| Objective To investigate TNF-a (tumor necrosis factor-a, TNF-a),IL-1β(interleukin 1-0, IL-10) expression and the number of activated microglia changes after rat bone marrow mesenchymal stem cells (BMSCs) transplantation in preterm white matter damage rats and evaluate the long-term neurobehavioral function of the rats at the same time. To discuss the mechanism of BMSCs transplantation in repairing WMD.Methods 1. Bone marrows were isolated from tibia and femur of healthy enhanced green fluorescent protein (EGFP)-transgenic rat of specific pathogen free (SPF) grade. Then, the whole bone marrow adherent method was used for isolation, culture and purification of BMSCs. The morphological change was noted by continuous observation under inverted fluorescence microscope. The growth curve of cells was drawn through the method of CCK-8 and the proliferation compared with wild type BMSCs. And the surface markers of BMSCs were detected by flow cytometry. The BMSCs were induced to differentiate into osteoblasts, adipocytes and chondrocytes lineages.2. Three-day-old SD rat pups were randomly divided into three groups:BMSCs transplantation group, namely establishing the model of WMD according to the classical Rice method (left common carotid artery ligation and hypoxia in 6%O2 and 94%N2 for two hours), and lateral ventricle injecting 2μL BMSCs (2x105); WMD model group, namely lateral ventricle injecting 2μL PBS; sham operation group, without any intervention.3. The contents and mRNA of TNF-a and IL-1β were determined by ELISA and RT-PCR. The histological changes of brain were detected by HE staining and the number of cells positive for ectodermal dysplasia-1 (ED-1) which is a specific marker for activated microglia staining was detected by immunohistochemistry.4. Evaluating the long-term neurobehavioral function of the rats in WMD model group, BMSCs transplantation group, and sham operation group.5.Statistical analysis:Statistical analysis was performed by SPSS 19.0 statistical software, measurement data was expressed by X±SD. The data were compared with single factor analysis of variance. Two sample mean compared with t test.Expressed as P<0.05 difference has statistics significance.Results 1.BMSCs with stably expressing green fluorescent protein gene can be obtained successfully, presented with the fusiform-shaped appearance and the form of circinate cell colony. The growth curve of EGFP-MSCs showed the characteristic of active proliferation, and no significant difference compared with the wild-type BMSCs. The expression rate of the surface markers of BMSCs CD29, CD90, CD34, CD49d, CD45 was 99.4%,96.4%,0.171%,0.049%,0.038%. The GFP can be detected in lung post-transplantation 3 days. After osteogenic, adipogenic, chondrogenic induction, oil red-0 and alizarin red positive signals, toluidine blue positive cells were detected.2. HE staining showed that cerebral white matter had not been observed with cellular edema or necrocytosis on post-transplantation 7th day in BMSCs transplantation group. However, in WMD model group, cellular degeneration and necrocytosis were observed. In sham operation group, cerebral white matter was also normal. The numbers of ED-1 positive cells in experimental group were significantly lower than those in WMD model group, higher than those in WMD model group. After 7d of intervention, engraftment of cells can plant in the brian. But after the intervention for 27d, engraftment of cells within the brain almost disappeared. The contents of TNF-a and IL-1β both in experimental group and WMD model group increased to a peak at 24h post-transplantation, then gradually decreased, still did not reach normal levels at 7d.The contents of TNF-α and IL-1β in brain tissue in WMD model group were significantly higher than those in experimental group at 6h,12h, 48h and 7d after BMSCs transplantation. The contents of TNF-α and IL-1β in WMD model group with no significantly difference in different time were significantly lower than those in experimental group. The mRNA of TNF-α and IL-1β at 24h post-transplantation in WMD model group were significantly higher than those in experimental group and WMD model group. In sham operation group, the mRNA of TNF-α and IL-1β were significantly lower than that in experimental group.3. Long-term neurobehavioral function and animal body weight of rats in BMSCs transplantation group are better than that in WMD model group.Conclusions BMSCs with high-purity and stably expressing green fluorescent protein gene can be cultured using the whole bone marrow adherent method. GFP does not affect the stem cell properties, and expresses stably after transplantation. BMSCs transplantation improved neural functional recovery in rats. BMSCs may play an important role in recovering preterm WMD and the mechanism may due to the inhibition of microglia activation and down regulation of inflammatory factors expressions in early stage of preterm WMD model. |
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