| Background and Aims:Neural stem cell is a kind of precursor of nerval cells with the ability of proliferation and multipotential to generate all kinds of embryonic layer cells, It has been isolated from different regions of brain and spinal cord of the embryo and adult of mammalian,such as infra-brain ventricle, hippocampus dentate gyrus, rhinencephalon, cerebellar cortex germinal layer et al. It has been cultured in vitro successfully. The discovery of neural stem cells provides a new tool for clinical therapy for dieases,aiming to replace, plerosis or enhance the biologic functions of damaged tissues and organs.The therapy of sensorineural deafness is always a puzzle for the otology doctors.With the discovery of neural stem cell and pretty therapeutic efficacy of neural stem cell on clinic,the otology scientists have transplanted neural stem cells into cochlea, hoping to help the sensorineural deafness patients. Former animal experiments have indicated that the neural stem cells transplanted into guinea pig model cochlea with sensorineural deafness have the differentiation tendency to hair cells. However,the injuries existed in ear or caused by surgical operation can induce inflammation response and enhance the level of IL-6 in blood. IL-6 plays a core role in inflammation response, Thus, IL-6 may affect the survival and differentiation oftransplanted neural stem cell. There are a few of reports about the effect of IL-6 on guinea pig neural stem cell on abroad, and few reports in China; We have known many reports about protecting effect of Tanshinone IIA on neural stem cells transplanting in department of cerebral surgery,but none of report in otology. The purpose of this research is to study the potential proliferation and differentiation of the cells isolated from the guinea pig hippocampus in culture medium adding IL-6 in vitro and the changes of IL-6 after transplantation surgery and the protective effect of tanshinone IIA on neural stem cells transplanted into cochlea. Methods:1. Hippocampus were removed from within 24 hour born guinea pig and separated to single cells, then collected and planted the single cells in another culture flask in which there was b-FGF and EGF containing medium, in order to obtain primary cultured neural spheres. We evaluated neural stem cells by anti-nestin immunocytochemistry stain. The proliferating ability of neural stem cells was proved by BrdU labeling analysis. Next, we investigated the effects of different culture medium on the differentiation of neural stem cells. The neural spheres generated from primary culture were put into different conditioned medium containing 10% serum, primary medium(no cytokine) and IL-6 of different concentrations separately. The different cells were identified by immunocytochemistry stain and the NF-200 and GFAP positive cells were analyzed.2. Selected white hair and red eyes male guinea pigs with sensitive auricle reflect and without contacting ototoxicity drugs which weight are between 250-350g. Gentamicin (lOOmg/kg) was injected everyday through nuchae subcutaneous delivery for 14 days to make sensorineural deafness model. The injuries of hair cells were observed through silver nitrate staining of cochlea and membranous spiral lamina stretching. The auditory threshold was tested with ABR instrument 2 days before gentamicin delivery and 2 days after cessation and the animals with auditory threshold increasing more than 20dB after injecting gentamicin were took as model animals. 32 model guinea pigs were put into 4 groups randomly. A groups: merely NSCs transplantion groups, 2 days after local application of gentamicin, a small hole wasthen made in the lateral wall of the cochlear first turn, The medium containing NSCs aggregates (5ul) was then injected through the hole in the cochlea and the hole was repaired with connective tissue and was covered with adherent agents. At 24h and 72h after transplantion operation, lml serum was drew from recipient animals to detecet the concentration of IL-6 by ELIASA. 7 days after transplantion, we took cochlea from recipient animals to do HE routine staining and anti-BrdU,anti-NF200 and anti-GFAP immunohistochemistry staining. B groups: merely NSCs transplantion + tanshinone IIA groups, tanshinone IlA(50mg/kg) was injected through intraperitoneal rout everyday 2 day before operation and for 7 days after operation. The evaluation methods were same to A groups.C groups: merely NSCs transplantion + toowen80(solvent) groups, toowen80(equivalent dosage) was injected through intraperitoneal rout instead of tanshine II A. The other deals were same to B groups. D groups: model groups, gentamicin sensorineural deafness guinea pig model without NSCs transplantion and tanshinone HA, The evaluation methods are same to A groups; each group had 8 animals. 8 white hair and red eyes male guinea pigs with sensitive auricle reflect and without contacting ototoxicity drugs were selected as E groups at the same time. Statistics were analysed by SPSS 10.0: paired t -test for dependent samples and LSD-test (the least significant difference). The significant level: #=0.05. Results:1 .The guinea pig hippocampus neural stem cells suspended in primary culture after cultured 48 hours in vitro,The majority of the cells suspend singly and few suspend as cells clone containing 2-3 cells. Five days later,the NSCs form NSCs clones containing lots of simple neural stem cells suspending in the culture medium, some of clones begin to pasting wall and differentiating. Anti-Nestin immunocytochemistry indicate neural stem cell. BrdU labelling experiment manifest 98.7% labelling rate of BrdU.2. The effects of different culture medium on the differentiation of neural stem cells: NSCs begin to differentiate in the conditioned medium containing IL-6 or serum. IL-6 and serum decrease the differentiating rate of neur: the differentiating rate ofneur in the conditioned medium containing IL-6 is lower than the rate in the conditioned medium containing 10% serum, moreover, the mount of neur decreased with the concentration of IL-6 increasing; On the other hand, IL-6 and serum induced NSCs to differentiate into astrocyteand the differentiating rate of astrocyte in the conditioned medium containing IL-6 is higher than the rate in the conditioned medium containing 10% serum and primary medium(no cytokine). Furthermore, the numbers of astrocyte increased with the concentration of IL-6 increasing. The numbers of positive cells of each sample were calculated and expressed as x ± s.Statistics were analysed by SPSS: paired t -test for dependent samples and LSD-test (the least significant difference). There was difference amone 4 groups (P< 0.05) .3.Ill waves detected through ABR showed that the auditory threshold of normal guinea pig is between 20~30dB and 50~80dB of sensorineural deafness guinea pig model; The silver nitrate staining of Cochlea and membranous spiral lamina stretching preparation revealed that the outer hair cells on the first turn of cochlea membranous spiral lamina had been damaged more severely than the second and third turn and the inner hair cells remained integrity.4. IL-6 was detected through enzyme-linked immunoadsordent assay(ELISA) : The concentration of IL-6 of the NSCs transplantion groups added tanshinone IIA is significantly lower than other NSCs transplantion groups without tanshinone IIA (P <0.05 ) at 24h or 72h after operation;At 24h and 72h after operation, the level of IL-6 of each group has statistical difference/* |
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