【作者】 刘勇；

【导师】 高毅；

【作者基本信息】 南方医科大学 ， 普通外科学， 2009， 博士

【摘要】 第一部分人工肝支持系统治疗肝功能衰竭的Meta分析研究背景:肝脏作为代谢器官,其功能是调节机体能量供给、合成多种基本物质、清除毒性物质,在维持全身器官稳态中发挥关键作用。肝脏至少有600多种酶,在临床医学中很少有比肝细胞的广泛坏死更有破坏性。重型肝炎中尤其急性重型肝炎所致暴发性肝功能衰竭（FHF）易引起脑、肾等多器官功能衰竭（MOSF）。FHF常规内科综合治疗尽管监护技术和支持疗法改善,但效果较差、病死率仍在90%左右。当前以原位肝移植（Orthotopic Liver Transplantation,OLT）治疗FHF效果最佳,病死率可降低到20%。但由于供体缺乏、费用昂贵和需长期使用免疫抑制剂等原因的限制,直到目前仍不能普遍应用,尤其在我国进行原位肝移植的病人更少。肝功能衰竭的内科治疗效果不佳,而肝移植需要复杂技术,且供肝来源稀少。人工肝或生物肝是肝功能衰竭的重要手段,十年来被广泛肝采用目前根据其组成和性质主要可分为三类:1、非生物型,又称物理型,主要通过物理或机械的方法进行治疗,包括血浆置换（plasmaexchange,PE）、胆红素、氨及药物灌流吸附（directhemoperfusion/plasmaabsorption,DHP/PA）、血液滤过（HF）等均属此类。2、生物型,将生物部分,如同种及异种肝细胞与合成材料相结合组成特定的装置,患者的血液或血浆通过该装置进行物质交换和解毒转化等。3、混合型,由生物与非生物型结合组成的具有两者功能的人工肝支持系统。尽管人工肝系统对于降低胆红素等具有确切效果,但是目前的研究结论认为人工肝支持系统对肝功能衰竭的预后改善意义尚不完全清楚。研究目的:本研究拟通过对近十年来发表的相关文献尤其近四年的文献进行Meta分析,探讨人工肝支持系统对死亡率的干预效果。研究方法:和检索国内外1970年1月-2008年6月公开发表的人工肝治疗肝功能衰竭的相关论文,提取生存率或出院时临床好转率等可以反映远期预后的资料,以风险比（RR）为效应量进行异质性检验和统计量合计分析。并进行Meta回归分析,探讨影响结论的相关因素研究结果:共有12例研究入选,共包含肝功能衰竭病例304例。治疗组给于人工肝或生物肝联合常规内科治疗。对照组均给于常规内科治疗.总体而言,人工肝支持系统对肝功能衰竭的预后有一定程度的改善（RR值0.65,可信区间0.43-0.96,P值0.03）。对慢性肝功能衰竭患者急性加重患者的预后则改善明显。（RR 0.265,可信区间0.103-0.682,P值0.006）研究结论:人工肝支持治疗对肝功能衰竭的生存率改善轻微,对慢性肝功能衰竭患者急性加重者明显降低死亡率。人工肝支持系统类型影响生存率,生物肝对急性肝衰竭或慢性肝功能衰竭患者急性加重都可以降低死亡率。进一步开发生物肝支持系统是将来的研发方向和重点。第二部分旋转生物反应器内微载体肝细胞大规模扩增研究研究背景:肝细胞是BAL的主要生物成分,在BAL中起核心作用,BAL的支持作用主要来源于肝细胞的生物代谢功能。制备一个具有相似功能的生物人工肝需要约200g肝细胞。临床使用的生物反应器内,细胞数量至少应该达到15%的人肝细胞数量,即约10⁹个数量级才有临床意义。大规模培养肝细胞是人工肝领域的一个重要课题。理想的生物反应器必须达到:双向物质传输;细胞密度达1×10⁷细胞/ml水平,总量达到2×10¹⁰个细胞;肝细胞代谢功能至少达单层培养的水平,并至少保持2周以上。目前人工肝反应器有四种模式:中空纤维型,单层平板培养型,灌流平板/支架型,包裹式微球/悬液或柱状反应器。但均无法完全满足人工肝的要求。最近模拟微重力反应器研究取得了明显进展,该反应器优点在于:反应器旋转可促进溶质交换,减少培养液溶质梯度差;肝细胞于模拟的微重力条件下聚集生长。体外试验猪肝细胞呈球状生长,乳酸脱氢酶（LDH）在14d观察期内始终保持正常范围,血氨12h后达到正常,凝血因子、蛋白、胆碱酯酶合成功能保持稳定。在动物实验中证实对猪爆发性肝衰模型肝支持治疗能延长其寿命达150%。但是其应用猪肝细胞作为细胞材料有先天性缺陷,且培养体系没有用支架材料,肝细胞呈球状聚集生长,仍然没有解决中心部位营养及细胞代谢产物交换的问题。模拟微重力细胞培养利于3D培养细胞生长,是目前不多的可进行细胞共培养的系统,利于细胞分化,是肝细胞大量扩增,高密度培养的最佳方式之一,并且细胞分化好,细胞活性维持时间长,细胞功能能达到单层细胞培养水平,有望成为人工肝生物反应器内理想的细胞装载模式。对肝生物反应器的各种因素进行优化,实现肝细胞大规模培养,是肝生物反应器的关键环节,是否具有临床可行性的决定因素。研究目的:探讨RCCS下肝细胞微载体大规模培养的方法,优化培养条件研究方法:本实验探索在旋转生物反应器内应用微载体技术快速扩增肝细胞的方法.将CL-1细胞和HepFMMU应用Cytodex-3微载体在旋转生物反应器（RCCS）内进行动态培养,观测肝细胞的生长情况和细胞代谢率,分析影响培养效率的因素,并予以优化研究结果:肝细胞在Cytodex-3微载体上生长迅速、生长倍增时间缩短.在培养第五天,细胞数量可达最初接种的23倍.细胞的数量在第五天左右达到最高,然后开始下降。功能学检查,倒置显微镜,扫描电镜及MTT均提示细胞功能良好。相对最佳条件为:细胞贴壁期,以静止为主,间断震荡为辅。细胞贴壁后初始速度为5-10RPM/分,随细胞生长增大到15RPM/分。初始细胞浓度为1-5×10⁵/ml。微载体浓度为5mg/ml。研究结论:RCCS微载体肝细胞大规模培养是一种可行的细胞快速扩增方法,但对于人工肝庞大的细胞数量和功能需求,应进一步研究,提高细胞培养规模和功能。第三部分旋转生物反应器内微载体活体细胞原位示踪荧光显像研究背景:组织工程的三大要素是:细胞、生物材料制备的细胞支架,以及组织和器官的构建。理想的细胞支架应具有一定的生物降解速度、良好的细胞亲和性、呈特定的三维多孔结构,且含有可控制释放的生长因子。组织工程中细胞支架起着为细胞增殖营造环境的作用,它应随着细胞的繁殖而逐渐降解、消失,以利于将空间让位于细胞,并使所生成的组织和器官具有同细胞支架相同的几何形状。因此作为细胞支架的高分子材料必须具有生物降解性,即在生理或体内环境下组成材料的分子链能自动断裂,并由此形成的小分子能逐渐被机体代谢或吸收。此外,还要求材料的降解速度与细胞的增殖速度相匹配、以及由降解所形成的小分子不会对细胞繁殖产生不利的影响。因此组织工程的细胞支架材料必须具有生物降解性和一定的降解速度、良好的生物相容性及细胞亲和性。此外,组织工程的细胞支架,不仅应具有在细胞培养过程中能保持形状、不会破碎、可以操作的力学强度外,从临床应用出发,细胞支架还应具有一定的柔韧性,使之能够同机体组织相缝合、并能同机体组织贴合,也不会对机体组织造成机械损伤的力学性能。因此,作为组织工程细胞支架的生物材料必须兼具有生物可降解性、良好的生物相容性、细胞亲和性、一定的力学性能、可加工性,以及易于消毒。微载体作为一种比较理想的细胞支架广泛被用于贴壁细胞的培养中。一系列新型的微载体如多孔微载体被研发,投入到细胞培养,疫苗生产等。但目前应用的微载体特别是多孔微载体,由于透光性,而难以在细胞培养时采用常规显微镜观察到细胞细胞分布,细胞形态和活力。多孔微载体呈黄褐色,因而不易将细胞同背景区分。支架内生长的细胞也难以观察。常规使用的细胞活力染色剂如台盼兰易将微载体一起染色,降低了区分度。MTT染色的黑色与背景黄褐色的区分度也不高。寻找一种具有良好区分度,便于随时观察活体细胞的示踪方法具有重要实际意义。活体细胞荧光染色已经有成熟的应用的经验。建立一种高分辨率的在线微载体肝细胞示踪方法重要的实用价值。研究目的:探讨微载体细胞培养中活体细胞原位示踪的方法研究方法:采用活细胞核酸染色剂（SYTO13）和溴化乙啶（EB）2种核酸染色剂对微载体生长的细胞进行双重染色,荧光显微镜下通过颜色不同判断细胞的活力状态并计算出活力百分比。以间接活力测定的MTT法同时对肝细胞进行活力的检测,并将结果进行比较。利用图像分析软件计算微载体的细胞生长数量。研究结果:SYTO 13/EB法可以直接观察到微载体上培养细胞的活力状态,染色时间短,操作简便,并可以定量分析,优于传统的MTT法。研究结论:SYTO 13/EB法可作为微载体上培养细胞活力状态的常规方法应用。第四部分灌注式RCCS肝细胞生物反应器的CFD模拟分析研究背景:RCCS（微重力旋转培养仪）广泛用于生物反应器,以提供低重力的理想的细胞生长环境,从而允许细胞聚集、三维生长和分化。微载体培养是目前公认的最有发展前途的一种动物细胞大规模培养技术,其兼具悬浮培养和贴壁培养的优点,放大容易。目前微载体培养广泛用于培养各种类型细胞,生产疫苗、蛋白质产品,如293细胞、成肌细胞、Vero细胞、CHO细胞。RCCS反应系统可针对上述限制性因素,为微载体细胞培养与扩增提供低剪切力、高氧传递效率、易于细胞传代等适宜的外部环境。尽管RCCS生物反应器中的营养供应和废物排除是细胞培养至关重要的因素,但目前缺乏微载体类型,大小,旋转速度等因素对500ml以上大规模RCCS培养效率的系统研究。如何优化RCCS培养条件,提高培养效率,是重要的研究方向之一。传统的实验方法具有耗时,成本昂贵等弊端。而利用计算机技术和现代流体力学理论,对RCCS进行数值仿真分析,有助于快速准确的分析影响RCCS培养效率的关键因素,寻找最佳培养条件。流体力学是连续介质力学的一门分支,是研究流体（包含气体及液体）现象以及相关力学行为的科学。可按研究对象的运动方式分为流体静力学和流体动力学,还可按应用范围分为水力学,空气动力学等等。理论流体力学的基本方程是纳维-斯托克斯方程,简称N-S方程。通用CFD软件包,用来模拟从不可压缩到高度可压缩范围内的复杂流动。由于采用了多种求解方法和多重网格加速收敛技术,因而FLUENT能达到最佳的收敛速度和求解精度。灵活的非结构化网格和基于解的自适应网格技术及成熟的物理模型,使FLUENT在转捩与湍流、传热与相变、化学反应与燃烧、多相流、旋转机械、动/变形网格、噪声、材料加工、燃料电池等方面有广泛应用。FLUENT软件具有以下特点:①FLUENT软件采用基于完全非结构化网格的有限体积法,而且具有基于网格节点和网格单元的梯度算法;②定常/非定常流动模拟,而且新增快速非定常模拟功能;FLUENT软件中的动/变形网格技术主要解决边界运动的问题,用户只需指定初始网格和运动壁面的边界条件,余下的网格变化完全由解算器自动生成。网格变形方式有三种:弹簧压缩式、动态铺层式以及局部网格重生式。其局部网格重生式是FLUENT所独有的,而且用途广泛,可用于非结构网格、变形较大问题以及物体运动规律事先不知道而完全由流动所产生的力所决定的问题;FLUENT软件具有强大的网格支持能力,支持界面不连续的网格、混合网格、动/变形网格以及滑动网格等。值得强调的是,FLUENT软件还拥有多种基于解的网格的自适应、动态自适应技术以及动网格与网格动态自适应相结合的技术研究目的:探讨RCCS生物反应器的仿真方法并阐明微载体旋转速度等对氧供的影响研究方法:联立不可压缩牛顿流体连续性方程和动量守恒方程,微载体内以及流动空间中氧的传递和反应方程,设置文献报道的参数并求解。首先以Gambit 2.2建立RCCS网格模型,并用Fluent 6.02软件在欧拉多相模式下进行生物反应器数值模拟及流体力学分析研究结果:基于欧拉-欧拉多相模型和氧运输公式建立RCCS数值分析模型。微载体的直径及RCCS旋转速度作为自变量。结果显示微载体直径和旋转速度均影响培养效率。直径200mm的微载体旋转速度应为10rpm。300mm的微载体为12rpm。400mm的微载体为14rpm;和旋转速度10rpm微载体直径200mm相比,18rpm微载体直径600mm条件下由于对流效应,旋转1小时后RCCS中间区平均氧浓度增加85%。研究结论:CFD模拟分析可以定性定量分析影响微载体及氧供的相关因素,并为优化培养条件提供参数以供参考。更多还原

【Abstract】 PARTⅠTitle:Artificial and Bioartificial Support Systems for Acute and Acute-on-Chronic Liver Failure:A Meta-analysisBackgroundLiver falure is characterized by hepatic encephalopathy jaundice,coagulopathy,and high mortality rates.Viral hepatitis,drugs,or toxins can precipitate acute liver failure in patients without chronic liver disease.Metabolic stress such as bleeding or infections can precipitate acute-on-chronic liver failure in patients with chronic liver disease.Liver transplantation cures approximately 90%of patients with liver failure, but there is a serious shortfall of donors and costs are considerable.Furthermore, some patients may recover spontaneously without liver transplantation.The objective of artificial and bioartificial support systems is to“bridge”patients with liver failure to transplantation or recovery.Liver support must include removal of toxins, synthesis of products,and treatment of inflammation.The first artificial support systems removed toxins through hemodialysis,hemofiltration,or hemoperfusion. More recent systems combine hemodialysis with adsorption to charcoal or albumin （hemodiabsorption）or use living hepatocytes,which add synthetic functions to the detoxification。（bioartificial support systems）.However the effect of bioartificial support systems treatment on survival of patients of ALF and AOCLF is not well known. ObjectivesEvaluate the effect of artificial and bioartificial support systems for acute and acute-on-chronic liver failure.MethodsRandomized trials on any support system vs standard medical therapy were included irrespective of publication status or language.Trials were identified through electronic searches through July 2008.Of 1134 references identified,12 randomized trials with 479 patients were included.Data were extracted and trial quality was assessed independently by 2 reviewers.The primary outcome measure was all-cause mortality.Results were combined on the risk ratio（RR） scale. Fix-effects models were used.Sources of heterogeneity were explored through meta-regression and stratified meta-analyses.ResultsOverall,support systems had slightly significant effect on mortality compared with standard medical therapy（RR,0.63;95%confidence interval[CI],0.43-0.94）.Meta-regression indicated that the effect of support systems depended on the type of liver failure（P=.00）.In stratified meta-analyses,support systems appeared to reduce mortality by 74%in acute-on-chronic liver failure（RR,0.26;95%CI,0.11-0.65）,but not in acute liver failure（RR,0.80;95%CI, 0.52-1.26）.ConclusionsThis review suggests that artificial support systems reduce mortality in acute-on-chronic liver failure compared with standard medical therapy.Artificial support systems did not appear to affect mortality in acute liver failure.But bioartificial support systems have great potential and should be the focus of further research.PARTⅡTitle:Rapidly large scale proliferation of liver cell line in vitro by microcarrier vehicle in rotating bioreactorBackground Hepatocytes are unrivaled by any other parenchymal cell type in functional diversity and complexity.These cells divide only one or two times throughout the normal lifespan of a mature liver.However,hepatocytes exhibit an extraordinary regenerative ability in response to liver injury.It has been exceedingly difficult to establish large scale cultures of hepatocytes in vitro.Previous efforts to do so have failed when either plastic or collagen-coated rigid plates were used Improved growth was achieved by culturing hepatocytes between two layers of collagen. However,all of these culture systems yield hepatocytes that are functionally limited, as shown by transient mitogenic responsiveness over a 2-3-wk period.Recent studies indicate that cell expansion and specific differentiation patterns can be stimulated in cultured ceils with a defined,growth factor-enriched medium and extracellular matrix. Rat hepatocytes have been maintained for up to 2 month.with a synthetic matrix of biodegradable polymeric scaffolds composed of polyglycolic acid（PGA）.These cells produced albumin and expressed cytochrome P450 enzyme activity that was measured by the ability of cells to transform the nonfluorescent compound ethoxy-fluoreseein ethyl ester to fluorescein.Hepatocyte cultures have also been maintained with an artificial capillary system and a hollow fiber binreactor with cells entrapped in collagen.While previously used systems support spheroid formation, they are labor-intensive and useful within only a narrow range of cell density.The advent of the Rotary Cell Culture System（RCCS）,developed at the Johnson Space Center,Houston,Texas,has enabled the growth of suspension cells as well as anchorage-dependent cells in a simulated microgravity environment.This system sinmlares microgravity by randomizing the gravitational vector while supporting cellular colocation in three-dimensional space in a low shear,high mass transfer environment.To configure a bioreactor for nurturing a high density of hepatocytes within a solid substrate known as a scaffold is one of the promising approaches for the development of a BAL assist device.Maintenance of specific functions of cultured hepatocytes at a higher level is greatly appreciated to support the hepatic patients and thus a highly essential requirement for a superior BAL.To date many researchers have examined maintenance of viability and functions of primary hepatocytes.From these investigations,the significance of the choice of culture conditions,such as medium components,cultural substratumand oxygen concentration,has been exhibited.ObjectivesTo establish the large scale culture of liver cell in RCCS and microcarrier and evaluate the effect of biological function of bioreactor consisting of cultivated liver cell line CL₁ and HepFMMU on microcarriers in RCCS.MethodsHigh concentration liver cell line CL₁ and HepFMMU were cultured on microcarriers.Meanwhile,RCCS was used as a bioreactor.Histomorphology and biological functions were evaluated accordingly in phase contrast observation,scanning electron microscope（SEM）,biological functions test and MTT test..ResultsLiver cell cultivated in the bioreactor in three dimensions were expanded more than twenty times in one generation.The expanded cells performed well in major functions examined by inverted microscope in phase contrast observation,scanning electron microscope（SEM）,biological functions test and MTT test.ConclusionsThe bioreactor used as the core of ABL system has functions of biosynthesis and biotransformation.It can become an effective bioartificial liver device,although further reseach need to minimized the cost and decrease the human cost by improve the supply of energy and oxygen and enhance the clearness of wastePARTⅢTitle:In situ assessment of liver cell viability on microcarrier by fluorescent staining. BackgroundMicrocarrier cell culture is widely used for the propagation of anchoragedependent cells in suspension.And the determination of cell viability is also important in liver cell culture.One disadvantage of the Cytodex 3 microcarriers is the property of these materials,which make microscopic examination of cell behavior difficult.Routine microscopic examination is important in microcarrier culture to observer cell distribution,morphology,and growth throughout the cultivation period. Polystyrene and glass beads have light-reflecting surfaces,which blur the image of the microcarriers and cells on them.The macroporous gelatin beads are amber colored when observed with ordinary light microscopy;discerning cells from the microcartier matrix is difficult on the external surface and impossible inside the beads.Nevertheless,Trypan blue exclusion and MTT are a common test for cell viability,but they also stains the supporting matrix when used with dextran-, collagen-,or gelatin-based microcarriers,making microscopic examination of the cells difficult.ObjectivesTo develop an assay for detection of viability of liver cell cultured on the Cytodex 3 in situ.MethodsTwo nucleic acid dyes,SYTO 13 and ethidium bromide（EB）,were used to assess the viability of liver cell.Cells were stained with SYTO 13\EB.The viability of cells was determined by different colors under fluorescence microscope.and compared with MTT assayResultsAlthough the cell number and morphology can be clearly seen with the Cytodex 3, but cells can not be discerned.No cell obviously visible inside the gelatin beads, although some cells are seen on the external surface in MTT assay.When cells were cultivated as aggregates,This SYTO/EB staining technique can also be applied to such a system.High viability can be seen in large clumps of cells. ConclusionsThis staining technique is simple,quick and reliable when used in microcarrier liver cell culture.This technique has been applied to a number of microcarrier.The use of this technique with macroporous microcarriers makes it a potential visualization technique in other immobilized mammalian cell systems.PARTⅣTitle:CFD analysis of rotating bioreactors for liver tissue engineering.BackgroundThe Rotary Cell Culture System^TM(RCCS^TM) is new technology for growing either anchorage dependent or suspension cells in the laboratory.The system enables the researcher to cultivate many types of cells to high densities.Cells that could not be grown easily by other methods have been grown without difficulty in RCCS vessels.The growth of co-cultures of differentiated 3D masses that mimic the structure and function of the parent tissues can be achieved in the RCCS^TM.The system provides a reproducible complex 3D in vitro culture system for the investigation of the structural processes and isolation of the growth and regulatory molecules that control differentiation in normal tissues and transformation in neoplastic tissues.Some articles in scientific journals have used the term“organoids”to describe the 3 dimensional differentiated tissue models and expanded surgical explants that have been grown in the RCCS^TM.The rotating wall vessel（RWV） bioreactor developed by NASA/JSC is one of the noble suspension-type reactors.The RWV bioreactor that is designed originally for simulating microgravity conditions on earth provides an adequate oxygen supply and mass transfer.In addition,it has characteristics of solid-body rotation and reduced shear force on the cells which are cultured on the surface of microcarrier beads of similar density with the culture medium The microcarrier bead is added to the vessel to promote cell attachments on its surface,especially in the case of anchorage-dependent cell lines.Various kinds of cells and tissues have been cultured using the RWV bioreactor not only to investigate the microgravity effects on the cell physiology,but also to exploit the vessel to generate engineered tissue equivalentsThe unique fluid mechanics and mass transfer in the RWV bioreactor have been studied by many researchers.The gravitational force that causes the sedimentation of cell aggregates in the vessel is on average vectorless because of the hydrodynamic forces that are created by rotation,including centrifugal,Coriolis,and shear forces. The maximum shear stress experienced by a microcarrier is as low as 0.07 dyne/cm2 in the RWV,as compared to 5 dyne/cm2 in a typical stirred vessel.It is difficult to decide the operating conditions of the bioreactor for the optimal suspension of cells through trial and error due to the dimensions of the cells and beads and the high costs of materials Therefore,the optimal flow conditions of the bioreactor need to be parametrically determined by calculations.The microcarrier dynamics and migration in simulated microgravity conditions have been studied numerically and experimentally by several groups of researchers, who found that decreasing the density difference between the microcarrier and the culture medium can reduce the maximum shear stress and increase the particle suspension time.But the previous studies have not included oxygen distribution and consumption by cells within the vessel in relation to the bead location.The broad physical modeling capabilities of FLUENT have been applied to industrial applications ranging from air flow over an aircraft wing to combustion in a furnace,from bubble columns to glass production,from blood flow to semiconductor manufacturing,from clean room design to wastewater treatment plants.The ability of the software to model in-cylinder engines,aeroacoustics,turbomachinery,and multiphase systems has served to broaden its reach.ObjectivesTo simulate the RCCS liver bioreactor model with computational fluid dynamic（CFD） software and analyze the characteristic of supply of nutrition and waste removal.MethodsA numerical analysis of the RWV bioreactor is conducted by incorporating the Eulerian-Eulerian multiphase and oxygen transport equations.The bead type,size and rotating speed are the control variables in the calculationsResultsThe present results show that the rotating speed for appropriate suspensions needs to be increased as the size of the bead/cell increases:10 rpm for 200 mm;12 rpm for 300 mm;etc.As the rotating speed and the bead size increase from 10 rpm/200 mm to 18 rpm/600 mm,the mean oxygen concentration in the 80%midzone of the vessel is increased by 85%after 1-h rotation due to the high convective flow for 18 rpm/600 mmcase as compared to 10 rpm/200 mm case.ConclusionsThe present results may serve as criteria to set the operating parameters for a RWV bioreactor,such as the size of beads and the rotating speed,according to the growth of cell aggregates.In addition,it might provide a design parameter for an advanced suspension bioreactor for 3-D engineered cell and tissue cultures.更多还原