| The death of a large quantities of cells and the serious neuraldysfunction syndrome are usually caused by the cerebral contusion.Generally speaking, the central nervous system lacks the ability ofregeneration. The study concludes that the key to treatment of braincontusion is to transplant the exogenous cells for supplying or replacingloss of the nerve cells inorder to promote the survival of the nerve cells andthe growth of axons and reconstruct the functional synapse. Thetransplantation of neural stem cells (NSCs) has good application prospectsto treat brain contusion. In this study, we investigated the methods ofisolating the NSCs of the fetal rats forebrain in vitro, the methods inprimary and subculture cultivation and the identification of the NSCs. TheNSCs characteristics, the mechanisms of differentiation and the conditionsof induction were observed. Furthermove, we explored survival, migrationand differentiation of NSCs transplanted into rat cerebral contusion focus.Part One: A study of cultivation and differentiation of fetal ratsforebrain NSCs in vitroThe experiment was completed at Experimental Center of Hebei NorthUniversity from November 2008 to October 2009. Cleanliness level,embryonic 16d and pregnant Wistar rats were provided by the AnimalLaboratory of the Medical Sciences Institute of the Chinese Academy.Under aseptic conditions, we obtained fetal rats. NSCs were isolated fromfetal rats forebrain to prepare single cell suspension. The cell density of 1×106·ml-1 and 1×108·ml-1 was inoculated in culture bottle containing theDMEM/F12 medium and N2 additives, with a final concentration of20ng·ml-1 epidermal growth factor (EGF) and 10ng·ml-1 basic fibroblastgrowth factor (bFGF) to stimulate clonal growth. The inducingdifferentiation experiment included two parts. A. The suspensioncultivation test was divided into three groups: the volume fraction of 10%fetal bovine serum (FBS) +20ng·ml-1 EGF +10ng·ml-1 bFGF, 10%FBS,20%FBS. In these conditions, NSCs were induced differentiation. B. Theadherent cultivation test was divided into three groups: deckingpoly-L-lysine group, decking gelatin group and non-decking group. NSCswere stimulated differentiation with the volume fraction of 20% FBS.Nestin, glial fibrillary acid protein (GFAP), neuron-specific enolase (NSE)and microtubule-associated protein 2 (MAP-2) were detected withimmunocytochemistry and indirect immunofluorescence staining. As aresult, NSCs were successfully isolated from fetal rats forebrain, and thenand showed nestin-positive antigen. In inducing conditions of thesuspension cultivation test, the promoting differentiation effect of 20% FBSgroup was slightly stronger than 10% FBS group (P>0.05); the promotingdifferentiation effect of 10% FBS group was stronger than 10% FBS+20ng·ml-1 EGF +10ng·ml-1 bFGF group (P<0.05). NSCs weredifferentiated into neurons and glial cells. In the adherent cultivation test,the anchorage-dependent ability of decking poly-L-lysine group anddecking gelatin group to differentiate into neurons and glial cells wasstronger than not-decking group (P<0.01). Decking poly-L-lysine groupwas a little stronger than decking gelatin group (P>0.05). GFAP, NSE andMAP-2 were detected positive by immunocytochemistry and indirectimmunofluorescence staining.Conclusion:NSCs were rich in the fetal rats forebrain, and they couldbe obtained in vitro cultivation. They had the proliferative capacity and theproficiency of multi-differentiation. At the same time, the effect of promoting differentiation was very strong in 20% FBS group. It was notsignificant in 10% FBS + EGF + bFGF group. In the inductingdifferentiation of NSCs, the poly-L-lysine and the gelatin, as the supportivematerial of adherent cultivation, played a role in raising the quantity of celldifferentiation, and NSCs were mostly differentiated into astrocytes.Part Two: The survival, migration and differentiation of NSCs aroundthe rat cerebral contusion focus region.The study used the serum-free cultivation technology, plus EGF andbFGF, to stimulate the growth and the proliferation of embryo-derivedNSCs. They were cultivated two passage in vitro, marked by 6μg·ml-15-bromodeoxyuridine (BrdU) and detected by immunocytochemistry andindirect immunofluorescence staining, to confirm the ability ofproliferation. Then we produced an improved model Fenney's free-fallcerebral contusion by Wistar rats. After transplanted 1d, NSCs weretransplanted into the edge of the rat cerebral contusion focus cortex bystereotactic localization. Transplanted NSCs were detected byimmunohistochemical and immunofluorescence double staining method,and the survival, migration and differentiation of them had been observedaround the rat cerebral contusion focus region in the post-transplant 1d, 7d,14d, 21d. Results: The number of BrdU-positive cells around the ratcerebral contusion focus region reduced gradually. BrdU-positive cellsscattered around the cerebral contusion focus and migrated to thesubcortical region. Besides, GFAP-positive cells increased with someregularity around cerebral contusion focus region after injury.Conclusion: The results of this study suggest that forebrain NSCs afterallogenic transplantation can survive and migrate around the rat's cerebralcontusion focus region, and they shows the characteristics ofmorphological integration with the brain.
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