Lentivirus载体对许旺细胞的转染效率

doi:10.3969/j.issn.2095-4344.2014.51.019

摘要/Abstract

摘要：

背景：近年来研究较多的LV载体，因其强大的转染能力以及较高的转染效率等特点，在基因转染的实验中应用越来越广泛。
目的：进一步验证Lentivirus载体在体外对原代许旺细胞的转染效率。
方法：以Lentivirus三质粒系统构建病毒载体，在体外分别以MOI值为1，5，10，20，40对原代大鼠许旺细胞进行转染，转染后第1，3，5，7，9天在荧光显微镜下观察Lentivirus携带的荧光表达情况，并在显微镜的计数方格内计算细胞的转染情况。发出绿色荧光的为转染成功的许旺细胞，否则认为没有转染病毒载体的，从而算出转染效率。
结果与结论：在病毒转染3 d后，在各不同MOI值的培养孔中，均能观察到极少量的荧光反应，在第5天时，荧光数量较前明显增多。第7天时达到高峰，第9天的荧光数量与第7天变化不明显。从细胞转染效率来看，不同的MOI值明显不同，MOI值为1时的转染效率约为45%，MOI值为5时为80%，MOI值为10时为90%，MOI值为20时为78%，MOI值为40时转染效率为70%。

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

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关键词: 组织构建, 组织工程, 许旺细胞, Lentivirus病毒, 病毒载体, 感染复数, 转染效率, 细胞培养, 脊髓损伤, 绿色荧光蛋白, 国家自然科学基金

Abstract:

BACKGROUND: The Lentivirus carrier studied in recent years has been widely used in the gene transfection experiment due to its strong transfection ability and high transfection efficiency.
OBJECTIVE: To study the transfection efficiency of Lentivirus carrier to primary Schwann cells in vitro.
METHODS: The virus vector was constructed with Lentivirus three-plasmid system. In vitro, the primary Schwann cells were transfected by recombinant virus with different-multiplicities of infection (1, 5, 10, 20 and 40). At 1, 3, 5, 7 and 9 days after transfection, fluorescent expression of Lentivirus was observed under fluorescence microscope, and the transfection efficiency was calculated in the counting squares of the microscope. Green fluorescence presented the transfected Schwann cells, and the others were untransfected Schwann cells. Then the transfection efficiency was calculated.
RESULTS AND CONCLUSION: At 3 days after transfection, the small amount of fluorescence could be seen in the culture dishes with different multiplicities of infection, and then the fluorescence amount was increased at 5 days after transfection. It reached peak after 7 days. There was no significant difference in the fluorescence amount between 7 and 9 days after transfection. Different multiplicities of infection could lead to different transfection efficiencies: the transfection efficiency was 45% when the multiplicity of infection was 1; the transfection efficiency was 80% when the multiplicity of infection was 5; the transfection efficiency was 90% when the multiplicity of infection was 10; the transfection efficiency was 78% when the multiplicity of infection was 20; and the transfection efficiency was 70% when the multiplicity of infection was 40.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

Key words: lentivirus, schwann cells, transfection, cells, cultured

中图分类号:

R318

连小峰，徐建广，曾炳芳，周蔚，孔维清，张涛，侯铁胜. Lentivirus载体对许旺细胞的转染效率[J]. 中国组织工程研究, 2014, 18(51): 8301-8304.

Lian Xiao-feng, Xu Jian-guang, Zeng Bing-fang, Zhou Wei, Kong Wei-qing, Zhang Tao, Hou Tie-sheng. Transfection efficiency of Lentivirus carrier to Schwann cells in vitro[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(51): 8301-8304.

图/表（结果） 3

参考文献

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引言

材料方法

讨论

3.1 许旺细胞基因转染的载体关于许旺细胞基因转染所应用的载体，主要有反转录病毒载体和腺病毒载体。Weidner等^[10]应用反转录病毒载体将人神经生长因子基因导入许旺细胞，应用ELISA定量分析发现经基因转染的许旺细胞可分泌神经生长因子36 ng/(10⁶·d)，而未转染人神经生长因子基因的许旺细胞只分泌神经生长因子 0.05 ng/(10⁶·d)。Sorensen等^[11]用反转录病毒转染许旺细胞，其转染率仅为18%，且目的基因的表达随时间的延长逐渐下降。虽然反转录病毒载体也可转染分裂细胞及非分裂细胞，但是它导入效率低，靶向性差，而且反转录病毒载体的转染率不高，因此它更适合于体外转染。目前，用反转录病毒载体转移基因的研究正在逐步减少。腺病毒载体为双链DNA病毒载体，也可转染分裂细胞及非分裂细胞，表达时间快，表达丰度高，但其基因组由于游离于宿主基因组外，只能瞬时表达外源基因，难于真正满足基因治疗及组织工程技术的应用要求。而近年来研究较多的LV载体，因其强大的转染能力以及较高的转染效率等特点^[12]，在基因转染的实验研究中应用越来越广泛。

3.2 慢病毒Lentivirus载体的特点慢病毒是反转录病毒的一种，具有反转录病毒的基本结构，但也有不同于反转录病毒的组份和特性，作为基因治疗载体发展起来^[13]，最近已用于转基因动物制备。利用LV构建的shRNA载体，与化学合成的siRNA和基于瞬时表达载体构建的shRNA相比，一方面可以扩增替代瞬时表达载体使用，另一方面，LV-shRNA克隆经过LV包装系统包装后，可用于转染依靠传统转染试剂难于转染的细胞系如原代细胞、悬浮细胞和处于非分裂状态的细胞，并且在转染后可以整合到受转染细胞的基因组，可以伴随细胞的增殖分裂而不丢失所携带的目的基因，进行长时间的稳定表达。因此，其对神经细胞如本实验的许旺细胞尤其适合^[12]。另外，慢病毒载体LV可以携带并稳定表达多基因，既能达到治疗目的，又能方便检测基因的转染情况及表达情况^[14]。实验所应用的是三质粒载体，其最大的益处是使序列重叠的机会明显减少, 减少载体重组过程中产生RCV的可能性^[15]，且病毒滴度可达10⁹TU/L。

3.3 Lentivirus载体对许旺细胞的体外转染情况 Hu等^[12]研究发现携带睫状神经营养因子的LV载体对许旺细胞的转染效率在48 h后即达90%以上，且能稳定、高效表达睫状神经营养因子达3个月。Hurtado等^[16]应用LV载体携带目的基因脑源性神经营养因子、NT-3和报告基因绿色荧光蛋白的多基因转染许旺细胞其转染效率可达95%以上，植入体内6周后仍能稳定高效地表达目的基因。在本实验中，应用LV载体的三质粒系统包装构建的LV载体，携带有报告基因绿色荧光蛋白转染许旺细胞。经报告基因绿色荧光蛋白的表达情况可检测本实验所构建的重组LV载体对许旺细胞的转染效率达90%以上，与上述文献报道的一致。

利用基因转移技术治疗脊髓损伤，目前主要是采用基因修饰种子细胞如许旺细胞、嗅鞘细胞或成纤维细胞后再将其植入鼠的脊髓损伤区，观察其轴突再生的情况^[17-19]。在应用神经生长因子基因修饰许旺细胞治疗脊髓损伤的实验研究中，Tuszysnki^[20]将神经生长因子基因导入培养的许旺细胞内，再将许旺细胞移植到损伤的脊髓，发现其在体内能稳定地表达神经生长因子并促进轴突的再生。同年Tuszynski等利用来源于莫洛氏鼠白血病的反转录病毒载体，将人的神经生长因子基因导入鼠成纤维细胞中，再将成纤维细胞移植到半横贯损伤的鼠脊髓中(T₇平面)。1年后成纤维细胞仍能表达神经生长因子，电镜及免疫组织化学检查，发现有大量的脊髓轴突再生(感觉纤维)。之后，基因修饰许旺细胞移植治疗脊髓损伤不断得到实验证实，但也存在一些需要解决的问题，主要有以下3个方面：一是免疫排斥反应，即宿主对移植细胞的免疫排斥。因为尽管中枢神经系统的免疫应答能力微弱，但对抗原较强的或种属相关较远的受体细胞仍具有免疫排斥反应。在以SD大鼠为动物模型的研究中，人们发现其免疫排斥极小，几乎可以忽略不计^[21-22]。二是移植细胞的长时程存活问题。目前在SD大鼠中移植异体许旺细胞已证实可以存活4个月以上^[23]。三是遗传修饰细胞移植后转移基因的表达可能会随着时间的延长而逐渐下降，以致失去治疗作用。而本实验构建的LV慢病毒载体可以很好地解决这一问题。

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

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