Effects Of Bone Marrow Mesenchymal Stem Cells And Their Bubbles On The Repair Of Chronic Renal Injury In

Objective: Mesenchymal stem cells are a class of self-renewing, multipotent differentiation and proliferation of cells. Micro-vesicles(MVs) from bone mesenchymal stem cells(MSCs) have been showed to contribute to the recovery of damaged kidney. The aim of the study was to investigate the biological effectsof MSCs and MVs in vitro.Bone marrow mesenchymal stem cells(mesenchymal stem cells, MSCs) are self-renewal, multilineage differentiation and high proliferation cells. Mesenchymal stem cells transplantation can obviously improve the renal function, it may be the main cell groups in the recovery of renal tubules and renal repair. From the bone marrow mesenchymal stem cells in the extraction of microvesicles(Micro-vesicles, MVs) can promote the repair of damaged kidney function. The purpose of this study was to observe the effect of MSCs, MVs on the repair of damaged kidney cells in vitro experiments.Methods: The C57BL/6 male,6 week mice were isolated and cultured by the whole bone marrow PCR purification of mouse mesenchymal stem cells, cultured to the third generation of MSC(P3-MSC) to detect the expression of cell surface markers by flow cytometry(CD45, CD105) and the adipogenic differentiation into identification of bone. MSC medium were collected in the process of cultivation, and then for speeding frozen centrifugal extraction of MVs. In vitro cultured renal proximal tubular epithelial cell line(HK-2), with a concentration of 6ng/ml TGF- β1(TGF: transforming growth factor, transforming growth factor) stimulate cell for 48 or 72 hours. Experimental groups:(1) HK-2,(2) HK-2+TGF-β1(6ng/ml),(3) HK-2+TGF-β1+MSCs(1*105cells/200ul),(4) HK-2+TGF-β1+MVs(30μg/ml). Acollection of cells were observed in morphology, and the detection of α-smooth muscle actin alpha(α-SMA), E- cadherin(E-cadherin) m RNA and protein expression levels.Results: The cells were cultured for 48 or 72 hours respectively with TGF-β1 stimulation. The cell apoptosis increased significantly, the intercellular space increased, cell and fibrosis significantly, fusiform, and with the passage of time, the degree of injury increased significantly under light microscope; MSCs, MVs reduce the apoptosis of HK-2 cells, alleviate the degree of fibrosis, a few obvious cell gap widened, the status of cells obviously improved than that in the injury group. RT-PCR showed after stimulation, E-cadherin protein level decreased, α-SMA expression increased, and the expression level of 72 h is more than 48h; and MSC, MV group, α-SMA expression decreased significantly, E-cadherin expression decreased slightly.Conclusions: Bone marrow mesenchymal stem cells can inhibited the TGF- beta 1induced renal tubular epithelial cell injury and fibrosis, play a certain role in repair, and stem cells extracted microbubbles can play a similar role in the process.AIMS Several studies have demonstrated administration of mesenchymal stem cells(MSCs) could reverse kidney injury by paracrine mechanisms rather than by MSC trans-differentiation. A few researchers found micro-vesicles(MVs) derived from MSCs might be a paracrine mechanism for cell-to-cell communication in recent study. The aim of the study was to investigate the repair effects of MVs in 5/6 subtotal nephrectomy(Nx) mice model.MATERIALS and METHODS The animals were randomly divided into 4 groups: Control, Nx, Nx+MSCs, and Nx+ MVs group. MSCs were injected(1×106/mouse) through caudal vein in Nx+ MSCs group at the second day after the surgery, and MVs were injected(30μg/mouse) through caudal vein in Nx+ MVs group on alternate days. Mice were sacrificed on day 7 after the first time of administration. BUN, Scr, UA and proteinuria were evaluated. Histopathology of kidney was analyzed.RESULTS In Nx mice, the levels of Scr, UA and proteinuria were significantly decreased with administration of MVs and MSCs(p<0.05). The remnant kidneys of MV and MSC-treated Nx mice showed less fibrosis, interstitial lymphocyte infiltrates, and less or absent tubular atrophy compared with untreated Nx group. The Histological Score of Kidney in untreated mice was 3.13±0.74, while in MSC-treated group 1.67±0.47, and in MV-treated group 1.80±0.44, nearly preserving normal morphology of the kidney(p<0.01).CONCLUSION This study showed MVs protect against renal injury induced by 5/6 Nx, which could mimic the role of MSCs in kidney repair. The research showed a newly potential therapeutic approach to kidney diseases.AIMS Micro-bubbles(MV) derived mesenchymal stem cells(MSC) may pass a variety of genetic information and bioactive substances, which play an essential role in the repair of renal injury. MV treatment is expected to become a new direction for the research of stem cell therapy. The aims of the study are to investigate the biological effects and repair mechanisms of MVs.METHODS The MSCs were harvested, dissociated, and digested. MVs were obtained from MSC supernatants. In vitro, the mice were divided into 4 groups: Control, unilateral ureteral obstruction(UUO), UUO+MSC, and UUO+MV. MVs and MSCs were injected after surgery. The mice were killed 7 or 14 days after surgery and handled for further tests. In addition, the mi-RNA expressions of MVs and MSCs were labeled using the mi RCURY? Hy3?/Hy5? Power labeling kit and hybridized on the mi RCURY? LNA Array.RESULTS In vivo, the level of Blood Urea Nitrogen(BUN) in the MV and MSC group was significantly lower than the UUO group(p< 0.01, 11.3±0.5 vs. 9.8±0.7 mmol/L; p< 0.01, 12.3±1.6 vs. 8.7±1.5 mmol/L). The serum creatinine concentration(Scr) level decreased after 7 days of MV treatment(p< 0.05, 107.1±10.1 vs. 57.0±10.8 mmol/L). Administration of MSC and MV reduced Scr level at day 14(p<0.05, 112.1±28.9 vs. 75.4±17.7 mmol/L, 112.1±28.9 vs. 64.2±9.8 mmol/L). The level of serum UA decreased with MV administration(day 7 and 14, p<0.01, 227.7±63.7 vs. 80.9±26.5 mmol/L; 237.0±16.1 vs. 78.0±18.9 mmol/L). Besides, a total of 503 expressed mi RNAs were detected, of which, 266 were in MSC, including237 in MVs.CONCLUSION MVs-MSCs can protect kidneys from damages of fibrosis. MVs-MSCs are superior to MSCs in some respects. MVs can be a potential therapy in treatment of kidney diseases.