Study On The Effects Of Macrophage And GM-CSF On Rat DIEP Flap Survival

DIEP flap has been widely used in clinical as a representation of perforator flaps in recent decades.With the merit of no rectus abdominis muscle sacrifice,DIEP flap has become the first choice of therapy for autogenous tissue breast reconstruction over TRAM flap.The other merits of DIEP flap for breast reconstruction are ample tissue quantity,similar skin color and texture to the recipient area,satisfaction about the appearance,color and handle of the reconstructed breast.It is easy for the flap plasticity by long flap vessel pedicle.The caliber of the inferior epigastric artery is similar to the thoracodorsal artery and internal thoracic artery which makes it easy for vessel anastomose.The biggest advantage of DIEP breast reconstruction is the decrease of donor site complications.This technique preserves the rectus abdominis anterior sheath,reduces the incidence of postoperational abdomen wall pain, weakness,abdomen wall bulge and hernia and protect the donor site farthest.It can gain function recovery and cosmetic results and reduce the recovery time.Yet there are still complications with DIEP breast reconstruction.Choosing DIEP will increase the complexity of the operation and decrease the blood supply of the flap by cutting down the quantity of the rectus abdominis muscle perforator.It will increase the rate of flap partial necrosis and fat liquefaction in theory.It is still a puzzle in plastic surgery and oncologic surgery how to increase the survival rate of DIEP flap in breast reconstruction.Now the focus on the research about promoting flap survival is the study around VEGF and other growth factor such as bFGF,PDGF,TGF-β.It has been considered that VEGF is the most important vessel growth factor and significant in therapeutic vessel formation.But VEGF also plays important role in pathological vessel formation.The study of oncology indicates that VEGF is known as endothelium mitogen with most effectivity and high selectivity at present and the important regulatory factor in tomor vessel formation.It is important in growth and metastasis of many solid tumor.We think it is not appropriate to promote the flap survival of patients received breast reconstruction with breast cancer by gene therapy which conducts VEGF with potential ability of tumor growth and metastasis promotion. Recombined human Granulocyte-macrophage colony-stimulating factor has been used in clinic for years,it is routine therapy for breast cancer assistant chemotherapy. In clinical works,we found that the wound of poor healing in the patients whose leucocyte is lower than normal caused by chemotherapy would heal quickly after receiving rhGM-CSF therapy.So we assume whether monocyte or macrophage or GM-CSF can promote vessel formation of flap.Literatures indicate that more and more research is about GM-CSF promoting wound healing recently and most of them show positive results.But there are few research about applying GM-CSF directly on flap and observing its effect on flap survival.So we design DIEP model with GM-CSF macrophage and research its influence on flap survival.It has special significance in clinic.In th first part of these experiment,we investigate the structure character of rat abdominal flap and improve the havesting process of rat-DIEP flap.We choose the perforator on the layer of rectus abdominis anterior sheath and cut down other blood supply without anatomy of rectus abdominis muscle,and it can shorten the operation time and process more operation in one day.And this will help us degrade the operation,makes the model stable and reproducible.In the first part of the experiment we succeed building a stable rat-DIEP model and form a relatively easy and easy handled process.So rat-DIEP can be a routine model for flap survival research. After model formation,we can record the flap survival progress through general observation and histological examination with HE staining and measure the MVD variety of different time point through immunohistochemistry identified by VWF.We also can analyse the collagen quantity of flap with method of picric-sirius red polarized light.We can trace out the relationship between MVD and collagen quantity in different time during the rat-DIEP flap formation without intervention.The objective of the second part of experiment is to study the influence of GM-CSF and Mψto the survival rate of rat flap.We directly lavaged the rat's abdomen and gained macrophage after separation and purification.The model rats were divided into 4 groups,which were subcutaneous injection recombined rat GM-CSF group,GM-CSF combining adoptive rat Mψgroup,adoptive rat Mψgroup and saline group.The samples were collected the 7th day after operation,the flap survival area were measured,immunohistochemistry MVD were detected and collagen quantity were examined by Masson staining.Activated Mψcan excrete more than 100 kinds of substance including massive cellular factor which can induce vessel growth directly.In theory,Mψshould promote flap survival,yet in our experiment it shows no increase of flap survival ratio in adoptive rat Mψgroup.In the examination of flap MVD,it has no statistical significance between MψT group and control group. In the collagen quantity examination,we found it was lower in the Mψgroup than the control group(p＜0.05).The reason may be Mψover activate metalloproteinase to lead to prolong the collagen decomposition.On the contrary,the flap survival rate is higher in GM-CSF combining adoptive rat Mψgroup than the control group(p＜0.05) and its MVD and collagen quantity is higher than the control group(p＜0.001).The conclusion from the second part of the experiment is subcutaneous injection recombined rat GM-CSF and GM-CSF combining adoptive rat Mψcan elevate the survival rate of rat DIEP flap.adoptive rat Mψcannot elevate the survival rate of rat DIEP flap.Recombined rat GM-CSF and GM-CSF combining adoptive rat Mψcan distinctively elevate the MVD and collagen quantity of rat DIEP flap the 7th day after operation.The objective of the third part of the experiment is to explore the molecular mechanism of recombined rat GM-CSF and GM-CSF combining adoptive rat Mψpromoting rat DIEP flap survival.In the experiment,we test multiple gene expression in the flap we used in the second part with the technique of RT-PCR,including EMMPRIN,MMP2,TIMP1,CollagenⅠ,CollagenⅢ,EGF,TGF-β1,VEGF,EGFR and VEGFR et al.We compared their expression in the flap and discuss the mechanism of promoting flap survival by GM-CSF and GM-CSF combining adoptive rat Mψin gene level.The result showed EMMPRIN,MMP2,CollagenⅠ,EGF和EGFR expression was higher in GM-CSF group than the control group.EMMPRIN,MMP2,TIMP1,CollagenⅠ,CollagenⅢ,EGF,TGF-β1 and EGFR expression in combining GM-CSF+Mψgroup was higher than the control group.EMMPRIN,MMP2,TGF-β1 and EGF expression was high in Mψgroup than the control group. The upregulation the expression of these gene on the molecular level can explain why the flap survival rate was higher in the experiment group.In conclusion,we find rat DIEP model is a stable and reliable flap study model and recombined rat GM-CSF and GM-CSF combining adoptive rat Mψcan elevate the survival rate of rat DIEP flap.In further research,we find it can upregulate the some gene expression of the flap to promote vessel formation and collagen modeling, it lead to promote flap survival eventually.