| Objective1.To establish rabbit models of hepatic fibrosis,and study their liver's function and histopathological change.2.To establish the method of transgenic labeling by autologous bone marrow mesenchymal stem cells(BMSCs).3.To observe the number,immigration,and distribution of labeling cells in rabbits after BMSCs transplantation,and detect the index change of liver's function in serum,and evaluate the therapeutic effect through studying hepatic tissue pathology.Methods1.Establish rabbit models of hepatic fibrosis and evaluate their attributes. Establishment of the hepatic fibrosis animal model:Totally 40 rabbits were randomly assigned into experimental group(n=30) and control group(n=10).Rabbits in the experimental group were injected with 40 g/L CCL4 and olive oil suspension into the abdominal cavity,whereas those in the control group were treated with the same volume of saline.At 1-3 weeks,the injection frequency was 0.2 mL/kg,twice every week.At 3-6 weeks,according from diet reaction and body variation of rabbits,the quantity was 0.4 mL/kg.At 6-8 weeks,the quantity was adjusted to 0.5 mL/kg.At 4, 8 and 12 weeks after drug injection,hepatic tissue and serum specimen was subjected to hematoxylin-eosin staining for pathological observation and biochemical index.2.Cultivate BMSCs and label them:24 model rabbits were randomly assigned into experimental group(n= 12) and control group(n=10).Rabbits in the experimental group were transplanted with BMSCs,and which in control group were transplanted with physiological saline.Bone marrow aspiration come from flank bone were done, and marrow mononuclear cell were extracted by density gradient centrifugation,then BMSCs were cultured,later green fluorescent protein(GFP) were used to labeled on BMSCs to observe.And the positive rate is 99%,cells labeled keep good shape.3.Evaluate therapeutic effect after BMSCs transplantation:3×10~6cells/ml BMSCs labeled with GFP were transplanted in rabbit through cranial mesenteric vein, after 0,4,8,12 weeks rabbit serum were taken to detect,and little hepatic tissue were cut to make paraffin section to observe the mumber,migration and distribution of fluorescence cell after deparaffinage by fluorescence microscope;and HE dyeing were done to observe pathology of hepatic fibrosis tissue.4.Statistical analysis:Experimental data was described as(?)±s,and was analyzed by method of duplicate measurement anova analysis with SPSS 13.0 software;If numerical data between groups have multiple level,use LSD analysis,if there are two kinds of level,use independent sampler t analysis to dispose that,size of test is that there are significant difference when P＜0.05.Result1.The histopathology change of experiment rabbits:The liver of the control group looks kermesinus.Pathological sections exhibited that the structure of hepatic lobule was integrity;central veins radiated arranging to hepatic cells.At 8 weeks,the liver in the experimental group developed purple,seems wrapping with millets. Pathological sections show punctiform or lamellar cellular necrosis,inflammatory cell infiltration around liver canaliculus,displaying early symptoms of liver fibrosis.At 12 weeks,the liver looks gray,seems wrapping with many types of millet.Pathological sections exhibited that the structure of hepatic lobule was destroyed,hepatic cord was in disorder,hepatic cells exhibited fatty degeneration,some of which showing interstitial fiber fibroplasias and inflammatory cell infiltration.These were significant hepatic fibrosis manifestation.2.Biochemical index of experiment rabbits:Accompany with time prolonged, albumin levels and ratio of albumin to globulin decreased rapidly from 1.26±0.90 to 0.93±0.17,globulin,and indirect bilirubin and direct bilirubin contents gradually increased.Alanine aminotransferase and glutamic oxalacetic transaminase significantly increased from 36.80±7.66,23.40±14.20 to 392.00±57.93,282.00±38.54 in prophase,but decreased later to normal.3.Morphology of BMSCs before transplantation:primary cell grows in whirlpool,distribute in direction and exhibit multilayer in whirlpool central;BNSCs looks like Fusiform shape,and cell boundary can't be identified.P1 cell growths much fast and homogeneous distribution.P3 cell exhibits fusiform shape manifest. Green fluorescence of BMSCs can be find in all field by fluorescence microscope, and positive rate is 99%.4.Morphology of BMSCs after transplantation:A lot of green fluorescence cell were seen in hepatic fibrosis tissue by fluorescence microscope,with intensive and homogeneous distribution,compatible with hepatic tissue in high power lens,without reject reaction.With time prolong,fluorescence cell migrate in large circle, concentrate to focus of infection,more and more assemble to central vein in 3 days, then distribute to fringe of liver.5.Hepatic tissue pathology after transplantation:Hepatic tissue pathology of 4 weeks in control group exhibit that hepatic cord in disorder,interstitium fiber in accrementition,some of which extend to folial,and inflammatory cell infiltrate. Hepatic tissue pathology in experiment group exhibit less of above-mentioned symptom,degeneration and dead cell decrease obviously.Hepatic tissue pathology of 12 weeks in experiment group exhibit very little degeneration and dead cell, collagenous fibrils deposit in central veins and portal area,structure of hepatic lobules is clear,basic to recover,and have no significant difference refer to control group.6.Biochemical indicator change in serum after transplantation:TP,ALB in serum after transplantation heighten gradually,and have no statistically significant (P＞0.05) refer to control group;GLO in experiment group degrade gradually,and have statistically significant(P＜O.05) compare to control group in 8 and 12 weeks; TBIL,DBIL in experiment group degrade gradually,and have statistically significant (P＜0.05) compare to control group in4,8 and 12 weeks;ALT,AST in experiment group degrade gradually,and have statistically significant(P＜0.05) compare to control group in4,8 and 12 weeks.Conclusion1.Rabbit model of the hepatic fibrosis was established by peritoneal injection of 40%CC14-olive oil for 12 weeks,and the change of liver pathohistology and liver function is consistent with standard of hepatic fibrosis.2.BMSCs were successfully labeled by GTP,and the positive rate is 99%.3.BMSCs transplantation can make part impaired hepatic tissue recover to normal structure and function,partle inhibit the development of liver fibrosis,and adjust their function to normal. |
