| Spinal cord injury (SCI) is a common disease which may lead disorders of somatesthesia and locomotion, paralysis and even death. For elder people, it is more likely to result from degeneration of intervertebral disc, but for younger people, it is more likely to result from violence or accidents.As the ability of spinal cord’s renewal is very limited, the results of SCI treatment are far from satisfactory. Nucleus pulposus in intervertebral disc mainly consists of chondrocytes and extracellular matrix such as type Ⅱ collagen. Like neurocytes, chondrocytes also has limited ability of self-proliferation. After intervertebral disc degenerates, it may constrict spinal cord. Surgical treatment, as a major treatment for SCI nowadays, still cannot be regarded as an ideal solution. Hence, the transplantation of stem cells as a new and promising treatment for SCI, draws more and more attention. Neural stem cells(NSCs) bear characteristics for proliferation and differentiation into three main neural cell type(s):neurons, astrocytes and/or oligodendrocytes, so NSCs can be the candidate of cell transplantation therapy in SCI。BMSCs can differentiate into chondrocytes and therefore it can be used on recovery of intervertebral disc degeneration.However, many uncertain factors could affect the outcome of stem cells’proliferation, differentiation and migration and consequently influence the results of the treatment. Our study explored Fprs effects on NSCs and Collagen Ⅱ effects on BMSCs, and hope that could provide new candidates for cell replacement therapies to cure SCI.Part 1. The roles of formylpeptide receptors in migration and differentiation of rat neural stem cells in vitroNeural stem cells (NSCs) bear characteristics for proliferation, migration and differentiation into three main neural cell type(s):neurons, astrocytes and/or oligodendrocytes. The expression of formylpeptide receptors (Fprs), belonging to the family of G protein-coupled receptors, have been detected in neurons in the central nervous system (CNS). Here, we report that Fpr1 and Fpr2 are expressed in NSCs tested with immunohistochemistry and WB assays. In addition, we preliminarily explored Fpr effects on NSCs. The data indicated that Fprl and Fpr2 could promote NSC migration and enhance neuronal specification. The results of this study strengthens understanding of Fpr1 and Fpr2 in NSCs as a promising candidate for activating endogenous NSC migration and differentiation, and open a unique window for cell replacement therapies to cure brain or spinal cord injury.Objective:To investigate the roles of formylpeptide receptors in migration, proliferation and differentiation of rat neural stem cells in vitro.Methods:NSCs, seeded with Fpr1’s agonist/antagonist, Fpr2’s agonist/antagonistand control group, were assessed via CCK8 assay to detect the proliferation of NSCs. Western Blots and immunofluorescence were used to detect the protein expression of DCX, GFAP and Olig2 in different intervention group. Morphological observation and transwell assay were used to detect the migration of NSCs in vitro.Results:(1) Both Fpr1 and Fpr2 are expressed in NSCs, (2) Fpr1 and Fpr2 promote Neuronal differentiation of NSCs; (3) Fpr1 and Fpr2 enhance NSC migration, (4)Fpr1 and Fpr2 have no effect on proliferation of NSCs.Part 2. Collagen Ⅱ promotes rat bone marrow mesenchymal stem cells differentiation into chondrocytes in vitroThe main reasons of intervertebral disc degeneration are cartilage extracellular matrix’s lossing and cellular degeneration. With the increase of the ages, the probability of getting cartilage injury also increased. Various factors such as bad living habits and metabolic disorder cause the injury. Moreover, mechanical agents such as trauma, disarticulation and joint instability may result in the injury. Unfortunately, once cartilage is injured, its ability of reborn and recovery are very limited due to lack of direct blood circulation.There are various treatments. The purposes of the treatments focus on maintaining basic function of cartilage and reducing pain. Most treatments are limited by their consequential side effects. Hence, the massive use of those treatments can hardly realize. In recent years, more and more attentions has been paid to tissue engineering technology, as it has made great progress on medical research and clinic application. Evidences show that Collagen Ⅱ could promote the BMSC differentiation into osteocytes in vivo and vitro. We preliminarily explored Collagen Ⅱ effects on BMSCs, which could promote BMSC differentiation into chondrocytes. We hope that our study in Collagen Ⅱ as biomimetic materials could cure the injury of cartilage.Objective:To investigate the promotion of type Ⅱ collagen (Collagen Ⅱ) on differentiation of rat bone marrow mesenchymal stem cells (BMSCs) into chondrocytes.Methods:BMSCs, seeded with different concentrations of type Ⅱ collagen (25jig/ml, 50μg/ml, 100μg/ml,200μg/ml) and control group, were assessed via CCK8 assay on day 7, 14 and 21 in vitro. qPCR and immunofluorescence were used to detect the mRNA and protein expression of Collagen Ⅱ, SOX9 and Aggrecan in different intervention group.Results:(1) CCK8 assay showed that the absorbance values at wavelength of 450nm had no significance on day 7,14 and 21 in different groups(p>0.05); (2) The mRNA expression of Collagen Ⅱ, SOX9 and Aggrecan was upregulated on day 21 (p<0.05), especially the group of 100μg/ml was obviously changed (p<0.01), while the mRNA expression in group of 100μg/ml was highly upregulated on day 14, but there was no significance on day 7, compared with control group; (3) immunofluorescence results show that the protein of Collagen Ⅱ, SOX9 and Aggrecan in group of 100μg/ml was obviously upregulated, compared with other groups (p<0.05).Conclusions:(1) Both Fprl and Fpr2 are expressed in NSCs, and both of them could promote Neuronal differentiation and enhance migration of NSCs. (2) Type Ⅱ collagen could promote rat bone marrow mesenchymal stem cells (BMSCs) into chondrocytes in vitro, and the group of 100μg/ml bears the best efficiency. |
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