【作者】 喻成波；

【导师】 李兰娟；

【作者基本信息】 浙江大学 ， 内科学， 2009， 博士

【摘要】 研究背景重型肝炎病情危重，预后，凶险，其治疗是临床上一大难题。生物型人工肝（bioartificial liver,BAL）理论上讲可以代偿肝脏的全部功能，构建BAL已经成为人工肝研究领域治疗重型肝炎的热点。生物反应器是生物人工肝的核心装置。目前应用最多的是中空纤维生物反应器，但是难以放大且半透膜致使物质弥散障碍等都限制了其应用。微囊肝细胞培养的流化床反应器是必然的选择。BAL欲得到临床应用，必须容易“放大”，约需10¹⁰数量级的肝细胞数。传统体外培养方法难以达到，另外还必须解决免疫排斥问题。生物微胶囊具有选择性物质通透性，允许营养成分及小分子物质自由通过，并且有免疫隔离效果。目前微囊技术已广泛用于生物人工肝研究。目前转瓶大规模微囊肝细胞培养国内外均未见报道，而适合微囊肝细胞的反应器为流化床式生物反应器，流化床的流化作用能使体外循环液充分接触微囊化肝细胞进行物质交换和物质代谢。传统的固化床液体流动时具有剪切力大，物质交换效率低，易于形成无效腔和死腔等缺点。并且其结构多为圆柱体状，液体流化时的不均一性，存在边际效应，导致无效腔形成。我们设计并研制了一种基于改善流化床反应器径向传递性能及混合效果和便于规模化应用的一套漏斗形新型结构流化床反应器。本研究旨在建立微囊永生化肝细胞大规模高活性培养体系；构建新型漏斗形流化床式反应器及对其结构参数的研究；使用体外培养基循环以及重型肝炎血浆模型对此构建的生物反应器进行体外评价，为今后进一步实验提供理论基础。第一部分微囊化永生化肝细胞的大规模转瓶培养及评价目的：本研究旨在建立一种大规模海藻酸／壳聚糖（AC）微囊化肝细胞的转瓶培养体系，并对大规模微囊化肝细胞进行功能评价研究。方法：AC微囊的表征测定（机械强度、物质渗透性免疫隔离作用）、一步法大规模包裹制备微囊永生化肝细胞，微囊肝细胞与裸细胞转瓶培养20天，动态评价微囊肝细胞增殖能力及物质代谢功能。结果：AC微囊能承受高速振荡剪切力的作用而保持形态完整；不仅能对小分子物质如白蛋白自由通过，而且阻止免疫球蛋白的释放。与裸细胞培养比较，大规模转瓶培养AC微囊肝细胞能改善细胞的生长代谢及功能（白蛋白合成、氨代谢以及利多卡因清除能力）。结论：建立了一种大规模AC微囊化肝细胞的转瓶培养体系；一步法大规模制作的AC微囊性能可靠；转瓶培养微囊化肝细胞功能在2周左右达到最佳状态，此培养模式的建立有应用生物人工肝支持系统的广阔前景。第二部分新型漏斗形流化床式反应器初步研制及结构参数研究目的：本研究旨在设计一种符合流体力学的漏斗形生物反应器，通过微囊置于反应器中在模拟体外循外灌流状态下摸索反应器的适宜流化参数。材料与方法：自行设计一定高度和锥度的漏斗形流化床式生物反应器，计算反应器结构参数；观测微囊在流化床流化状态的变化情况。排液体积法测定循环液气含率，测定流化床表观速率、最佳流化高度、观察微囊流化8小时后的完整性。结果：自行设计的漏斗形反应器容积约550ml，高度25cm，锥度92达到最佳流化状态大致分四个过程：固化床、起始流化床、循环床、完全循环流化床。流化床的气含率随着微囊的固含率增加，气含率随之增加，300g／L微囊固含率时气含率达到最大，而后固化率增加气含率缓慢下降。最佳流化高度为23 cm时，泵速为85ml／min，微囊的流化速率为1．19cm／s。流化8小时，微囊完整性在97％以上。结论：流化床式反应器作为承载微囊化人工肝适宜的生物反应器，流化床的流化参数直接影响到流化效果，对这些参数的探索对今后微囊流化床生物反应器的应用于生物人工肝研究具有指导意义。第三部分新型微囊漏斗形流化床式反应器体外初步评价目的：应用培养基循环验证流化床反应器流化作用对肝细胞功能代谢的影响；应用废弃重型肝炎血浆模型对新构建的生物反应器进行评价，观察流化床微囊化肝细胞对重肝血浆的代谢作用以及二者的相互影响。为下一步动物实验奠定基础。材料与方法：正确建立体外循环，将培养2周的微囊化肝细胞分为两组（培养基体外培养组和流化床组），实验组微囊肝细胞置于流化床式生物反应器中，DMEM高糖完全培养基1000ml体外循环12小时，动态检测上清中的ALT、LDH以及白蛋白的水平；重型肝炎血浆置换术后的被置换血浆，作为微囊生物人工肝治疗的血浆模型。将总量约5×10⁹活率在90％以上的微囊肝细胞置于流化床反应器，稀释50％的血浆体外循环6小时，实验前后测定血浆中ALT、TBi、DBi、ALB变化以及微囊肝细胞的活性以及电镜观察肝细胞的微观结构变化情况。结果：体外培养基循环12小时的评价中ALT、LDH水平均有上升，静止培养组ALT上升更为明显，两组相比有显著性差异（H4，p＜0．01；H6，p＜0．05；H12，p＜0．01）。两组白蛋白的合成均有不同程度增多，流化床组升高更为明显（H6，p＜0．01；H12，p＜0．05），与此同时，静止培养组和流化床组从6小时后白蛋白合成量较前比较明显增多，差异有统计学意义（p＜0．05或者p＜0．01）。微囊流化床组实验前肝细胞活率平均为93．5±3．2％，循环12小时后肝细胞活率为90．6±6．5％（P＞0．05）。而培养基静止培养组实验前肝细胞活率平均为93．9±4．4％，实验结束后微囊肝细胞活率略有下降，平均为87．8±3．8％，但没有统计学意义。废弃血浆体外循环功能评价显示：微囊流化床流化6小时后ALT水平无明显上升，静止培养组ALT有明显的升高，两组相比有显著性差异（H6，p＜0．05），而静止培养组中ALT水平较实验前有明显上升（p＜0．05）。体外静止培养组治疗前后总胆红素及直接胆红素无明显下降；而漏斗形流化床组则6小时体外循环后相比静止培养组有明显的下降（p＜0．05）。此外，漏斗形流化床组在治疗前后胆红素也有一定程度下降（p＜0．05），具有统计学意义。两组中白蛋白无明显变化。微囊流化床组循环6小时后肝细胞活率有明显下降（p＜0．05）；静止培养组实验结束后微囊肝细胞活率明显下降，有显著性差异（p＜0．01）。两组在实验结束后活率比较，静止培养组肝细胞活率下降明显，具有显著性差异（p＜0．05）。废弃血浆静止培养的微囊化肝细胞实验结束后肝细胞有明显的损害，出现了空泡性结构。而反应器流化组在实验结束后肝细胞细胞器清晰可见，生长状态尚可，无明显的空泡形成。结论：新构建的漏斗形流化床式生物反应器能使微囊化肝细胞在体外有效地保持活性和功能。载有以大规模微囊化肝细胞接种的漏斗型流化床式生物反应器为基础的的生物人工肝对重肝血浆中的胆红素等有害物质有效清除作用，同时流化床的流化作用对肝细胞有保护作用。建立了评价反应器的体外完整的评价体系，为今后进一步动物实验打好基础。更多还原

【Abstract】 BackgroudSevere hepatitis is a critically ill disease, which is still a worldwide challenge tophysicians. Bioartificial liver （BAL） has been increasingly playing the key role intreatment of severe hepatitis, which can can compensate for the functions of an entireliver. So construction of a BAL has been hot spots of the study on artificial liver.It is well known that a bioreactor is the key device in a BAL. Currently, the hollowfiber bioreactor performs the most application, but it is difficult to magnify and the massdiffuses handicapped by its semipermeable membrane. Also the immunogenicity ofheterologous hepatocytes exists. Based on these, the fluidized-bed bioreactor forcultivated microencapsulated hepatocytes is the optimal selection.About 10¹⁰ hepatocytes are required in a typical bioartificial liver-assisted device.Such a large quantity of hepatocytes, it is very difficult to abtain in conventionalmonolayer culture. Moreover, immunological rejection is also to be solved.Encapsulated biomicapsule has solved the problem. Selective permeability permitsnutrients and micromolecules secreted from hepatocytes to traverse membrane freely,but the macromolecules such as immunoglobulin are isolated by the microcapsules.Presently, microencapsulation has been used for the research on BAL extensively.Spinner cultivation of microencapsulated hepatocytes takes the advantages ofmicrocapsule and spinner Cultivation. Microencapsulation is advantageous tometabolism, high-density culture rapid growth, sythesis, and secretion. Currently, thecultivation of encapsulated hepatocytes in spinners has scarcely been demonstrated. Fluidized-bed bioreactor is a proper circulation device for encapsulatedhepatocytes. In fixed bed, the beads were unable to withstand high stresses caused byhigh perfusion velocity. The fluid often inclines to mobile by preferential channels inthe space devoid of beads. In view of the traditional fluidized bed bioreactors, most ofthem are cylindrical configuration, they still existed the ununiformity of the fluidizationand marginal effect, which led to void volum or dead space. Based on these, wedeveloped a choanoid bioreactor configuration allowing the fluidization of AC beads.This new type fluidized-bed bioreactor was designed to improve the radial mass transferand the mix efficiency so to be convenient for scale-up.In this study, we established the scaling-up encapsulated hepatocytes with spinnercultivation; also constructed a new type of choanoid bioreactor and studied itsconfiguration parameters; The systematic evalution of our choanoid bioreactor byculture media and abandoned plasma of plasma exchange was given in our research.PartⅠIn vitro large-scale cultivation and evaluation ofmicroencapsulated immortalized human hepatocytes in spinnersObjective: To develop a large-scale and high-production AC microcapsule spinnerculturefor encapsulation of immortalized human hepatocytes. In this study, the efficacyof encapsulated cells with spinner cultivation was evaluated in vitro.Methods: Microencapsulted immortalized human hepatocytes with high producibilityusing a single-stage procedure grew in large-scale spinner-culture system, free cellscultured in spinners served as controls. The mechanical stability, permeability, andimmuno-isolation of AC microcapsules were investigated, and the growth, metabolismand function of encapsulated cells were also evaluated.Results: The mechanical stability of the microcapsules to withstand the shear stressinduced by high agitation rate was well presented; the microcapsules were not onlypermeable to small molecules up to albumin, but prevented the release of immunoglobulins. AC microcapsules were advantageous to improve the growth,metabolism, and functions （albumin synthesis, ammonia elimination and lidocaineclearance） of immortalized human hepatocytes during different periods of spinnercultivation compared with that of free hepatocytes of spinners.Conclusions: We developed a large-scale and high-production AC microcapsulespinner- culture system for the encapsulation of immortalized human hepatocytes. Onestep procedure is a reliable method for microencapsulation; Large-scale spinnercultivatedencapsulated hepatocytes are a promising candidate for use in future BAL.PartⅡDevelopment of a novel choanoid fluidized bed bioreactor andresearch on its fluidization parameters.Objective: We developed a novel choanoid fluidized bed bioreactor configurationconsistent with hydromechanics, which allowed the fluidization of AC beads in whichcells can be immobilized. In the study, we also researched on the appropriatefluidization parameters of this bioreactor for BAL.Materials and methods: A novel type choanoid bioreactor was designed with a certainheight and coning, and the configuration parameters were calculated. We also observedthe changes of fluidized bed from the initial fluidization till to the optimal fluidization.We performed the determination of the gas holdup, apparent rate, and height of theoptimal fluidization. The intact rate of the microcapsules was also observed.Results: The designed choanoid bioreactor is 550 milliliters volume with effectiveheight of 25 centimeters. Process of fluidization is mainly categorized as: fixed bed,fluidized bed, circulating fluidized bed, fully circulating fluidized bed. The gas holdupof this fluidized bed increases accompanied by the solid holdup. The gas holdup reached the maxium of 1.4 percent with 300 gram per liter. So when we took 300 gramper liter solid holdup, optimal fluidization required by pump speed was 85 milliliter perminute and thus the height of optimal fluidization was 23 centimeters. Takingcontinuous fluidzation of 8 hours, the ratio of intact capsules was more than 97 percent.Conclusion: Fluidized bed bioreactor is eligible for artificial liver based onmicroencapsulation, the fluidzation parameters directly infuenced the fluidization effect.So exploring these parameters, it would instructive to microcapsule fluidized bedbioreactor applicable for the research on BAL.PartⅢPreliminary evaluation of the novel choanoid fluidized bedbioreactor （CFBB） in vitroObjective: To investigate the fluidization effect of CFBB on microencapsulatedhepatocyte metabolism through cultural media circulation. The exchanged plasmaserved as the plasma model which was used for evaluation of the newly constructedbioreactor. To observe the influence of the encapsulated hepatocyte metabolism infludized bed on the severe hepatitis plasma, and also to observe the interaction betweensevere hepatitis plasma and microencapsulated hepatocytes.Materials and methods: The 2nd week cultural hepatocytes （total numbers 5×10⁹） putinto the fluidized bed bioreactor and DMEM circulated through the bioreactor for 12hours, the 200ml same proportional encapsulated hepatocytes （control, total numers 1×10⁹） cultivated under static conditions in spinner flask during 12h. The samples werecollected for measurement of alanine aminotransferase （ALT）, lactate dehydrogenase（LDH）, and albumin. The exchanged plasma of severe hepatitis patients was used as theplasma model. 5×10⁹ encapsulated hepatocytes （the viability was above 90%） werecirculated in fluidized bed bioreactor through the half-diluted plasma（1000ml） for 6h, and 1×10⁹ encapsulated hepatocytes were cultured under static conditions for 6h ascontrol. The samples were collected for measurement of ALT, total total bilirubin （TBi）,direct bilirubin （DBi）, and albumin before and after the experiments. Also, viability ofmicroencapsulated hepatocytes was measured and the microstructure was observed.Results: The level of ALT rose in the 12h circulation of extracorporeal medium. Thestatic culture group rose obviously （H4, p<0.01; H6, p<0.05; H12, p<0.01）. The releaseof LDH was higher in static group than that in fluidized bed group after 6h （H6, p<0.05;H12, p<0.01）. Whereas, although the albumin synthesis increased in both groups,albumin secretion by fluidized bed was higher than that in static group （H6, p<0.01;H12, p<0.05）. The viablity of hepatocytes decreased from 93.5±3.2% to 90.6±6.5% influidized bed compared with that 93.9±4.4% to 86.8±3.8% in static group（p>0.05）.In both groups, the level of ALT rose in the severe hepatitis plasma within 6h. Thestatic group rose obviously （p<0.05）. After circulation, TBi concentration decreasedobviously in fluidized bed group （p<0.05）, there were no obvious changes of TBiconcentration after 6h static culture in the plasma. The albumin concentration was notsignificant difference in both groups after 6h. Although the hepatocyte viablity in bothgroups decreased, the static group decreased obviously （p<0.05）. Scanning electronmicroscope showed that cells in static group were damaged with vacuole-likearchitecture. The construction of encapsulated cells in fluidized bed was almost nomal.Conclusions: The newly designed fluidized bed bioreactor facilitated encapsulated cellskeep their viability and function in vitro; The BAL based on a fluidized bed bioreactorwith microencapsulated hepatocytes appeared to be effective in treatment of severehepatitis plasma, with protection of hepatocytes and bilirubin elimination. The efficacyof this novel bioreactor seemed to be promising for human clinical care.更多还原