摘要
背景:动物研究显示,自体髓核细胞移植能有效修复椎间盘退变。然而髓核细胞体外增殖能力差,这就限制了其作为种子细胞在椎间盘退变性疾病治疗中的研究及应用。目的:构建包含外源性人端粒酶反转录酶基因的腺相关病毒2载体,观察其转染人髓核细胞后人端粒酶反转录酶基因的表达。方法:构建pSNAV2.0-pRSV-hTERT质粒并鉴定,采用AAVMaxTM包装系统进行重组腺相关病毒2-人端粒酶反转录酶载体的构建,以PCR及酶切方法验证构建的质粒,构建成功后扩增,并纯化。利用腺相关病毒2-增强型绿色荧光蛋白载体转染第1代人髓核细胞,测定最佳感染复数。参照此感染复数值,确定腺相关病毒2-人端粒酶反转录酶对人髓核细胞转染的相关感染复数;对照组采用不含外源性人端粒酶反转录酶基因的腺相关病毒2进行转染。转染后1,2,4周分别采用RT-PCR对人端粒酶反转录酶基因mRNA水平进行半定量检测。结果与结论:实验成功构建了腺相关病毒2-人端粒酶反转录酶载体;并获得了滴度达2×1011 v·g/mL的腺相关病毒2-人端粒酶反转录酶载体。测得腺相关病毒2-人端粒酶反转录酶载体对人髓核细胞的最佳感染复数为5×104 v·g/cell。在以1×104,5×104,1×105 v·g/cell转染人髓核后,均可检测到人端粒酶反转录酶基因mRNA的高量表达。采用RT-PCR半定量检测方法,发现以转染后2周时人端粒酶反转录酶mRNA表达量相对最高(P<0.05),4周时仍可见人端粒酶反转录酶基因mRNA的稳定表达。而对照组无论在何时间点均未能检测到人端粒酶反转录酶mRNA的表达。提示利用腺相关病毒2可以成功构建包含外源性人端粒酶反转录酶基因的病毒载体,腺相关病毒2-人端粒酶反转录酶能有效转染人髓核细胞并稳定表达人端粒酶反转录酶基因mRNA,此结果可能为增强髓核细胞性能提供新的策略。
BACKGROUND：In animal experiments, transplantation of autologous nucleus pulposus cellscan effectively repair the intervertebral disk degeneration. However, nucleus pulposus cells have a poor ability of proliferation in vitro, which limits its application as seed cells in treatment of intervertebral disk disease.
OBJECTIVE：To construct recombinant adeno-associated virus type-2 vector carrying human telomerase reverse transcriptase and observe the human telomerase reverse transcriptase mRNA expression in human nucleus pulposus cells in vitro.
METHODS：After the plasmid pSNAV2.0-pRSV-hTERT was constructed and identified, recombinant adeno-associated virus type-2 vector carrying human telomerase reverse transcriptase were constructed, amplified and purified by AAVMaxTM package and purification system. The optimal multiplicity of infection for human nucleus pulposus cells was detected by recombinant adeno-associated virus type-2 vector carrying enhanced green fluorescent protein. According the optimal multiplicity of infection （5 × 104 v·g/cell）, three different multiplicity of infection （1×104, 5×104, 1×105 v·g/cell） of recombinant adeno-associated virus type-2 vector carrying human telomerase reverse transcriptase were determined to transfect the first passage human nucleus pulposus cells in vitro. In control group, the cells were transfected with adeno-associated virus type-2 vector without human telomerase reverse transcriptase. At 1, 2, 4 weeks after transfection, mRNA expression of human telomerase reverse transcriptase in human nucleus pulposus cells were semi-quantitatively detected by RT-PCR.
RESULTS AND CONCLUSION：The recombinant adeno-associated virus type-2 vector carrying human telomerase reverse transcriptase was successful y constructed, and the titer of the obtained vector was more than 2×1011 v·g/mL. The optimal multiplicity of infection was 5×104 v·g/cell. The mRNA expression of human telomerase reverse transcriptase in human nucleus pulposus cells could be detected in different multiplicity of infection （1×104, 5×104, 1×105 v·g/cell）. At 2 weeks post-transfection, mRNA expression of human nucleus pulposus cells was the highest （P〈0.05）, as detected by semi-quantitative RT-PCR. Moreover, the stable and high mRNA expression of human telomerase reverse transcriptase could be detected at 4 weeks post-transfection. In control group, no human telomerase reverse transcriptase mRNA expression was found. The recombinant adeno-associated virus type-2 vector carrying human telomerase reverse transcriptase can be successful y constructed, and can mediate a stable mRNA expression of human telomerase reverse transcriptase in human nucleus pulposus cells. Our findings provide a novel strategy of enhancing the properties of nucleus pulposus cells.
出处
《中国组织工程研究》
CAS
CSCD
2014年第15期2409-2414,共6页
Chinese Journal of Tissue Engineering Research
