亚甲蓝光化学法对血浆中水疱口膜炎病毒灭活效果的研究

目的 评价亚甲蓝光化学法(MBP)对血浆中水疱口膜炎病毒(VSV)的灭活效果.方法 将7.12 logTCID50·mL-1 VSV加入血浆中,采用不同的参数组合[亚甲蓝(MB)浓度(0.8、1.3 μmol·L-1)、光照强度(20 000、50 000 LUX)、光照时间(10、20、30 min)]灭活处理后,分别将样品加入非洲绿猴肾细胞(Vero)单层细胞中培养,观察细胞病变(CPE)状况,评价病毒灭活的效果.结果 经过30 min灭活处理,可将病毒彻底灭活,病毒滴度下降6 log值以上.实验设置的MB浓度和光照强度对血浆病毒灭活的影响不明显.结论 在MB浓度为0.8 μmol·L-1和光照强度20 000 LUX的条件下,处理血浆30 min以上就能有效灭活血浆中的VSV病毒.     

幼年与成年兔椎间盘髓核细胞的形态学差异及其意义

目的探讨幼年与成年兔髓核细胞的形态学差异。方法利用激光共聚焦显微镜和透射电镜,对幼年和成年兔的髓核组织进行组织细胞水平和超微结构的观察。结果幼年兔的髓核细胞呈圆形,簇状分布,其直径为(266±167)μm,细胞簇的密度为每个高倍(×40)视野(14.9±4.3)个,细胞内含有大量空泡状的包含体。成年兔的髓核细胞呈圆形或类圆形,细胞簇小或呈单个散在分布,细胞簇的直径为(94±42)μm,细胞簇密度为每个高倍(×40)视野(8.0±2.4)个细胞,其中包含体数目少且不含大的包含体。结论幼年与成年兔的髓核细胞具有明显的形态结构差异。椎间盘成年期发生的这种形态结构上的变化可能是随年龄增加而椎间盘生物学功能逐渐降低的原因之一。     

杆状病毒介导的GFP转染与退变的兔椎间盘髓核细胞

目的:检测杆状病毒转染正常与退变的椎间盘髓核细胞的效率以及杆状病毒介导的GFP基因(Ac/CMV/GFP)在正常与退变细胞中的表达水平。方法:取新西兰大白兔的髓核细胞进行离体培养,第1组:健康幼兔;第2组:椎间盘退变模型兔;第3组:椎间盘发生自然退变的成年兔。以200MOI杆状病毒介导的绿色荧光蛋白(GFP)转染离体培养的细胞,于第48h采用流式细胞仪检测转染效率,同时采用HPIAS2000图像分析软件半定量分析外源性GFP基因的表达水平。结果:3组细胞被转染的百分率分别为84.4±3.4,82.9±5.7,81.8±5.5(P>0.05)。外源性基因表达荧光蛋白的光密度值分别为0.0054±0.0029,0.0052±0.0032,0.0056±0.0031(P>0.05)。结论:杆状病毒转染椎间盘髓核细胞的效率与椎间盘退变程度无关,杆状病毒介导的外源性基因能在正常与退变的髓核细胞中高水平的表达。     

Construction of recombinant baculovirus Ac-CMV-hSox9 for gene therapy of intervertebral disc degeneration

Objective： To construct the recombinant baculovirus Ac-cytomegalovirus （CMV）-hSox9 for gene therapy of intervertebral disc degeneration. Methods： Bac-to-Bac system was used for the construction of baculovirus Ac-CMV-hSox9. The cDNA of hSox9 was first cloned into a plasmid vector under the control of CMV promotor to generate the donor plasmid pFastBacDul-green fluorescene protein （ GFP ）-CMV （pgGC）-hSox9. The resultant plasmid was transformed into DH10Bac cells and then the transformation mixture was spread on Luria-Bertani （LB） agarose culture medium containing isopropyl-β-D-thiogalactoside （IPTG）, X-gal, gentamicin, kanamycin and tetracycline. The white colonies were selected and cultured for amplification, and the hSox9 Bacmid DNA was extracted. After verification, recombinant baculovirns Ac-CMV-hSox9 was obtained through transfecting Sf 21 cells. The expression of hSox9 gene in the intervertebral disc cells in rabbits was determined by Western blotting and immunohistochemical staining. Results： Polymerase chain reaction （ PCR ） confirmed the presence of hSox9 gene in the recombinant baculovirus and the Sf 21 cells transfected by the baculovirus showed the expression of fluorescence protein. Western blotting and indicated that exogenous hSox9 disc cells. staining analysis Conclusions： The successful construction of the recombinant baculovirus Ac-CMV-hSox9 and the confirmation of the target gene expression provides a novel expression vector system for basic research and clinical treatment of intervertebral degenerative disc disease.