逆转录病毒介导的人CNTF在嗅神经鞘细胞中的表达及其对神经细胞存活和突起生长的影响  

EXPRESSION OF HUMAN CNTF MEDIATED BY RETROVIRAL VECTOR IN OLFACTORY ENSHEATHING CELLS in vitro AND EFFECTS ON NEURONAL SURVIVAL AND NEURITE OUTGROWTH

作　　者：杨浩[1] 金卫林[1] 范明[2] 游思维[1] 鞠躬[1] 

机构地区：[1]第四军医大学神经科学研究所,西安710032 [2]军事医学科学院基础医学研究所,北京100850

出　　处：《解剖学报》2003年第4期337-343,共7页Acta Anatomica Sinica

基　　金：国家自然科学基金资助项目 ( 3 0 0 70 799)

摘　　要：目的　研究含有睫状神经营养因子 (CNTF)表达调控系统修饰的嗅神经鞘细胞 (OECs)对神经细胞存活和突起生长的影响。　方法　通过基因克隆 ,用KpnⅠ +XbaⅠ将pcDNA3 S(NGF信号肽 ) hCNTF质粒中含有NGF信号肽的CNTF切下 ,插入逆转录病毒表达载体pRev TRE中。酶切鉴定后 ,将其与本系统的调控质粒pRev Tet On转入Ecopack 2 93细胞进行病毒包装 ,制备重组缺陷型hCNTF和Tet On逆转录病毒感染原代培养的大鼠OECs,用不同浓度的强力霉素诱导 ,以Western blot法对hCNTF的表达培养上清进行检测。将转染hCNTF的OECs与新生 2d大鼠DRG联合培养 ,用其上清培养视网膜节细胞 (RGCs)。经 β tubulin免疫组织化学染色后 ,分别测量DRG神经突起的长度并统计阳性RGCs数目。　结果　 1 经HindⅢ和BamHⅠ酶切鉴定 ,pRev TRE S hCNTF重组体分别被切下 6 30bp和 4 0 0bp片段 ,插入子的方向和完整性经确认与预期相符。 2 以不同浓度强力霉素诱导hCNTF修饰的OECs ,其培养上清均有分子量约 2 4kD的hCNTF蛋白质的显著特异表达 ,此表达与强力霉素的浓度呈正相关。未诱导组和对照组未见明显蛋白带。 3 同单纯OECs(2 3 15± 4 7)、空载 (2 4 5 5± 5 8)、空白 (16 8± 6 5 )等对照组相比 ,经hCNTF转染的OECs上清组的存活RGC数显著?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 β\|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±67μm) and denser in CNTF\|modified OECs group,as compared with unmodified OECs(418±45μm),Mock+OECs(400±65μm) and blank (0μm) control groups.No process migrated and grew from the tissue mass in blank group.Conclusion\ hCNTF can be e

关 键 词：逆转录病毒介导 CNTF 嗅神经鞘细胞 表达 神经细胞 存活 突起生长 背根神经节 细胞培养 

分 类 号：R322.81[医药卫生—人体解剖和组织胚胎学] R329.28[医药卫生—基础医学]