| Objectives:To investigate the protective effects of Rhiizoma Dioscoreae pretreatment and Bone Marrow Mesenchymal Stem Cells in easing renal ischemia-reperfusion injury and promoting the Endothelial cell regeneration in renal ischemia-reperfusion in rat.Methods:In Vitro Experiment:after isolation and purification,Rat MSCs were induced by VEGF,bFGF. The morphological and ultra-structure changes of the MSCs were observed with the inverted microscope and transmission electron microscope on the 0th,3d,5th,7th day respectively.In Vivo Experiment:1. 60 Spargue-Dawley rats (4-6 week-old) were randomly assigned into 4 group: (1) normal control group (N, n=6),(2) the I/R group(n=18),rats were subjected to 60 min of the left renal ischemia-reperfusion injury with microvascular clips ,after 60 min of reperfusion they were injected saline intravenously and then 6h, 48h, and 2w after reperfusion six rats each were randomly euthanized for blood sampling and kidney harvesting, (3)the MSCs group(n=18), rats were subjected to 60 min of the left renal ischemia-reperfusion injury with microvascular clips, after 60 min of reperfusion they were injected in BrdU positive MSCs ( 1×10~6/ml )intravenously and then 6h, 48h, and 2w after reperfusion six rats each were randomly euthanized for blood sampling and kidney harvesting, (4) the Rhiizoma Dioscoreae and MSCs group(n=18), the rats with pretreated with Rhiizoma Dioscoreae for five days before being subjected to 60 min of the left renal renal ischemia and reperfusion then the step is the same as the third group.2. The blood urea nitrogen(BUN) and serum creatinine(Scr) were measured with automatic biochemistry analyzer. The renal tissue homogenate content of endothelin (ET) was determined with radio immunoassay methods. Renal morphologic changes were scored with Paller's criterion on hematoxylin and eosin(H&E) stained sections. PCNA positive cells of renal tubular epithelia and glomerulus were detected immunohistochemically as proliferation index. The distribution of BrdU positive MSCs and FⅧ-RAg positive cells were identified by immunohistochemically.Results:In Vitro Experiment: within days of induction, MSCs changed shape from spindle alike cells into slabstone-like cells. Weibel-Palade bodies can be found in the cytoplasm of some cells by transmission electron microscope.In Vivo Experiment:1.Histological examination:6h,48h,2w after renal ischemia-reperfusion injury, the renal morphologic score in the Rhiizoma Dioscoreae and MSCs group and the MSCs group were (5.10±0.47,5.06±0.37,4.93±0.53), (6.63±0.54,6.20±0.28,5.48±0.49) respectively, significantly lower than that in the I/R group (7.35±0.56,7.50±0.93,6.23±0.33)(P < 0.05);they were also significantly difference between the the Rhiizoma Dioscoreae and MSCs group and the MSCs group at 6h,48h after renal ischemia-reperfusion injury(P < 0.05),but there were no difference between them at two weeks after renal ischemia-reperfusion injury(P>0.05).2. The change of serum creatinine and urea nitrogen level: the Scr and BUN value in N group were 43.24±2.58μmol/L, 6.48±0.64mmol/L. 6h after renal ischemia-reperfusion injury, the Scr and BUN value in each group were increased, the I/R group (75.38±4.53μmol/L,18.72±0.59 mmol/L) were significantly higher than that in the MSCs group (61.93±5.05μmol/L, 11.05±1.32 mmol/L)and the Rhiizoma Dioscoreae and MSCs group(50.40±3.38μmol/L,8.82±0.49 mmol/L) (P < 0.05),and there were also significantly difference between the MSCs group and the Rhiizoma Dioscoreae and MSCs group. 48h after renal ischemia-reperfusion injury,there was only the I/R group (79.09±3.32μmol/L,15.37±1.69 mmol/L) have significantly higher than that in the N group(P < 0.05). The Scr and BUN value were no difference among the I/R group (45.63±2.09μmol/L,6.10±0.53 mmol/L), the MSCs group(42.55±1.70μmol/L,5.88±0.52 mmol/L) and the Rhiizoma Dioscoreae and MSCs group ( 47.23±1.95μmol/L,5.80±0.59 mmol/L)(P>0.05) at two weeks after renal ischemia-reperfusion injury.3. The change of endothelin (ET) : the ET value in N group was 158.25±15.13 pg/ml. 6h after renal ischemia-reperfusion injury, the ET value in the I/R group, the MSCs group and the Rhiizoma Dioscoreae and MSCs group were 258.30±25.04 pg/ml,203.40±6.01pg/ml, 168.56±12.12pg/ml;The I/R group and the MSCs group were significantly higher than that in the N group(P < 0.05), but there were no difference between the Rhiizoma Dioscoreae and MSCs group and the N group. The ET value were no difference among the I/R group (154.61±16.12 pg/ml), the MSCs group(144.48±21.34pg/ml) and the Rhiizoma Dioscoreae and MSCs group(158.25±22.91pg/ml)(P>0.05) at 48h after renal ischemia-reperfusion injury.4.Cell proliferation and distriburion of the BrdU and the FⅧ-RAg positive cells in the kidney: 6h,48h,2w after renal ischemia-reperfusion injury, the PCNA-positive cells in the Rhiizoma Dioscoreae and MSCs group and the MSCs group were ( 30.17±0.63/HP,36.52±1.17/HP,32.01±0.42/HP ) , (22.42±0.68/HP,24.60±0.84/HP,22.36±0.85/HP) respectively, significantly higher than that in the I/R group (7.06±0.28/HP,17.64±0.72/HP,19.03±0.41/HP)(P < 0.05);there were also significantly difference between the the Rhiizoma Dioscoreae and MSCs group and the MSCs group at 6h,48h, 2w after renal ischemia- reperfusion injury(P < 0.05).After 6h and 48h of reperfusion, BrdU-positive cell were located diffusely at the renal interstitium. After 2w of reperfusion, a few BrdU-positive cells were located at the renal capillary and glomerulus.6h,48h,2w after renal ischemia-reperfusion injury, the FⅧ-RAg positive cells in the Rhiizoma Dioscoreae and MSCs group and the MSCs group were (34.96±0.56/HP,30.19±0.54/HP,27.42±0.77/HP),(23.98±0.44/HP,22.94±1.50/HP,22.10±0.78/HP)respectively, significantly higher than that in the I/R group (17.82±0.58/HP,18.23±0.49/HP,20.40±0.66/HP)(P < 0.05);there were also significantly difference between the the Rhiizoma Dioscoreae and MSCs group and the MSCs group at 6h,48h, 2w after renal ischemia-reperfusion injury(P < 0.05).Conclusions:A proportion of MSCs can be induced to vascular endothelial cells in vitro in culture medium including of VEGF, bFGF. MSCs is of therapeutical value to renal ischemia-reperfusion injury,ameliorating the tissue damage,contributing to renal function recovery,and MSCs may also differentiate into renal vascular endothelial cells in vivo. Rhiizoma Dioscoreae and MSCs have synergistic action in easing renal ischemia-reperfusion injury and promoting the Endothelial cell regeneration in renal ischemia-reperfusion.
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