| Summary: | 碩士 === 中原大學 === 奈米科技碩士學位學程 === 101 === Abstract
Mesenchymal stem cells (MSCs) play an important role in tissue regeneration which involves the processes of mobilization of stem cells from the bone marrow, homing of these cells to the site of injury, and differentiation of the stem cell into a functional cell of the injured tissue. Stroma-derived factor-1α (SDF-1α) plays a critical role in stem cell migration towards areas of tissue injury and hypoxia. The aim of this study was to examine the influence of low level light irradiation (LLLI) using red and near infrared (NIR) at energy dose on 4 J/cm2 on migration of rat bone marrow MSCs in the absence and presence of SDF-1α (SDF). Results of transwell migration assay showed that the migration of MSCs after Red and NIR light irradiation was greater than control. The migration was further enhanced with SDF-1α treatment. The gene and protein expression of C-X-C chemokine receptor type 4 (CXCR4) and phosphorylation of focal adhesion kinase (FAK) were also enhanced. The expression of FAK and CXCR4 was inhibited by transfecting MSCs with FAK and CXCR4 small interfering RNA (siRNA). MSCs transfected with FAK siRNA and CXCR4 siRNA exhibited decreased cell migration by 41% and 58%, respectively. LLLI or SDF treatment could not recover cell migration capability to the level comparable to that of untransfected control group. MSCs transfected with FAK siRNA and CXCR4 siRNA exhibited decreased pFAK expression by 36% and 78%, respectively. The expression of pFAK was also inhibited in the groups of Red, NIR, SDF, SDF + Red, and SDF + NIR. However, the level of CXCR4 expression was not affected by FAK siRNA transfection, LLLI or SDF treatment. The level of phospho-Nuclear factor-κappa B (pNF-κB) p65 expression was found to increase as shown by western blotting, and nuclear translocation of pNF-κB was observed in the groups of Red, NIR, SDF, SDF + Red, and SDF + NIR, especially in SDF + NIR group. LLLI or SDF treatment did not influence pNF-κB expression in FAK siRNA transfected MSCs. But pNF-κB expression was inhibited in all treatment groups of CXCR4 siRNA transfected cells. As demonstrated by gelatin zymography and quantitative real-time polymerase chain reaction (QPCR) analysis, the activity and gene expression matrix metalloproteinase (MMP)-2 were both elevated in the groups of Red, NIR, SDF, SDF + Red, and SDF + NIR. The levels of MMP-9 and MMP-14 expression of SDF, SDF + Red, and SDF + NIR groups were higher than that of control group, whereas the level of tissue inhibitor of matrix metalloproteinase (TIMP)-2 was lower than that of control group. MSCs transfected with FAK siRNA and CXCR4 siRNA exhibited decreased MMP-2 activity by 36% and 78%, respectively. LLLI or SDF treatment further suppressed MMP-2 activity, especially in SDF + NIR group. β1-integrin expression was upregulated in LLLI and SDF treatment but inhibited by CXCR4 siRNA transfection. There was no significant difference of β1-integrin expression in FAK siRNA transfected cells. The results of fluorescence staining showed that F-actin polymerization accumulated in the cortex of MSCs as well as Rac/Cdc42 and Rho protein expression in Red, NIR, SDF, SDF + Red and SDF + NIR groups. Furthermore, F-actin polymerization was inhibited after transfection of FAK siRNA and CXCR4 siRNA. QPCR analysis found that the expression of HSP27 gene of SDF, SDF + Red and SDF + NIR group decreased by 37, 25 and 22 %, respectively. The expression of HSP90 gene of Red, NIR, SDF, SDF + Red and SDF + NIR group decreased by 62, 58, 58, 71 and 79 %, respectively, compared to that of control group. This suggests that decreased levels of HSP27 and HSP90 might help LLLI and SDF-1α induced cell migration. In summary, LLLI with red and NIR light and SDF-1α treatment could stimulate MSCs motility in vitro through CXCR4 activation, FAK and NF-κB phosphorylation, increasing MMPs secretion, and upregulating F-actin polymerization as well as Rac/Cdc42 and Rho protein expression.
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