The Proliferative Status Of Liver Oval Cells In The Burned Mice And The Effects Of Liver Oval Cells Transplantated In The Radiational Injuried Mice

The Proliferative Status of Liver Oval Cells in the Burned Mice and the Effects of Liver Oval Cells Transplantated in the Radiational Injuried MicePart1The Meeh Constant to Estimate the Body Surface Area of a C57BL/6Mouse in a Thermal Injury Researching.Objective:To investigate the Meeh constant (k) which could be used in the Meeh Formula to estimate the body surface area (BSA) of the C57BL/6mice when making thermal injury models. Methods:24normal C57BL/6mice of6-12weeks age were randomly collected and fed with normal diet until the experiment day. On the3rd day before the experiment day, each mouse was weighted per4hours in the24hours. The next day, the24mice were randomly divided into3groups which contained4male and4female mice. On the experiment day, the1st group was anaesthetized, weighed and skinned at8AM, while the2nd group at4PM and the3rd one at0AM. The BSA and k value of each mouse was recorded.Results:The average weight of the24C57BL/6mice reached its bottom at4PM and its top at0AM. The k values were9.617±0.104at8AM,9.802±0.110at4PM, and9.570±0.244at0AM. There was statistical difference not only between the k values at8AM and4PM, but also between4PM and0AM (P<0.05). The popular k value was often quoted as9.1,9.5,10or11.4when the literature dealing with the Meeh Formula of mice was reviewed. There were statistical differences not only between the popular k value and the k values at8AM, but also between the popular k value and the k values at4PM (P<0.05).Conclusions:In the procedure of making the thermal injury model of C57BL/6mice, it was reliable to use9.617as the k value to estimate the BSA at8AM, but use9.802at4PM.Part2The Proliferative Status of Liver Oval Cells in the Burned MiceObjective:To investigate the proliferative status of liver oval cells in the burned mice.Methods:58of C57BL/6mice which included29male and29femal were devided into4groups. There were4mice in the positive control group,6mice in the sham group,42mice in the thermal injury group, and6mice in the thermal injury complicated with sepsis group. The quantity of male mice was equal to the femal in each group. The positive control group had been fed with the food containing DDC for2weeks until experiment, while the other3groups had been fed with the normal food. A25%of total body surface (TBS) third-degree burn of skin was made in the thermal injury group. A25%of TBS third-degree burn of skin combined with cecal ligation and puncture was made in the thermal injury complicated with sepsis group. The mice of the positive control group, the sham group, and the thermal injury complicated with sepsis group were killed at the24th hour after the thermal injury, and the livers were fixed in the5%formaldehyde solution. The mice of the thermal injury group were devided into7sub-groups which contained3male and3female in each sub-group. The7sub-groups were named as the time points at which they were killed. The7time points were the12th hour after burn, the24th hour after burn, the2nd day after burn, the3rd day after burn, the7th day after burn, the14th day after burn, and the21st day after burn. The livers of the mice in the thermal injury group were fixed in the5%formaldehyde solution. The paraffin sections of the fixed livers of all the4groups were made. HE staining and CK19immunohistochemistry stain were applied to the liver paraffin section of each mouse.Results:In the positive control group, the small proliferative cells were distributed in the liver of each mouse. In the positive control group, the small proliferative cells were positive to the CK19immunohistochemistry stain and had the portrait of oval cells which was basophilicly stained in the cytoplasm and nucleus, and had a high ratio of nucleus to toplasm. In the positive control group, some of the small proliferative cells formed as the small bile ducts in the portal area. In the thermal injury complicated with sepsis group, there was a mouse in whose liver the small proliferative cells ramdomly ocured in the portal area. So was in the24th hour sub-group of the thermal injury group. In the sham group, the thennal injury group and the thermal injury complicated with sepsis group, there was little small cell proliferation in all the livers except the2small-cell-proliferating mice. In the sham group, the thermal injury group and the thermal injury complicated with sepsis group, there were few CK19positive cells in all the livers.Conclusions:When the C57BL/6mouse suffered a25%of TBS third-degree burn of skin, the proliferation of CK19positive oval cells was negative in the liver.Part3The Effects of Liver Oval Cells Transplantated in the Radiational Injuried Mice Objective:To investigate the effects of liver oval cells (OCs) transplantated in the irradiational injuried C57BL/6mice.Methods:12female C57BL/6mice of6weeks had been fed with food containing DDC for2weeks until experiment, while152female mice of6weeks with normal food. There were2types of transplantated liver stem cells, one was mixed cells containing OCs and the other was pure OCs. The mixed cells containing OCs were collected from the liver of the12DDC-feeding mice. The pure OCs was a gift from Professor Hu YP. The152mice fed with normal food were irradiated, and the radiation dose varied from5Gy to9Gy as the experiment design. In the experiment of mixed cells transplantation,40mice irradiated9Gy were equally divided into the control group and the mixed cells group. In the experiment of OCs transplantation,40mice irradiated9Gy were equally divided into the control group and the OCs group,40mice irradiated6Gy were equally divided into the control group and the OCs group, and20mice irradiated5Gy were equally divided into the control group and the OCs group. In the experiment of Hoechst-staining OCs transplantation,12mice irradiated9Gy were equally divided into the control group and the OCs group.200μl of PBS was injected into each mouse of the5control groups. In the experiment of mixed cells transplantation,200μl of the single cell suspention which contained1.0×107/ml mixed cells was injected into each mouse of the mixed cells group. In the experiment of OCs transplantation,200μl of the single cell suspention which contained1.0×105/ml OCs was injected into each mouse of the3OCs groups. In the experiment of Hoechst-staining OCs transplantation,200μl of the single cell suspention which contained1.0×105/ml Hoechst33342-staining OCs was injected into each mouse of the OCs group. Among the80mice, which were irradiated9Gy in the experiment of mixed cells transplantation and the experiment of OCs transplantation, and the40mice which were irradiated6Gy in the experiment of OCs transplantation, the survival time of each mouse was recorded, the gross appearance of the liver, the spleen and the gastrointestine of the dead mice was observed, and the quantity of the peripheral blood cells was counted on the3rd day before irradiation as well as the7th day after irradiation. Among the20mice which were irradiated5Gy in the experiment of OCs transplantation, the quantity of the peripheral blood cells was counted on4time points which were the3rd day before irradiation, the7th day after irradiation, the14th day after irradiation and the21st day after irradiation. In the experiment of Hoechst-staining OCs transplantation, the12mice were killed on the4th day post-radiation, the Hoechst-staining cells were searched in the blood cells, gastrointestine, liver, pancreas, spleen, kidneys, lungs, heart, muscles, skin, brain and bone marrow. In the experiment of Hoechst-staining OCs transplantation, the irradiational intestinal injury of the OCs group was valued by the pathological score as well as the contol group.Results:Among the mice irradiated9Gy, the occurence of local hepatic hemorrhage varied from45%to50%, the intestine atrophy happened in all, the count of WBC decreased to the0.55%to5.65%of the normal level. In the experiment of mixed cells transplantation, there was no statistical difference between the servival time of the mixed cell group and the control goup. In the experiment of OCs transplantation, there was statistical difference between the servival time of the OCs group and the control goup among the mice irradiated9Gy, but no difference among the mice irradiated6Gy. In the experiment of OCs transplantation, the median survival time of the mice in the OCs group irradiated9Gy was8days which was longer than the control group (P<0.05), while there was no statistical difference between the servival time of the OCs group and the control goup among the mice irradiated6Gy. In the experiment of OCs transplantation, the quantity of the peripheral blood cells was no statistical difference between the OCs group and the control goup. In the experiment of Hoechst-staining OCs transplantation, a few of Hoechst-staining cells were observed in the liver and the spleen, the Hoechst-staining cell was negative in the blood cells, gastrointestine, liver, pancreas, spleen, kidneys, lungs, heart, muscles, skin, brain and bone marrow. In the experiment of Hoechst-staining OCs transplantation, the pathological score of the irradiational intestinal injury of the OCs group was less than the contol group, but there was no statistical difference between the2groups (P=0.14).Conclusions:The liver, intestine and hematopoietic system were injuried in the C57BL/6mice irradiated9Gy. The transplantation of2.0×104OCs showed the effect of prolonging the survival time of the C57BL/6mice irradiated9Gy. The irradiational intestinal injury seemed to be improved in the C57BL/6mice irradiated9Gy when the OCs were transplanted, but the improvement was not statistically different to the control group. In the experiment, the quantity of the peripheral blood cells was no statistical difference between the OCs group and the control goup. In the experiment of Hoechst-staining OCs transplantation, a few of transplanted oval cells were localized in the liver.