Zhang Jing, Wang Dongmei, Hu Na, Wang Qian, Yu Shanice, Wang Jun
Department of Pathophysiology, College of Basic Medical Sciences, Dalian Medical University, Dalian, People's Republic of China, 116044.
Department of Experimental Functionality, College of Basic Medical Sciences, Dalian Medical University, Dalian, People's Republic of China.
Tumour Biol. 2016 May;37(5):5847-55. doi: 10.1007/s13277-015-4405-z. Epub 2015 Nov 19.
This study aims to design and generate recombinant lentiviral vector containing the complete coding sequence (CDS) region of human serine protease inhibitor Kazal type 1 gene (SPINK1) and establish a human pancreatic cancer cell line (AsPC-1) stably overexpressing SPINK1. Then, to assess the proliferative and oncogenic effects of upregulated SPINK1 gene on pancreatic cancer AsPC-1 cells using different methods. Initially, the target coding sequence (CDS) of SPINK1 was amplified by polymerase chain reaction (PCR) and the synthesized product was subsequently subcloned into the lentiviral vector. The construction of recombinant SPINK1 gene was verified by the restriction digestion and sequencing analysis. The lentiviral particles carrying SPINK1 gene were produced by co-transfection of the recombination lentiviral vector and the packaging mix plasmids into 293 T cells and filtered and concentrated before AsPC-1 cells were infected by the virus particles. The cells transduced were differentially selected with puromycin, and the clones that highly expressed SPINK1 were chosen by real-time PCR and confirmed by Western blot after 7 weeks. The stably transduced AsPC-1 cell line showed significantly increased metabolic and proliferative capability using CCK-8 and Trypan Blue assays (P < 0.001). Cell cycle and DNA content analysis by flow cytometry showed that upregulated SPINK1 elicited significant increase in the percentage of AsPC-1 cells in the S and G2/M phase (P < 0.001). Clone formation assay demonstrated that the number of the colonies formed in the experimental group was greater than that in the control parental cells (P < 0.001). It was concluded that a stable AsPC-1 cell line capable of overexpressing SPINK1 had been successfully created, and that the proliferative capacity of AsPC-1 pancreatic cancer cells was significantly raised by SPINK1 upregulation as well as the ability of a single AsPC-1 cell to grow into a colony.
本研究旨在设计并构建含人1型 Kazal 型丝氨酸蛋白酶抑制剂基因（SPINK1）完整编码序列（CDS）区域的重组慢病毒载体，并建立稳定过表达SPINK1的人胰腺癌细胞系（AsPC-1）。然后，采用不同方法评估上调SPINK1基因对胰腺癌AsPC-1细胞的增殖和致癌作用。首先，通过聚合酶链反应（PCR）扩增SPINK1的目标编码序列（CDS），随后将合成产物亚克隆至慢病毒载体。通过限制性酶切和测序分析验证重组SPINK1基因的构建。将重组慢病毒载体与包装混合质粒共转染至293 T细胞，产生携带SPINK1基因的慢病毒颗粒，在病毒颗粒感染AsPC-1细胞之前进行过滤和浓缩。用嘌呤霉素对转导的细胞进行差异筛选，7周后通过实时PCR选择高表达SPINK1的克隆，并通过蛋白质免疫印迹法进行确认。使用CCK-8和台盼蓝试验检测，稳定转导的AsPC-1细胞系显示出代谢和增殖能力显著增强（P < 0.001）。通过流式细胞术分析细胞周期和DNA含量表明，上调SPINK1导致AsPC-1细胞在S期和G2/M期的百分比显著增加（P < 0.001）。克隆形成试验表明，实验组形成的集落数量多于对照亲代细胞（P < 0.001）。得出结论，已成功创建能够过表达SPINK1的稳定AsPC-1细胞系，并且上调SPINK1显著提高了AsPC-1胰腺癌细胞的增殖能力以及单个AsPC-1细胞生长成集落的能力。
