Experimental Study On The Contribution Of Bone Marrow Mesenchymal Stem Cells To Renal Repair Of Crush-induced Acute Kedney Injury (Failure)

Objective:(1) To culture rabbit Bone Marrow Mesenchymal Stem Cells (BMSCs) by the density gradient centrifugation and plastic adhering method.(2) To search for a better and stabilizing pattern of Crush-induced Acute Kedney (Failure).(3) To identify the contribution of Bone Marrow Mesenchymal Stem Cells (BMSCs) to Renal Repair of Crush-induced Acute Kedney Injury (Failure) in vivo.Methods:1. Experiment on the isolation, culture and identification of rabbit BMSCsa) BMSCs from bone marrow of rabbit were isolated, purified using the density gradient centrifugation with rabbit Lydroxypropylmethyl Cellulose, and cultured by plastic adhering method.b) The third passage BMSCs were cryopreserved by the slow freezing method.c) The growth curve of BMSCs was measured by MTT.d) The third passage BMSCs were induced with adipogenic inductive medium in vitro.e) The third passage BMSCs were induced with osteogenic inductive medium in vitro.f) The surface markers of the third passage BMSCs were identified with flow cytometry after amplification.2. Experiment on Crush-induced Acute Kedney Injury (Failure) Rabbits were randomly divided into 3 groups to induce Acute Kedney Injury (Failure) by crush: A group(6 rabbits), 27.5kg, 5h; B group(10 rabbits), 30kg, 5h; C group(42 rabbits), 30kg, 3h, after 24h crush again by the same way. Then measured the serum creatinine (Scr), blood urea nitrogen (BUN), creatine kinase (CK).And observe the incidence rate of AKI.3. Experiment on the Contribution of BMSCs to Renal Repair of Crush-induced Acute Kedney Injury (Failure)Rabbits were crushed by 30kg, 3h, after 24h crush again by the same way. Then the rabbits that occured Acute Kedney Injury (failure) were divided into 2 groups (experimental group and control group) randomly. Experimental group were injected BMSCs 10ml (1×106/ml) intravenously(n=20); Control group were injected PBS 10ml intravenously(n=15). Blood sample were harvested to measure the Scr and BUN by automatic biochemistry analyzer at1d, 2d, 4d, 7d, 14d and 28d after injection. At same time, two rabbits (one from experimental group, one from control group) were sacrificed to get the kidneys.Renal morphologic changes were compared on hematoxylin and eosin (HE) stained sections.Results:1. Experiment on the isolation, culture and identification of rabbit BMSCs BMSCs grew in the forms of colonies dispersely, and showed stable proliferative capability in primary and passage cultures, and can be induced into adipocytes and osteocytes in vitro.The result of flow cytometry showed that the third passage BMSCs positively expressed CD29 and CD90 surface markers, and negative expressed CD34 surface marker.2. Experiment on Crush-induced Acute Kedney Injury (Failure) The Crush-induced Acute Kedney Injury (Failure) experiment showed: Two rabbits were induced to Acute Kedney Injury (failure) in A group; five rabbits were induced to Acute Kedney Injury (failure) in B group; thirty-five rabbits were induced to Acute Kedney Injury(failure) in C group.The creatine kinase (CK) increased evidently in three groups.3. Experiment on the Contribution of BMSCs to Renal Repair of Crush-induced Acute Kedney Injury (Failure)According to the Scr and BUN value, there was no evident difference at 1d, 2d after injection. 4d, 7d, 14d after injection, the Scr and BUN value of experimental group were 208.00±9.46umol/L,155.12±16.13umol/L,98.38±9.51umol/L respectively, lower than that of Control group (222.69±18.52 umol/L, 173.50±13.69umol/L, 110.45±14.71 umol/L) ( P<0.05). 4d, 7d, 14d after injection, the value of BUN of experimental group were lower than that of Control group (P<0.05) too. 4d after injection, the renal injury reached the pear in two groups, but the renal morphologic change of experimental group was lower than that of control group. 7, 14d after injection, the renal repair in experimental group was faster than that in control group.Conclusions:1. Rabbit BMSCs can be cultured by the density gradient centrifugation and plastic adhering method.2. It is a better and stabilizing pattern of Crush-induced Acute Kedney (Failure) through crushing rabbit by 30kg, 3h, after 24h crush again by the same way.3. BMSCs may lessen the tissue damage, and contribute to renal repair of Crush-induced Acute Kedney Injury.