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背景:骨肉瘤OS-9607细胞是一种从人骨肉瘤组织中培养出的细胞株。构建该细胞系cDNA文库,以便从中克隆目的基因。目的:通过SMART技术(一种以RNA转录时5’末端转换机制为原理的技术)构建骨肉瘤OS-9607细胞cDNA文库并进行质量分析。设计:验证性的实验研究。单位:解放军第二军医大学长海医院骨科。材料:实验于2005-05在解放军第二军医大学长海医院骨科完成。骨肉瘤OS-9607细胞在CO2培养箱中培养。细胞先用DM处理30min,然后在标准培养基中恢复24h。骨肉瘤OS-9607细胞的全部RNA可通过RNAease试剂盒分离。全部RNA的数量及完整性通过紫外线光谱分析器和溴乙锭染色的琼脂糖凝胶电泳检测。方法:从人骨肉瘤OS-9607细胞中提取全部RNA并分离出mRNA,通过原位聚合酶链反应和长距离聚合酶链反应技术获得OS-9607细胞的cDNA,最后将cDNA与去磷酸化的λ噬菌体连接。重组物是用SMARTcDNA文库试剂盒构建的。插入的cDNA长短和文库的多样性通过聚合酶链反应检测。主要观察指标:总RNA的数量和完整性,未扩增文库和扩增文库的滴度及重组指数,分析文库中cDNA长度。结果:①总RNA的浓度测得其吸光谱位于260～280nm,其吸光度大约为1.9A,可清晰的显示其为28SrRNA和18SrRNA,其亮度比值为2∶1(28S/18S)。②在试管内与λ噬菌体连接并转染宿主细菌后,未扩增文库的滴定度达到2.2×107pfu/mL,而扩增文库达到4.5×1010pfu/mL;重组指数分析,在一个培养基中蓝色斑点有8个,无色斑点达到160个,重组指数为99.5%。③获得的cDNA文库由1.6×106个重组噬菌体组成,重组物中平均外源插入物达到1.5kb。结论:本研究获得的骨肉瘤OS-9607细胞cDNA文库质量优良,为今后骨肉瘤OS-9607细胞方面的相关研究及骨肉瘤相关基因的筛选奠定了坚实的基础。
BACKGROUND: Osteosarcoma OS-9607 cells are a cell line cultured from human osteosarcoma tissue. The cell line cDNA library was constructed for cloning of the gene of interest. OBJECTIVE: To construct and characterize osteosarcoma OS-9607 cell cDNA library by SMART technique, a technique based on the 5 ’end transformation mechanism of RNA transcription. Design: Confirmatory Experimental Research. Unit: Changhai Hospital, Second Military Medical University. Materials: The experiment was performed in Department of Orthopedics, Changhai Hospital, Second Military Medical University, 2005-05. Osteosarcoma OS-9607 cells were cultured in CO2 incubator. Cells were first treated with DM for 30 min and then resumed for 24 h in standard medium. Osteosarcoma OS-9607 cells total RNA can be isolated by RNAease kit. The amount and integrity of total RNA was detected by UV spectroscopy and ethidium bromide-stained agarose gel electrophoresis. Methods: Total RNA was extracted from human osteosarcoma OS-9607 cells and mRNA was isolated. CDNA of OS-9607 cells was obtained by in situ polymerase chain reaction and long-distance polymerase chain reaction. Finally, cDNA was combined with dephosphorylated λ Phage attachment. Recombinants were constructed using the SMART cDNA Library Kit. The length of the inserted cDNA and the diversity of the library were detected by polymerase chain reaction. MAIN OUTCOME MEASURES: Quantity and integrity of total RNA, titer and recombination index of unexpanded and amplified libraries, and analysis of cDNA length in the library. Results: (1) The absorbance of total RNA was 260 ~ 280nm and its absorbance was about 1.9A. It clearly showed 28SrRNA and 18SrRNA with a brightness ratio of 2: 1 (28S / 18S). (2) The titer of the non-amplified library reached 2.2 × 107pfu / mL and the amplified library reached 4.5 × 1010pfu / mL after being ligated with lambda phage in a test tube and transfected into the host bacteria. Recombinant index analysis showed that in a medium of blue There are 8 color spots, 160 colorless spots, and the recombination index is 99.5%. ③ The obtained cDNA library consisted of 1.6 × 106 recombinant phage and the average foreign insert in the recombinant reached 1.5 kb. Conclusion: The osteosarcoma OS-9607 cell cDNA library obtained in this study is of good quality, which lays a solid foundation for the future research on osteosarcoma OS-9607 cells and the screening of osteosarcoma related genes.