| BACKGROUND:The specific mechanism of vena caudalis transplantation of stem cells through the blood-brain barrier to reach the host brain damage region is not clear.OBJECTIVE:To evaluate the safety of vena caudalis transplantation of human umbilical cord mesenchymal stem cells (hUC-MSCs) for the treatment of cerebral ischemia injury in Sprague-Dawley (SD) rats and to explore the therapeutical and potential mechanism. This will provide more therapy foundation in clinical application on ischemia disease and help to discover effective methods.METHODS:hUC-MSCs were isolated using density gradient centrifugation combined with adherent screening method,1Omg/L BrdU was used for labeling before hUC-MSCs transplantation. SD rats were used to establish the middle cerebral artery ischemia-reperfusion model with modified suture method, and then divided them into control group、transplantation group A and transplantation group B randomly. Rats of transplantation group A at7days after injury and group B at14days were given hUC-MSCs through the vena caudalis. Rats in the model group were left intact. The moving ability of rats were evaluated with modified Neurological severity scores. Rats were sacrificed after2months. Observe the immunohistochemistry double-staining cells (Brdu+Nestin、Brdu+MAP-2、Brdu+GFAP、Brdu+FⅧ, Brdu+VEGF) and the immunohistochemistry single-staining cells(Nestin、MAP-2、GFAP、FⅧ、VEGF), to find the situation of hUC-MSCs after transplation.RESULTS:By the way of density gradient centrifugation combine with adherence screening method to separate human umbilical cord mesenchymal stem cells, subsequently transfer of culture and amplification and differentiate in vitro. The separated cells was detected by flow cytometer, result display the cells express CD29、CD44、CD105and HLA-DR, accord with the characteristics of hUC-MSCs, such as the form and cell-surface marker.The cells possess favourable self-renewal ability and multi-directional differentiation potential, confirm the cells we separated is hUC-MSCs. The MCAO model was successful,and provide eligible conditions for the transplantation of hUC-MSCs in ischemia rats. The mNSS score of transplantation group A and group B was lower than that of model group after transplanted for7,14,28,35,42,49and56days (P<0.05), and the mNSS score of transplantation group A was lower than that of transplantation group B after transplanted for7,14,28,35,42and49days (P<0.05). The mNSS score was reached to plateau phase at35days in model group,42days in transplantation group A and49days in transplantation group B. Double-staining cells of immunohistochemistry could be found at the center of damage site(bilateral Hippocampus) and the surrounding site in rats of the group. Brdu+Nestin, Brdu+microtubule-associated protein2, Brdu+glial fibrillary acidic protein, Brdu+factor Ⅷ and Brdu+vascular endothelial growth factor immunohistochemical double staining positive cells could be seen at the center of damage site in rats of the transplantation group A and transplantation group B, there were no CD11b and MPO immunohistochemical single staining positive cells.CONCLUSION:human umbilical cord mesenchymal stem cells could separated and amplification in vitro. hUC-MSCs transplanted through vena caudalis is able to survive in the host brain damage region and improve the recovery of the neural function of ischemic brain injury rats. The mechanism may be related to the transplanted cells differentiating into neuron-like and glial-like cells, and promote the angiogenesis and secret the neurotrophic factors. In this study, no obvious immune rejection and tumor cells were found, it has the high safety.Transplantation of hUC-MSCs is effective, suggesting it can be a new way for ischemia dieases. |
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