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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 investigate the effects of olfactory ensheathing cells (OECs) containing ciliary neurotrophic factor (CNTF) expression and regulation system on the survival and neurite outgrowth of neurons. Methods CNTFs containing NGF signal peptide in pcDNA3 S (NGF signal peptide) hCNTF plasmid were excised by using KpnⅠ + XbaⅠ gene and inserted into retrovirus expression vector pRev TRE. After restriction enzyme digestion, the recombinant plasmid pRev Tet On was transferred into Ecopack 2 93 cells for virus packaging. Recombinant defective hCNTF and Tet On retrovirus were used to infect primary cultured rat OECs with different concentrations of Doxycycline induction, hCNTF expression supernatant was detected by Western blot. OECs transfected with hCNTF were co-cultured with neonatal 2-d rat DRGs and their retinal ganglion cells (RGCs) were cultured with their supernatants. After β tubulin immunohistochemical staining, the lengths of DRG neurites were measured and the number of positive RGCs was counted. Results1 After digestion with Hind Ⅲ and BamHⅠ, the 6 30bp and 400 bp fragments of pRev TRE S hCNTF were excised respectively. The orientation and integrity of the insert were confirmed as expected. 2 OECs modified with different concentrations of doxycycline induced hCNTF, the culture supernatant of molecular weight of about 24kD of hCNTF protein was significantly specific expression, the expression was positively correlated with the concentration of doxycycline. There was no obvious protein band in the non-induced group and the control group. 3 Survival of hCAMF-transfected OECs supernatants compared with those of control OECs (23 15 ± 4 7), no-load (24.5 ± 5 8) and blank (16 8 ± 6 5) RGC number significant?