【作者】 郭素真；

【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】 单核趋化因子蛋白1（Monocyte chemoattractant protein1，mcp-1）属于趋化因子CC亚族成员，能通过与单核类趋化因子受体（CCR2）结合并诱导淋巴细胞等的激活、分化、发育等，参与血管再生过程，对肿瘤的抑制起作用；脂质运载蛋白-2（lipocalin-2, lcn2）为脂质运载蛋白家族，被多种类型的细胞表达，参与多种生理活动，如细胞凋亡、增殖以及分化，免疫炎症反应等。mcp-1和lcn2基因均能通过调控凋亡相关基因（如Bax、Bcl-2等）诱导细胞凋亡，且可作为急性肾脏损伤的生物标记物。为了研究mcp-1和lcn2基因在肝癌细胞BEL-7402中的作用，在本实验室成功克隆mcp-1和lcn2基因并构建其表达载体和干涉载体的基础上，本文通过采用脂质体法将构建的表达载体pEGFP-N1-mcp-1、 pEGFP-N1-lcn2及其对照pEGFP-N1、干涉载体pGenesil-1.0-mcp-1(255)、pGenesil-1.0-lcn2(292)及其对照pGenesil-1.0-HK转染肝癌细胞BEL-7402，并用G418筛选出稳定转染的单克隆细胞，并进行下列检测，采用活体观察和HE染色进行细胞形态观察；通过细胞计数和MTT法进行生长曲线的测定；通过PCNA免疫组化检测细胞增殖；通过Hoechst33258染色检测细胞凋亡；通过甲基纤维素成集落率实验检测细胞的恶性增殖程度；药物MTT法检测细胞的耐药性。结果表明：转染pEGFP-N1-mcp-1的细胞形态与BEL-7402细胞无明显变化，但细胞核质比变小；生长曲线显示转染pEGFP-N1-mcp-1的细胞增殖比转染pEGFP-N1的细胞慢；PCNA检测显示mcp-1抑制细胞增殖；Hoechst33258凋亡染色显示mcp-1不引起细胞凋亡；甲基纤维素成集落率检测显示mcp-1能抑制BEL-7402细胞的恶性增殖程度。转染pEGFP-N1-lcn2的细胞细胞核固缩变小，核仁减少；生长曲线显示转染pEGFP-N1-lcn2的细胞生长明显降低；PCNA检测显示lcn2明显抑制细胞增殖；Hoechst33258凋亡染色显示lcn2能引起细胞凋亡；甲基纤维素成集落率检测显示lcn2明显抑制BEL-7402细胞的恶性增殖程度；药物MTT检测则发现，lcn2基因对氨基酸衍生物L-4-氟苯丙氨酸（Fp）和L-4-氯苯丙氨酸（Clp）无明显的耐药性。综上所述，mcp-1基因能抑制肝癌细胞BEL-7402的细胞增殖，降低其恶性增殖程度，但对肝癌细胞BEL-7402的细胞凋亡作用不明显；lcn2基因同样能抑制肝癌细胞BEL-7402的细胞增殖，降低其恶性增殖程度，同时又对肝癌细胞BEL-7402起细胞凋亡作用。更多还原

【Abstract】 Monocyte chemoattractant protein1(mcp-1), a member of the CC chemokine family, could combinewith chemokine(C-C motif) ligand receptor2, and then induce lymphocyte activation, differentiation anddevelopment, and play a vital role in tumor growth or inhibition. Lipocalin-2(lcn2), a member of thelipocalin family, can be expressed and secreted by various types of cells, and participates in manyphysiological activities, such as cell proliferation, apoptosis, differentiation and immune inflammatoryresponse. mcp-1and lcn2genes could induces cell apoptosis by regulated apoptosis related genes, such asBax, bcl-2etc, and have potential as a biomarker of acute kidney injury. In order to clarify the impact ofmcp-1and lcn2on the hepatic cancer cell BEL-7402. Based on the successful cloning of mcp-1and lcn2and establishment of their kinds of vectors, the expression vectors pEGFP-N1-mcp-1, pEGFP-N1-lcn2andits control pEGFP-N1, interference vectors pGenesil-1.0-mcp-1(255), pGenesil-1.0-lcn2(292)and its controlpGenesil-1.0-HK were stably transfected into hepatoma cell line BEL-7402in vitro by lipofectamine, andtheir stably transfected cell lines were selected by G418. Then, the stably transfected cell lines and theircontrol were detected by using the following methods: cell morphology by HE staining; cell growth curvedrew by cell counting and MTT assay; cell proliferation detected by PCNA immunocytochemistry; cellapoptosis detected by Hoechst33258staining; cell colony formation rate detected by methyl cellulosesemi-solid medium; cell resistance to drugs detected by MTT assay.The results detected were as following. The cells stably transfected with pEGFP-N1-mcp-1were notsignificantly different in morphology compared with BEL-7402, but the ratio of uncleus and cytoplasmbecame smaller, and nucleolus was reduced. The growth curve showed that the cells withpEGFP-N1-mcp-1growed slower than those with pEGFP-N1; PCNA immunocytochemistry showed thatmcp-1inhibited cell growth; Hoechst33258staining showed that mcp-1had no significant effect oninducing cell apoptosis; The detection results of methyl cellulose solid medium showed that mcp-1inhibited the degree of malignant proliferation of BEL-7402cells. The uncleus of the cells withpEGFP-N1-lcn2became smaller and condensed, and the nucleolus were reduced; The growth curveshowed that he cells with pEGFP-N1-lcn2growed obviously slower than others; PCNAimmunocytochemistry showed that lcn2dramatically inhibited cell growth; Hoechst33258staining showedthat lcn2had significant effect on inducing cell apoptosis; The detection results of methyl cellulose solid medium showed that lcn2inhibited the degree of malignant proliferation of BEL-7402cells obviously; TheMTT assay of drug found that lcn2had no obvious resistant to amino acid derivative L-4-fluorophenylalanine (Fp) and L-4-fluoro phenylalanine (Clp).In summary, mcp-1could inhibite cell proliferation and malignant phenotypes of the BEL-7402cells,but had no significant effect on inducing the apoptosis of BEL-7402cells; Lcn2could inhibite cellproliferation and malignant phenotypes of the BEL-7402cells obviously, but could promote the apoptosisof BEL-7402cells.更多还原