The Experimental Research Of Sucrose Protective Effect On Rabbit Corneal Endothelium During Vacuum Freeze-drying

PrefaceFreeze - drying is defined that the water of cornea is freezed, then sublimated under vacuum pressure. Freeze - dried cornea cant be oxidized and contaminated , it can be preserved under 4t without consuming lots of energy, short distance conveniently transported under environmental temperature.There are a lot of factors that affect the quality, such as protective agent, freezing temperature, freezing rate, freeze - drying conditions. That most important factors of them is cryoprotective agent, has already confirmed by internal and external reserachs.The damage of freeze - drying cell can be avoided in great degree by using suitable protective agent. To date, people has discovered a-bout more than forty kinds of low temperature protective agent, which are divided into extracellular protective agent and intracellular protective agent according to penetrating cell membrane ability. Other factors affect freeze - drying quality are fixed, the protective effect of sucrose on freeze - dried rabbit cornea is explored and the mechanism is analyzed by adding variable concentration sucrose to constant DMSO and HSA in this paper.Experimental materials and methods. Experimental materials1. sucrose: analytical simple produced by Shenyang the third reagent factory.2. DMSO: SIGNA CHMICAL CO. P. 0 GERMANY.3. 20% HAS Shanghai RAAS Blood Products CO. Ltd.4. experimental reagents: alizarin produced in American, departed load in Shanghai; trypan blue produced in American, departed load in Shanghai.5. experimental animals: 2 -3Kg albino rabbits.6. experimental instruments; extra clean workingtable, maded by Beijing the first semiconductor equipment factory; electron balance, type; AP250D; OLYMPUS optics microscope; ultra low temperature measure instrument; foil resistance:Pt -100; freeze-drying machinery: ZLG - 0. 05 , produced by northeast university and Shenyang three loop vacuum factory; refrigeration ; Hquid nitrogen, refrigerator; simple controlled rate freezer.Experimental methods1. Making up protective agent: the protective agent consists of three ingredients, 20% HSA, DMSO, and sucrose, the number of control group, experimental group I^IINIII are 60 shares respectively, and each share is 2.5ml. 30 shares are used in freezing experiment, and 30 shares are used in freeze - drying experiment.2. Experimental groups; Capella four steps freezing protective a-gent is used as the control group in the experiment.Fig 1 the protective agent composition of control groupProtective agentControl group (60 shares, each is 2. 5ml)Number 1Number 2Number 3Number 420% HSA2.45ml2.40ml2.35ml2.3125mlDMSO0.05mlO.lml0.15ml0. 1875mlSucrose g/v2.5%5%7.5%10%Fig 2 the protective agent composition of experimental group IProtective agentExperimental group I (60 shares, each is 2. 5ml)Number 1Number 2Number 3Number 420% HSA2.45ml2.40ml2.35ml2.3125mlDMSO0.05mlO.lml0.15ml0. 1875mlSucrose0000Fig 3 the protective agent composition of experimental group IIProtective agentExperimental group E (60 shares, each is 2. 5ml)Number 1Number 2Number 3Number 420% HSA2.45ml2.40ml2.35ml2.3125mlDMSO0.05mlO.lml0. 15ml0. 1875mlSucrose g/v7.5%15%22. 5%30%Fig 4 the protective agent composition of experimental group IIIProtective agentExperimental group Iff (60 shares, each is 2.5ml)Number 1Number 2Number 3Number 420%'HSA2.45ml2.40ml2.35ml2.3125mlDMSO0.05ml ?O.lml0.15ml0. 1875mlSucrose g/v25%50%75%100%3. Experiment approach: freeze - drying consists of freezing and drying. Firstly, the protective effect of sucrose on endothelium is studied on the freezing stage. Secondly, the protective effect of sucrose on endothelium is studied on freeze - drying stage.3.1 Freezing experiment3. 1. 1 The protectant component; the number of protective a-gent in control group ^ experimental group I ,JI^ III is 30 share respectively, and each share i...