| Part one:Evolving trends of indications and surgical techniques for keratoplasty in Chinese PLA General Hospital during the last ten yearsObjective:To report the evolving of the indications for keratoplasty and the shift of the type of keratoplasty performed in Chinese PLA General Hospital from 2004 to 2015.Methods:A retrospective data review was carried out to all the records of keratoplasty in XX Hospital from January 2004 to December 2015,and patient's demographics,indications and types of transplantation performed were analyzed.Results:Toally 721 keratoplasties were performed on 631 patients.Four hundred and thirty patients(68.15%)were male(489 eyes),and 201 cases(31.85%)were female(232 eyes).Keratoleukoma(135 cases,18.72%),regraft(122 cases,16.92%),purulent keratitis(90 cases,12.48%)and keratoconus(89 cases,12.34%)were the leading indications for keratoplasty,and transplantations for purulent keratitis,regraft and keratoconus increased year by year.In 2004,keratoplasties were performed in 3 cases of purulent keratitis,9 cases of regraft and 3 cases of keratoconus,and the frequencies reached to 16,15 and 20 respectively in 2015.Of all keratoplasties performed,414(57.42%)were penetrating keratoplasty(PKP),161(22.33%)were deep lamellar keratoplasty(DLKP),134(18.59%)were lamellar keratoplasty(LKP),and 12(1.66%)were endothelia keratoplasty(EKP).DLKP(2010,n=6;2015,n=20)and LKP(2010,n=8;2015,n=18)increased in recent 5 years,while PKP had been decreasing since 2012(2012,n=60;2015,n=34).Conclusion:The keratoleukoma,regraft,purulent keratitis and keratoconus were the top 4 indications for keratoplasty.DLKP demonstrated an increasing trend while PKP showed a decrease.EKP has been applied in clinical practice.Part two:Preliminary research of subconjunctivally injection of MSCs during the acute stage of cornea alkali burn.Objective:MSC-based therapy have been proposed for the repair of tissue damage,autoimmune diseases,solid organs transplantation and the treatment of metabolic diseases.Moreover,MSCs are promising for the treatment of cornea injury and limbus stem cells shortage.The aim of this study was to investigate the effects of subconjunctivally administered MSCs on corneal wound during the acute stage of an alkali burn.Methods:We builted 18 models of alkali burn rabbits using lmmol/L NAOH.The rabbits reiceived a subconjunctival injection of(i)BMSCs(2 ×106/0.1ml),(ii)0.1ml HBSS or(iii)without any treatment immediately after injury.The deficiencies of the corneal epithelium,histological and molecular biological assays with PCR were performed toevaluate the inflammation and the effect of MSCs treatment.Resuts:We found that the inflammatory infiltration and the swelling were significantly ameliorated in cornea treated with BMSCs.Furthermore,the levels of TNF-?,IL-1?,MMP-9,MMP-2 were down-regulated by MSCs.However,Corneal levels of VEGF and TSG-6 were up-regulated in BMSCs-treated rabbit.Conclusion:The subconjunctivally administered BMSCs could reduce inflammatory reaction in the cornea and significantly acceleratecorneal healing by prohibiting the secretion of pro-inflammatory cytokine and promoting the secretion of anti-inflammatory cytokine.Part three:The isolation and identification BMSC-derived exosomesObjective:MSC-based therapies have been widely applied in clinical trials.Theeffectiveness of MSC-based therapies is largely attributed to the function of paracrine secretion,particularly the exosomes.The aim of this study was to isolate and identify the exosomes.Methods:Exosomes were isolated from BMSC-CM and purified.The protocol inculded several centrifugations to remove dead cells and cell debris.Then the supernatants were ultracentrifugatd to discard protein and to harvest the exosomes.Results:Exosomes were observed by transmission electron micrographs.Their diameter ranged from 30 to 150nm.Western blot found these exosomes expressed CD9 and CD81.Conclusion:The exosomes from BMSC-CM can be isolated by ultracentrifugation.These exosomes expressed typical indicators such as CD9 and CD81. |
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