| Nerve functional impairment caused by nervous system injury and neurodegenerative diseases has been a problem that troubled the human health. It is also a focus that scientists dedicated to study. For a long time, it is generally believed that the nerve cells of the nervous system lack the ability to regenerate. To date, drug treatment is only symptomatic treatment, but cannot fundamentally solve the lack of nerve cells.With the development of stem cells technology, it provided a chance to treat nerve injury and neurodegenerative diseases. The stem cells in specific microenvironmental conditions can be induced to differentiate into a variety of tissues and cells. Through transplantation to recipients, stem cells participate in tissue regeneration and repair. It provides a new promising therapeutic means for clinical medicine.Mesenehymal stem cells(MSCs) seem to be a more promising one regarding their advantages over ESC and other type of adult stem cells. A number of studies have shown, mesenchymal stem cells in neurodegenerative diseases, nervous autoimmune disease, neurological impairment and cerebral vascular disease have a positive therapeutic significance. For with convenient collection, low immunogenicity and viral contamination rate, the umbilical cord can be used as an ideal source of mesenchymal stem cells.At present, the neural differentiation methods of umbilical cord mesenchymal stem cells in vitro are main chemical molecules and neurotrophic factor induction method. But, chemical molecules such as dimethyl sulfoxide, β-mercaptoethanol and butylated hydroxyanisole are more or less toxic. Neurotrophic factors with large molecular weight, such as FGF and BDNF, do not easily passing the blood-brain barrier and causing untoward side effects. Thus those methods are limited for use in vivo.Progesterone (PROG) is a hormone secreted by ovaries and placenta. PROG is also synthesized in the brain. PROG is an important hormone and has a variety of beneficial effects. PROG has been shown to improve behavioral and functional recovery and to reduce inflammation, oxidative damage, cerebral edema, and neural cell death. PROG is required for the maintenance and the differentiation of primary hippocampal/cortical/striatal neurons in vitro.Objective:In the present study, human umbilical cord mesenchymal stem cells (hUMSCs) are derived form the umbilical cords by tissue culture method. hUMSCs was cultured in rat brain tissue extrects to Investigate if hUMSCs could have neural differentiation ability in mimic microenvironme-nts of brain. On this basis, HPLC-MS assay was applied to investigate PROG, PREG and AP levels. Afterwards, we explore the effect of PROG on neuronal differentiation of hUMSCs in mimic microenvironments of brain, urthermore explore its mechanism. The purpose of this paper is to provide new ideas and basis for treatment ervous system injury and neurodegenerative diseases.Methods:1. Isolation, culture, and identification of hUMSCsHuman umbilical cords were obtained from full-term caesarian section births and were rinsed twice by phosphate-buffered saline (PBS) to wash off blood. After removal of blood vessels (one vein and two arteries), the Wharton’s jelly was stripped carefully and cut into pieces of1mm3, which were then cultured in DMEM/Ham’s F12medium (1:1) supplemented with10%FBS plus100U/mL penicillin and streptomycin. The cultures were incubated at37℃with5%CO2. The medium was changed every3days after the initial seeding. When the cultures reached80-90%confluence, the cells were subcultured.To analyze MSC surface markers, hUMSCs from the third passages were incubated with antibodies against human CD105, CD44, CD29, CD45, CD34and HLA-DR. The cells were then assessed by the FACSCalibur flow cytometer.Assay of adipogenic and osteogenic differentiation were performed after plating the cells into6-well plates at a density of2x104cells. For adipogenic differentiation,10-6M dexamethasone,100mg/L isobutylmethylxanthine,50mg/L ascorbic acid were added to the growth medium. For osteogenic differentiation,10-8M dexamethasone,50mg/L ascorbic acid and10mmol/L β-glycerophosphate were added to the growth medium. After two weeks of induction, cells were fixed with4%paraformaldehyde and stained with Oil red O to view lipid vacuoles in adipocytes and VonKossa staining to view calcium deposition in osteocytes.2. Influence of brain tissue extracts on hUMSCsSD rat brains were obtained immediately following craniotomy and were placed on ice. The brains were measured and homogenized in DMEM/F12(150mg/mL). After homogenization, the samples were incubated on ice for10min. The homogenates were centrifuged twice at5000×g at4℃for15min. The supernatants were filtered with a0.22μm filter and stored at-70℃.hUMSCs from the third passages were cultured in gelatine-coated24-well plates at a density of1×105cells in DMEM/F12medium containing10%FBS. When the cultures reached80-90%confluence, fresh medium with brain tissue extracts was added. Phase-contrast microscope was used to record morphology changes. After cultured72h, immunocytochemistry was used to detect the expression of nestin、NSE and GFAP. Toluidin blue staining was carried out for detection of Nissl body.3. The level changes of main neurosteroid and3β-HSD of hUMSCs cultured in brain tissue extractshUMSCs from the third passages were cultured in gelatine-coated24-well plates at a density of1×105cells in DMEM/F12medium containing10%FBS. When the cultures reached80-90%confluence, change the medium. Control group:(DMEM/F12+10%FBS), test group:brain tissue extracts. The culture media were collected at0,1,3,7d, centrifugate5minutes at1000r·min-1. The supernatant were mixed with methyltesterone(MT). Then, samples were isolated using ethylacetate/hexane and wre further purified by solid phase extraction(SPE). Thereafter, samples were derivatized with2-nitro -4-trfluo-romethylphenylhydrazine(2NFPH) and determinated by high performance liquid chromatography-mass spectrometry(HPLC-MS). Immu-nocytochemistry was used to detect the expression of3β-HSD.4. Influence of progesterone treatment on hUMSCs cultured in brain tissue extractshUMSCs from the third passages were cultured in6-well plates at a density of1×106cells in DMEM/F12medium containing10%FBS. When the cultures reached80-90%confluence, change the medium. Control group: brain tissue extracts, test group:0.0μM、1μM、10μM progesterone in brain tissue extracts. Phase-contrast microscope was used to record morphology changes. After cultured72h, PT-PCR was carried out to detect the expression of NSE. Flow cytometry method was used to verify the yesukts.5. Influence of progesterone treatment on BDNF level of hUMSCs cultured in brain tissue extractshUMSCs from the third passages were cultured in24-well plates at a density of1×105cells in DMEM/F12medium containing10%FBS. When the cultures reached80%confluence, change the medium. The experiment was divided into3groups:①DMEM/F12+10%FBS②brain tissue extracts③PROG(1μM) in brain tissue extracts. The culture media were collected at0,1,3,7d, centrifugate5minutes at1000r·min-1. The supematant was determinated using Enzyme-Linked ImmunoSorbent Assay (ELISA).6. Data and statisticsSatatistic was performed with SPSS13.0for windows software, and data were expressed in mean±SD. Significance between the groups was analyzed by independent samples t test or ANOVA. Significance was considered when P<0.05.Results:1. Isolation, culture, and identification of hUMSCsThree to five days after primary culture, adherent cells with a homogenous fibroblastic morphology came out of fragments of Wharton’s jelly. Two weeks after plating, when the cells reached80%-90%confluence, they were detached with0.25%trypsin/EDTA solution and were passaged at a ratio of1:3. After subcultured, the cells proliferate rapidly. The cells can reach80%-90%confluence in4to5days. FACS revealed that hUMSCs expressed putative markers of MSCs, which included CD29, CD44and CD105, but not hematopoietic andendothelial cell markers, such as CD34, CD45. They also did not express human leukocyte antigen HLA-DR. After being exposed to adipogenic differentiation medium for two weeks, most of fibroblastic cells changed into large, flattened appearance with accumulated lipid vacuoles, which stained red with Oil Red O. In osteogenic differentiation medium for two weeks, these cells changed into multi-angular or irregular appearance with calcium in cytoplasm detected by VonKossa.2. Neuronal differentiation of hUMSCs in brain tissue extractsAfter cultured in brain tissue extracts for12h, we observed that some cells had undergone morphological changes. Initially, the cytoplasm retracted towards the nucleus, and then cells bodies became increasingly retractil. After72h incubation, most cells had a simple bipolar or multipolar shape, similar to neuron. A simple cross-linking between cells. At72h, neuron specific enolase (NSE)-positive cells、nestin-positive cells and GFAP-positive cells were observed in the cultures. We observed that deep blue granular Nissl body in cytoplasm after Toluidin biue staining.3. The changes of main neurosteroids and3β-HSD in neuronal differentiation of hUMSCsWith the increasing time of culture, the level of PROG gradually increased, Od:1.78±0.50ng/mL; Id:2.88±0.29ng/mL;3d:7.51±0.65ng/mL;7d:10.56±0.65ng/mL (P<0.05). The level of PREG gradually decreased, Od:14.74±0.63ng/mL; Id:12.34±0.56ng/mL;3d:10.62±0.32ng/mL;7d:8.61±0.50ng/mL (P<0.05). The level of AP was lower than LLOQ.Compared with control group, the cells cultured in brain tissue extracts showed expression of3β-HSD. With the increasing time of culture, expression enchanced.4. The effect of progesterone on neuronal differentiation of hUMSCs in brain tissue extractsBy analyzing the expression of NSE by PT-PCR, we found that PROG could increase the hUMSCs’differentiation into neuron-like cells and1μM of PROG has the best effect on the differentiation. This result was also confirmed by flow cytometry analysis.5. The effect of progesterone on BDNF level in neuronal differentiation of hUMSCsCompared with control group, the level of BDNF was significantly increased in brain tissue extracts group and PROG group(P<0.05).Compared with brain tissue extracts group, the level of BDNF was significantly increased after treatment with PROG (P<0.05).Conclusion:1. MSCs derived from human umbilical cord could be easily isolated cultured and passaged in vitro. hUMSCs could differentiate into fat and osteoblast under permissive conditions. HUCMSCs expressed putative markers of MSCs, which included CD29, CD44and CD105, but not hematopoietic andendothelial cell markers, such as CD34, CD45. They also did not express human leukocyte antigen HLA-DR. So, umbilical cord was a rich source of MSCs.2. Brain tissue microenvironment can promote hUMSCs differentiation into neuron-like cells. The level of PROG and PREG changed during differentiation. PROG production significantly increased and PREG levels reduced. It showed that the cells had mature nerve cell function. The cells could synthesis and secret neurotransmitters.3. PROG treatment could improve the neural differentiation rate of hUMSCs in brain tissue microenvironment and promot hUMSCs to differentiate into neurons4. During hUMSCs cultured in brain tissue microenvironment and PROG treatment, the level of BDNF in cell culture medium improved. After PROG treatment, the level of BDNF improved more significantly. It suggested that increase of BDNF level maby one of the mechanisms of neural differentiation of hUMSCs cultured in brain tissue microenvironment. It also maybe one of the mechanisms that PROG promoted neural differentiation. |
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