| Head injury is the common and frequently-occurring disease, Clinical and experimental research have been accomplished by neurosurgeon. Neural stem cells (NSCs) have the ability of self-renewal and multiple differentiation in the central nervous system. In the past, neurons or axons were thinked not to regenerate after the injury of central nervous system. But it has been confirmed that there are NSCs with the ability of self-renewal and multiple differentiation in the central nervous system. They could proliferate and be induced to differentiate in the specific condition and there could be the implementation of the transplantation of NSCs in order to therapy the central nervous system injury and nerve degenerative diseases. In the recent years it has been found that NT-3 has the ability of promoting NSCs to differentiate into neurons and glias, but it is very difficult that transplanted NSCs continue to survive anddifferenciate in the transplanting site perhaps because of the deficiency of neurotrophins in the microenvironment. So we investigated the effect of NT-3 on the transplantation of neural stem cells to brain injury .1 To establish the methods of harvest and culture of NSCs of SD embryonic rat and observe the characteristics of proliferation, induce differentiation and identification of NSCs. Telencephal cortex of SD embryonic rat were dissociated mechanically and enzymatically. The suspension culture technique was performed to acquire cell clones. Immunocytochemical staining was used to identify the NSCs. We have found that cell clones could be acquired from cerebral cortex of SD embryonic rat after primary and passage culture. There clones were Nestin-positive and possessed the ability of proliferation and self-renewal. The differentiatied cells express neurons, astrocytes and oligodendrocytes specific characteristics.2 To establish a cryopreservation method for NSCs from SD embryonic rats and to study of viability and biological properties of these cells after cryopreservation. NSCs in 10% BSA +10%DMS0 were stored in ultra low temperature refrigerator (-150* C). Cells culture and immunocytochemical staining were used to identify the viability, morphology, differentiation and specific antigen expression of NSCs after cryopreservation. We have found that different time of cryopreservation and different passages do not affect survival rate of NSCs aftercryopreservation (P>0. 05). And NSCs could survive and expansionin vitro for many passages.3 To explore the effect of NT-3 on the differentiation of NSCs of SD embryonic rat. NT-3 lng/ml, 5ng/ml and lOng/ml were respectively added into NSCs culture solution. NSCs of SD embryonic rat continued to be cultured in the incubator with constant temperature and 5%C02 and growth and differentiation of NSCs were observed every day. The result is that NSCs cultured in NSCs culture solution containing NT-3 mainly differentiated to many of neurons and oligodendrocytes and small amounts of astrocytes. They were respectively stained positively by the antibody of NSE, GalC and GFAP. NT-3 chiefly promoted NSCs of SD embryonic rat into many of neurons and oligodendrocytes and small amounts of astrocytes.4 To majorly observe the therapeutic effect of transplantation of NSCs of SD embryonic rat after head injury and the survival, differentiation and immigration of transplanted NSCs in vivo in the condition of dministration of NT-3 lng/ml, 5ng/ml and lOng/ml at the same time; NSCs of telencephal cortex of SD embryonic rat were cultured in vitro and transfected by BrdU. The model of head injury was founded; NSCs were transplanted into the rats with head injury in the NSCs group and at the same time NT-3 lng/ml, 5ng/ml and lOng/ml were administered respectively in the NT-3 group. The rats were tested in nerve functional and behavioral score after 1, 2, 3 weeks and then were sacrificed by dislocationin order to carry out the examination of fluid cell test,HE staining, hybridization in situ of Tunel and immunohistochemistry staining of antibody of Nestin, GFAP, NSE, GalC and BrdU . There is statistical difference (P<0. 05) in the nerve functional and behavioral score between in control group and in NSCs group and NT-3 group in the 1, 2, 3 week. There is lower positive rate of immunohistochemistry staining of antibody of Nestin, GFAP, NSE, GalC and BrdU and higher positive rat in control group than in NSCs group and NT-3 group and There is statistical difference (P<0. 05). There is higher positive rate of immunohistochemistry staining of antibody of Nestin, GFAP, NSE, GalC and BrdU and lower positive rat in NSCs group than in NT-3 group and There is statistical difference (P<0. 05), and there is most significant difference in NT-3 lOng/ml group. NSCs of telencephal cortex of SD embryonic rat could survive, proliferate, and differentiate into neurons, astrocytes and oligodendrocytes in the cerebral region after transplantation. A few NSCs could immigrate, differenciate in the vicinity of injury region. NT-3 could promote transplanted NSCs to survive, proliferate, and differentiate into neurons, astrocytes and oligodendrocytes in the cerebral region, especially NT-3 lOng/ml.Conclusion ? The NSCs of telencephal cortex of SD embryonic rat isolated and cultured by using the present methods have the ability of self—renewal and differentiation. (2)Cryopreservation and resuscitation have not changed biological properties of NSCs,including their morphologyand capability for expansion and differentiation. (3) NT-3 lng/ml, 5ng/ml and lOng/ml could all make NSCs of telencephal cortex of SD embryonic rat differentiate to many of neurons and oligodendrocytes and small amounts of astrocytes, especially NT-3 lOng/ml. ?NSCs of telencephal cortex of SD embryonic rat were early transplanted into rat with head injury and could survive, proliferate and differentiate into neurons, astrocytes and oligodendrocytes in the cerebral region and immigrate, differenciate in the vicinity of injury region. It was in favor of recovery of head injury. When NT-3 lng/ml, 5ng/ml and lOng/ml were administered at the same time after head injury, there was more survival rate and differentitated rate of transplanted NSCs of telencephal cortex of SD embryonic rat and a smaller quantity of Tunel postive cells.
|
PDF Full Text Request