| Objective1. To find a new type of corneal perfusion culture system, thus improve the survivable environment of keeping cornea in the traditional organ culture and predict the feasibility in cornea preservation.2. To find how this new type of perfusion culture system affect the survival environ- ment of keeping hare cornea in traditional organ culture system and the activity of corneal endothelial cells, and detect the application in cornea preservation.3. Do research on PKP using hare cornea kept by this culturing system, and evaluate the reliability on keeping activity of corneal endothelial cells for a long time.Methods1. Design a culturing devices with a pedestal and cornea fixing structure, and build up a cornea perfusion culture system and animal mordal using a precise peristaltic pump. According to the culturing speed, 20 hare corneas were put into 4 groups randomly and were cultured for 4 weeks. Detect the pH value, glucose, lactic acid and microorganisms after 4 weeks. Using trypan blue associated with chinalizarin vital staining to calculate the CEC density of corneas in all groups, and observe the affect of different culturing speed towards cornea keeping to choose the best culturing speed.2. 60 rabbit cornea were randomly divided into three groups by perfusion culture system, as well as the traditional liquid confined and save regularly. Each group was subdivided into four subgroups by the time of preservation (1 weeks, 2 weeks, 3 weeks and 4 weeks) with each subgroup having five cornea. In addition two conrea were kept with perfusion culture system for 2 month. After preservation detect the pH value, glucose, lactic acid and microorganisms.All corneal buttons were examined by trypan blue combined with alizarin red staining and calculate the CEC density, the death rate and percentage of hexagonal cells. Sample from the longest time vibration group was examined by Na+-K+ATPase and SDH enzymohistochemical staining and transmission electron microscopy.3. 15 New Zealand rabbits were obtained with 5 (10 corneas) as donors and 10 as receptors.According to culturing item, donor corneas were divided into 2 groups( 4 weeks and 8 weeks culturing, 5 corneas each group). PKP were done in two groups. The thickness and clarity of grafts were measured and observed after PKP in different periods. The viability of grafts CEC was examined by trypan blue combined with alizarin red staining at the 30th days after PKP.Results1. Through perfused cultured,in the groups of the animal models constructed by laboratory, except the major component of group after perfused with flow rate of 5ul/min changed obviously,there were no significant deviation among the other 3 groups. Consided the effect and the dose of culture solution generally,the flow rate of 10ul/min was the best choice.2. We find,compared with different conserving methods,perfused culture system could provide more stable culture environment,and could prevent pollution effectively. The cornea kept By this perfused culture system had higher CEC density, more complete histologic appearance and ultramicrostructure.3. The rabbit cornea were subjected to PKP after perfusion culture. Examinations showed that thickness of grafts from each group diminished gradually. The implanted buttons remained transparent 30 days later after operation. The results of viability staining showed that CEC had high rate of vital endothelium and normal shape cell.Rate of transparence, thickness, vital endothelium of CEC between two group were not significantly different.Conclusions 1. Research results show that the corneal perfusion culture system is reliable.Its has the feasibility in the long-term preservation of the CEC.2. Animal model results show that perfusion can optimize training environment in corneal organ culture.This system can preserve CEC activity for a long time. Perfusion culture has more advantages.3. The animal experiments showed that perfusion culture can ensure the long-term preservation of the cornea.The effect of PKP is very definitely.
|
PDF Full Text Request