Basic And Clinical Application Of H-1α Gene Modified Adipose Stem Cells To Construct Vascularized Tissue Engineering Fat Flap

Objective:To explore the ideal way to achieve the effect of CD54+/rASCs carrier complex in vivo on adipocytes and the transfection of fat stem cells(Adipose-derived stem based on Hypoxia-inducible factor-1α(HIF-1α)gene cells,ADSCs)suitable technical methods for constructing vascularized tissue-engineered fat flaps,which provide experimental basis and theoretical support for the current clinical transplantation of autologous adipose tissue and adipose stem cells to repair soft tissue organ defects and breast reconstruction after breast cancer surgery.Methods:1.In vitro cell experiment.Separate and purify the inguinal subcutaneous adipose tissue of adult New Zealand white rabbits,and take the third-generation rabbit adipose stem cells(rASCs)with good growth for digestion,then proceed into adipogenesis,osteogenesis and Induce differentiation into cartilage.Using immunomagnetic beads to sort out rASCs that highly express CD54+and induce adipogenic differentiation.Recombinant lentiviral vector-mediated green immunofluorescent protein labeled rASCs in vitro,Ad-HIF-1α transfected CD54+/rASCs in vitro according to the MOI value determined in the experiment,to obtain HIF-1α highly expressed seed cells(HIF-1α/CD54+/rASCs)and in vitro amplification.2.In vivo animal transplantation experiment.The third-generation CD54+/rASCs cells were mixed with the scaffold and the scaffold biocompatibility was tested.The prepared cell scaffolds were divided into four groups,Group A:HIF-1α/CD54+/rASCs,implanted in the left anterior back muscle fascia of rabbit;Group B:CD54+/rASCs,implanted in the right anterior back muscle of rabbit Intramembrane;Group C:HIF-1α/CD54-/rASCs,implanted in the rabbit right posterior back muscle fascia;Group D:CD54-/rASCs,implanted in the rabbit left posterior back subcutaneous muscle fascia,12 weeks later,samples were taken to obtain graft tissue for histological,gene,and protein detection.3.Clinical application and curative effect evaluation.Combination treatment of fat-derived stromal cell gel(Stromal Vascular Fraction Gel,SVFG)and autologous platelet-rich fibrin gel(PRFG),and both with autostructural granulated fat the efficacy and the postoperative complications of mixed transplantation were evaluated for safety and effectiveness,and compared with cases of traditional treatment methods.Results:1.The primary rASCs isolated and digested by type Ⅰ collagenase can grow adherently and form typical fusiform cells.RASCs can induce multi-directional differentiation of adipogenesis,osteogenesis and chondrogenesis.High-purity CD54+/rASCs were obtained from the first generation of rASCs by magnetic bead sorting,and the positive expression rate of CD54 was as high as 99.2%.After sorting,they were passed to the third generation of CD54+/rASCs to induce adipogenesis for 2 weeks.Fat droplets aggregated and oil red O staining was positive;the analysis showed that CD54+/rASCs,rASCs and CD54-/rASCs induced fat formation after 2 weeks,and the density of mature adipocytes was(39.60±4.33)cell/mm2,(18.63±2.78)cell/mm2,(7.20±1.91)cell/mm2,the lipid droplet content is(3.82±0.55)U,(1.54±0.31)U,(0.65±0.20)U;the differences are statistically significant(P<0.01).2.The rASCs transfected with the immunofluorescence protein lentivirus carrying the HIF-1α gene maintain a good normal shape,and the cells are stable and can be passaged continuously.Transfect CD54+/rASCs with a complex infection value of 50 and continue to culture for 3d and 7d.The content of cytokines or growth factors in the culture supernatant detected by enzyme-linked immunosorbent assay was significantly increased.(*P<0.05,**P<0.05).3.The results showed that the wet weight,mature fat cell number,intracellular lipid droplet content,and microvessel number of the neonatal tissue in the four groups were statistically analyzed.The difference between the group A and the groups B,C,and D was significant(P<0.01),group B and group C,group B and group D,group C and group D,the difference is significant(P<0.01).Using q-PCR and immunoblotting techniques to quantitatively analyze the cytokines or growth factors in the four groups of new tissue flaps,the mRNA and protein were highly expressed in groups A and C,and low in groups B and D.Group A was compared with groups B and D,group C was compared with groups B and D,and the difference was significant(*P<0.01,#P<0.01);mRNA test results of adipogenic differentiation-related gene PPARy showed that group B was high expression,but low expression in group A and group C,group B compared with groups A,C,D,group A compared with groups B and D,group C compared with groups B and D,the difference has significant significance;The results of mRNA detection of adipogenic differentiation-related genes C/EBPαand ADD1 showed that groups A,B,and C were all highly expressed,compared with the control group(group D),and group A was compared with the three groups B,C,andD.All have significant significance(*P<0.01,**P<0.01,#P<0.01).4.The clinical results show that SVFG combined with PRFG can treat various refractory wounds.They significantly promote the healing speed,improve the quality of healing,and reduce the incidence of dysfunction due to scar hyperplasia after wound healing.In the treatment of facial soft tissues the concave deformity can not only significantly improve the survival rate and long-term efficacy of granular adipose tissue,reduce the incidence of postoperative complications such as liquefaction necrosis,cysts,and nodules,but also greatly improve the patient’s cosmetic effect and satisfaction.Breast reconstruction after breast cancer,breast dysplasia or postpartum breast atrophy and deformation can significantly improve the survival rate of granular adipose tissue and reduce the incidence of complications such as liquefaction necrosis,cysts and nodules.Conclusions:HIF-1α gene-modified CD54+/rASCs combined with human-like collagen scaffold can construct a larger volume of vascularized tissue engineering fat flap,which is widely used in tissue engineering,regenerative medicine research and clinical application of fat stem cells promote wound repair,tissue regeneration,organ reconstruction to provide a reliable theoretical basis.