| Objectives:1. To establish the methods of isolating and purifying tilapia hepatocytes.2. To investigate the immunoisolation effect of the microencapsulation in the hepatocytes xenotransplantation between donor-recipient combination of tilapia to rat.3. To observe the treatment effect of microencapsulated tilapia hepatocytes graft to rats with acute liver function failure.4. To approach the possibility of xenotransplantation between fish and mammal.Methods:1. Tilapia hepatocytes were isolated by the method of collagenase digestion, purified by the two-step low-speed centrifugation. The viability of cells were measured by Trypan-blue exclusion, and the morphology of tilapia hepatocytes were observed by inverted light microscope and the ultrastructure study of tilapia hepatocytes were observed by electron microscope. The hepatocytes with vitality rate exceeding 90% were encapsulated by alginate-chitosan-alginate membrane and grafted into rat intraperitoneal by means of injection.2. All the SD rats with acute liver function failure were induced by D-GalN.3. The ALF rats were divided randomly into 4 groups: microencapsulated tilapia hepatocytes group (microencapsulated group, n=35): injection of microencapsulated tilapia hepatocytes into rat abdominal cavity; free tilapia hepatocytes group(hepatocytes group, n=35) : injection of free tilapia hepatocytes into rat abdominal cavity; free capsule group(free group, n=28): injection of free microcapsules into rat abdominal cavity; normal saline group (NS group, n=28): injection of normal saline into rat abdominal cavity.4. One-week survival rate of the recipients was observed; the liver function of the recipients were tested and compared at 24 hours and 48 hours after transplantation; the pathological changes of grafts were checked by light microscope with HE staining at the 24h,48h,7d and 14d after transplantation respectively.Results:1. The mean viability of tilapia hepatocytes was 90.1±0.79% with the mean hepatocytes yield 0.93±0.22×10~8 cells per tilapia and the purity 89.75±1.92% as well. The fresh isolating hepatocytes were round and bigger than the other types of cells of liver. Under the light microscope, most of cells were translucent circular and in the similar sizes, few of them were gathered. Under the electron microscope, the membrane was covered by the abundant microvillus; there were many large round or oval mitochondria and glycogen granules in the cytoplasm; there were also existed many rough and smooth endoplasmic reticulum.2. The one-week survival rates of microencapsulated group was higher than that of hepatocytes group (57.9% vs 21.1%, P < 0.05); and also higher than that of free capsule group and of NS group(57.9% vs 16.7 %,16.7%, P < 0.05) respectively. There were no significant difference statistically among that of hepatocytes group,free capsule group and NS group(P>0.05).3. Recipient liver function examination at the 48h after transplantation:(1) ALT of microencapsulated group was lower than that of hepatocytes group (1103.9±132.4U/L vs 2188.3±185.4U/L, P < 0.01); and also lower than that of free capsule group and of NS group(1103.9±132.4U/L vs 2257.8±283.3U/L,2379.2±168.7U/L, P < 0.01) respectively. There were no significant difference statistically among that of hepatocytes group,free capsule group and NS group(P>0.05).(2) TBIL of microencapsulated group was lower than that of hepatocytes group (6.21±0.86μmol/L vs 9.18±0.96μmol/L, P < 0.05); and also lower than that of free capsule group and of NS group(6.21±0.86μmol/L vs 8.82±0.93μmol/L,8.76±0.74μmol/L, P < 0.05) respectively. There were no significant difference statistically among that of hepatocytes group,free capsule group and NS group(P>0.05).(3) ALB of microencapsulated group was descended more slowly than that of hepatocytes group (23.2±1.14g/L vs 21.0±1.98g/L, P < 0.05); and also more slowly than that of free capsule group and of NS group(23.2±1.14g/L vs 20.6±1.34g/L,19.3±1.22g/L, P < 0.05) respectively. There were no significant difference statistically among that of hepatocytes group,free capsule group and NS group(P>0.05).4. Pathological examination of the grafts:At the 48h after transplantation, in free tilapia hepatocytes group in the recipients abdominal cavity, there were lots of white blobs adhered to the peritoneum with fibrosis formation in which there were many hepatocytes, and most of the hepatocytes were degenerate and necrosis; at the same time in the microencapsulated group, all the microcapsules were intact and most of hepatocytes in it were alive and the viability of the hepatocytes was more than 80%.At the 7 days after transplantation, in the microencapsulated group, all the microcapsules remained intact and no adhesion. There were a lot of hepatocytes in the microcapsules, which were still alive, and the viability of the hepatocytes was 45.3±5.8% (Trypan-Blue exclusion). Under the TEM, the tilapia hepatocytes were normal and there were also abundant organelles found in cell cytoplasm. At the 14 days after transplantation, most of microcapsules adhered to intestine,omentum and lateral abdominal wall and few of them were gathered. The viability of the hepatocytes was 34.7±4.0% (Trypan-Blue exclusion). The histological characteristics of the grafts were observed by light microscope, the microcapsules were elliptical in shape and the membrane was integrity, the hepatocytes in the microcapsules were still alive, and the angiogenesis could be seen.Conclusions:1. Tilapia hepatocytes obtained by the collagenase digestion method were intact and in a good viability. The cold collagenase digestion method was feasible for isolation of tilapia hepatocytes.2. The tilapia hepatocytes could be alive in the microcapsules for longer time, with the characteristic of hypoxia and hyperthermal tolerance.3. The ACA microcapsules possessed good biocompatibility; could provide immune protection to xenografts and prolong the survival of tilapia hepatocytes grafted.4. Xenotransplantation of microencapsulated tilapia hepatocytes provided sufficient metabolic support to damaged recipient liver and increase the survival rates of ALF rats.
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