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BACKGROUND: Present studies have verified that Baicalin has protective effects on various brain damage in the nervous system.OBJECTIVE: To study the possibility of intravenous transplantation of human umbilical blood mesenchymal stem cells (hUBMSCs) and Baicalin after hypoxic-ischemic brain damage (HIBD) in neonatal rats.DESIGN, TIME AND SETTING: The randomized controlled animal study was performed at the Laboratory of the Department of Neurology, First Affiliated Hospital, Nanchang University from February 2007 to January 2008.MATERIALS: Totally 10 umbilical blood samples from healthy full-term pregnant women were obtained from the Department of Obstetrics, First Affiliated Hospital, Nanchang University. A total of 85 clean Sprague Dawley neonatal rats aged 7 days were randomly assigned to a normal control group (n=15), a model group (n =20), a cell transplantation group (n =25), a cell transplantation + Baicalin group (n =25).METHODS: The umbilical blood mononuclear cells were isolated by the gelatin sedimentation + density gradient centrifugation method, and amplified in vitro. Cells at the fifth passage were used for transplantation. Cells were labeled by DAPI at 6-12 hours before use. Neonatal rats in the model, cell transplantation and cell transplantation + Baicalin groups were used to establish HIBD models. Rats in the blank control group were left intact. At 2, 3,4, 5 weeks following model induction, rats in the cell transplantation and cell transplantation + Baicalin groups were injected with DAPI-labeled hUBMSCs (5-10 μL/g) via caudal vein at the density of 1 ×10~9/L. From the first day of transplantation, rats in the cell transplantation + Baicalin group were injected with 120 mg/kg Baicalin via intraperitoneal injection, once a day, for three successive days.MAIN OUTCOME MEASURES: The following parameters were measured: brain tissue lesion, DAPI-positive cell number, location of hUBMSCs following transplantation.RESULTS: Lesion rate of brain tissue was significantly lower in the cell transplantation + Baicalin group compared with the model and cell transplantation groups at 4 weeks following transplantation (P < 0.05). Compared with the cell transplantation group,DAPI-positive cell number was significantly increased in the cell transplantation + Baicalin group at 1, 2, 4 weeks (P < 0.01). From the 3~(rd) week following model induction, abundant DAPI-labeled cells were found surrounding the lesion site, without obvious boundary integrated with the host brain. Few DAPI-positive hUBMSCs were found in non-ischemic region. At 4 and 5 weeks following model induction, DAPI-positive cells were significantly decreased in the lesion site.CONCLUSION: The third week following HIBD is an optimal time for cell transplantation. Baicalin can make a large number of hUBMSCs across the blood-brain barrier to distribute and scatter around the disease focus integrated with host brain tissue.
RESUMEN
BACKGROUND: Both antioxidant and cytokine can induce the differentiation of umbilical cord blood-derived mesenchymal stem calls (UCB-MSCs) towards neuron-like cells in vitro. It remains unclear regarding how to select an inductor that has the ability of either neuro-protection like cytokines or powerful antioxidation. After wide screening, baicalin has been included in this study. OBJECTIVE: To observe the effects of baicalin on in vitro purification, amplification, and differentiation towards neuron-like cells of hUCB-MSCs.DESIGN, TIME AND SETTING: A randomized, controlled, cytological in vitro observation was performed at the laboratory of Department of Neurology, First Affiliated Hospital of Nanchang University between February and April 2005. MATERIALS: Ten portions of UCB were collected from healthy full-term normal delivery pregnant women aged 23-25 years old. Baicalin with purity > 95% was purchased from Department of Pharmaceutics, Xiangya Medical College of Central South University. Antioxidant additive J -mercaptoethanol was provided by Sino-American Biotechnology Company, China. METHODS: The collected UCB was anticoagulated with heparin to separate mononuclear cells. After concentration adjustment (1 ×109/L), UCB mononuclear cells were purified and amplified with dulbecco's modified eagle's medium containing fetal bovine serum (0.2 volume fraction), glutamine, B27, granulocyte colony-stimulating factors, and stem cell factors. According to antioxidant additive application, 4 groups were set: baicalin, blank control, β -mercaptoethanol, and baicaUn+ β -mercaptoethanol. Cells in each group were cultured for a total of 4 consecutive weeks. MAIN OUTCOME MEASURES: (1) Detection of CD 34 and CD 29 immunoreactive expression on days 7, 14, 21, and 28 after cryopreservation. (2) Cellular morphology observation. (3) Detection of surface antigen expression of MSCs by flow cytometry. (4) Detection of neuron-specific enolase (NSE), microtubule associated protein 2 (MAP-2), and glial fibrillary acidic protein (GFAP) expression after 4 weeks of culture by immunocytochemistry. RESULTS: O Compared with prior to cryopraservation, trypan blue exclusion rate of UCB-MSCs was significantly reduced on days 7, 14, 21, and 28 after cryopreservation (P < 0.05). (2) Morphological observation: UCB mononuclear cells adhered to the wall 2-3 days after culture, reached a peak level at 2 weeks, and formed a confluence of approximately 80%-90% 3 weeks after culture; at this time, all UCB-MSCs displayed a spindle shaped appearance. Four weeks later, in the baicalin group, some spindle-shaped UCB-MSCs began to present shrinkage, with slender processes on the cell edge, and some UCB-MSCs tended to be spherical-, conical-, and triangle-shaped appearance, with many slender processes on the pseudopodia. In the β-mercaptoethanol and baicalin+β -mercaptoethanol groups, an increasing number of cells defoliated and died with culture time in addition to above-mentioned appearances. (3) Four weeks after culture, cells were positive for CD45 in the blank control group, while cells in the remaining groups were positive for CD29 and CD 83, in particular in the baicalin+ β -mercaptoethanol group, followed by the baicalin group, and lastly the β -mercaptoethanol group. Significant difference in CD29 and CD 83 immunoreactivity exhibited between groups (P < 0.01). No CD34 immunoraactive calls were found in each group. (4) Four weeks after culture, NSE and MAP-2 immunoreactive expression was significantly lower in the blank control and β -mercaptoethanol groups than in the baicalin group (P < 0.01). The percentage of cells expressing GFAP was lower than 1% in each group. CONCLUSION: 100 μmol/L baicalin can promote the in vitro amplification of UCB-MSCs in a time-dependent manner and also can induce the differentiation of UCB-MSCs towards neuron-like cells in vitro to some extent.