene that was first identified using the SEREX technique, and named by our laboratory. Using conventional RT-PCR and real time RT-PCR, we tested the expression profile of Biot2 in normal tissues and various murine tumor cell lines. Using RNA interference, we studied the biological function of Biot2 in tumorigenesis. We applied various types of growth assay, such as the in vitro MTT, colony-forming and BrdU incorporation assays, along with in vivo tumorigenicity assays, to reveal its inhibition of tumor cell proliferation. The results revealed that the Biot2 transcript was detected only and strongly in the testis tissues and abundantly in five types of murine cancer cell line. Treating B16 murine melanoma, LL/2 murine Lewis lung carcinoma and CT26 murine colorectal adenocarcinoma with special shRNA targeting Biot2 can significantly reduce the proliferation rate of these three tumor cell lines in vitro, as measured by the MTT, colony-forming and BrdU incorporation assays. The tumorigenicity of the CT26 cells transfected with special shRNA targeting Biot2 was also decreased distinctly in vivo compared with the control. It was therefore concluded that Biot2 plays a key role in tumorigenesis and could be a potential target for biotherapy.
Keywords RNA interference, Biot2, Testis-specific, Proliferation
Address and Contact Information 1State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Keyuan Road 4, Chengdu, Sichuan, 610041, P. R. China,
2Department of Radiation Oncology, Sichuan Cancer Hospital, Chengdu, Sichuan, 610064, P. R. China,
3Department of Thoracic Oncology of Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Guoxuexiang No. 37, Chengdu, Sichuan, 610041, P. R. China
+These authors contributed equally to this research
* Author for correspondence; e-mail address: evenforeven@yahoo.com.cn or yhansh@scu.edu.cn, tel: +86 28 85164063, fax: +86 28 85164060
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-009-0007-3 Volume 14 (2009) pp 377-394
Title AN EVALUATION OF A NEW APPROACH TO THE REGENERATION OF Helichrysum italicum (ROTH) G. DON, AND THE MOLECULAR CHARACTERIZATION OF THE VARIATION AMONG SETS OF DIFFERENTLY DERIVED REGENERANTS
Authors Rosaria Perrini1,§, Vittorio Alba2,§, Claudia Ruta1, Irene Morone-Fortunato1, Antonio Blanco2 And Cinzia Montemurro2,*
Abstract A protocol for the induction of regeneration from leaves of Helichrysum italicum was established. Calli were found to form on the basal medium only when it was supplemented with thidiazuron (TDZ) alone or in combination with naphthalene acetic acid (NAA), with a percentage ranking of at least 80%. The hormone-free medium showed the highest percentage of shoot regeneration (62%) even though no callus formed. AFLP markers were employed to verify tissue culture-induced variation in the regenerated plantlets obtained by direct shoot regeneration or the indirect shoot regeneration process (callus formation). Seven out of the eleven AFLP primer pairs yielded polymorphic patterns. The average number of fragments per primer pair was 64.1. Singletons were represented by 12 (2.7%) fragments. Student’s T-test was performed both on the average number of shared fragments and on the nucleotide diversity, and no significant statistical difference was observed between the two regeneration treatments.
Keywords Helichrysum italicum, In vitro culture, Tissue culture-induced variation, AFLP markers, Nucleotide diversity
Address and Contact Information 1Department of Plant Production, University of Bari, Via Amendola 165/A, Bari, 70125 Italy,
2Department of Agro-Forestry and Environmental Biology and Chemistry, section of Genetics and Breeding, University of Bari, Via Amendola 165/A, Bari, 70125 Italy
§These authors contributed equally to this work.
* Author for correspondence; e-mail: c.montemurro@agr.uniba.it, tel.: 00390805443558, fax: 00390805442200
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-009-0010-8 Volume 14 (2009) pp 395-410
Title INTERACTIONS BETWEEN CANTHAXANTHIN AND LIPID MEMBRANES – POSSIBLE MECHANISMS OF CANTHAXANTHIN TOXICITY
Authors Agnieszka Sujak
Abstract Canthaxanthin (ß, ß-carotene 4, 4’ dione) is used widely as a drug or as a food and cosmetic colorant, but it may have some undesirable effects on human health, mainly caused by the formation of crystals in the macula lutea membranes of the retina. This condition is called canthaxanthin retinopathy. It has been shown that this type of dysfunction of the eye is strongly connected with damage to the blood vessels around the place of crystal deposition. This paper is a review of the experimental data supporting the hypothesis that the interactions of canthaxanthin with the lipid membranes and the aggregation of this pigment may be the factors enhancing canthaxanthin toxicity towards the macula vascular system. All the results of the experiments that have been done on model systems such as monolayers of pure canthaxanthin and mixtures of canthaxanthin and lipids, oriented bilayers or liposomes indicate a very strong effect of canthaxanthin on the physical properties of lipid membranes, which may explain its toxic action, which leads to the further development of canthaxanthin retinopathy.
Keywords Canthaxanthin, Retinopathy, Macula lutea, Model lipid membranes, Molecular interactions
Address and Contact Information Department of Physics, University of Life Sciences, 20-950 Lublin, Poland
* Author for correspondence; e-mail: agnieszka.sujak@up.lublin.pl, tel.: +48 81 445 6564, fax: +48 81 533 3549
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-009-0008-2 Volume 14 (2009) pp 411-423
Title THE EFFECTS OF MARKEDLY RAISED INTRACELLULAR SPHINGOSINE KINASE-1 ACTIVITY IN ENDOTHELIAL CELLS
Authors Vidya Limaye1,2,3*, Mathew A. Vadas4, Stuart M. Pitson2,5 and Jennifer R. Gamble4
Abstract The enzyme sphingosine kinase-1 (SK1) promotes the formation of sphingosine-1-phosphate (S1P), which is an important survival factor for endothelial cells (EC). Modest increases in intracellular SK1 activity in the EC are known to confer a survival advantage upon the cells. Here, we investigated the effects of more dramatic increases in intracellular SK1 in the EC. We found that these cells show reduced cell survival under conditions of stress, enhanced caspase-3 activity, cell cycle inhibition, and cell-cell junction disruption. We propose that alterations in the phosphorylation state of the enzyme may explain the differential effects on the phenotype with modest versus high levels of enforced expression of SK1. Our results suggest that SK1 activity is subject to control in the EC, and that this control may be lost in conditions involving vascular regression.
Keywords Sphingosine kinase-1, Endothelial cells, Cell survival
Address and Contact Information 1Royal Adelaide Hospital, Department of Rheumatology, North Tce, Royal Adelaide Hospital, Adelaide, SA 5000, Australia
2Division of Human Immunology, Institute of Medical and Veterinary Science, Frome Road, Adelaide, SA 5000, Australia,
3Department of Medicine, University of Adelaide, Frome Rd, Adelaide, SA, Australia,
4Centenary Institute, University of Sydney, NSW, Australia, 5School of Molecular and Biomedical Science, University of Adelaide, Australia
* Author for correspondence; e-mail: vidya.limaye@health.sa.gov.au, tel.: 61-8-82225190, fax: 61-8-82225895
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-009-0009-1 Volume 14 (2009) pp 424-441
Title CHRONIC INCREASES IN SPHINGOSINE KINASE-1 ACTIVITY INDUCE A PRO-INFLAMMATORY, PRO-ANGIOGENIC PHENOTYPE IN ENDOTHELIAL CELLS
Authors Vidya Limaye1, 2, 3,*, Pu Xia6, Chris Hahn2, Malcolm Smith4, Mathew A. Vadas6, Stuart M. Pitson2, 5 and Jennifer R. Gamble6
Abstract Sphingosine kinase-1 (SK1) promotes the formation of sphingosine- 1-phosphate (S1P), which has potent pro-inflammatory and pro-angiogenic effects. We investigated the effects of raised SK1 levels on endothelial cell function and the possibility that this signaling pathway is activated in rheumatoid arthritis. Human umbilical vein endothelial cells with 3- to 5-fold SK1 (ECSK) overexpression were generated by adenoviral and retroviralmediated gene delivery. The activation state of these cells and their ability to undergo angiogenesis was determined. S1P was measured in synovial fluid from patients with RA and OA. ECSK showed an enhanced migratory capacity and a stimulated rate of capillary tube formation. The cells showed constitutive activation as evidenced by the induction of basal VCAM-1 expression, and compared with empty vector control cells (ECEV). These changes had functional consequences in terms of enhanced neutrophil binding in the basal and TNFstimulated states in ECSK. By contrast, over-expression of a dominant-negative SK inhibited the TNF-induced VCAM-1 and E selectin and inhibited PMN adhesion, confirming that the observed effects were specifically mediated by SK. The synovial fluid levels of S1P were significantly higher in patients with RA than in those with OA. Small chronic increases in SK1 activity in the endothelial cells enhance the ability of the cells to support inflammation and undergo angiogenesis, and sensitize the cells to inflammatory cytokines. The SK1 signaling pathway is activated in RA, suggesting that manipulation of SK1 activity in diseases of aberrant inflammation and angiogenesis may be beneficial.
Keywords Inflammation, Angiogenesis, Endothelial cells, Sphingosine kinase, TNF
Address and Contact Information 1Royal Adelaide Hospital, Department of Rheumatology, North Tce, Royal Adelaide Hospital, Adelaide SA 5000, Australia,
2Division of Human Immunology, Institute of Medical and Veterinary Science, Frome Road, Adelaide, SA 5000, Australia,
3Department of Medicine, University of Adelaide, Frome Rd, Adelaide, SA, Australia,
4Repatriation General Hospital, Daw Park, SA Australia,
5School of Molecular and Biomedical Science, University of Adelaide,
6Centenary Institute, University of Sydney, NSW, Australia
* Author for correspondence; e-mail: vidya.limaye@health.sa.gov.au, tel.: 61-8-82225190, fax: 61-8-82225895
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-009-0011-7 Volume 14 (2009) pp 442-456
Title THE EXPRESSION OF COX-2, hTERT, MDM2, LATS2 AND S100A2 IN DIFFERENT TYPES OF NON-SMALL CELL LUNG CANCER (NSCLC)
Authors Mojca Strazisar, Vid Mlakar and Damjan Glavac*
Abstract Several studies have reported different expression levels of certain genes in NSCLC, mostly related to the stage and advancement of the tumours. We investigated 65 stage I-III NSCLC tumours: 32 adenocarcinomas (ADC), 26 squamous cell carcinomas (SCC) and 7 large cell carcinomas (LCC). Using the real-time reverse transcription polymerase chain reaction (RT-PCR), we analysed the expression of the COX-2, hTERT, MDM2, LATS2 and S100A2 genes and researched the relationships between the NSCLC types and the differences in expression levels. The differences in the expression levels of the LATS2, S100A2 and hTERT genes in different types of NSCLC are significant. hTERT and COX-2 were over-expressed and LATS2 under-expressed in all NSCLC. We also detected significant relative differences in the expression of LATS2 and MDM2, hTERT and MDM2 in different types of NSCLC. There was a significant difference in the average expression levels in S100A2 for ADC and SCC. Our study shows differences in the expression patterns within the NSCLC group, which may mimic the expression of the individual NSCLC type, and also new relationships in the expression levels for different NSCLC types.
Keywords COX-2, hTERT, MDM2, LATS2, S100A2, RT-PCR, Expression
Address and Contact Information Department of Molecular Genetics, Institute of Pathology, Faculty of Medicine, University of Ljubljana, Korytkova 2, 1000 Ljubljana, Slovenia
* Author for correspondence; e-mail: damjan.glavac@mf.uni-lj.si, tel.: 386 1 543 7180, fax: 386 1 543 7101
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-009-0012-6 Volume 14 (2009) pp 457-465
Title THE EFFECTS OF DISODIUM PAMIDRONATE ON HUMAN POLYMORPHONUCLEAR LEUKOCYTES AND PLATELETS: AN in vitro STUDY
Authors Eleonora Salvolini1*, Monia Orciani1, Arianna Vignini2, Roberto Di Primio1 and Laura Mazzanti2
Abstract Recent reports have indicated that, as well as having antiresorptive effects, bisphosphonates could have an application as anti-inflammatory drugs. Our aim was to investigate whether this anti-inflammatory action could be mediated by the nitric oxide (NO) released by the leukocytes migrating to the site of inflammation. In particular, we investigated in vitro the intracellular calcium concentration ([Ca2+]i), the level of NO released by PMN and platelets, and the PMN myeloperoxidase activity after incubation with disodium pamidronate, since there was a postulated modulatory effect of this aminosubstituted bisphosphonate on leukocytes both in vitro and in vivo. Our data shows that the pamidronate treatment provoked a significant increase in the [Ca2+]i parallel to the enhancement in NO release, suggesting a possible activation of constitutive nitric oxide synthase, while the myeloperoxidase activity was significantly reduced. In conclusion, we hypothesized that treatment with pamidronate could stimulate NO-production by cells present near the bone compartment, thus constituting a protective mechanism against bone resorption occurring during inflammation. In addition, PMN- and platelet-derived NO could act as a negative feed-back signal to restrict the inflammatory processes.
Keywords Pamidronate, Polymorphonuclear leukocytes, Platelets, Nitric oxide, Intracellular calcium, Myeloperoxidase activity
Address and Contact Information 1Department of Molecular Pathology and Innovative Therapies – Histology and
2Institute of Biochemistry, Polytechnic University of Marche, Via Tronto 10/A, 60020 Ancona, Italy
* Author for correspondence; e-mail: e.salvolini@univpm.it, tel.: +39 071 2206078, fax: +39 071 2206078
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-009-0013-5 Volume 14 (2009) pp 466-480
Title THE EXPRESSION OF PROTEIN KINASE B IN GASTRIC CANCER CELL APOPTOSIS INDUCED BY 12-O-TETRADECANOYLPHORBOL- 1,3-ACETATE
Authors Bing Zhang1 and Chun Xia2*
Abstract The plant nuclear genome is largely composed of mobile DNA, which can rearrange genomes and other individual gene structure and also affect gene regulation through various promoted activities: transposition, insertion, excision, chromosome breakage, and ectopic recombination. Ty1-copia-like retrotransposon is a widespread class of transposable elements in the plant kingdom, representing a large part of the total DNA content. Here, a novel retrotransposon-like sequence was isolated and identified as the Ty1-copia-like reverse transcriptase domain (named here CLCoy1), based on the homology of known elements. Fluorescence in situ hybridization, revealed that CLCoy1 was mainly located in telomeric and sub-telomeric regions along the Citrus chromosomes. CLCoy1 composes 3.6% of the genome and, interestingly, while transposons are mostly specific to a species, this element was identified in other Citrus species such as Citrus aurantium, Fortunella margarita and Citrus paradisi, but undetected in Poncirus trifoliata. We also determined that wounding, salt and cell culture stress produced transcriptional activation of this novel retroelement in Citrus limon. The novel Ty1-copia-like element CLCoy1 may have played a major role in shaping genome structure and size during Citrus species evolution.
Keywords TPA, Cell apoptosis, PKB/Akt, Gastric cancer BGC-823 cell line
Address and Contact Information 1Medical School, Xiamen University, Xiamen 361005, Fujian Province, P.R. China,
2Zhongshan Hospital, Xiamen Univeisity, Xiamen 361004, Fujian Province, P.R. China
* Author for correspondence: e-mail: chunxia99@yahoo.com.cn, tel.: +86 5925921461, fax: +86 5922188680
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-009-0014-4 Volume 14 (2009) pp 481-496
Title THE TRANSCRIPTIONAL CASCADE ASSOCIATED WITH CREATINE KINASE DOWN-REGULATION AND MITOCHONDRIAL BIOGENESIS IN MICE SARCOMA
Authors Soumen Bera* and Manju Ray
Abstract The tissue-specific expressions of creatine kinase (CK) isoforms are regulated by the coordinated action of various transcription factors. The myogenic differentiation factor D (MyoD) family of proteins and the myocytespecific enhancer binding factor 2 family of transcription factors are important in regulating the muscle-specific expression of cytosolic muscle-type CK (MCK) and mitochondrial CKs. As reported in some related studies, TNF-α mediated degradation of MyoD and myogenin mRNA may lead to severe muscle wasting and cachexia, which is characterized by a low transcript level of MCK and myosin heavy chain proteins. In our previous study, we reported on a complete loss of total CK activity and expression when sarcoma was induced in mouse skeletal muscle (Patra et al. FEBS J. 275 (2008) 3236-3247). This study aimed at investigating the transcriptional cascade of CK down-regulation in carcinogen-induced sarcoma in mouse muscle. Both CK deficiency and enhanced nitric oxide synthase (NOS) were known to augment mitochondrial biogenesis, so we also explored the activation of the transcriptional cascade of mitochondrial biogenesis in this cancer. We observed the activation of the TNF-α-mediated nitric oxide production pathway with NFκB activation and concomitant degradation of MyoD and myogenin mRNA. Exploration of mitochondrial biogenesis revealed high cytochrome c oxidase activity and mitochondrial DNA content in sarcoma. The PGC-related co-activator seems to have a major role in regulating mitochondrial biogenesis by upregulating nuclear respiratory factors and mitochondrial transcription factor A. From the above findings, it can be concluded that severe muscle degeneration leads to CK downregulation in sarcoma, and that the stimulation of mitochondrial biogenesis indicated a scenario representing both CK deficiency and NOS overexpression on the one hand, and altered bioenergetic profiling on the other.
Keywords Sarcoma, Creatine kinase, Nitric oxide synthase, Muscle degeneration, Mitochondrial biogenesis
Address and Contact Information Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata - 700 032, India
* Author for correspondence: e-mail: bcsb@iacs.res.in, tel.: + 91 33 2499 0282, fax: + 91 33 2473 2805
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-009-0016-2 Volume 14 (2009) pp 497-510
Title CAPSAICIN INDUCES APOPTOSIS BY GENERATING REACTIVE OXYGEN SPECIES AND DISRUPTING MITOCHONDRIAL TRANSMEMBRANE POTENTIAL IN HUMAN COLON CANCER CELL LINES
Authors Kyung Min Yang1, Jong Ok Pyo2, Gyu-Yeol Kim3, Rina Yu4, In Seob Han5, Seong A. Ju6, Won Ho Kim1 and Byung-Sam Kim5*
Abstract Although genetic factors are a well-known cause of colorectal cancer, environmental factors contribute more to its development. Despite advances in the fields of surgery, radiotherapy and chemotherapy, the cure rates for colon cancer have not substantially improved over the past few decades. Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide), the principal pungent ingredient of hot chili pepper, has exhibited an anti-tumor effect in many cell types. However, the mechanisms responsible for the anti-tumor effect of capsaicin are not yet completely understood. In this study, we investigated whether capsaicin induces apoptosis in colon cancer cell lines. Capsaicin decreased cell viability in a dosedependent manner in Colo320DM and LoVo cells. In addition, capsaicin produced cell morphology changes and DNA fragmentation, decreased the DNA contents, and induced phosphatidylserine translocation, which is a hallmark of apoptotic cell death. We showed that capsaicin-induced apoptosis is associated with an increase in ROS generation and a disruption of the mitochondrial transmenbrane potential. A possible mechanism of capsaicin-induced apoptosis is the activation of caspase 3, a major apoptosis-executing enzyme. Treatment with capsaicin induced a dramatic increase in caspase 3 activity, as assessed by the cleavage of Ac-DEVD-AMC, a fluorogenic substrate. In conclusion, our results clearly showed that capsaicin induced apoptosis in colon cancer cells. Although the actual mechanisms of capsaicin-induced apoptosis remain uncertain, it may be a beneficial agent for colon cancer treatment and chemoprevention.
Keywords Capsaicin, Colon cancer cell line, Apoptosis, Mitochondrial transmembrane potential, Reactive oxygen species, Caspase 3
Address and Contact Information 1Department of Internal Medicine, Institute of Gastroenterology, Brain Korea 21 for Medical Science, Yonsei University College of Medicine, Seoul, Korea,
2School of Biological Science/Bio-MAX Institute, Seoul National University, Seoul, Korea,
3Department of Surgery, Ulsan University Hospital, College of Medicine, University of Ulsan, Ulsan, Korea,
4Department of Food Science and Nutrition, University of Ulsan, Ulsan, Korea,
5Department of Biological Sciences, University of Ulsan, Ulsan, Korea,
6Biomedical Research Center, Ulsan University Hospital, College of Medicine, University of Ulsan, Ulsan, Korea
* Author for correspondence: e-mail: bskim@ulsan.ac.kr, tel.: (82)-52-259-2748, fax: (82)-52-258-2740
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-009-0019-z Volume 14 (2009) pp 511-527
Title THE EXPRESSION OF INDUCIBLE NITRIC OXIDE SYNTHASE (iNOS) IN THE TESTIS AND EPIDIDYMIS OF RATS WITH A DIHYDROTESTOSTERONE (DHT) DEFICIENCY
Authors Agnieszka Kolasa1*, Mariola Marchlewicz1, Rafał Kurzawa2, Wojciech Gł±bowski1, Grzegorz Trybek1, Lidia Wenda-Różewicka1 and Barbara Wiszniewska1*
Abstract In our previous studies, we showed that a finasteride-induced DHT deficiency may cause changes in the morphology of the seminiferous epithelium without any morphological alteration of the epididymis. In this study, we demonstrated the constitutive immunoexpression of inducible nitric oxide synthase (iNOS) in the testis and epididymis of Wistar rats treated with finasteride for 28 days (the duration of two cycles of the seminiferous epithelium) and 56 days (the duration of one spermatogenesis). We noted that a 56-day finasteride treatment mainly caused a decrease in the level of circulating DHT, as well as a statistically insignificant decrease in the level of T. The hormone deficiency also led to a change in the iNOS immnoexpression in the testis and epididymis of the finasteride-treated rats. In vitro, DHT did not modify NO production by the epithelial cells of the caput epididymis even when stimulated with LPS and IFNγ, but it did give rise to an increase in NO production by the epithelial cells of the cauda epididymis without the stimulation. DHT did not have a statistically significant influence on estradiol production by cultured, LPS- and IFNγ-stimulated epithelial cells from the caput and cauda epididymis. In conclusion, our data clearly indicates that a finasterideinduced DHT deficiency intensifies the constitutive expression of iNOS in most rat testicular and epididymal cells, so it can be expected that the expression of inducible nitric oxide synthase (iNOS) could be regulated by DHT. On the other hand, the profile of the circulating DHT and T levels strongly suggests that the regulation of constitutive iNOS expression is complex and needs more detailed study.
Keywords iNOS immunoexpression, DHT-deficiency, Testis, Epididymis, Rat
Address and Contact Information 1Department of Histology and Embryology,
2Department of Reproductive Medicine and Gynecology, Pomeranian Medical University, Powstanców Wlkp. 72, 70-111 Szczecin, Poland
* Authors for correspondence. Tel./fax: + 48 91 466 1677, e-mails: ak1712@sci.pam.szczecin.pl, barbwisz@sci.pam.szczecin.pl
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-009-0015-3 Volume 14 (2009) pp 528-536
Title THE DIFFERENTIATION OF HUMAN PLACENTA-DERIVED MESENCHYMAL STEM CELLS INTO DOPAMINERGIC CELLS in vitro
Authors Li Chen, Dong-Mei He* and Yuan Zhang
Abstract Mesenchymal stem cells (MSCs) constitute an interesting cellular source to promote brain regeneration after Parkinson’s disease. MSCs have significant advantages over other stem cell types, and greater potential for immediate clinical application. The aim of this study was to investigate whether MSCs from the human placenta could be induced to differentiate into dopaminergic cells. MSCs from the human placenta were isolated by digestion and density gradient fractionation, and their cell surface glycoproteins were analyzed by flow cytometry. These MSCs were cultured under conditions promoting differetiation into adipocytes and osteoblasts. Using a cocktail that includes basic fibroblast growth factor (bFGF), all trans retinoic acid (RA), ascorbic acid (AA) and 3-isobutyl-1-methylxanthine (IBMX), the MSCs were induced in vitro to become dopamine (DA) neurons. Then, the expression of the mRNA for the Nestin and tyrosine hydroxylase (TH) genes was assayed via RT-PCR. The expression of the Nestin, dopamine transporter (DAT), neuronal nuclear protein (NeuN) and TH proteins was determined via immunofluorescence. The synthesized and secreted DA was determined via ELISA. We found that MSCs from the human placenta exhibited a fibroblastoid morphology. Flow cytometric analyses showed that the MSCs were positive for CD44 and CD29, and negative for CD34, CD45, CD106 and HLA-DR. Moreover, they could be induced into adipocytes and osteocytes. When the MSCs were induced with bFGF, RA, AA and IBMX, they showed a change in morphology to that of neuronal-like cells. The induced cells expressed Nestin and TH mRNA, and the Nestin, DAT, NeuN and TH proteins, and synthesized and secreted DA. Our results suggest that MSCs from the human placenta have the ability to differentiate into dopaminergic cells.
Keywords Mesenchymal stem cell, Human, Placenta, Differentiation, Dopamine
Address and Contact Information Institute of Hematology, Medical College of Jinan University, Guangzhou 510632, P. R. China
* Author for co rrespondence: e-mail: thedm@jnu.edu.cn, fax: 86-20-85221343
[Rozmiar: 1332 bajtów]