| ObjectiveTo observe the impact of the MSCs carrying BMP-7 gene on the reconstruction of Ischemia reperfusion injury kidney and reveal the effects of BMP-7 on the directional differentiation of the kidney mesenchymal cells to the epithelial cells in vivo.MethodsRat MSCs were separated and purified by density gradient centrifugation.In vitro, rat MSCs were transfected with BMP-7 gene by slow virus vectors. The expression of the BMP-7 in the supernatant was detected by PCR. MSCs expressing BMP-7 gene were isolated and continued to be cultured for proliferation. Rat models of kidney ischemia reperfusion injury were established, then injected with BMP7-MSCs through inferior vena cava.At the same time,the controlling groups were set up.In the scheduled time (3,5,7 and 30 days after transplantation), rats in each group were kindly sacrificed 4 hours after injection of BrdU(50mg/kg)transabdominally. Bilateral kidneys of the rats were removed, then prepared for tissue slices. Analysis of the cell proliferation were taken through BrdU immunohistochemical staining, and cell types were identified through E-cadherin, vimentin and a-SMA immunohistochemical assay. Renal fibrosis was revealed through collagen staining, and the source of the donor cells was confirmed by the expression of green fluorescent proteins through a fluorescence microscope. Quantitative analysis of the expression of vementin, a-SMA, BMP-7 protein and E-cadherin were conducted through PCR methods.Results1. Rat MSCs had been separated and purified by density gradient centrifugation successfully.2. Rat MSCs had been transfected with BMP-7 gene by slow virus vectors successfully. The expression of the BMP-7 in the supernatant had been detected by PCR.3. 160 rat models of kidney ischemia reperfusion injury had been established successfully.4. Early increase in serum Cr and BUN could be suppressed by the MSCs transplantation. Compared with the controlling groups, serum Cr and BUN levels decreased more rapidly in MSCs transplantation groups(P<0.05).5. Synergistic inhibiting effects on the serum Cr and BUN levels between exogenous MSCs and BMP-7 could be observed in the early stage of the ischemic reperfusion injured rat kidneys. Compared with the MSCs transplantation groups, early inhibiting effects in the BMP7-MSCs transplanting groups were more significantly (P<0.05).6. BrdU positive cells were more in the MSCs transplantation groups than that in the controlling groups (P<0.05).7. BrdU positive cells were more in the BMP7-MSCs transplantation groups than that in the MSCs transplantation groups (P<0.05).8. Compared with the MSCs transplantation groups, E-cadherin+/GFP+ positive cells increased in the BMP7-MSCs transplantation groups (P<0.05).9. Post operation increasing expression of E-cadherin mRNA could be seen in each group with significant differences between every two time points (P<0.05). Increasing expression of E-cadherin mRNA was more obvious in the MSCs transplantation groups than that in the blank controlling groups, and as it was in the BMP7-MSCs transplantation groups compared with the MSCs transplantation groups.10. Expression of SMA was similar between the MSCs and BMP7-MSCs transplantation groups, with their peak at the 3 days post- transplantation, and decreased thereafter. However, at each time point, there was a more decreased expression of SMA in the C groups (BMP7-MSCs transplantation group) than that in the B groups (MSCs transplantation group) (P<0.05).11. Post operation decreased expression of Vimentin mRNA was more obvious in the MSCs transplantation groups than that in the blank controlling groups, and as it was in the BMP7-MSCs transplantation groups compared with the MSCs transplantation groups. 12. Post operation increasing expression of BMP-7 mRNA was more obvious in the MSCs transplantation groups than that in the blank controlling groups, and as it was in the BMP7-MSCs transplantation groups compared with the MSCs transplantation groups(P<0.05).ConclusionRat MSCs could be separated and purified by density gradient centrifugation successfully.Rat MSCs could be transfected with BMP-7 gene by slow virus vectors efficiently.Exogenous MSCs and BMP7-MSCs could suppress the early increase of serum Cr and BUN, promote the regeneration of the renal cells and differentiate into the renal tubular epithelium, and also alleviate the fibrosis of the renal interstitial in the ischemic reperfusion injured kidney in the rat models. BMP-7 has a promoting effect on reparation of the injured kidney via MSCs.
|
PDF Full Text Request