人白介素-12蛋白在人源骨肉瘤细胞U2-OS中的表达及抗肿瘤活性研究

Research on the Expression of hIL-12 in U2-OS Cells and the Research of its Antitumor Activity

为研究人骨肉瘤细胞(U2-OS)表达人白介素12(hIL-12)的可行性,利用Lipofectamine 2 000将重组质粒转染至U2-OS细胞中,采用实时荧光定量PCR法及Western-Blot法检测人白介素(hIL-12)在细胞的表达,并对产物活性进行了验证。将hIL-12成熟肽基因序列5'端添加Hind Ⅲ酶切位点,3'端添加6×His标签及Bam HI酶切位点,再将基因通过5'Hind Ⅲ和3'Bam HI克隆至载体pc DNA3.1上,最后转化大肠杆菌DH5α,挑取转化子并提取质粒进行酶切及测序验证。获得重组表达质粒pc DNA3.1-hIL-12,利用Lipofectamine 2 000将重组质粒转染至U2-OS细胞中,采用实时荧光定量PCR法及Western-Blot法检测hIL-12的表达。High affinity Ni-charged resin纯化hIL-12,动物体内验证其抗肿瘤活性。实验研究显示,通过基因转染技术可以将hIL-12基因导入到人U2-OS细胞,组成hIL-12的人源表达系统,经验证,hIL-12基因在U2-OS细胞内表达相应蛋白。活性研究显示,荷瘤小鼠体内IFNγ水平显著升高,脾脏细胞毒T细胞(CTL)活性及自然杀伤性细胞(CK)活性均显著升高,肿瘤重量也明显降低。

To study the expression of the feasibility of interleukin 12（hIL-12） in bone sarcoma cells（U2-OS） is the purpose of the research. In this study,human interleukin 12（hIL-12） was expressed in human-derived cancer cells by gene engineering technique using the rapid proliferation of human cancer cells,infinite passage,easy in vitro culture and human glycosylation modification system. And the activity of the product is verified. To analyze whether hIL-12 can be expressed in U2-OS cells,connect hIL-12 mature peptide gene with plasmid pc DNA3. 1 through T4 DNA ligase,transform Escherichia coli DH5α,select transformants and extract plasmids for digestion and sequencing verification. The recombinant plasmid pc DNA3. 1-hIL-12 was transfected into U2-OS cells by Lipofectamine 2 000. The expression of hIL-12 was detected by real-time fluorescence quantitative PCR and Western-blot method. The purification of hIL-12 is completed by High Affinity Ni-charged resin and its antitumor activity is verified in vivo. The results of this study show that hIL-12 can be expressed in U2-OS cells. The efficiency of hIL-12 mature peptide was analyzed by the expression of hIL-12-mRNA and hIL-12-protein. The expression of hIL-12 in U2-OS cells was observed. The expression of hIL-12 protein in U2-OS cells transfected with hIL-12 was the highest in the process of expressing protein. The level of IFN-γ in the mice was higher compared with the control. The cytotoxicity of the CTL from the splenocytes and the NK activity were also significantly higher in the mice treated with hIL-12.