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The Preliminary Study on the Effect of Transplanting BM-MSCs to Renal Subcapsule on Obstructive Nephropathy in Rats

Author: ChengQing
Tutor: BaiZhiMing
School: Central South University
Course: Surgery
Keywords: BM-MSCs R-UUO subcapsular injection interstitial fibrosis macrophage
CLC: R692.2
Type: Master's thesis
Year: 2011
Downloads: 16
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Abstract


Objective:Isolating and culturing high-grade mesenchymal stem cells from rats bone marrow, then characterizing its property of plastic-adherent and cell surface markers. Consummating the protocol of cells injection to the subcapsule of caco-renal of rats with intact capsule. We preliminary assessed the efficacy and safety of bone marrow-derived mesenchymal stem cells (BM-MSCs) subcapsular injection in reversable unilateral ureteral obstruction (R-UUO)model of rats through the level of macrophage infiltration and collagen deposition.Method:Isolating and culturing of mesenchymal stem cells(MSCs) from male rats bone marrow with reforming plastic-adherent method and observing its morphology and performance with phase-contrast microscopy. Analyze the cell surface markers by flow cytometry in passage 2. Female rats underwent R-UUO model with V-shape tube compression method for 5days and then were removed the compressive tube. Follow that,delivered MSCs or its vehicle to the subcapsule after the fulfillment of the subcapsular injection protocol which includes micro syringe;a very fine needle and uniformly arrangement of injection point. Immunohistochemical staining was performed with mouse anti-macrophage monoclonal antibody (ED-1),The 4μm sections of kidney were stained with the Masson trichrome technique.Iamge Pro Plus(IPP) software were adopted to analysis the mean integration optical density (IOD) of positive staining of ED-1 and the per area of collagen deposition. All data were expressed as mean±SEM. Comparisons among the three groups were made by one-way analysis of variance (ANOVA) if the data consistent with test of normality(Shapiro-Wilk test),if not, Kruskal-Wallis test is applied. A value of p<0.05 was considered statistically significant except Shapiro-Wilk test whose signifcance up toα=0.10. SPSS software for Windows was used for analyses.Results:1. These cell populations are comprised of morphologically homogenous fibroblast-like cells and present plastic-adherent property. Flow cytometry analysis shows that these cells are homogenously positive for the mesenchymal markers CD29, CD44 and CD90, but negative for hematopoietic and endothelial cell marker CD34,and the pan-leukocyte marker CD45.2. V-shape tube compression method is an effective operational procedure to make R-UUO model in rats.The total achievement ratio is 80%.3. The effective protocol of deliverd cells to the caco-renal subcapsule content three element:micro syringe;a very fine needle and uniformly arrangement of injection site.4. MSCs delivered at the subcapsule significantly reduced the mean IOD of ED-1 expression after 14 days of transplantation(P< 0.05), but failed to attenuated the collagen deposition in each survey point.5. Injecting caused a significantly rise in ED-1 mean IOD contrast with no injection sham group in 14 days after pricking the renal capsule(P<0.05), but not augmented the interstitial fibrosis.6. After relieving the obstruction, collagen deposition was declined gradually over time(P<0.05),as well as the level of ED-1 expression in interstitium in sham group(P< 0.05). While the other two groups did not show any statistical significantly decreased in ED-1 level in any time-point.Conclusion:1.Reforming plastic-adherent method is an effective and convenient procedure to isolate and culture high-purity MSCs from rats bone mairrow.2. Micro syringes and a very fine needle accompany with uniformly arranged the inject point are composed a feasible protocol of transplanting cells to the caco-renal subcapsule.3. Delivered MSCs at the renal subcapsul in R-UUO rats could ameliorate the macrophage infiltration in interstitium, but not improved the interstitial fibrosis within 2 weeks.4. Subcapsular injection aggravate inflammation of obstructive nephropathy in 14 days after transplantation, but not contribute to the interstitial fibrosis.5. Relieving the obstruction is a definite fruitful treatment to obstructive nephropathy.

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CLC: > Medicine, health > Surgery > Urology ( urinary and reproductive system diseases) > Kidney disease > Renal infarction ( hydronephrosis)
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