Stromal Cell-derived Factor 1 Promoted Migration Of Adipose-derived Stem Cells To The Wounded Area

Backgroud:Tissue defect is a widely clinical phenomenon mainly caused by tumor resection, deep burn and radiation therapy. Currently, the mainstreams of repair proposal are used in clinic but with obvious flaws. For example, wound covering combined with cytokines is unsatisfied for its limited curative effect as well as high medical costs. Tissue flaps transferred to the wound are prone to induction of secondary tissue injury and cicatrices, while prosthetic filling usually leads a complication of spasm. Presently, it is well known that repair in trauma are mainly affected by stem cells (SCs). SCs are multi-potent cell type that enables them to readily differentiate into several different cell types (osteoblasts, chondrocytes, adipocytes and myocytes) under specific culture conditions. To date, SCs have been widely used in treatment of several diseases. Adipose-derived stem cells (ADSCs) are the most commonly used SCs, mainly because of easy and frequently employed acquisition procedure. ADSCs therapy has been made in the treating wound injury successfully. In 2012, CoUawn SS et al confirmed ADSCs accelerate wound healing in an organotypic raft culture model, and later, the other study also concluded that ADSCs transplantation promotes regeneration of expanded skin. It is well accepted that transferred process of the cells to cutaneous wound involved migration,of ADSCs from normal residential area to circulatory blood, the adhesion to endothelial cells near the injured area oriented by chemotactic molecules, and the adhesive cells transfer through vessel wall to reach the injured tissue. As we known, the efficacy of extraneous ADSCs is mainly affected by its delivery routes as well as final distribution.In comparison with subcutaneous, intravenous and intramuscular injection, topical administration of ADSCs has several drawbacks, such as complex process of injection and deficient cell activity induced by initiative environment. In 2001, Lee K et al confirmed transfer of mesenchymal stem cells (MSC) to NOD/SCID mice by subcutaneous injection had a better transfer efficiency than those of intramuscular injection and intravenous injection (IV). Additionally, Baug F et al noticed IV and intraperitoneal injection (IP) MSC transplantation exerted positive effects on UFF in a chronic rat model of PD. However, healing effect of small solute transport in MSC-IP group was better than MSC-FV group. Recently, several papers suggested intra-arterial (LA) of SCs was an alternative way for SCs transplantation. Du S et al noticed LA route is a safe and effective way to deliver hBMSCs. Makela T further disclosed SCs delivered by IV were almost uptake and captured by lung, while IA administration appears to have the potential to allow more cells to reach systemic vasculature and target organs of cellular therapy, such as the brain, heart and kidneys. Therefore, in this paper, IA was used to deliver ADSCs. And we want to find a way to improve the migratory efficiency of ADSCs to home to the wounded area. A number of studies have suggested SCs migration and distribution are controlled and regulated by chemokines and its mediated-signaling pathways.Stromal cell-derived factor 1 (SDF-1) is produced by stromal cells and usually called C-X-C motif chemokine 12 (CXCL12). SDF-1 is a potent chemotactic cytokine with various biological functions such as stem cell mobilization, inflammatory cell infiltration and angiogenesis. Previous studies pointed out SDF-1 mediated trafficking and homing of endothelial progenitor cells to injury microenvironment, and increased expression of SDF-1 at wound margin contributed to accumulation of endothelial progenitor cells and acceleration of neovascularization. Importantly, the later published papers demonstrated that systemically transplanted ADSCs homed to the injured liver after transplantation, possibly based on the mechanisms of SDF-1/CXCR-4 axis,and analogically, combination of SDF-1 with collagen-glycosaminoglycan scaffold accelerates re-epithelialization of dermal wounds in wild-type mice. These results indicate curative effects of ADSCs on wound are affected and controlled by SDF-1. However, the detailed reason as well as its mechanism is still unclear. Here, we firstly showed migratory and distributional process of ADSCs in wound repair is promoted by SDF-1.Objective:The purpose of our research is to verify whether SDF-1 has a positive adjustment in migration of ADSCs to the wounded area. And we want to show to whether it is a feasible way to improve the migratory efficiency of ADSCs homing to the wounded area through regulating the local concentration of SDF-1.To find a new strategy to fully mobilize ADSCs for the benefit of wound-repair at large. It would provide important clinic direction for stem cell therapy, worthing the further research.Method:1.ADSCs of SD rats were isolated by collagenase I digestive method, and then cultured with DMEM/F12 containing 10% fetal bovine serum.The cells morphological characteristics was observed by inverted microscopy. The cell surface antigens(CD29,CD34,CD45 and CD90) were detected by the flow cytometer. ADSCs were induced to differentiate into adipose cells and osteoblasts, in order to identify the multiple differentiation potential of ADSCs.2. Lentiviral vectors encoding a gene for the enhanced green fluorescent protein (lentivirus-eGFP) were used to transfect the P3 ADSCs with MOI=40. 48h,72h,72h,96h after transtection, eGFP expression was observed by fluorescence microscope. And one week after transtection, cell transfection rate was measured by flow cytometry technology. eGFP-ADSCs were cultured and passaged 9 times, eGFP expression was observed by fluorescence microscope. Cell activity of eGFP-ADSCs was detected by MTT method. eGFP-ADSCs were induced to differentiate into adipose cells and osteoblasts, in order to identify the multiple differentiation potential of eGFP-ADSCs.3. P3 ADSCs were cultured with complete medium containing 500ug/L SDF-1 for 2 days. After that, the expression of SDF-1 receptor (CXCR4,CXCR7) was detected by western blot and flow cytometer. Then, compare the treated group with the nomal group.4. Transwell migration assays was used to examine the effect of SDF-1 on ADSCs migration. We set a series of SDF-1 concentration (0ug/L,1ug/L,10ug/L,100ug/L), to check the correlation between ADSCs migratory efficiency and the concentration of SDF-1.5. Rat dorsal full-thickness skin defects were made and randomly divided into three groups:Group 1 (traumatic rats) received intradermal injection of SDF-1 in wound sites at Id,2d,3d, while Group 2(traumatic rats) and Group 3(normal rats) received PBS with the same manner. Meanwhile, all Groups were treated with eGFP-ADSCs by carotid artery injection. The levels of SDF-1 in traumatic tissues were assayed by ELISA. Also, the eGFP gene expression was detected by RT-PCR. The eGFP-ADSCs distributed in injured tissue were observed by laser confocal microscopy. The plasma levels of eGFP-ADSCs dujing wound healing were detected by flow cytometry.6. The wounds were observed during the experiment. The rates of wound closure were calculated on ld,3d,7d,14d,21d post operation. The expression of CD31 was assayed in traumatic tissues by immunohistochemical staining between two groups to evaluate the microvessel density.Result:1. ADSCs were fusiform or polygon observed by inverted microscopy. ADSCs tested positive for the homogeneous markers of surface proteins CD29, CD90; negative for hematopoietic lineage marker of CD45 and CD34. Oil red O staining demonstrated positive reaction after 14 days adipogenic induction, alizarin red staining also demonstrated positive reaction after 28 days osteogenic induction. It proved that ADSCs had the ability of multipotent differentiation.2. The lentivirus-eGFP infected P3 ADSCs expressed strong green fluorescent after 96 hours, then maintained at a higher level. The eGFP-ADSCs grew well after 9 times of passages, and steadily expressed green fluorescent protein. One week after infection with lentivirus, the cells were tested by FCM, which showed a high positive rate above 80%.The outcomes of MTT value suggested that the difference of proliferation activity have no statistics significance between eGFP -ADSCs and nomal ADSCs. Oil red staining and alizarin red staining both demonstrated positive after induced differentiations. It proved that infection did not affect the multipotent differentiation ability of ADSCs.3. Data from FCM showed that after processing 3 times, ADSCs almost negative for CXCR4 or CXCR7. However, two days after treated with SDF-1, both CXCR4 and CXCR7 expression was significantly upregulated in P3 ADSCs. Western blot analysis showed the similar results. It proved that SDF-1 stimulated both CXCR4 and CXCR7 expression in ADSCs.4. In the transwell migration assay, four different concentration of SDF-1 (0 ug/1,1 ug/1,10 ug/1, and100 ug/l)induced the migration of ADSCs by 9.77%> 13.45%> 28.14%、49.14% respectively, in a concentration-dependent manner.5. The results of ELISA demonstrated that, SDF-1 is less expressed in normal skin tissue, but is more expressed in wounded tissue. SDF-1 level reached to a small peak value on 24h after injury and decreased during 24h-72h.Then the SDF-1 level increased continuously until attained maxium on the 7th day, and following gradually decreased. The SDF-1 level in the wounded tissue treated by SDF-1 increased steadily during ld-7d after wounded.6. To examine ADSCs alteration in injured tissue, a number of frozen sections of traumatic tissue were made at different time points after supplementation of SDF-1, and then was viewed with a confocal laser fluorescence. eGFP-ADSCs began to be found in the wounded tissue on the 1st day after cell transplantation. And the number of eGFP-ADSCs increased gradually as the time pass. We found that mainly distributed among epidermal tissue, around blood vessels and glands. The SDF-1-treated rats exhibited a significantly increased number of ADSCs in injured skin as compared with PBS-treated rats. RT-PCR analysis showed the similar results.7. In order to test effects of SDF-1 on migration of eGFP-ADSCs in circulation, the blood samples of injected rats were made at different time point, then the percentage of eGFP-ADSCs was assayed by flow cytometer, and the gene expression of eGFP was assayed by RT-PCR. The results showed that at each point during the wound healing, levels of GFP-ADSCs in the blood presents the following tendency:SDF-1 treated traumatic rats> traumatic rats>normal rats.8. After ADSCs transplantation, the healing time in SDF-1 treated traumatic rats were shorter than that in traumatic rats without SDF-1, and the healing rate was increased in SDF-1 treated traumatic rats on the 7m day,14m day after injury. Immunohistochemistry sections of traumatic tissue were made on the 7th day after injury. The result showed that the microvessel density of SDF-1 treated group were higher than the control group.Conclution1. ADSCs were transfected with lentiviral particles containing eGFP gene.Then, the cells were successfully marked by eGFP. eGFP-fluorescent-labeled cells were able to proliferate and differentiate, maintaining their eGFP expression and increasing their cell numbers. So, the eGFP-ADSCs Can meet the need of animal experiment.2. P3 ADSCs almost negative for CXCR4 or CXCR7. SDF-1 can stimulat both CXCR4 and CXCR7 expression in ADSCs.SDF-1 can promote ADSCs migration in vitro. The inducing effect was in a concentration-dependent manner.3. SDF-1 levels were up-regulated after injury. The mobilization and homing of ADSCs can be promoted by injury. What’s more, the effect can be reinforced by ectogenic SDF-1. The ADSCs we transplanted played a role in the wound healing,and SDF-1 can also play an important role in this process by recruiting ADSCs.