The Research Of Bone Marrow Stromal Cells Differentiation In Vitro And The Cilinic Observation Effect Of Autologous BMSCs Transplantation On The TBI Patients

Traumatic brain injury happens frequently in neurosurgery clinic,and its fatality rate and disability rate is the highest of the organ injury on human's body. Currently,the clinic therapy methods are tried to promote the neuron to grow and repair the synaptic connection through neurotrophy and neural physics therapy renovation.These methods can't achieve a good effect because the human brain can't recover the injuried neuron and make new functional neuron itself.So cell transplant brings a new way to treat the TBI,which can supply new neuron or repair the injuried neuron.Because the neural stem cell is difficult to draw the material from human and it's potential to differentiate to tumor cell,the NSCs transplant is hard to applied to clinic.But bone marrow stem cell was discovered differentiate to neural cell which includes NSC and neuron in particular condiction,on the other hand it has the characteristic of convenience to draw the material and steady passage.So BMSC transplantation is the hope to the therapy of TBI. Blood serum is important in the cell culture system,and it effects on the cell division growth and vital movement.But the cell differentiation is very sensitive to the microenvionmentespecially in the cell differentiation test in blood serum culture system,the different serum desity will make the result of differentiation different.We had contrasted the rate of differentiation of hBMSCs to neural cells under different density serum,to research the impact effect of microenvironment on the BMSCs differentiation,and we had discovered the low densty serum can promot the differentiation though high desty serum inhibit.So this research can provide important message to the BMSCs differentiation in vitro and its clinic application.1 The primary culture and identification of hBMSCsObjective to get a great quantity of hBMSCs through steady and reliable BMSCs culture model in vitro.Methode to obtain the bone marrow of volunteer and isolate it with 1.073g/ml Percoll separating medium in vitro,suck the middle flocc cell layer,then culture it with DMEM/F12+15%FBS,the cells were discovered to adherence after 48h,change the liquid and will see the cells bespread all the bottle bottom.Result The cells were discovered to aggregation, arrange like bars and whirlpool,the cell shape is fusiform,astro-shape or triangle. Immunochemistry stain the cells with CD34 and CD105,the result showed that CD34 negative but CD105 positive.Conclusion The cells in primary culture were BMSCs.2 Test of microenviroment'influence on the differentiation of BMSCs to neural cellsObjective To investigate how to raise the differentiation rate of BMSCs to neural cells through inducing hBMSCs to neural cells in vitro in different serum density.Method Get the primary BMSCs and take the 3th cells after identification,pre-inducing 72h with 10ng/mlbFGF+10ng/mlEGF in fetal bull blood serum with concentration of 1%,2%,5%,10%andl5%separately,then induce the cells with 0.1umol/L RA,10ng/ml GDNF+10ng/ml BDNF in different concentration serum,and set the none serum group.Immunohistochemistry technique was used to identify the differentiated BMSCs with mouse anti-nestin,rabbit anti-NSE,rabbit anti-GFAP.Result After 1 day of culture,some BMSCs in grupe A appeared to contract and the cells turns round,the cells in FBS groups grows faster than none FBS group,the disintegrating cell nucleus increased and part of these cells turned into typical morphological neuronal traits.Cell bodies appeared refractile,cone like or spherical.The 2%FBS group cells were discover expressed NSE and Nestin more than other groups after Fluorescence immunohistochemistry test and expressed GFAP least.Conclusion BMSCs can be induced into neural like cells by basic fibroblast growth factor(bFGF),epidermal growth factor(EGF),retinoic acid (RA),brain-derived neurotrophic factor(BDNF)and gliocyte-derived neurotrophic factor(BDNF)and the 2%FBS can promote this course,with the differentiation rate of neuron was the highest.3 The clinic observation effect of autologous BMSCs transplantation on the patients with neural afunction after TBIObjective To analyse the seculity and effect of cell transplant for the TBI treatment through observe effect of autologous BMSCs transplantation on the the patients with neural afunction after TBI,thus to promote the cell transplant job to generalize.Methode culture the BMSCs in vitro,take the 3th generation cells after identification.Inject the cells which had been tested to be asepitic with the density 1×10e5个/ml into the lateral cerebral ventricle of a PVS through the Ommaya reservoir and the other two patients received the cells through intrathecal injection,all this jobs had been permited by hospital Ethics Committee and the family member had signed on the operation permission.The patients receive relate examination and test before transplantation.Result The PVS patient who accept cells transplant had not occur epilepsy and get fever 1 week later,the high muscular tension of limbs turned low,but lasted for only several days and turn high.3 months later the high muscular tension of limbs degraded and never fluctuated,the patient's eyeball can tracing stare though it can't do it before transplant,the cranium CT and PET-CT exam show that the injured lateral temporal lobe had growed new brain tissue and emerge the brain metabolism.The other two patients who receive BMSCs were found different degree improvement in symptom,such as high muscular tension of limbs dropping and the myodynamia of limbs increasing.The BDNF density in ncurolymph of 3 patients steped up gradually in 1st,3rd,5th,7th day after transplant,and it begin to drop after 14th day.Conclusion Self BMSCs transplant for therapy on the patien with neural afunction after TBI has the theoretically feasibility and the clinic practice result support this view.