本文選題：可逆性永生化　切入點(diǎn)：肝細(xì)胞　出處：《浙江大學(xué)》2012年博士論文　論文類型：學(xué)位論文

【摘要】：研究背景： 肝衰竭發(fā)生時,肝臟會喪失其正常的解毒、生物合成和生物轉(zhuǎn)化功能,其臨床特征包括：凝血酶原時間延長、肝性腦病和黃疸。根據(jù)是否存在已知的或未知的慢性肝病病史,不同致病因素所引起的肝衰竭可被劃分為兩大類：急性肝衰竭和慢加急性肝衰竭,兩者均伴隨著高病死率。迄今為止,肝臟移植仍然是治療終末期肝衰竭公認(rèn)的最終解決方案,但世界性的供體短缺使其在臨床實(shí)際應(yīng)用中受到限制。在這種背景下,有研究者致力于體外人工肝支持系統(tǒng)的研發(fā),以期為肝衰竭患者提供一種肝移植前的過渡治療手段,或者為自體肝臟的恢復(fù)贏得機(jī)會。 根據(jù)是否裝載具有代謝活性的肝細(xì)胞,人工肝支持系統(tǒng)可分為非生物人工肝和生物人工肝。盡管非生物人工肝有改善肝衰竭患者的生化指標(biāo)和臨床癥狀的作用,但其在改善患者預(yù)后方面尚未達(dá)到預(yù)期的效果。目前公認(rèn)的觀點(diǎn)是,僅具備解毒功能的非生物人工肝不足以對肝衰竭患者提供全面的支持治療,而在理論上,以載細(xì)胞生物反應(yīng)器為核心的生物人工肝可提供大部分乃至全部的肝功能支持[7,8,10-16]。然而,我們不得不承認(rèn),生物人工肝的發(fā)展還不成熟,距離常規(guī)臨床應(yīng)用仍有距離。迄今,只有兩篇文獻(xiàn)報道了生物人工肝治療治療的隨機(jī)對照臨床試驗,結(jié)果不如人意。在這種背景下,有學(xué)者認(rèn)為理想的體外人工肝支持系統(tǒng)應(yīng)該是載細(xì)胞生物反應(yīng)器和多種非生物人工肝治療手段的有機(jī)整合。 為研發(fā)基于這種理念的體外人工肝支持系統(tǒng),我們必須從以下三方面著手：1)選擇合適的細(xì)胞源；2)設(shè)計制造能夠為肝細(xì)胞提供近似體內(nèi)環(huán)境的生物反應(yīng)器；3)研發(fā)新的體外循環(huán)裝置,該裝置不僅可以控制載細(xì)胞的生物反應(yīng)器,還可控制現(xiàn)有的大多數(shù)非生物人工肝治療模式。 最近,本研究團(tuán)隊設(shè)計制造了一種以裝載海藻酸鈉-殼聚糖(AC)微球包裹的原代豬肝細(xì)胞的漏斗形流化床式生物反應(yīng)器為核心的生物人工肝支持系統(tǒng),并證明該系統(tǒng)可顯著延長D-氨基半乳糖誘導(dǎo)的急性肝衰竭豬的生存時間。然而,原代豬肝細(xì)胞有使患者罹患動物源性傳染病的潛在風(fēng)險。這促使我們?nèi)ふ乙环N更加適用于生物人工肝臨床治療的肝細(xì)胞源。近年來,本實(shí)驗室致力于永生化肝細(xì)胞的研究。盡管這類細(xì)胞具有體外擴(kuò)增能力,但猿猴病毒40大T抗原基因(SV40LT)的持續(xù)表達(dá)具有潛在的致瘤風(fēng)險。為了解決這個問題,本團(tuán)隊?wèi)?yīng)用他莫西芬介導(dǎo)的Cre/LoxP位點(diǎn)特異性重組,建立了一株可逆性永生化人肝細(xì)胞系(HepLi-4), SV40LT可以被程序性的切除。在此基礎(chǔ)上,以可逆性永生化人肝細(xì)胞為細(xì)胞源對大型動物進(jìn)行生物人工肝治療的首次試驗得以開展。 困擾我們的另一個問題是,研究所用的人工肝治療用體外循環(huán)裝置大多依賴進(jìn)口。例如,評價漏斗形流化床式生物反應(yīng)器和HepLi-4細(xì)胞所用的體外循環(huán)裝置是日本旭化成株式會社制造的Plasauto-iQ。而且,目前國外相關(guān)設(shè)備的功能較單一。在這種背景下,本團(tuán)隊進(jìn)行人工肝治療用體外循環(huán)裝置的研發(fā),該裝置應(yīng)不僅能夠控制血漿置換(PE)、血漿吸附(PA)、血液透析(HD)等目前臨床常規(guī)應(yīng)用的非生物人工肝治療模式和尚處于科研階段的生物人工肝治療(BAL)模式,而且,以上各種模式均可便捷的進(jìn)行聯(lián)合或序貫應(yīng)用。本研究擬初步構(gòu)建具備上 述功能的原理機(jī),為下一步的人工肝治療研究提供一個可靠的平臺。 第一部分基于可逆性永生化人肝細(xì)胞的生物人工肝系統(tǒng)的動物實(shí)驗評估 目的： 通過中國實(shí)驗小型豬急性肝衰竭模型,對以可逆性永生化人肝細(xì)胞(HepLi-4)為肝細(xì)胞源的生物人工肝系統(tǒng)進(jìn)行評估。 方法： 表達(dá)他莫昔芬依賴性Cre重組酶的HepLi-4細(xì)胞在轉(zhuǎn)瓶中擴(kuò)增后,通過在含有500nM的4-羥基他莫昔芬的培養(yǎng)液中培養(yǎng)5～7切除SV40LT基因。豬急性肝衰竭模型通過在無麻醉情況下靜脈推注D-氨基半乳糖(1.5g/kg)誘導(dǎo)。包裹HepLi-4細(xì)胞的海藻酸鈉-殼聚糖(AC)微球通過一步法制備。15只急性肝衰竭豬分為以下三組：1)生物人工肝治療(BAL)組：實(shí)驗動物接受裝載微囊化的HepLi-4細(xì)胞的生物人工肝支持系統(tǒng)治療(n=5)；2)假治療(Sham BAL)組：即設(shè)備對照,實(shí)驗動物接入生物人工肝支持系統(tǒng)進(jìn)行體外循環(huán),但反應(yīng)器內(nèi)僅裝入無細(xì)胞AC微囊(n=5)；3)急性肝衰竭(ALF)組：即基線對照,實(shí)驗動物僅接受監(jiān)護(hù)但不接受治療(n=5)。記錄以上三組實(shí)驗動物的生存時間以及三種干預(yù)過程前后實(shí)驗動物的血液生化參數(shù)。生物人工肝治療前后,進(jìn)行微球完整性和細(xì)胞活力檢測,同時,進(jìn)行微囊化HepLi-4細(xì)胞內(nèi)肝臟特異性基因的轉(zhuǎn)錄水平分析,正常成人肝組織作為對昭 結(jié)果： 干預(yù)后,相對于Sham BAL組和ALF組,BAL組動物的Fischer指數(shù)較高,血清間接膽紅素相對低,差異具有統(tǒng)計學(xué)意義。BAL組動物的平均生存時間長于兩個對照組,但差異不具有統(tǒng)計學(xué)意義。經(jīng)歷人工肝治療后,進(jìn)行微球完整性和細(xì)胞活力沒有出現(xiàn)顯著的下；同時,微囊化HepLi-4細(xì)胞內(nèi)肝臟特異性基因的轉(zhuǎn)錄水平得以保持,但其相對于正常成人肝組織存在明顯的變異。 結(jié)論： 盡管HepLi-4細(xì)胞在生物人工肝治療過程中發(fā)揮了一些對急性肝衰竭豬有益的代謝功能,但HepLi-4細(xì)胞尚不能作為人工肝治療用的理想細(xì)胞源。我們需要在維持肝細(xì)胞分化的研究中付出更多的努力。 第二部分人工肝體外循環(huán)裝置的研發(fā) 目的： 構(gòu)建可以控制現(xiàn)有的大部分非生物人工肝治療模式和生物人工肝治療模式的人工肝體外循環(huán)裝置的原理機(jī)。 方法： 人工肝體外循環(huán)裝置的原理機(jī)包含非生物部分和生物部分。體外循環(huán)裝置的控制中樞由的工業(yè)用個人計算機(jī),兩個串行通訊端口和一個外設(shè)部件互連標(biāo)準(zhǔn)(PCI)數(shù)字輸入/輸出(DIO)卡組成。體外循環(huán)裝置的大多數(shù)部件可通過RS485總線得到整合。該裝置的軟件為用戶提供了操作指導(dǎo)和人機(jī)交流界面。同時,基于硬件和軟件設(shè)計,該裝置在運(yùn)行過程中可實(shí)現(xiàn)實(shí)時狀態(tài)監(jiān)測和調(diào)節(jié)。此外,體外循環(huán)裝置運(yùn)行過程中的所有參數(shù)值都會被自動記錄,便于事后分析。為了驗證我們的設(shè)計中,我們對該原理機(jī)進(jìn)行了體外測試和動物實(shí)驗測試。 結(jié)果： 在體外測試和動物實(shí)驗測試中,體外循環(huán)裝置原理機(jī)的硬件和軟件在所有治療模式下均運(yùn)行正常。該裝置中所有部件的功能都得到了驗證。此外,該裝置可以實(shí)時監(jiān)測到治療中的異常情況。 結(jié)論： 體外循環(huán)裝置的原理機(jī)具有良好的安全性和人性化的設(shè)計。可以為這種醫(yī)療設(shè)備的商品化提供可靠的研究平臺,也可為培養(yǎng)相關(guān)技術(shù)人員提供一個模擬培訓(xùn)的平臺。
[Abstract]:Research background:
Liver failure, liver will lose its normal detoxification, biosynthesis and biotransformation function, including their clinical features: prolonged prothrombin time, hepatic encephalopathy and jaundice. According to the history of chronic liver disease are known or unknown, liver failure caused by various pathogenic factors can be divided into two categories: acute liver failure and acute on chronic liver failure, both with high mortality rate. So far, liver transplantation is still the ultimate solution for the treatment of end-stage liver failure admitted, but the shortage of donor worldwide in the clinical application is restricted. In this context, researchers have devoted to the extracorporeal artificial liver support system development, in order to provide a bridging therapy before liver transplantation for patients with liver failure, or autologous liver recovered to win opportunities.
According to whether the loading with the metabolic activity of the liver cells, artificial liver support system can be divided into non biological artificial liver and bioartificial liver. Although non bioartificial liver could improve the biochemical index of patients with liver failure and clinical symptoms, but in the aspect of improving the prognosis of patients have not yet reached the expected effect. At present, the accepted wisdom is that only with the non biological artificial liver detoxification function is not enough to provide support for comprehensive treatment of patients with liver failure, but in theory, is a carrier of biological artificial liver cell bioreactor as the core can provide all or most of the liver can support [7,8,10-16]. however, we have to admit that the development of bio artificial liver is not mature, the distance routine clinical application is still distance. So far, only two articles reporting randomized controlled clinical trials for treatment of biological artificial liver treatment, the result is not satisfactory. Under this kind of background, Some scholars believe that the ideal artificial liver support system in vitro should be an organic integration of the cell bioreactor and a variety of abiotic artificial liver treatments.
For the development of in vitro artificial liver support system based on this concept, we must start from the following three aspects: 1) select the appropriate source of cells; 2) can provide design and manufacture for hepatic cell bioreactor to approximate the in vivo environment; 3) the development of a new extracorporeal circulation device, the device can not only control the load cell bioreactor and can also control the majority of non biological artificial liver in the treatment of the existing mode.
Recently, the research team designed a alginate chitosan (AC) to load a choanoid fluidized bed bioreactor in primary hepatocytes microsphere biological artificial liver support system as the core, and proves that this system can be significantly prolonged induced by D- galactosamine in acute liver failure pigs survival time. However, primary porcine hepatocytes have the potential risk of patients with animal infectious diseases. This prompted us to search for a more suitable for the clinical treatment of biological artificial liver cell source. In recent years, the laboratory is committed to immortalized hepatocytes. Although these cells have proliferated in vitro, but simian virus 40 large T antigen gene (SV40LT) potential oncogenic risk has sustained expression. In order to solve this problem, the team used tamoxifen mediated Cre/LoxP site-specific recombination, established a reversible In the immortalized human hepatocyte line (HepLi-4), SV40LT can be resected by program. On this basis, the first experiment of bioartificial liver treatment for large animals was carried out by reversible immortalization of human hepatocytes as a cell source.
Another puzzling problem is, for the study of artificial liver treatment with extracorporeal circulation device mostly dependent on imports. For example, extracorporeal circulation device for evaluation of a choanoid fluidized bed bioreactor and HepLi-4 cells is Japan's Asahi Kasei Corporation made Plasauto-iQ. and the foreign related equipment has single function in this. Under the background, the team with the development of in vitro artificial liver circulation device, the device can not only control the plasma exchange (PE), plasma adsorption (PA), hemodialysis (HD) and the clinical routine application of non bioartificial liver in treatment model and bioartificial liver therapy research stage (BAL) Moreover, model, combined or sequential application of various convenient mode or longer. In this study we have constructed on
The functional principle machine provides a reliable platform for the next step of artificial liver treatment.
Part I animal experimental assessment of bioartificial liver system based on reversible immortalization of human hepatocytes
Objective:
The biological artificial liver system with reversible immortalized human hepatocyte (HepLi-4) as the source of hepatocyte was evaluated through the model of acute liver failure in Chinese experimental miniature pigs.
Method錛，

本文編號：1561515