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Study on Human Wharton’s Jelly-derived Mesenchymal Stem Cells Promote Schwann Ceils Secrete Neurotrophic Factors

Author: LiuYan
Tutor: GongXu
School: Jilin University
Course: Surgery
Keywords: Wharton’s jelly Schwann cell Purification mesenchymal stem cells Peripheral nerveregeneration
CLC: R651.3
Type: PhD thesis
Year: 2013
Downloads: 73
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Abstract


Autologous nerve grafting is the current golden standard for peripheral nerve defectrepair, this method inevitably results in complication at donor sites and only a limited amountof nerve grafts can be harvested. Hence, Great efforts have been devoted to createtissue-engineered nerve grafts.A number of basic and clinical trials provide accumulatingevidence that functional outcome of peripheral nerve repair is improved by scaffold seededwith Bone marrow mesenchymal stem cells (BMSCs). The reason is that BMSCs promoteperipheral nerve regeneration not only via their direct release of neurotrophic factors, but alsothrough indirect modulation of cellular behaviors of Schwann cells (SCs). But the clinicalapplication is limited because of donor site morbidity, the invasive procedure, and thedecreased number of BMSCs. Human Wharton’s jelly-derived mesenchymal stemcells(HWJ-MSCs)can be easily accessible and noncontroversial, as well, can effectivelysuppress mitogen-induced T-cell proliferation. These findings suggest that HWJ-MSCs couldbe promising alternative to BMSCs. Our study is to investigate the influences of HWJ-MSCson proliferation of and neurotrophic factor expression by SCs and to provide a new cells seedsource for peripheral nerve regeneration.Methods:(1) HWJ-MSCs were cultured, drawing growth curve and cryopreservationand thawing. Characteristic of cell phenotype were confirmed by flow cytometry. We usedRT-PCR to detect the neurotropic factors from the HWJ-MSCs. We used ELISA method toquantity the content of growth factors secreted by HWJ-MSCs.(2) Schwann cells wereisolated from sciatic nerve segments of postnatal SD rat, aliquots were seeded onto dishes andtreated with2rounds of purification by differential cell attachment and detachment. Controlgroups that did not undergo purification were setted. The cells were observed underphase-contrast microscopy. The purity of the SCs was obtained by morphological analysis.The final SCs were identified with immunocytochemistry and flowcytometricanalysis.(3)HWJ-MSCs and SCs were cocultured in the Transwell system served asco-cotured group. However, SCs on both the layers were served as the controls. SCs werecounted for total four times. MTT assay and bromodeoxyuridine/Hoechst33342doublestaining were performed to assess Cell viability. The Western blot was performed to assess theexpression of the nerve growth factor (NGF), and the brain-derived neurotrophic factor(BDNF) in SCs.Result:(1) HWJ-MSCs showed immunopositive for MSC surface markers andimmunonegetive for the haemopoietic cell surface markers. RT-PCR results showed thatHWJ-MSCs expressed BDNF, GDNF, HGF, NT-3, VEGF, IGF-1and NT-4. We furtherobtained from ELISA results the content of BDNF, GDNF, HGF and NT-3in the conditionedmedium.(2)within96h, after two rounds of attached and detached purification, the number ofcells reached about112.2±3.6×10~4per dish and Schwann cell purity of more than98.49±0.94%was achieved in experimental group.The finally SCs were all immuno-positivefor P75NTR but contaminated fibroblasts were negative for the staining. The flowcytometricresults showed that P75NTR positive cells accounted for99.87%in experimental group. Theresults were consistent with the above morphology calculation under phase-contrastmicroscopy. There was a significant difference in the cell yield and Schwann cell puritybetween experimental group and control group(p <0.01).(3)The values of SCs in theco-culture groups were significantly higher than values of control group. The Western blotindicated that NGF was expressed obviously in the co-cultured group and was less visible inthe control cells. The expressions of BDNF in the cocultured SCs were much stronger than inthe controls.Conclusion:(1) HWJ-MSCs not only contain a plenty of neurotrophic factors, but alsocan significantly promote SCs secrete neurotrophic factors when they are co-cultured.(2)Alarge number of purified Schwann cells can be obtained by our simple, fast and safe method within only96h, and it might be valuable for studies related to basic science of peripheralnerve injury and regeneration.Our results help to elucidate the mechanisms by whichHWJ-MSCs function as a cell therapy agent in peripheral nerve repair.

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CLC: > Medicine, health > Surgery > Of surgery > Head and Neurosurgery > Peripheral nerve
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