Therapeutic Effect And Mechanisms Of Derived Mesenchymal Stem Cells In Intracerebral Hemorrhage Rat

Objective: Bone mesenehymal stem cells(BMSCs) are stem cells which are lying in marrow can be separated from the marrow of age-appropriate rates.Recent studies have shown that transplantation of BMSCs into the central nervous system can successfully survive in the environment can be differentiated into neural cells and associated supporting cells, accordingly nerve diseases can have a therapeutic effect.Vitro expansion of BMSCs can be extracted and transplanted into the central nervous system, thus the treatments of cerebral hemorrhage, trauma, ischemia, and even Parkinson's disease and other nervous system injury and degenerative diseases are possible. The purpose of this experiment is to study the rat bone marrow mesenchymal stem cells extracted in vitro, and identified using phenotypic characteristics of stem cells, then transplantation in the rat brain around the hematoma, observe the improvement of neurological function of rats ,the survival, differentiation, and endogenous apoptosis after stem cell transplantation central nervous system in rats.Methods: 1. Rat bone marrow mesenchymal stem cells (BMSCs) extraction, identification, and in vitro markers. Extraction 4-6 weeks rat bone marrow under sterile conditions, separation nuclear cell by density gradient centrifugation , using the characteristics of BMSCs adherent propertiesto remove the remaining nucleated cells,passage amplification with low sugar medium containing 10% fetal calf serum to be more pure BMSCs, using flow cytometry CD90, CD29, CD45, CD11bc expression to detect cell surface types , the purity and nature of the BMSCs were identified, labeled BMSCs which were within 6 generations with Brdu (5 - bromodeoxyuridine nuclear glycosides) , identify markers for successful transplantation. 2. Modeling intracerebral hemorrhage in rats and transplantation BMSCs around hematoma . Select 192 adult male SD rats which are weigh in 180-250 grams, injection anesthesia the intraperitoneal of rat with 10% chloral hydrate (4ml/kg), determine the success of SD rats were anesthetized and were fixed prone position on the rat stereotaxic apparatus, then exposure of the skull make fontanelle is the origin, right 3.5mm, back 0.5mm, drilling skull with a bone drill with a diameter of about 1.0mm and keep dura completed, microinjector which was pumping wellⅣcollagenase was fixed on rat with positioning system into the needle slowly about 5.5mm (part of the caudate nucleus) along the skull drilled holes, slow injection of collagenaseⅣ(0.2ul, each containing collagenaseⅣ1.0ul 0.2U), determine the modeling upon completion with anatomy and nerve function , transplante BMSCs around the hematoma under the guidance of the locator . 3. Experimental animal groups. The successful model of the rats were randomly divided into two groups,each group was 96 rats , a total of 192. Group 1 Control group: After successful modeling cerebral hemorrhage 48h, use a hamilton syringe around the hematoma saline 3ul under the assistance of the locator . Group 2 Experimental groups: After cerebral hemorrhage 48h using a hamilton syringe under the assistance of cells injected with saline solution 3ul with the positioning system (containing BMSCs 1×10 ~6unit). 4. Neurological function score, immunohistochemistry to detect cell proliferation and survival (Brdu) and the NF-200 (neurofilament protein), Caspase-3 (endogenous caspase) expression around hematoma. Motor function: All the animals survived within 28 days. After transplantation, 1d, 3d, 7d, 14d, 21d, 28d, respectively, degree of neurological deficit score were observed that the nerve function of experimental group have significantly improved compared with the control group, assess whether the nerve function have significant statistically differences.Brdu, NF-200, Caspases-3 immunohistochemistry, expression of positive staining cells Brdu observed survival and the number of changes were observed around the hematoma NF-200, Caspases-3 to observe the proliferation and apoptosis of neurofilament staining and endogenous cells.Western blot (Western blot) detected Bcl-2 (B cell lymphoma/leukemia-2) and ART (Artemin glial cell line-derived neurotrophic factor) situation.ELISA enzyme-linked immunosorbent assay G-CSF (granulocyte colony stimulating factor) and BDNF (neurotrophic factor) of the situation.Features observed on the secretion of BMSCs apoptosis and proliferation of neurons.Results: Intravenously transplanted MSCs migrated to the perihematomal areas and differentiated into neurons and astrocytes glial cells. The MSC-transplanted group demonstrated better neural function on mNSS test at 7, 14, 21, and 28 days after ICH compared with the control group (P<0.05), though the difference at 1 and 3 days were insignificant. The MSC-transplanted group also showed reduced hemorrhage volume at 24h and 72h after ICH. In the perihematomal regions of rat brain with ICH, a substantial number of Brdu labeled MSCs were observed, and high protein expression of caspase 3, NF200 and GFAP were found in MSCs transplanted group. The protein content of Akt, Bcl-2, G-CSF and BDNF were all elevated by MSCs transplantation.Conclusions: Intravenously transplanted MSCs can improve functional recovery and restore neurological deficits in experimental ICH. The mechanisms are related to enhanced survival and differentiation of neural cells, increased expression of anti-apoptotic proteins and trophic factors.