中文摘要：

生长激素（growth hormone,GH）是由垂体分泌的一种促进动物机体生长、肌肉发育、调节代谢等重要生理功能的多肽激素。本研究根据小鼠（Mus musculus）GH m RNA序列设计1对m GH小发卡RNA（short hairpin RNA,sh RNA）并构建了整合型可控表达m GH sh RNA载体p Single-t TS-m GH sh RNA-RFP。重组载体转染小鼠垂体瘤At T-20细胞,在培养液中添加诱导底物强力霉素（doxycyclin,DOX）,借助荧光显微镜观察红色荧光蛋白（red fluorescent protein,RFP）的表达,利用实时荧光定量PCR（quantitative Realtime PCR,qRT-PCR）及Western blot检测细胞内m GH的表达变化。结果表明,经DOX诱导后,转染含GH sh RNA重组载体细胞组GH m RNA相对表达水平比未经DOX诱导组及其他对照组细胞中降低70%左右,差异极显著（P〈0.01）,GH蛋白也下降90%左右,其他各组间GH m RNA及蛋白表达量差异不显著（P〉0.05）。本研究成功构建了可控表达GH sh RNA载体,并在细胞水平证明有很好的可控基因沉默效率,后续配合可控过表达GH基因系统,通过控制GH基因表达水平研究其在生长发育及相关疾病发生中的机制。

英文摘要：

Growth hormone（GH） screened by the pituitary, is a polypeptide hormone which promotes the growth of the animal body, muscle development, and regulates metabolism and other important physiological functions. In this study, a pair of Mus musculus GH（m GH） short hairpin RNA（sh RNA） was designed based on the 340bp-abasic site of m GH m RNA sequence, and the m GH sh RNA sequence was linked to a integrated tetracycline induced expression vector with sequences syntheses method, the recombinant vector named as p Singlet TS-m GH sh RNA-RFP（red fluorescent protein）. The recombinant vector was transfected to mice pituitary tumor cell lines At T-20 and screened with G418 two weeks to enrich cell clones which integration of exogenous genes. The RFP expression was observed under a fluorescent microscope and the GH expression levels were detected with qRT-PCR and Western blot methods. The results showed, little RFP signal could be observed in control cell groups（cell groups transfected with p Single-t TS-RFP plasmid） and experimental cell groups（cell groups transfected with p Single- t TS- m GH sh RNA- RFP plasmid） induced with doxycyclin（DOX）, the RFP signal was obviously enhance in the above 2 cell groups. The m GH m RNA in control cell groups, control cell groups adding DOX, experimental groups and experimental groups induced with DOX was 1 ± 0.17, 1.03 ±0.12, 0.758±0.19 and 0.204±0.07, respectively. Compared with the other 3 groups, the GH m RNA expression in experimental groups induced with DOX（working concentration, 600 ng/μL） difference between them was extremely obvious（P〈0.01）, whereas the difference among the other 3 groups was not obvious（P〈0.05）.The average GH protein expression level in control cell groups, control cell groups adding DOX, experimental groups and experimental groups induced with DOX was 1, 1.07, 0.88 and 0.32, respectively. An effective m GH sh RNA sequence was designed in this study and a controllable m GH sh RNA overexpression vector was constructed