Effect Of Periostin On Proliferation Of Keloid Mesenchymal Stem Cells In Vitro

BackgroundKeloid is the result of abnormal healing after skin damage,which results of excessive deposition of fiber matrix.It has following features: always growing out of the injury zone,growing as an invasive tumor and accompanied with other symptoms such as pain and itching,which seriously affects the appearance and causes serious physical and mental problems.At present,studies on the mechanism of keloid formation and development haven't got any breakthrough.Therefore,the effect of treatments such as surgical resection,drugs and radiation on keloid can not achieve the expected results.The high recurrence rate is a serious problem which is an urgent issue to be solved.In recent years,researches related to stem cells involves a wide range of fields.Due t o the multi-directional differentiation of stem cells,they can be induced to differentiate into the purpose cells under specific conditions in order to participate in wound repair,which makes it be a hot topic in clinical field.The previous researches have demenstrated that some stem cells involve in pathogenesis of keloid,furthermore,Moon et al [1]had isolated a kind of multipotent cells from keloid,named keloid-derived mesenchymal-like stem cells,and these cells can regulate the pathogenesis of keloid through self-renewal,cell proliferation and drug-resistance.Though Moon's research had proved that there are stem cells in keloid.But how they mediate the specific process of keloid formatio n is still unknown.Osteoblast-specific factor2(periostin)is an extracellular protein which has axial function in individual's development.Previous studies have found that local stromal periostin which is vital for maintaining the stemness of stem cells,once been blocked,the metastasis of tumor is inhibited.Previous studies found that the expression of periostin in keloid is significantly higher than that of normal skin,which might suggest that periostin is a kind of keloid-associated protein.Its ectopic expression can cause some biological behaviors of MSCs,such as migration,invasion and adhesion.Related studies on various tumors,such as gastric cancer,lung cancer and breast cancer,have demonstrated that there is close correlation between periostin and tumor malignance and metastasis.All of these results suggested that periostin is a key element in tumorgenesis.At the same time,studies have shown that periostin could promote the proliferation of adipose-derived stem cells,human limbal stem cells and human meural stem cells,inhibite cell apotosis and involve in canceration process of some tissues.However,whether periostin is able to regulate the proliferation of mesenchymal stem cells in keloid microenvironment and suppress the keloid pathogenesis is unclear.Therefore,explore the function and mechanism of periostin in keloid formation is in needed.ObjetiveTo explore the effect of periostin on proliferation of keloid mesenchymal stem cells in vitroMethods1.Clinical specimens selection criteria1)Age in 5-30 years old,no gender limitation.2)Clarly diagnosed as“keloid”,in our department.3)No diseases in cardiocascular system,repiratory systerm and so on.4)No fiber diseases in oter organ systern.5)First time to cure keloid.2.To establish keloid stem cells libraryThe sterile keloid tissues and corresponding normal skin obtained from Southwest Hospital were divided into two groups,namely “KELOID” and “SKIN”.Then keloid cells and normal cells were separated by using tissue culture method and digestion method under the same condition.After that,human mesenchymal stem cells specific medium was added in culture dish to end digestion and conduct cell culture.After cultured,we observed the morphology under the electronic microscope.Furthermore,we continued subculture by enzyme digestion method,and the third passage cells were identified by immunofluorescence and flow cytometry.Finally,the stem cells were sorted out by flow cytometry and then expanded.We used Western blot and immunohistochemistry technique to detect the difference of the expression of periostin between keloid and skin tissues.Furthermore,western blot,q PCR,and immunocytochemical assay were used to determine whether the expression in tissues was consistent with that in stem cells.Then we constructed the sh Periostin lentiviral vector and infected KMLSCs,suppressing the expression of periostin in KMLSCs.We used q PCR,Western blot assay to detect cell interference efficiency.Finally,the cell proliferation,cycle,apoptosis were determined by MTT and flow cytometry.The expression changes of the downstream related proteins were detected by cell immunofluorescence assay and laser confocal technology.Strength of cell proliferation,cycle,apoptosis and the changes of the downstream of related proteins expression were also conducted by above technology,then the data were integrated and analyzed to clarify Periostin's influence on keloid stem cell proliferation,as well as the relevant mechanisms.Results1.We had establish the keloid and keloid mesenchymal stem cells library successfully,and used flow cytometry to sort out the cell populations with CD73+,CD90+,CD105+?CD34-,CD45-from cultured cells of keloid and corresponding skin.The rate of KMLSCs was as high as 95.1% and the rate of SKPs was 94.9%,which was fully consistent with international criteria in mesenchymal stem cells.2.After isolation of stem cells,the results of immunofluorescent staining and western blot showed that the expression of periostin in KMLSCs was stronger than that in SKPs(P<0.05);3.Western blot and q PCR results showed that the expression of periostin gene was significantly interfered by sh Periostin lentiviral vector.4.After gene silence,the expression of periostin was obviously attenuated,.Low-level of periostin could inhibit the proliferation of KMLSCs by MTT assay,promote the apoptosis and extend the cell cycle significantly by flow cytometry.5.Silencing periostin gene in KMLSCs could downregulate the expression of LRP5 in Wnt signaling pathway.Conclusion1.There is high proportion of KMLSCs withCD73+,CD90+,CD105+,CD34-,CD45-in keloid microenvironment.3.Study on keloid stem cells proliferation in vitro2.The expression of periostin in KMLSCs is remarkably higher than that in SKPs.3.shPeriostin lentiviral vector can interfere the gene expression of periostin effectively.4.After silencing of periostin gene,the proliferative ability of KMLSCs become weaker,while stronger in cell apoptosis and longer in cell c ycle.5.Silence of periostin gene will attenuate the level of LRP5.