Effects And Mechanism Of Transplantation Of Human Amniotic Mesenchymal Cells On Hypoxic-ischemic Brain Injury In Neonatal Rats

Part I Neurobiologic characteristics of human ammiotic mesenchymal cells cultured in vitroObjective To evaluate the neurobiologic characteristics and neural nutrition function of human amnion mesenchymal cells (hAMCs) cultured in vitro. Methods Immuocytochemistry was used to detect the expression of mesenchymal stem cell markers (STRO-1, vimentin), neural stem cell markers(nestin, PSA-NCAM), neural cell markers (β-tubulin-Ⅲ, TH) and the proteins related to neuron differentiation (Math-1, Mash-1) in hAMCs. The expression of PCNA, BrdU incorporation assay and CellTiter 96(?) AQueous Non-Radiotive Cell Proliferatim Assay kit (MTS) were applied to evaluate the self-proliferation of hAMCs.β-NGF, human-NT3 and human-BDNF were detected by ELISA in hAMCs extract and their culture medium. We homogenized the brain of healthy 10-day-old Sprague-Dawley (SD) organization and induced hAMCs to neuron-like cells by co-culture, Western Blot was used to detect the expression of vimentin, NSE and NF. Results①HAMCs express the markers of mesenchymal stem cells (STRO-1, vimentin) and neural stem cells (nestin, PSA-NCAM) when cultured in vitro.②HAMCs express the markers of neuron (β-tubulin-Ⅲ, TH), and markers related to neural differentiation (Math-1 and Mash-1) when cultured in vitro.③PCNA and BrdU were positive in hAMCs dectected by immunofluorescence.④MTS assay shows that hAMCs proliferate fastest from the 6th day to the 8th day. In contrast, from the 8th day to the 24th day, the amount of hAMCs live cells was relatively stable.⑤A subpopulaton of hAMCs manifest coexpression of TH/BrdU andβ-tubulin-Ⅲ/BrdU.⑥The level ofβ-NGF, human-NT3 and human-BDNF was detectable in both hAMCs and their culture medium.⑦The expression level of vimentin, NSE and NF was greatly increased in hAMCs when cocultured with rat brain extract.Conclusion hAMCs not only express specific markers of neural stem cells and neurons but also have the capacity of self-proliferation and potential of differentiating into neural cells. The hAMCs cultured in vitro can differentiate into neural-like cells spontaneously. Math-1 and Mash-1, which are related to neuron differentiation, may partly contribute to the differentiation. HAMCs can synthesize and secrete neural nutrition factors, which may be protective and reparative for neuron. Something which can drive differentiation of hAMCs to neuron exists in the brain of neonatal rat, as implicates that the microenvironment in neonatal brain could be inductive to differentiation of hAMCs to neural cells. PartⅡStudies on the directional differentiation of human amnion mesenchymal cells into vascular endothelial cellsObjective To investigate the potential of hAMCs differentiating into vascular endothelial cells in vitro and to evaluate the possibility of their transplatation for hypoxic-ischemic brain injury (HIBI) treatment. Methods①HAMCs were isolated from abandoned human amnion obtained in Cesarean Section by 0.25% trypsin and 0.2% collagenase V and cultured in endothelial media to induce the endothelial differentiation in vitro. Then related biological characteristics are validated.②After hAMCs were induced by endothelial media containing VEGF and bFGF for 12 day, morphological observation, immunocytochemistry and Western Blot were used to study the cellular morphology and expression of CD31, vWF and VEGFR-2. Results①Primary cultured hAMCs are long spindle shape or irregular shape and evenly distributed. They could grow into larger cells after passage which is about 5-7 days once.②In vitro cultured hAMCs express markers for MSCs, such as STRO-1 and vimentin. After inductive culture with vascular endothelial media for 12 days, some hAMCs express CD31, vWF and VEGFR-2. Conclusion HAMCs can be easily obtained, cultured in vitro. HAMCs have potential to differentiate into vascular endothelial-like cell, which can be used as seed cells for neonatal hypoxic-ischemic brain injury treatment. Part III Effects of human amniotic mesenchymal cells transplantation for hypoxic-ischemic brain injury in neotatal ratObjective To investigate the effect of transplantation of human amniotic mesenchymal cells (hAMCs) on neonatal rats with hypoxic-ischemic brain injury, and its mechanism of recovering neural function. Methods HAMCs were isolated from abandoned human amnion in Cesarean Section and purified for in vitro culture. The left carotid artery of healthy 7-day-old Sprague-Dawley (SD) was ligated and exposed to hypoxic condition for 4h to produce HIBI model. After 72h,2×104 hAMCs were injected into rats cerebral tissue for the transplantation group (n=33). Cell culture medium was injected as control group (n=23). The weight, behavioral ability and neuropathologic changes were observed to evaluate HIBI model. The effects of hAMCs transplantation on HIBI rats were observed. HNA and HMA positive cells were detected by immune histochemistry.β-NGF, human-NT3 and human-BDNF within brain tissue in all groups were compared in d14. Results①The weight in the HIBI group increased obviously slower than that in the normal control group and sham operated group (p<0.01). All the rats of the HIBI group behavior abnormally. HE staining showed the neuron damage and hyperpiasm of neuroglial cells at left hemisphere in the HIBI group.②There were no difference of livability and activity between transplant group and control group.③HNA and HMA positive cells were found in hypophloeodal tissue.④The levels of NT3 and BDNF in transplantation group were higher than that in control group on d 14 after transplantation, except forβ-NGF. Conclusion The model of HIBI is successful and is accorded with the course of neonate HIBI much more in our experiment. The hAMCs injected into the cerebral of HIBI rats can survive in cerebral tissue and promote the synthesis and secretion of cerebral NT3 and BDNF to protect and repair the damaged neural system.