Study On The Source Of Cell Factors Of Promoting Vascularization In The Paracrine Mechanism Via Adipose-derived Cell Therapy

Objective：To investigate the origin of the angiogenic cytokines in angiogeniccell therapy through a nude mice random flap model. Provide a theoretical basis forclinical translational research of cell therapy.Methods:1. Cell culture and characterizationAdipose tissues were harvested from the inguinal fat pads of inbred male Wistarrats and digested with collagenase.Subconfluent ASCs of passage3were culturedwith specific induction media to demonstrate their multilineage differentiationcapacities toward osteogenic, adipogenic and chondrogenic lineages.2. Nude mice dorsal random pattern flap model.Dorsal random pattern flap was made on male nude mice. Briefly, a1cm×2.5cm rectangle random flap based cranially was elevated. Rat ASCs of1×107wereinjected subcutaneously on the flap after CM-Dil labeling. The flaps were thensutured back to their native configuration.3. investigation of the angiogenic cytokines in the flap.3.1cell sorting.At postoperative day1,3and7, the skin flap tissue were harvested, cut intosmall pieces and then digested with0.2%collagenase with vigorous shaking for anhour Cell pellet was resuspended and processed with FACS to sort out the engraftedDil-labelled ASCs from the flap tissueflaps of the control group were also digested inthe same manner3.2After cell sorting, the engrafted cells and the flap tissue cells were processedwith the quantitative real-time PCR assay respectively to determined the expressionlevel of several angiogenic factorsResults:1. Cultured rat ADSCs displayed a fibroblast-like morphology, with strongproliferative ability in vitro. After cultured in lineage-specific medium, adipogenic,chondrogenic differentiation could be observed by relevant histological stainings. 2. The flap survival rate of the the ADSCs treatment group was significantlyhigher than that in the control group, p <0.001, statistically significant.3. After surgery1、3and7days, the number of implanted cells (positive cells withfluorescently labeled) showed a clear downward trend.4. quantitative PCR results suggest that: cell therapy intervention flaps, theexpression levels of HIF-1α and its downstream molecules compared with the controlgroup, there is an increasing trend over time.Conclusion: The cytokines promote revascularization of injured tissues derivedfrom nude mice, but is not implanted in the ADSCs the secretion. Confirmed that theimplantation of the cells by up-regulating organization of the vascular factor level toindirectly accelerate angiogenesis.