环境因素诱导卵圆细胞增殖的机制研究

发布时间：2018-01-07 12:33

  本文关键词：环境因素诱导卵圆细胞增殖的机制研究　出处：《华中科技大学》2009年博士论文　论文类型：学位论文

  更多相关文章： 卵圆细胞 诱导 分离 培养 鉴定 黄曲霉素B1 卵圆细胞 增殖 Toll样受体2 细胞因子 卵圆细胞 黄曲霉素B1 乙型肝炎病毒 增殖 基因芯片

【摘要】： 第一部分小鼠卵圆细胞增殖模型的建立及卯圆细胞的分离、培养和鉴定 目的建立一种稳定的成体小鼠肝脏卵圆细胞的诱导、分离和培养方法，并对分离培养的卵圆细胞加以鉴定。 方法喂饲2-乙酰氨基芴(2-AAF)+部分肝切除(PH)方法诱导小鼠肝脏卵圆细胞的增殖。改良的经门静脉灌注胶原酶消化法和等密度离心法分离纯化卵圆细胞，并在体外进行长期培养。采用免疫荧光、RT-PCR和糖原染色等方法对培养的卵圆细胞加以鉴定。 结果此模型中肝脏有明显的卵圆细胞增殖。分离的卵圆细胞为大小不一、为异质性的，代表了肝脏卵圆细胞的所有亚群。体外培养的卵圆细胞呈集落样生长，稳定传代并已培养至3个月。免疫荧光、RT-PCR和糖原染色证实培养的细胞为卵圆细胞，并显示该细胞具有分化潜能。 结论2-AAF+2／3肝切除方法同样可以诱导小鼠肝脏卵圆细胞的增殖。本文所用方法是一种稳定的成体小鼠卵圆细胞的分离和培养方法，为原发性肝癌的卵圆细胞源性相关研究奠定基础。 第二部分Toll样受体2信号通路在AFB1诱导卯圆细胞增殖过程中的作用 目的研究AFB1诱导卵圆细胞增殖的情况，探讨Toll样受体2(TLR2)信号通路在AFB1诱导卵圆细胞增殖中的表达变化和意义。 方法采用腹腔注射AFB1的方法建立AFB1诱癌的小鼠模型。检测血清谷丙转氨酶(ALT)和谷草转氨酶(AST)浓度变化；免疫组化方法检测肝脏组织内卵圆细胞增殖的情况；使用实时定量PCR检测不同时间点肝组织TLR2、髓样分化因子(MyD88)、肝细胞生长因子(HGF)和AFP mRNA的表达情况；Western blot方法检测肝组织TLR2、MyD88和核因子-κBp65(NF-κBp65)的表达情况；酶联免疫吸附试验(ELISA)方法检测细胞因子肿瘤坏死因子-α(TNF-α)、白介素(IL)-1、IL-6和HGF的表达情况。 结果在此模型中，3小时时ALT和AST即有明显升高，随后逐渐升高，30天时达到高峰，随后有所下降，但明显高于对照组(P＜0.05)。对照组肝组织内未见卵圆细胞的增殖，AFB1组肝组织7天时卵圆细胞开始增殖，一直持续到60天。TLR2、MyD88和NF-κB及其下游细胞因子在此过程中表达明显增高(P＜0.05)。 结论AFB1可以引起明显肝脏损伤，进而诱导小鼠肝脏卵圆细胞的增殖。TLR2信号通路可能通过启动下游细因子的释放在卵圆细胞的增殖过程中起着重要作用。深入研究TLR2信号通路可能为卵圆细胞的增殖和肝癌发生的研究提供新的理论依据和新的治疗靶点。 第三部分环境因素诱导卵圆细胞增殖过程中肝细胞和卵圆细胞基因的表达变化 目的建立HBV、AFB1和两者协同诱导肝癌的动物模型，监测肝脏卵圆细胞的增殖。研究此过程中肝细胞和卵圆细胞基因表达的差异，为肝癌的干细胞理论寻找新的实验依据。 方法40只Balb／c小鼠和40只HBV转基因小鼠分为四组：对照组(A组)、AFB1组(B组)、HBV组(C组)和AFB1+HBV组(D组)。AFB1150mg／kg，腹腔注射。检测HBsAg定量、HBeAg半定量和乙肝五项，明确HBV转基因鼠转基因是否有效。检测各组血清ALT和AST的浓度变化；免疫组化检测肝脏卵圆细胞增殖情况。6个月后剖杀动物，采用本试验室改良的方法分离和培养四组的肝细胞和卵圆细胞。抽提总RNA，Illumina小鼠基因芯片检测四组内肝细胞和卵圆细胞的基因表达变化。 结果(1)转基因小鼠的检测：血清HBsAg250.00IU／mL(阳性≥0.05IU／mL)，HBeAg1.66S／CO(阳性≥1.00S／CO)，HBsAg抗体(—)，HBeAg抗体(—)，HBcAg抗体(—)。(2)各组死亡率：正常组为0％(0／20)，AFB1组为30％(6／20)，HBV组为10％(2／20)，AFB1+HBV组为60％(12／20)。后三组明显高于正常组，AFB1+HBV组明显高于AFB1和HBV组(P＜0.05)。(3)肝功能的变化：正常组肝功能无明显变化，AFB1、HBV组出现明显肝脏损伤，两者协同引起肝脏损伤进一步加重(P＜0.05)。(4)卵圆细胞的增殖：正常组肝组织中未发现卵圆细胞增殖；后三组肝组织中有明显卵圆细胞增殖，呈团索状，主要分布于门脉区。(5)与正常组比较，AFB1组卵圆细胞有1577个基因表达出现变化；肝细胞出现了805个。HBV组卵圆细胞有3467个基因表达出现变化；肝细胞出现1196个。AFB1+HBV组卵圆细胞，有4846个基因表达出现变化；肝细胞出现2542个。其中涉及Notch、Wnt、PI3K-AKT等数十个信号通路。AFB1、HBV均引起卵圆细胞和肝细胞癌基因和抑癌基因的表达变化，其中卵圆细胞表达升高的癌基因明显多于肝细胞，而抑癌基因明显少于肝细胞；两者协同导致这种效果更加明显。 结论(1)转基因小鼠体内有稳定、高滴度的HBV病毒合成。(2) AFB1、HBV和两者协同作用于小鼠均可引起明显的肝脏卵圆细胞增殖，并可导致死亡率升高和引起严重肝损伤。(2) HBV转染虽然无法引起机体的免疫反应，，但可以通过其它途径引起肝脏损伤，刺激卵圆细胞增殖。(3) AFB1、HBV及两者协同作用均引起肝细胞和卵圆细胞基因的异常表达，其中对卵圆细胞的影响更为显著；表达异常的癌基因和抑癌基因可能在卵圆细胞转化为肝癌过程中起着重要作用。(5)增殖的卵圆细胞可能是肝癌的起源细胞。
[Abstract]:The first part of the mouse oval cell proliferation model and the isolation, culture and identification of the sockets
Objective to establish a stable induction, isolation and culture method of oval cells in the liver of adult mice, and to identify the isolated oval cells.
Method of feeding 2- acetylaminofluorene (2-AAF) + partial hepatectomy (PH) murine hepatic oval cell proliferation induced by modified method. The portal vein perfusion with collagenase and density gradient centrifugation method for the separation and purification of oval cells, and cultured in vitro. By immunofluorescence, RT-PCR and glycogen staining method identify of oval cells in culture.
The results of this model in hepatic oval cell proliferation obviously. Oval cells isolated for size, heterogeneity, on behalf of the hepatic oval cells of all subsets. In vitro cultured oval cells showed colony like growth, and has been passaged cultured for 3 months. Immunofluorescence. RT-PCR and glycogen staining showed that the cultured cells were oval cells, and the cells have the potential of differentiation.
Conclusion 2-AAF+2 / 3 hepatectomy can also induce the proliferation of hepatic oval cells in mice. The method used in this paper is a stable method for isolation and culture of adult oval cells, which lays the foundation for the study of oval cell related origin of primary liver cancer.
The role of the second part of Toll like receptor 2 signaling pathway in the proliferation of rounded cells induced by AFB1
Objective to investigate the proliferation of oval cells induced by AFB1, and to explore the changes and significance of Toll like receptor 2 (TLR2) signaling pathway in the proliferation of oval cells induced by AFB1.
Methods to establish a mouse model of AFB1 induced cancer by intraperitoneal injection of AFB1. The detection of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) concentration; immunohistochemical method to detect liver oval cell proliferation; using real-time quantitative PCR detection of liver tissue at different time points (TLR2, myeloid differentiation factor MyD88), hepatocyte growth factor (HGF) expression of AFP and mRNA; Western blot method TLR2 liver tissue detection, MyD88 and nuclear factor kappa Bp65 (NF- K Bp65) expression; enzyme linked immunosorbent assay (ELISA) method for detection of cytokine tumor necrosis factor alpha (TNF- alpha), white interleukin (IL) -1, the expression of IL-6 and HGF.
The results in this model, 3 hours of ALT and AST increased significantly, then increased gradually, reached the peak at the 30 day, then decreased, but significantly higher than the control group (P < 0.05). The control group were not found in liver oval cell proliferation, liver tissue AFB1 group 7 days of oval cells began the proliferation lasted until 60 days.TLR2, significantly increased the expression of MyD88 and NF- kappa B and its downstream cytokines in this process (P < 0.05).
Conclusion AFB1 can induce liver injury and proliferation induced by.TLR2 signaling pathway in murine hepatic oval cells may activate the downstream through the release of cytokines plays an important role in the proliferation of oval cells. Further study of TLR2 signal pathway may occur for proliferation and liver oval cells provide a new theoretical basis and new therapeutic targets.
Third environmental factors induced changes in the gene expression of hepatocyte and oval cell during the proliferation of oval cells
Objective to establish animal models of hepatocellular carcinoma (HCC) induced by HBV, AFB1 and their synergistic effects, and to monitor the proliferation of hepatic oval cells, and to study the difference of gene expression between hepatocytes and oval cells during the process, and to find new experimental evidence for stem cell theory of liver cancer.
Methods 40 Balb / c mice and 40 HBV mice were divided into four groups: control group (A group), AFB1 group (B group), HBV group (C group) and AFB1+HBV group (group D).AFB1150mg / kg, intraperitoneal injection. Detection of HBsAg quantitative, semi quantitative HBeAg and hepatitis B five. Clear the HBV transgenic mouse, gene transfer is effective. To detect the change of concentration of serum ALT and AST were observed; immunohistochemical detection of hepatic oval cell proliferation.6 months after killing the animal, using the method of laboratory modified isolation and culture of liver cells and oval cells in the four groups. Total RNA was extracted and the expression change of Illumina the mouse gene chip detection in the four groups of liver cells and oval cell gene.
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