摘要
目的构建及鉴定携带沉默人端粒酶逆转录酶基因(human telomerase reverse transcriptase,h TERT)慢病毒(lentivirus,LV)表达载体,并观察其在子宫颈癌caski细胞的表达情况。方法以Designer3.0(Genepharma)软件设计靶向h TERT基因特异性的小片段RNA(sh RNA)干扰序列,将h TERT-sh RNA基因片段插入重组慢病毒p GLV3/H1/GFP+Puro,构建慢病毒表达质粒LV3-sh RNA-h TERT,酶切、DNA测序验证h TERT片段准确性。LV3-sh RNA-h TERT与包装质粒共转染293T细胞,浓缩上清液并测定病毒滴度获得重组慢病毒后感染caski细胞。将细胞分为空白对照组(未感染病毒的caski细胞)、阴性对照组(感染空载病毒LV3-sh NC的caski细胞)和h TERT干扰组(感染携带LV3-shh TERT慢病毒的caski细胞)。绿色荧光蛋白(GFP)的表达判断转染结果并估计转染效率,流式细胞术仪检测病毒感染率,实时荧光定量PCR法(q RT-PCR)检测转染后基因h TERT m RNA的表达情况,CCK-8法检测caski细胞增殖情况。结果将目的序列成功连接到载体上,并经测序分析证实载体构建成功;成功包装成高滴度的慢病毒,且能有效感染子宫颈癌caski细胞。荧光定量PCR检测结果证实构建的h TERT-小片段干扰RNA慢病毒表达载体可显著抑制h TERT基因的表达。h TERT干扰组细胞增殖受抑制,生长速度较阴性对照组生长缓慢。结论成功构建了携带h TERT-sh RNA慢病毒表达载体LV3-sh RNA-h TERT,并稳定转染子宫颈癌caski细胞。该载体能够有效抑制h TERT的表达,使子宫颈癌caski细胞增殖缓慢,为进一步探讨h TERT基因在子宫颈癌的发病机制和体外基因干预治疗奠定基础。
Objective To construct a plasmid expressing a small hairpin RNA （shRNA） to knockdown expression of human telomerase reverse transcriptase （hTERT）,and to observe its effects on hTERT expression in caski cervical cancer cells. Methods Designer 3.0 software （Genepharma） was used to design an shRNA targeting hTERT. The shRNA was cloned into the pGI,V3/Hi/GFP＋Puro vector and confirmed by sequencing. Cells were divided into blank control group,negative control group and hTERT interference group. The resulting plasmid was transfected into easki cervical cancer cells and expression of the GFP reporter was analyzed. In addition,expression of chromosomal hTERT mRNA was quantified using RT-PCR. Cell proliferation in the presence and absence of shRNA was measured using the cck-8 assay. Results A plasmid expressing an shRNA targeting hTERT was constructed and used to generate recombinant lentivirus. Lentiviral infection of caski ceils led to lower expression of hTERT mRNA and slower cell proliferation than in controls. Conclusion A plasmid expressing shRNA targeting hTERT can effectively knockdown endogenous hTERT expression. This reagent may prove useful for understanding the role of telomerase in the pathogenesis of cervical cancer.
出处
《中国癌症防治杂志》
CAS
2014年第4期342-347,共6页
CHINESE JOURNAL OF ONCOLOGY PREVENTION AND TREATMENT
基金
广西自然科学基金资助项目(2013GXNSFAAO19254)
关键词
子宫颈肿瘤
人端粒酶逆转录酶基因
慢病毒载体
小片段RNA
CASKI细胞
Cervical neoplasm
Human telomerase reverse transcriptase gene （hTERT）
Lentivirus-based vectors
Small hairpin RNA
Caski cell
