丝氨酸蛋白酶抑制剂基因spink3对肝癌细胞株BEL-7402的作用研究
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摘要
丝氨酸蛋白酶抑制剂Kazal Ⅲ型（serine protease inhibitor, Kazal type3, SPINK3）是一种胰蛋白酶抑制剂，在胰腺癌、大肠癌，乳腺癌、前列腺癌、肝癌等多种肿瘤中高表达，但其对肝癌细胞株的作用研究尚未见报道。
为此，本文构建了spink3基因的表达载体（pEGFP-N1-spink3），干涉载体（pGenesil-1.0-spink3(123)和pGenesil-1.0-spink3(143)）和检验载体（pGenesil-1.0-spink3(123)-spink3、spink3(143)-spink3和-HK-spink3）。将各检验载体进行尾静脉液压转基因至小鼠肝脏，用荧光显微技术观察与SPINK3融合表达的增强型绿色荧光蛋白（enhanced greenfluorescent protein, EGFP）的阳性细胞率，筛选出最佳干涉载体。采用脂质体介导法将4种质粒（表达载体pEGFP-N1-spink3及其对照pEGFP-N1，最佳干涉载体pGenesil-1.0-spink3(123)及其对照pGenesil-1.0-HK）转染肝癌细胞株BEL-7402，并用G418选择性培养基筛选出稳定转染细胞株。对正常BEL-7402细胞株和上述筛选出来的四种稳定细胞株用下列方法进行检测：通过HE染色观察细胞形态结构；荧光显微镜下观察细胞形态以及EGFP分布；通过计数法和普通MTT法绘制细胞的生长曲线；通过PCNA细胞免疫化学检测细胞中PCNA的表达变化；通过Hoechst33258染色检测细胞凋亡；通过甲基纤维素半固体培养基检测集落形成率；通过药物MTT法检测细胞的耐药性。结果表明： SPINK3-EGFP融合蛋白均匀分布在细胞质中，细胞核无荧光，并且在细胞核外侧核膜周围表达量较高，为典型的分泌性表达模式；spink3能抑制肝癌细胞株BEL-7402增殖，但不引起细胞凋亡；spink3能够降低肝癌细胞株BEL-7402恶性程度，但对其耐药性无明显影响。
The serine protease inhibitor, Kazal type3(SPINK3) is a trypsin inhibitor which is highly expressed inpancreatic cancer, colorectal cancer, breast cancer, prostate cancer, liver cancer and other tumors, but theresearch on the effect of spink3on hepatic cancer cell strain has not been reported.
Therefore, the expression vector (pEGFP-N1-spink3), interference vectors (pGenesil-1.0-spink3(123) and pGenesil-1.0-spink3(143)) and test vectors (pGenesil-1.0-spink3(123)-spink3, pGenesil-1.0-spink3(143)-spink3and pGenesil-1.0-HK-spink3) were established. The constructed test vectors wereadministrated using a hydrodynamics-based procedure in the tail vein of little rat to select the bestrecombinant interference plasmid using fluorescence microscopy technique to observe enhanced greenfluorescent protein (EGFP)-positive cell rate. Next, the expression vector pEGFP-N1-spink3and its controlpEGFP-N1, interference vector pGenesil-1.0-spink3(123) and its control pGenesil-1.0-HK were stablytransfected into hepatic cancer cell strain BEL-7402in vitro by lipofectamine and their stably transfectedcell strains were selected by G418. Then, the hepatic cancer cell strain BEL-7402and the four kinds ofstably transfected cell strains above were detected using the following methods, including cell morphologyobserved by HE staining, cell morphology and EGFP distribution observed under a fluorescencemicroscope, cell growth curve drew by cell counting and MTT assay, PCNA expression changes detectedby PCNA immuneocytochemistry, cell apoptosis detected by Hoechst33258staining, cell colony formationrate detected by Methyl cellulose semi-solid medium, and cell resistance to drugs detected by MTT assay.The results show that the SPINK3-EGFP fusion protein was evenly distributed in the cytoplasm, nofluorescence in the nucleus, more expression surrounding the outer nuclear membrane which is a typicalsecretory expression pattern, that spink3can inhibit hepatic cancer cell strain BEL-7402proliferation, butnot cause apoptosis, and that spink3can reduce the degree of malignancy of cells, but had no significanteffect on the resistance to drugs.
为此，本文构建了spink3基因的表达载体（pEGFP-N1-spink3），干涉载体（pGenesil-1.0-spink3(123)和pGenesil-1.0-spink3(143)）和检验载体（pGenesil-1.0-spink3(123)-spink3、spink3(143)-spink3和-HK-spink3）。将各检验载体进行尾静脉液压转基因至小鼠肝脏，用荧光显微技术观察与SPINK3融合表达的增强型绿色荧光蛋白（enhanced greenfluorescent protein, EGFP）的阳性细胞率，筛选出最佳干涉载体。采用脂质体介导法将4种质粒（表达载体pEGFP-N1-spink3及其对照pEGFP-N1，最佳干涉载体pGenesil-1.0-spink3(123)及其对照pGenesil-1.0-HK）转染肝癌细胞株BEL-7402，并用G418选择性培养基筛选出稳定转染细胞株。对正常BEL-7402细胞株和上述筛选出来的四种稳定细胞株用下列方法进行检测：通过HE染色观察细胞形态结构；荧光显微镜下观察细胞形态以及EGFP分布；通过计数法和普通MTT法绘制细胞的生长曲线；通过PCNA细胞免疫化学检测细胞中PCNA的表达变化；通过Hoechst33258染色检测细胞凋亡；通过甲基纤维素半固体培养基检测集落形成率；通过药物MTT法检测细胞的耐药性。结果表明： SPINK3-EGFP融合蛋白均匀分布在细胞质中，细胞核无荧光，并且在细胞核外侧核膜周围表达量较高，为典型的分泌性表达模式；spink3能抑制肝癌细胞株BEL-7402增殖，但不引起细胞凋亡；spink3能够降低肝癌细胞株BEL-7402恶性程度，但对其耐药性无明显影响。
The serine protease inhibitor, Kazal type3(SPINK3) is a trypsin inhibitor which is highly expressed inpancreatic cancer, colorectal cancer, breast cancer, prostate cancer, liver cancer and other tumors, but theresearch on the effect of spink3on hepatic cancer cell strain has not been reported.
Therefore, the expression vector (pEGFP-N1-spink3), interference vectors (pGenesil-1.0-spink3(123) and pGenesil-1.0-spink3(143)) and test vectors (pGenesil-1.0-spink3(123)-spink3, pGenesil-1.0-spink3(143)-spink3and pGenesil-1.0-HK-spink3) were established. The constructed test vectors wereadministrated using a hydrodynamics-based procedure in the tail vein of little rat to select the bestrecombinant interference plasmid using fluorescence microscopy technique to observe enhanced greenfluorescent protein (EGFP)-positive cell rate. Next, the expression vector pEGFP-N1-spink3and its controlpEGFP-N1, interference vector pGenesil-1.0-spink3(123) and its control pGenesil-1.0-HK were stablytransfected into hepatic cancer cell strain BEL-7402in vitro by lipofectamine and their stably transfectedcell strains were selected by G418. Then, the hepatic cancer cell strain BEL-7402and the four kinds ofstably transfected cell strains above were detected using the following methods, including cell morphologyobserved by HE staining, cell morphology and EGFP distribution observed under a fluorescencemicroscope, cell growth curve drew by cell counting and MTT assay, PCNA expression changes detectedby PCNA immuneocytochemistry, cell apoptosis detected by Hoechst33258staining, cell colony formationrate detected by Methyl cellulose semi-solid medium, and cell resistance to drugs detected by MTT assay.The results show that the SPINK3-EGFP fusion protein was evenly distributed in the cytoplasm, nofluorescence in the nucleus, more expression surrounding the outer nuclear membrane which is a typicalsecretory expression pattern, that spink3can inhibit hepatic cancer cell strain BEL-7402proliferation, butnot cause apoptosis, and that spink3can reduce the degree of malignancy of cells, but had no significanteffect on the resistance to drugs.
引文
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[2] Kazal LA, Spicer DS, Brahinsky RA. Isolation of a crystalline trypsin inhibitor-anticoagulant proteinfrom pancreas[J]. J Am Chem Soc,1948,70(9):3034-3040.
[3]郑青亮,盛清,张耀洲. Kazal型蛋白酶抑制剂结构与功能研究进展[J].生物工程学报,2006,22(5):695-700.
[4]李海深,曹云,钱朝南. SPINK家族与人类疾病[J].基础医学与临床,2010,30(5):550-553.
[5] Komiyama T, Bigler TL, Yoshida N, et a1. Replacement of P1Leul8by Glul8in the reactive site ofturkey ovomucoid third domain converts it into a strong inhibitor of Glu-specific Streptomycesgriseus proteinase(GluSGP)[J]. J Biol Chem,1991,266(17):10727-10730.
[6] Laskowski M Jr, Kato I. Protein inhibitors of proteinases[J]. Annu Rev Biochem,1980,49:593-626.
[7] Krowarsch D, Cierpicki T, Jelen F, et a1. Canonical protein inhibitors of serine proteases[J]. Cell MolLife Sci,2003,60(11):2427-2444.
[8] Bode W, Huber R. Natural protein proteinase inhibitors and their interaction with proteinases[J]. EurJ Biochem,1992,204(2):433-451.
[9] Laskowski M, Qasim MA. What can the structures of enzyme-inhibitor complexes tell us about thestructures of enzyme-substrate complexes?[J]. Biochim Biophys Acta,2000,1477(1):324-337.
[10] Jackson RM. Comparison of protein-protein interactions in serine protease-inhibitor andantibody-antigen complexes: Implications for the protein docking problem[J]. Protein Science,1999,8(3):603-613.
[11] Lu SM, Lu W, Qasim MA, et al. Predicting the reactivity of proteins from their sequence alone:Kazal family of protein inhibitors of serine proteinases[J]. Proc Natl Acad Sci USA,2001,98(4):1410-14l5.
[12] Lu W, Apostol I. Qasim MA, et al. Binding of Amino Acid Sidechains to S1Cavities of SerineProteinases[J]. J Mol Biol,1997,226(2):441-461.
[13] Read RJ, Fujinaga M, Sielecki AR, et a1. Structure of the complex of Streptomyces griseus proteaseB and the third domain of the turkey ovomucoid inhibitor at1.8-A resolution[J]. Biochemistry,1983,22(19):4420-4433.
[14] Laskowski MJ, Kato I, Ardelt W, et a1. Ovomucoid third domains from100avian species: isolation,sequences, and hypervariability of enzyme-inhibitor contact residues[J]. Biochemistry,1987,26(1):202-221.
[15] Qasim MA, Lu W, Lu SM, et a1. Testing of the additivity-based protein sequence to reactivityalgorithm[J]. Biochemistry,2003,42(21):6460-6466.
[16] Horii A, Tomita N, Yokouchi H, et al. On the cDNA's for two types of rat pancreatic secretory trypsininhibitor[J]. Biochem Biophys Res Commun,1989,162(1):151-159.
[17] Iwai K, Fukuoka S, Fushiki T, et al. Purification and Sequencing of a Trypsin-sensitiveCholecystokinin-releasing Peptide from Rat Pancreatic Juice[J]. J Biol Chem,1987,262(19):8956-8959.
[18] Hirota M, Ohmuraya M, Baba H, et al. Genetic background of pancreatitis[J]. Postgrad Med J,2006,82(974):775–778.
[19] Fukuda M, Fujiyama Y, Sasaki M, et al. Monitor peptide (rat pancreatic secretory trypsin inhibitor)directly stimulates the proliferation of the nontransformed intestinal epithelial cell line, IEC-6[J].Digestion,1998,59(4):326-330.
[20] Miyasaka K, Funakoshi A, Nakamura R, et al. Differences in stimulatory effects between ratpancreatic secretory trypsin inhibitor-61and-56on rat pancreas[J]. Jpn J Physiol,1989,39(6):891-899.
[21] Fukuoka S, Scheele GA. Rapid and selective cloning of monitor peptide, a novelcholecystokinin-releasing peptide, using minimal amino acid sequence and the polymerase chainreaction[J]. Pancreas,1990,5(1):1-7.
[22] Tsuzuki S, Fushiki T, Kondo A, et al. Effect of a high-protein diet on the gene expression of atrypsin-sensitive, cholecystokinin-releasing peptide (monitor peptide) in the pancreas[J]. EurBiochem,1991,199(1):245-252.
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