Experimental Study Of Treating Serious Traumatic Brain Injury With G-CSF Mobilized MSCs In Mice And Its Mechanism

Traumatic brain injury (TBI) is a kind of serious injury happened in war time and peace time. Its treatment remains a great challenge, and the casualty and disable rate is the highest among serious trauma. At least 10 million patients with TBI worldwide are serious enough to result in death or hospitalization annually. In the United States, an average of 1.4 million TBI occur each year, and 50,000 deaths. There are about 1 million TBI patients every year in our country, and 100,000 of them can not survive the injury, and the most survivals are disabled, causing big burden on individuals and society as well. How to decrease the death rate and disable rate is a long-lasting tough problem in traumatic medicine. There are some advancement in the manipulation of TBI patients recent years, especailly by the combination of different countermeasures, which can lessen or prevent the further systemic damage and lower the death rate. However, the progress on promoting the regeneration of encephal tissue and the recovery of nerve function because nerve cells are difficult in regeneration and the stem cells only are confined to certain areas in brain.Neurons were considered previously a terminal differentiated functional cell, and loss of reproductive activity. Brain and spinal cord injury were repaired main by glial cell replacement, but not by neurons. This unsatisfied reapir would result in neural functional incapacity, and disable in intellegence or motion. Pliles of Studies have confirmed that neural stem cells can be isolated from brain tissue, amplified in vitro, and induced to differentiate into neurons. So it is possible for neural stem cells in brain tissue to differentiate into neuron in vivo and promote the histological construction repair and functional rehabilitation in injured tissue. After amplification and committed differentiation induction in vitro, neural stem cells can be transplanted into injury site to promote damage repair. In fact, it is almost impossible to get human neural stem cells for thraputic amplification and differntiation induction in vitro. The idea of adopting embryonic stem cells has evoked contradiction in ethics in addition to the technique problems such as allograft-caused immunologic rejection and the risk of neoplastic tramsformation during in vitro amplification. These remianed problems need to be solved before the application of stem cell transplantation in clinic.However, the multippotential differentiation, the plasticity, of adult stem cells has laid the foundation for new strategy and methods in the research interest of regeneration medicine and traumatic medicine. Increasing studies have reported that mesenchymal stem cells (MSCs) posses the characteristics, such as easy acquirement, (autoallergic or variant), feasible amplification in vitro and multipotential differentiation (to differentiate into osteocytes, muscle cells, adipocytes and neural cells). In our previous study, bone marrow cells from green fluorescent protein(GFP) transgenic mice were transplanted intravenously into homogenic mice with total-body irradiation of 10 Gyγray, and GFP-positive cells were found in intestine, liver, brain, skin of the recipients, confirming that the transplanted cells have differentiated or de-differntiated into enterocytes, hepatic cells and neurons. We also demonstrated that MSCs treated by supernatants of primary neural cell cultures expressed markers of neurons. From above findings, we drew the conclusion that MSCs can be recruited to the injury site through blood circulation and indued by the microenvironment (niche and humoral factors) to differentiate into specific tissue cells. Under physiological condition, the quantity of circulating MSCs that derived from bone marrow is too small to show theraputic effect. It is possible to increase the amount of MSCs recruited to injury site if take measures to mobilize MSCs from bone marrow to peripheral blood, and subsequently promote trauma tissue (include TBI) repair and function recovery. To explore the new way for trauma tissue repair and its mechanism will reveal the differentiation mechanism of MSCs regulated by microenvironment and facilitate clinic trauma care.In first part of this study, peripheral blood MSCs from rat and human were isolated and cultured. Colony forming units-fibroblastc (CFU-F) assay was performed to evaluate the mobilization effect on MSCs by granulocyte colony-stimulating factor (G-CSF). The morphology, differentiation potential, especially to neuron, and immunophenotype of PB-derived adherent cells were detected. In second part of this study, the experimental mouse model of serious TBI was established (impact force 50g in weight and 30cm in high of free fall). the effect of MSCs mobilized by G-CSF on the trauma recovery of different severities was evaluated by recording neuroethology scores (reference Bederson), motor function scores (reference Shapira) and the animal mortality rate. As G-CSF could not only mobilize bone marrow MSCs but also haemopoietic stem cell, which population of cells participate trauma repair need to be clearified. What was the main factor to attracte circulating cells to the injury site? Could the cells recruit to injury site of brain transform to neuron? So we designed third part study, the expression of SDF-1 was detected by Western blot, bone marrow cells of GFP mice were fractionated into MSCs, HSCs and non-MSCs/HSCs cells by adherent culture and magnetic bead cell sorting. The fractionated cells were transplanted into TBI mice to find out which cell population of cells participate in trauma repair. The differentiation/transdifferentiation of MSCs labeled by GFP in situ in trauma site were detected by double immunofluorescence. Main results and conlusion were as follow:1. PB MSCs were isolated and cultureed successfully or unsuccessfully from different genus, it concerned with many factor, especially a small number of circulating MSCs in BP. Under our experiment condition, rat's PB MSCs could been isolated and cultured steadily.2. we confirmed that G-CSF mobilized BM-derived MSCs, especially PB-derived MSCs (almost 4-fold increase) by CFU-F assay. PB-derived adherent cells formed a homogeneous cell layer that closely resembled BM MSCs. These cells were positive for marker molecule of MSCs: CD44, CD73, CD90, and CD106, but were negative for marker molecule of systema haemale: CD31 and CD45. These cells could be induced to differentiate into bone, lipoids and nerve cells. PB-derived adherent cells were considered bona fide MSCs in light of their morphology, differentiation potential, and immunophenotype.3. MSCs mobilized by G-CSF were induced to differentiate into neurons withβ-mercap- toethanol, supernatants of nerve cell cultures and con-antileptic (contain growth factor and chemical molecular), and express nerve cell markers NF, NSE, and NeuN. These MSCs could appear introvert sodium current after inducing with supernatants of nerve cell cultures.4. The experiment mouse model of serious TBI was established (impact force: 50g×30cm). It provided a experiment platform for research. 5. By research neuroethology scores, motor function scores and pathological section, result confirmed that G-CSF could significantly promote injury repair of serious TBI mice, and the mortality rate of mice after trauma was significantly degraded. Under our experiment condition, neuroethology and motor function scores were determined at different phase point after G-CSF mobilization, it was confirmed that the optimization mobilization dose was 20μg/kg.d.6. We fractionated bone marrow cells of GFP mice into MSCs, HSCs, and non-MSCs/HSCs cells by adherent culture and using magnetic beads, detect result by flow cytometer showed separation effect was good. Transplanted these cell types into trauma mice, We confirmed no HSCs or non-MSCs/HSCs cells labeled by GFP planted in injury sites, but MSCs labeled by GFP could plant in injury sites on 5d, 10d, 15d and 20d after trauma. Hence, MSCs mobilized by G-CSF promoted trauma repair.7. The expression of SDF-1 was detected by Western blot at trauma sites on different phase point. Result confirmed the expression of SDF-1 at trauma sites was significantly higher than that of normal brain tissue, and the expression of SDF-1 approached peak after trauma on 7d. The expression of SDF-1 was detected by Western blot in different tissue on 7d. Result confirmed the expression of SDF-1 at trauma sites was significantly higher than that of other tissue, and the expression of SDF-1 was lowest in heart. It could result in MSCs migrated to and planted in trauma sites.8. As the idio-marker molecule of neuron, NeuN is express on neuron nucleus. So we carried out fluorescence immunocytochemically, and got the disposition condition of neuron at brain tissue. MSCs labeled by GFP planted in trauma sites were found by double immunofluorescence to express the neuron marker NeuN on 10d and 20d. These findings confirmed that MSCs could directly differentiate into neurons at brain trauma sites to promot trauma repair.9. MSCs labeled by GFP planted in trauma sites were found by double immuno- fluorescence to express the glial cell marker GFAP on 7d, 12d and 20d. These findings suggest that MSCs can directly differentiate into glial cell at brain trauma sites to promot trauma repair.10. In view of above study, we considered G-CSF could mobilize TBI patients'MSCs from bone marrow to PB, SDF-1 high expressed at trauma sites could result in MSCs migrated to and planted in trauma sites, and planting MSCs could be induced by microenvironment factor to directly differentiate into neuron and glial cell for promot trauma repair, especial for functional recovery. This method is very practical, and has a favourable prospect for clinic application.