| Objectives: (1) To investigate the probability and rejection mechanisms ofxenogeneic hepatocytes transplantation between tilapia and rat. (2) To establish themethods of isolating and culturing tilapia hepatocytes. (3) To investigate the effect oftemperature on primarily cultured tilapia hepatocytes.Methods: (1) Tilapia hepatocytes were isolated by the method of collagenasedigestion and cultured in DMEM medium with some supplementing factors. Theywere divided into three groups: group A was cultured at 28℃, group B at 37℃, groupC at 39℃. The viability of the cells was assessed by trypan blure exclusion. Themorphologic change of cultured hepotocytes was observed by light microscope; theultrastructure study was conducted by electron microscope. The concentrations ofalbumin, urea, GPT and LDH in supernatant at different culture time were examined.(2) The outbred SD rats were divided randomly into two groups, the control groupand transplantation group. NS was injected into the spleen of the rat(recipient) in thecontrol group, and the tilapia(donor) hepatocytes(2×10~7) were transplanted into thespleen of the rat(recipient) in the transplantation group. The spleen specimens wereobtained at different time after transplantation and their immunohistochemistrical andpathological examinations were done. The histological characteristics of transplantedtilapia hepatocytes were observed by HE staining. The CD4 and CD8 lymphocytes,IgM and IgG around the grafts were detected by immunohistochemistrical method.Results: (1) The averagely yielded tilapia hepatocytes was 1.13±0.17×10~8cells per tilapia with the viability 92.05±0.66%, and the purity 92.70±0.83%respectively. The fresh isolating hepatocytes were round or elliptic. Under the lightmicroscope, the morphologic change of hepatocytes was almost same in group A andB: After 12 hours, hepotocytes were found to extend and attach to the bottle wall,their shape were polygon. After 24 hours, almost all cells attached to the bottle wall,and some of them were binuclear. After 3 days they were aggregated and arrangedlike insland. After 5 days, the hepotacytes increased in number. After 7 days, the cells covered 80% of bottle wall. After 11 days, some of them had been dead and almost allbeen dead after 3 weeks. Under the electron microscope, the abundant organelles offresh isolated hepatocytes could be found, even the bile canaliculus and tight junctionof particular ultrastructure of hepatocytes were found at the seventh day. Group C:After 12 hours, hepotocytes were extended and attached. After 3 days, they werealso aggregated and arranged like insland, but the island was fewer. After 5 days,some of them had been dead. (2)In group A, the concentrations of albumin and ureain the supernatant rose and then dropped gradually with the peak point at the seventhday, on the contrary, the concentrations of GPT and LDH dropped and then rosegradually with the valley point at the seventh day. The change of the concentrations ofalbumin, urea, GPT and LDH in group B was same as in group A. In group C, theconcentrations of albumin and urea in the supernatant dropped gradually duringculture; on the contrary, the concentrations of GPT and LDH rose gradually. (3)At thesame culture time, the concentrations of albumin, urea, GPT and LDH in group A wassame as them in group B (P＞0.05), but in group C they were significantly differentfrom that of group A and B (P＜0.05). (4) Grafted hepatocytes disappeared quicklyafter transplantation. Only a few living hepatocytes could be found in spleens after 8hours with a lot of inflammatory cells infiltrating. The IgM was founded around thegrafts at 1 hour post-graft and the IgG was founded at 18 hours as well. The CD4,CD8 lymphocytes were congregated around the grafts at 4 hours post-graft.Conclusions: (1) Tilapia hepatocytes obtained by the collagenase digestion wereintact and in a good viability. The cold collagenase digestion method is feasible forisolation of tilapia hepatocytes. (2) The tilapia hepatocytes cultured at 28℃and 37℃have a good function at the seventh day, but the growth and function of them is not sogood when cultured at 39℃. The tilapia hepatocyte has the characteristic ofanti-hyperthermia. (3) The transplanted tilapia hepatocytes could survive in the ratspleen in a short time. (4) Both humoral and cellular immune respones participated inthe rejection of xenogeneic hepatocytes transplantation between tilapia and rat. |
PDF Full Text Request