| Objective:To master the establishment of acute renal tubular necrosis model in rabbits and bone marrow mesenchymal stem cells acquisition, preparation and training.Autologous bone marrow mesenchymal stem cells were transplanted into rabbits by renal artery perfusion,and compared with the untreated control group,in order to observe the effects of transplanted cells on the repair of renal tubular necrosis after ischemia reperfusion injury and recovery of renal function.Differences were compared between the two groups.To provide experimental basis for clinical application of bone marrow mesenchymal stem cell transplantation in the treatment of acute tubular necrosis.Methods:1.The acquisition and cultivation of autologous bone marrow mesenchymal stem cells in rabbits:8 healthy Japanese flap eared white rabbits,2±0.5 Kg,they were randomly divided into two groups (arterial transplantation group and untreated control group),4 rabbits in each group.Before the model was established,blood samples were collected from all the experimental animals, and the serum creatinine and blood urea nitrogen were detected and recorded.1 rabbit from transplantation group and untreated control group was sacrificed respectively at 24h after the acute renal tubular necrosis model was established and nephrectomy was operated for pathological examination.Under aseptic conditions,5ml bone marrow of other 3 rabbits from transplantation group were collected,and autologous bone marrow stem cells were isolated by adherent culture method and carried out primary and passage culture.Morphological observation carried out during the culture.A bottle of P2 generation cells were randomly selected and marked by the 4’,6-diamidino-2-phenylindole DAPI and observed under fluorescence microscope.2.Establishment of experimental rabbit acute tubular necrosis model:Using ischemia-reperfusion injury replication model.Under sterile intravenous anesthesia,laparotomy blunt dissection bilateral renal artery,and double renal arteries were occluded by vascular injury-free clamp within 90mins.To observe the changes of the kidney (including size, color, and appearance), and then go to the folder recovery of renal artery perfusion.Each group selected a rabbit was sacrificed and fixed in kidney tissue, renal tubular necrosis was observed pathological sliced, HE staining.Through blood biochemistry and pathological changes, determine tubular necrosis model successfully copied.Blood creatinine and urea nitrogen values were measured at 3 hours and 24 hours after the model establishment.3.Targeting arterial transplantation group and control group comparative observation:(1) Artery transplantation group (3 rabbits):Separating the abdominal aorta under anesthesia laparotomy (position about double common iliac arteries above the bifurcation),taking Seldinger puncture technique,insert 3F Progreat microcatheter about 5cm to renal artery, slow infusion of autologous BM-MSCS suspension 2ml/ kg.Unplug the microcatheter and catheter sheath, sutures puncture site using resorbable without injury, and wait no bleeding, and then the abdomen was closed layer by layer. (2) Control group (3 rabbits):Compared with the transplantation group at the same time point, the normal saline was injected slowly into the inside vein of the indirect inguinal vein.(3) comparison between the two groups:After the transplantation of BM-MSCs, the blood samples were collected from two groups at 3 days,5 days and 7 days to detect the serum creatinine and urea nitrogen levels in all the experimental rabbits.The blood biochemical differences were compared and analyzed between the two groups.7 days after transplantation drawn the kidney tissues with 10% formaldehyde bilateral renal perfusion fixation under anesthesia,and the frozen sections of specimens and observe stem cell homing under fluorescent microscope.Result:1.Expansion cultures of BM-MSCs:After inoculation 24hours Cells were few adherent cells, and some cells colony were formed after 3～4days.After 5～ 7days, cells covered the bottom of the bottle, achieved the fusion state and subcultured.After the third generation, BMSCs division were active,and the cell morphology was changed into a fibrous shape,and most of them were long spindle shaped.2.After renal ischemia and reperfusion injury in rabbits,pathological observation showed that renal tubular epithelial cells were degeneration and necrosis in the two groups.7d after BM-MSCs artery transplantation,under the fluorescence microscope, the DAPI labeled BM-MSCs was observed to be deposited in the renal tissue.3.Before the model establishment, the mean blood creatinine level was 103.88± 8.11 μmol/L, and the mean value of urea nitrogen was 8.79±0.75mmol/L.After the model was copied, blood samples were collected at 3h,24h respectively.Serum creatinine and urea nitrogen levels were significantly doubled at 3h after model establishment, the mean values were 211.75±14.52μmol/L and 18.71±1.20 mmol/L;The mean value of serum creatinine was 339.63±26.36 μmol/L, and the mean value of urea nitrogen was 24.06±2.56mmol/L after model establishment at 24h.Comparison of model replication before and after model replication at 3h,24h, the difference of mean serum creatinine and blood urea nitrogen was statistically significant (P<0.05).4.After BM-MSCs transplantation through the blood test analysis,compared with the control group, the levels of serum creatinine and urea nitrogen of artery transplantation group were relatively small difference on the 3d after BM-MSCs transplantation(P> 0.05). The levels of serum creatinine and urea nitrogen of artery transplantation group were less than the control group difference on the 5d after BM-MSCs transplantation(P<0.05).On the 7d after BM-MSCs transplantation, blood creatinine and urea nitrogen in artery transplantation group were 116.33±12.50umol/L and 10.13±2.78mmol/L respectively;blood creatinine and urea nitrogenthe in control group was 243.67±17.21 μmol/L and 17.62±1.47mmol/L,there was a significant statistical difference between the two groups(P<0.05).Conclusion:1.Using cliping renal artery occlusion 90min can quickly establish a model of acute tubular necrosis in experimental rabbits.2.By intra-arterial catheter implanted BM-MSCs can be homing to the damaged kidney tissue, and DAPI labeled BM-MSCs still keeps the fluorescence characteristics after 7 days of transplantation.3.By intra-arterial catheter targeting implanted BM-MSCs can effectively improve the recovery of renal function after acute tubular necrosis. |
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