【作者】 申亚慧；

【导师】 徐存拴；

【作者基本信息】 河南师范大学 ， 细胞生物学， 2012， 硕士

【摘要】 胰腺炎相关蛋白(PAP)，是从胰腺炎患者的胰液中发现的一种炎症蛋白，有促进有丝分裂、抗细胞凋亡、抗炎症和促进细胞与胞外基质粘附等作用。为揭示其在肝再生中的作用，本文根据大鼠pap基因的mRNA序列，采用分子克隆的方法克隆出pap基因。在此基础上设计干涉片段并构建出pap基因的两个干涉载体pGenesil-1.0-pap(289)和pGenesil-1.0-pap(485)，三个检验载体pGenesil-1.0-pap(289)-pap、pGenesil-1.0-pap(485)-pap和pGenesil-1.0-HK-pap，并通过尾静脉液压转基因方法检测各检验载体的作用，筛选出最佳干涉载体pGenesil-1.0-pap(485)。采用脂质体转染法将pEGFP-N1-pap及对照pEGFP-N1，pGenesil-1.0-pap(485)及对照pGenesil-1.0-HK等4种质粒转入肝癌细胞BEL-7402内，再用G418筛选出稳定转染细胞株，并进行以下检测：采用活体观察和HE染色观察细胞形态结构；细胞计数和MTT法测定细胞的生长曲线；PCNA细胞免疫化学检测细胞增殖；Hoechst33258染色检测细胞凋亡；甲基纤维素半固体培养基检测集落形成率；药物MTT法检测细胞的耐药性。为进一步研究pap对肝再生的作用奠定基础，也为揭示肝再生的分子机制提供信息。体内实验采用液压转基因技术将pEGFP-N1-pap及对照pEGFP-N1﹑pGenesil-1.0-pap(289)和pGenesil-1.0-pap(485)及对照pGenesil-1.0-HK等5种质粒分别注入2/3肝切除后的大鼠肝脏内，采用常规组织学方法、荧光显微镜技术以及统计学方法等检测转染这些质粒后大鼠的死亡率﹑组织显微结构、肝系数以及肝再生率，以判断pap对肝再生的影响。体外实验结果表明，转染pEGFP-N1-pap的细胞明显成梭形，细胞核仁增多，与pap融合的绿色荧光蛋白(EGFP)主要分布在细胞核核膜周围，其它转染细胞株形态无明显变化，EGFP主要分布均在细胞核中；生长曲线检测显示转染pEGFP-N1-pap质粒的细胞增殖最快；PCNA细胞免疫化学和hoechst33258染色检测表明稳定转染pEGFP-N1-pap质粒的细胞可能促进细胞增殖、抑制凋亡；集落形成率结果显示稳定转染pEGFP-N1-pap的细胞恶性程度升高；药物MTT检测发现pap基因能促进BEL-7402细胞生长，但对其耐药性无明显影响。体内实验结果表明，转入pEGFP-N1-pap质粒后，有炎症反应发生；在120小时，pEGFP-N1-pap组比对照组的肝系数和肝再生率高，且与对照组相比pEGFP-N1-pap(289)组要比pEGFP-N1-pap(485)组的肝系数和肝再生率高。综上所述，pap基因可能有促进细胞增殖，增加肝癌细胞恶性程度的作用，而对肝癌细胞抗药性没有明显耐药性；pap可能参与促进肝再生进程。更多还原

【Abstract】 Pancreatitis-associated protein(PAP) is an inflammatory protein what was found in pancreatic juicefrom pancreatitis, to promote mitosis, the anti-apoptotic, anti-inflammatory and promote cell adhesion toextracellular matrix and so on. In order to study its role in liver regeneration, According to the mRNAsequence of rat pap gene, pap gene was cloned and constructed into the vector. On this basis, interferefragments were designed and two interference vectors pGenesil-1.0-pap(289) and pGenesil-1.0-pap(485),three test vectors pGenesil-1.0-pap(289)-pap，pGenesil-1.0-pap(485)-pap and pGenesil-1.0-HK-pap wereestablished, and pap(485) was slected by methods of the tail vein hydraulic testing the role of each vector.The expression vector pEGFP-N1-pap and its control pEGFP-N1, interference vector pGenesil-1.0-pap-(485) and its control pGenesil-1.0-HK transfected into hepatoma cell BEL-7402with lipofectamine, andselection of stably transfected cell lines to the use of G418. Then, all kinds of cell lines above were detectedusing the following methods: cell morphology observed in vivo and by HE staining; cell growth curve bycell counting and MTT assay; cell proliferation detected by PCNA immunocytochemistry; cell apoptosisdetected by Hoechst33258staining; cell colony formation rate detected by Methyl cellulose semi-solidmedium; cell resistance to drugs detected by MTT assay. These laid the foundation for further study of papin liver regeneration and provided information to reveal the mechanism of liver regeneration molecules. Invivo experiments, the pEGFP-N1-pap, pGenesil-1.0-pap(289) and pGenesil-1.0-pap(485) employed thetechnique of hydrodynamics-based transgening were injected into the remnant livers of rat after2/3partialhepatectomy(PH), and the mortality of rats, the morphological structure of regenerating livers, livercoefficient and liver regeneration rate were analyzed using statistical methods.The results of vitro experiments showed the nucleus of BEL-7402cell transfected with expressionvectors pEGFP-N1-pap significantly increased and looked like spindle; EGFP fusion proteins with papwere mainly distributed around the nucleus nuclear membrane, while no significant changes were observedin other transfected cell lines with EGFP mainly distributed in the nucleus; Growth curve and MTT assayshowed that the proliferation of cells transfected with pEGFP-N1-pap was obvious; PCNAimmunocytochemistry and Hoechst33258staining showed that plasmid pEGFP-N1-pap could promote cell proliferation and inhibit apoptosis; The degree of malignancy of cell lines transfected with pEGFP-N1-papwas increased; MTT assay showed that pap gene can promote BEL-7402cell growth, but no significanteffect on its resistance to drugs. The results of vivo experiments showed that liver coefficient and liverregeneration rate of rats transfected with pEGFP-N1-pap were higher than PH control at120h, but that ofrats transfected with siRNA vectors were lower than the control. In short, pap gene may promote cellproliferation, increase the role of malignancy of hepatoma cell, but has no apparent resistance on hepatomacell as anticancer agents; pap may be involved in promoting the process of liver regeneration.更多还原