本文關(guān)鍵詞：環(huán)境因素誘導(dǎo)卵圓細(xì)胞增殖的機(jī)制研究　出處：《華中科技大學(xué)》2009年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 卵圓細(xì)胞 誘導(dǎo) 分離 培養(yǎng) 鑒定 黃曲霉素B1 卵圓細(xì)胞 增殖 Toll樣受體2 細(xì)胞因子 卵圓細(xì)胞 黃曲霉素B1 乙型肝炎病毒 增殖 基因芯片

【摘要】： 第一部分小鼠卵圓細(xì)胞增殖模型的建立及卯圓細(xì)胞的分離、培養(yǎng)和鑒定 目的建立一種穩(wěn)定的成體小鼠肝臟卵圓細(xì)胞的誘導(dǎo)、分離和培養(yǎng)方法，并對(duì)分離培養(yǎng)的卵圓細(xì)胞加以鑒定。 方法喂飼2-乙酰氨基芴(2-AAF)+部分肝切除(PH)方法誘導(dǎo)小鼠肝臟卵圓細(xì)胞的增殖。改良的經(jīng)門(mén)靜脈灌注膠原酶消化法和等密度離心法分離純化卵圓細(xì)胞，并在體外進(jìn)行長(zhǎng)期培養(yǎng)。采用免疫熒光、RT-PCR和糖原染色等方法對(duì)培養(yǎng)的卵圓細(xì)胞加以鑒定。 結(jié)果此模型中肝臟有明顯的卵圓細(xì)胞增殖。分離的卵圓細(xì)胞為大小不一、為異質(zhì)性的，代表了肝臟卵圓細(xì)胞的所有亞群。體外培養(yǎng)的卵圓細(xì)胞呈集落樣生長(zhǎng)，穩(wěn)定傳代并已培養(yǎng)至3個(gè)月。免疫熒光、RT-PCR和糖原染色證實(shí)培養(yǎng)的細(xì)胞為卵圓細(xì)胞，并顯示該細(xì)胞具有分化潛能。 結(jié)論2-AAF+2／3肝切除方法同樣可以誘導(dǎo)小鼠肝臟卵圓細(xì)胞的增殖。本文所用方法是一種穩(wěn)定的成體小鼠卵圓細(xì)胞的分離和培養(yǎng)方法，為原發(fā)性肝癌的卵圓細(xì)胞源性相關(guān)研究奠定基礎(chǔ)。 第二部分Toll樣受體2信號(hào)通路在AFB1誘導(dǎo)卯圓細(xì)胞增殖過(guò)程中的作用 目的研究AFB1誘導(dǎo)卵圓細(xì)胞增殖的情況，探討Toll樣受體2(TLR2)信號(hào)通路在AFB1誘導(dǎo)卵圓細(xì)胞增殖中的表達(dá)變化和意義。 方法采用腹腔注射AFB1的方法建立AFB1誘癌的小鼠模型。檢測(cè)血清谷丙轉(zhuǎn)氨酶(ALT)和谷草轉(zhuǎn)氨酶(AST)濃度變化；免疫組化方法檢測(cè)肝臟組織內(nèi)卵圓細(xì)胞增殖的情況；使用實(shí)時(shí)定量PCR檢測(cè)不同時(shí)間點(diǎn)肝組織TLR2、髓樣分化因子(MyD88)、肝細(xì)胞生長(zhǎng)因子(HGF)和AFP mRNA的表達(dá)情況；Western blot方法檢測(cè)肝組織TLR2、MyD88和核因子-κBp65(NF-κBp65)的表達(dá)情況；酶聯(lián)免疫吸附試驗(yàn)(ELISA)方法檢測(cè)細(xì)胞因子腫瘤壞死因子-α(TNF-α)、白介素(IL)-1、IL-6和HGF的表達(dá)情況。 結(jié)果在此模型中，3小時(shí)時(shí)ALT和AST即有明顯升高，隨后逐漸升高，30天時(shí)達(dá)到高峰，隨后有所下降，但明顯高于對(duì)照組(P＜0.05)。對(duì)照組肝組織內(nèi)未見(jiàn)卵圓細(xì)胞的增殖，AFB1組肝組織7天時(shí)卵圓細(xì)胞開(kāi)始增殖，一直持續(xù)到60天。TLR2、MyD88和NF-κB及其下游細(xì)胞因子在此過(guò)程中表達(dá)明顯增高(P＜0.05)。 結(jié)論AFB1可以引起明顯肝臟損傷，進(jìn)而誘導(dǎo)小鼠肝臟卵圓細(xì)胞的增殖。TLR2信號(hào)通路可能通過(guò)啟動(dòng)下游細(xì)因子的釋放在卵圓細(xì)胞的增殖過(guò)程中起著重要作用。深入研究TLR2信號(hào)通路可能為卵圓細(xì)胞的增殖和肝癌發(fā)生的研究提供新的理論依據(jù)和新的治療靶點(diǎn)。 第三部分環(huán)境因素誘導(dǎo)卵圓細(xì)胞增殖過(guò)程中肝細(xì)胞和卵圓細(xì)胞基因的表達(dá)變化 目的建立HBV、AFB1和兩者協(xié)同誘導(dǎo)肝癌的動(dòng)物模型，監(jiān)測(cè)肝臟卵圓細(xì)胞的增殖。研究此過(guò)程中肝細(xì)胞和卵圓細(xì)胞基因表達(dá)的差異，為肝癌的干細(xì)胞理論尋找新的實(shí)驗(yàn)依據(jù)。 方法40只Balb／c小鼠和40只HBV轉(zhuǎn)基因小鼠分為四組：對(duì)照組(A組)、AFB1組(B組)、HBV組(C組)和AFB1+HBV組(D組)。AFB1150mg／kg，腹腔注射。檢測(cè)HBsAg定量、HBeAg半定量和乙肝五項(xiàng)，明確HBV轉(zhuǎn)基因鼠轉(zhuǎn)基因是否有效。檢測(cè)各組血清ALT和AST的濃度變化；免疫組化檢測(cè)肝臟卵圓細(xì)胞增殖情況。6個(gè)月后剖殺動(dòng)物，采用本試驗(yàn)室改良的方法分離和培養(yǎng)四組的肝細(xì)胞和卵圓細(xì)胞。抽提總RNA，Illumina小鼠基因芯片檢測(cè)四組內(nèi)肝細(xì)胞和卵圓細(xì)胞的基因表達(dá)變化。 結(jié)果(1)轉(zhuǎn)基因小鼠的檢測(cè)：血清HBsAg250.00IU／mL(陽(yáng)性≥0.05IU／mL)，HBeAg1.66S／CO(陽(yáng)性≥1.00S／CO)，HBsAg抗體(—)，HBeAg抗體(—)，HBcAg抗體(—)。(2)各組死亡率：正常組為0％(0／20)，AFB1組為30％(6／20)，HBV組為10％(2／20)，AFB1+HBV組為60％(12／20)。后三組明顯高于正常組，AFB1+HBV組明顯高于AFB1和HBV組(P＜0.05)。(3)肝功能的變化：正常組肝功能無(wú)明顯變化，AFB1、HBV組出現(xiàn)明顯肝臟損傷，兩者協(xié)同引起肝臟損傷進(jìn)一步加重(P＜0.05)。(4)卵圓細(xì)胞的增殖：正常組肝組織中未發(fā)現(xiàn)卵圓細(xì)胞增殖；后三組肝組織中有明顯卵圓細(xì)胞增殖，呈團(tuán)索狀，主要分布于門(mén)脈區(qū)。(5)與正常組比較，AFB1組卵圓細(xì)胞有1577個(gè)基因表達(dá)出現(xiàn)變化；肝細(xì)胞出現(xiàn)了805個(gè)。HBV組卵圓細(xì)胞有3467個(gè)基因表達(dá)出現(xiàn)變化；肝細(xì)胞出現(xiàn)1196個(gè)。AFB1+HBV組卵圓細(xì)胞，有4846個(gè)基因表達(dá)出現(xiàn)變化；肝細(xì)胞出現(xiàn)2542個(gè)。其中涉及Notch、Wnt、PI3K-AKT等數(shù)十個(gè)信號(hào)通路。AFB1、HBV均引起卵圓細(xì)胞和肝細(xì)胞癌基因和抑癌基因的表達(dá)變化，其中卵圓細(xì)胞表達(dá)升高的癌基因明顯多于肝細(xì)胞，而抑癌基因明顯少于肝細(xì)胞；兩者協(xié)同導(dǎo)致這種效果更加明顯。 結(jié)論(1)轉(zhuǎn)基因小鼠體內(nèi)有穩(wěn)定、高滴度的HBV病毒合成。(2) AFB1、HBV和兩者協(xié)同作用于小鼠均可引起明顯的肝臟卵圓細(xì)胞增殖，并可導(dǎo)致死亡率升高和引起嚴(yán)重肝損傷。(2) HBV轉(zhuǎn)染雖然無(wú)法引起機(jī)體的免疫反應(yīng)，，但可以通過(guò)其它途徑引起肝臟損傷，刺激卵圓細(xì)胞增殖。(3) AFB1、HBV及兩者協(xié)同作用均引起肝細(xì)胞和卵圓細(xì)胞基因的異常表達(dá)，其中對(duì)卵圓細(xì)胞的影響更為顯著；表達(dá)異常的癌基因和抑癌基因可能在卵圓細(xì)胞轉(zhuǎn)化為肝癌過(guò)程中起著重要作用。(5)增殖的卵圓細(xì)胞可能是肝癌的起源細(xì)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.
緇撴灉(1)杞熀鍥犲皬榧犵殑媯

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