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

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【摘要】： 第一部分小鼠卵圓細胞增殖模型的建立及卯圓細胞的分離、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胞的誘導(dǎo)、分離和培養(yǎng)方法，并對分離培養(yǎng)的卵圓細胞加以鑒定。 方法喂飼2-乙酰氨基芴(2-AAF)+部分肝切除(PH)方法誘導(dǎo)小鼠肝臟卵圓細胞的增殖。改良的經(jīng)門靜脈灌注膠原酶消化法和等密度離心法分離純化卵圓細胞，并在體外進行長期培養(yǎng)。采用免疫熒光、RT-PCR和糖原染色等方法對培養(yǎng)的卵圓細胞加以鑒定。 結(jié)果此模型中肝臟有明顯的卵圓細胞增殖。分離的卵圓細胞為大小不一、為異質(zhì)性的，代表了肝臟卵圓細胞的所有亞群。體外培養(yǎng)的卵圓細胞呈集落樣生長，穩(wěn)定傳代并已培養(yǎng)至3個月。免疫熒光、RT-PCR和糖原染色證實培養(yǎng)的細胞為卵圓細胞，并顯示該細胞具有分化潛能。 結(jié)論2-AAF+2／3肝切除方法同樣可以誘導(dǎo)小鼠肝臟卵圓細胞的增殖。本文所用方法是一種穩(wěn)定的成體小鼠卵圓細胞的分離和培養(yǎng)方法，為原發(fā)性肝癌的卵圓細胞源性相關(guān)研究奠定基礎(chǔ)。 第二部分Toll樣受體2信號通路在AFB1誘導(dǎo)卯圓細胞增殖過程中的作用 目的研究AFB1誘導(dǎo)卵圓細胞增殖的情況，探討Toll樣受體2(TLR2)信號通路在AFB1誘導(dǎo)卵圓細胞增殖中的表達變化和意義。 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；免疫組化方法檢測肝臟組織內(nèi)卵圓細胞增殖的情況；使用實時定量PCR檢測不同時間點肝組織TLR2、髓樣分化因子(MyD88)、肝細胞生長因子(HGF)和AFP mRNA的表達情況；Western blot方法檢測肝組織TLR2、MyD88和核因子-κBp65(NF-κBp65)的表達情況；酶聯(lián)免疫吸附試驗(ELISA)方法檢測細胞因子腫瘤壞死因子-α(TNF-α)、白介素(IL)-1、IL-6和HGF的表達情況。 結(jié)果在此模型中，3小時時ALT和AST即有明顯升高，隨后逐漸升高，30天時達到高峰，隨后有所下降，但明顯高于對照組(P＜0.05)。對照組肝組織內(nèi)未見卵圓細胞的增殖，AFB1組肝組織7天時卵圓細胞開始增殖，一直持續(xù)到60天。TLR2、MyD88和NF-κB及其下游細胞因子在此過程中表達明顯增高(P＜0.05)。 結(jié)論AFB1可以引起明顯肝臟損傷，進而誘導(dǎo)小鼠肝臟卵圓細胞的增殖。TLR2信號通路可能通過啟動下游細因子的釋放在卵圓細胞的增殖過程中起著重要作用。深入研究TLR2信號通路可能為卵圓細胞的增殖和肝癌發(fā)生的研究提供新的理論依據(jù)和新的治療靶點。 第三部分環(huán)境因素誘導(dǎo)卵圓細胞增殖過程中肝細胞和卵圓細胞基因的表達變化 目的建立HBV、AFB1和兩者協(xié)同誘導(dǎo)肝癌的動物模型，監(jiān)測肝臟卵圓細胞的增殖。研究此過程中肝細胞和卵圓細胞基因表達的差異，為肝癌的干細胞理論尋找新的實驗依據(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HBsAg定量、HBeAg半定量和乙肝五項，明確HBV轉(zhuǎn)基因鼠轉(zhuǎn)基因是否有效。檢測各組血清ALT和AST的濃度變化；免疫組化檢測肝臟卵圓細胞增殖情況。6個月后剖殺動物，采用本試驗室改良的方法分離和培養(yǎng)四組的肝細胞和卵圓細胞。抽提總RNA，Illumina小鼠基因芯片檢測四組內(nèi)肝細胞和卵圓細胞的基因表達變化。 結(jié)果(1)轉(zhuǎn)基因小鼠的檢測：血清HBsAg250.00IU／mL(陽性≥0.05IU／mL)，HBeAg1.66S／CO(陽性≥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)同引起肝臟損傷進一步加重(P＜0.05)。(4)卵圓細胞的增殖：正常組肝組織中未發(fā)現(xiàn)卵圓細胞增殖；后三組肝組織中有明顯卵圓細胞增殖，呈團索狀，主要分布于門脈區(qū)。(5)與正常組比較，AFB1組卵圓細胞有1577個基因表達出現(xiàn)變化；肝細胞出現(xiàn)了805個。HBV組卵圓細胞有3467個基因表達出現(xiàn)變化；肝細胞出現(xiàn)1196個。AFB1+HBV組卵圓細胞，有4846個基因表達出現(xiàn)變化；肝細胞出現(xiàn)2542個。其中涉及Notch、Wnt、PI3K-AKT等數(shù)十個信號通路。AFB1、HBV均引起卵圓細胞和肝細胞癌基因和抑癌基因的表達變化，其中卵圓細胞表達升高的癌基因明顯多于肝細胞，而抑癌基因明顯少于肝細胞；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胞增殖，并可導(dǎo)致死亡率升高和引起嚴重肝損傷。(2) HBV轉(zhuǎn)染雖然無法引起機體的免疫反應(yīng)，，但可以通過其它途徑引起肝臟損傷，刺激卵圓細胞增殖。(3) AFB1、HBV及兩者協(xié)同作用均引起肝細胞和卵圓細胞基因的異常表達，其中對卵圓細胞的影響更為顯著；表達異常的癌基因和抑癌基因可能在卵圓細胞轉(zhuǎn)化為肝癌過程中起著重要作用。(5)增殖的卵圓細胞可能是肝癌的起源細胞。
[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

本文編號：1392575