Experimental Study On The Possibility Of Hypoxic-ischemic Encephalopathy Treated By Bone Marrow-derived Mesenchymal Stem Cells

Objective To establish a method of culture and expansion of human bone marrow-derived mesenchymal stem cells (MSCs) in vitro and to explore their biological properties. To observe whether mesenchymal stem cells could break through the blood brain-barrier (BBB) of newborn Wistar rats and arrive in their brain parenchyma by intraperitoneal infusion. To observe the best times when MSCs could penetrate the BBB and when to inject MSCs in order to cure HIE. To observe the characteristics of both distribution and differentiation of mesenchymal stem cells in the brain tissues, and then explore the effects of both penetration and differentiation of MSCs to hypoxic ischemic brain damage. To observe the characteristics of both distribution and differentiation of mesenchymal stem cells in the brain tissues with the intervention of drug.Methods Mononuclear cells were obtained from 30ml adult human bone marrow by density gradient centrifugation method. The 3th passage MSCs were continuously prelabed with 5-bromo-2-deoxyuridine (Brdu) for 48 hours before transplantation. Animal models of HIE were built in 7-days-postnatal Wistar rats according to Rice method. Two hours after hypoxia-ischemia, rats with HIE group(n=8) were intraperitoneally infused MSCs 0.4ml(3 × 10~6) and Concentration of Salvia Miltorrhiza 0.4ml (n=2) and tagged fluid of Brdu 0.4ml(n=5) separately. In contralgroupe(n=8) 7-days-postnatal normal Wistar rats were intraperitoneally infused with the same amount of MSCs ( n=8 )and tagged fluid of Brdu( n=5 ). In 24 hours, 3 days, 14 days, 25 days after transplantation, all rats were sacrificed and their cerebra were sectioned by cryomicrotome Immunohistochemical staining with chromogen diaminobenzidine (DAB) was used to detect and measure the cells derived from MSCs, and to study the best time of penetrating the blood brain barrier and characters of distribution. To determine the differentiation of the Brdu positive cells entering the brains, we observed the ability of MSCs differentiating to neural cells by detecting expression of NSE, NF, GFAP. We can prove the cells coming from human by detecting them positive for CD44. Afer 48 hours of building animal models of HIE, HIE group (n=2) were intraperitoneally infused MSCs 0.4ml (3X 106) separately. In 25 days after transplantation, the groups of rats were sacrificed and their cerebra were sectioned by cryomicrotome. Immunohistochemical staining with chromogen diaminobenzidine (DAB) was used to detect and measure the cells derived from MSCs. We observed the ability of MSCs differentiating to neural cells by detecting expression of NSE, NF, GFAP. We can prove the cells coming from human by detecting them positive for CD44. We observed characters of distribution of MSCs and the best time of treatment by stem cells.Results MSCs harvested in this study were homogenous population and exhibited a spindle-shaped fibroblastic morphology. The cell growth curve showed that MSCs had a good ability of proliferation. The cells were positive for CD44, while negative for hematopoietic cell surface marker such asCD3>CD4^CD7^D13XD14XD^ CDl^ CD22^ CD33^ CD34> CD45 and HLA-DR, which was closely related to graft-versus-host disease. Above 90% cells of MSCs were found at G0/G1 phase. Theultrastructure of MSCs indicated that there were plenty of rough endoplasmic reticula. MSCs were distributed throughout the cerebra in both groups at the 24 hours after transplantation. With the different substance injected and different time, the penetration ratio of HIE group was higher than that of control group (X2=236.296^ 7.244^ 40.838^ 89.380, P (0.01), which had statistical significance. There were more cells which passed through the BBB in HIE group. In the HIE group, MSCs distributed more extensively, and some focus aggregations of MSCs were found. We compared the result, in HIE group, X2=18.873,P <0.01;in control group, X2=0.113, P>0.05. More Brdu reactive cells were observed in the right ischemic hemisphere than in the contralateral hemisphere, No significant difference was proved while comparing both cerebral hemispheres of the control group. Many MSCs expressed NSE, NF, GFAP, which were detected at the 25th day after transplantation. We compared NSE, NF, GFAP in defferent group, the positive ratio of HIE group using Concentration of Salvia Miltorrhiza was 70.90%,46.80%, 18.80%, the positive ratio of HIE group was 54.60%, 30.00%, 17.10%, while contral group, 15.00%, 4.00%, 9.30%, all P<0.01, which has statistical significance. The highest positive ratio is HIE group using Concentration of Salvia Miltorrhiza. We compared the effect of different time transplantation after building HIE models. X2=0.183, Perdu) 0.05, which has no statistical significance about the amount of passed cells. We compared the penetrating ratio in different time after transplantation, X2=562.132, P <0.01, which has statistical significance. The ability of MSCs penetrating BBB in 24 hours is the best after transplantation. We compared MSCs expressing NSE, NF, GFAP in both of cerebral hemispheres. X2=6.179, Pnf <0.05;X2=12.549, Pnse <0.01, which has statistical significance of differentiating in both of cerebral hemispheres. It had no statistical significance in GFAP group (X2=0.460, Pgfap) 0.05).Conclusion The method for culture and expansion of adult human bone marrow-derived MSCs in vitro has been successfully established in this study. MSCs are a homogenous population that has unique growth phenotype and multilineage potential and can act as seed cell of tissue engineering. MSCs can enter the cerebral parenchyma through BBB and migrate throughout the brain by intraperitoneal infusion. Hypoxic-ischemic brain damage could promote penetration and migration of MSCs. The part of the ischemic is better. Transplanted MSCs could differentiate to neuron and astrocytes. Concentration of Salvia Miltorrhiza injection is superiority to inducing MSCs to differentiate into neural-like cells. It is useful of MSCs to differentiate to neuron in early transplantation. Within 24hours of transplantation, the ability of MSCs breaking through BBB is the best. Hypoxic-ischemic brain damage could improve MSCs differentiate to neuron cell. But the effect of differentiating toastrocytes is not obvious.