RESUMO
Objective To explore the effects of expressing human ciliary neurotrophic factor (hCNTF) mediated by retroviral vector in olfactory ensheathing cells(OECs) on the survival and neurite outgrowth of cultured neurons. Methods S\|hCNTF fragment was digested with endonucleases(Kpn I and Xba I) from pcDNA\-3\|S\|hCNTF plasmid and cloned into pRev\|TRE vector.The harvested pRev\|TRE\|hCNTF was identified and transfected with pRev\|Tet\|On into ecotropic Ecopack\|293 cells,resulting in 2 retroviral supernatants(pRev\|TRE\|hCNTF and pRev\|Tet\|On).Primarily cultured rat olfactory ensheathing cells(OECs) were co\|infected with the 2 retroviruses,and induced to secrete hCNTF with different concentrations of doxycline.The secreted hCNTF in OEC culture supernatant was detected with Western\|blot.Dorsal root ganglion (DRG) from a postnatal rat of 2 days was co\|cultured with CNTF\|modified OECs,and the supernatant was used to culture retinal ganglion cells(RGCs).Following ?\|tubulin immunocytochemical staining,the length of DRG neurites were measured,while the numbers of surviving RGCs were counted. Results 1.Individual 630bp and 400bp fragments were digested from pRev\|TRE\|S\|hCNTF expression vector with endonucleases(Hind Ⅲ and BamH Ⅰ),and respected direction and integration of hCNTF cDNA which inserted pRev\|TRE vector were identified; 2.The expression of 24kD CNTF proteins in CNTF\|modified OEC culture supernatant was positively\|correlated with the concentration of doxycline,while no such protein expression was detected in the control groups; 3.The number of surviving RGCs in CNTF\|modified OECs group(41^34?5^4) was significantly higher than those in unmodified OEC(23^15?4^7),OECs(24^55?5^8) and blank(16^8?6^5) groups;and 4^The neurites of DRG were longer (660?67?m) and denser in CNTF\|modified OECs group,as compared with unmodified OECs(418?45?m),Mock+OECs(400?65?m) and blank (0?m) control groups.No process migrated and grew from the tissue mass in blank group.Conclusion\ hCNTF can be expressed in OECs with a doxycline concentration\|dependent manner after transfected via pRev\|TRE\|S\|hCNTF vector,and possesses a marked enhancing effect on the survival and neurite outgrowth of cultured neurons.[
RESUMO
Objective To compare the growth property of the stem cells taken from different brain regions at the same developmental stage. Methods Mice embryos at the same development stage were isolated under sterile conditions, cortex, striatum, diencephalon, mid-hind brain and spinal cord were collected and pooled separately, after single-cell suspension obtained, different regions' cell suspensions were seeded in FGF supplemented serum-free culture medium. Followed the neural stem cell clone(neurospheres) fromation, immuno-cytochemistry method was utilized to identify the cell characteristics, all these clones were passaged under same conditions, clone formation and cell migration were observed under phase-contrast microscope. Results In the FGF added serum-free medium, neural cells experienced a large scale death within 48h after being seeded, then few single cells began to proliferate and formed the floating cell clones in the medium. These clones (neurospheres) could form new clones when seeded as single cell suspension. If these clones were seeded on poly-orithine, they could differentiate into neurons and glia cells. Compare the clone formation and cell migration, we found that: cortex, striatum, diencephalon all could form floating clones with different rate, the cortex formed clones at the highest rate, striatum and diencephalon at lower rate; few neurospheres formed from cortex adhered to the culture plate substrate and few cells were found migrating out from the adhered clones, striatum and diencephalon derived neurospheres adhered the plate more easily, and there were apparent cell migration. Mid-hind brain and spinal cord formed clones at the lowest rate, floating clones were scarce, and the clones adhered to the substrate readily, there were large amount of cell migrating out from these adhered clones. Conclusion Neural stem cells could proliferate and be passaged in vitro in serum-free medium, and they could be induced to differentiate under certain conditions into major cells types of CNS, there were differences in clone forming rate and cell migration between neural stem cells derived from different CNC regions, nonetheless they were at the same development stage, this may reflect that, in some degree, these cells can keep some of their region-specified developmental intrinsic property in vitro.