纤维支架循环灌注式生物反应器规模化培养肝细胞的初步探讨

发布时间：2018-06-03 10:39

本文选题：纤维支架 + 生物反应器　；参考：《南方医科大学》2017年硕士论文

【摘要】：前言:急性肝衰竭(acute liver failure,ALF)是指无基础肝脏疾病患者,在肝脏受到各种创伤后(物理、化学、生物性等),发生的肝功能损害或衰竭的急症,其发病急,病程进展迅速,未加治疗自然病程死亡率高达80%[1,2]。目前尚无特效治疗方法,内科保守治疗,患者死亡率仍高达50%以上,临床最有效的治疗方法是肝移植,但由于供肝的短缺,限制了肝移植的广泛应用[3]。大部分急性肝衰竭的患者未能等到可移植肝源就已死亡,因此急切需要一种暂时可以替代肝脏功能的机器,给予急性肝功能衰竭患者充足的时间过渡到肝移植状态或肝细胞再生状态,人工肝是治疗肝衰竭患者的有效途径,可在短时间内达到部分或全部替代肝脏功能的目的,为急性肝衰竭患者赢得宝贵时间[4]。近十年来,人工肝研究取得了长足进展,从早期简单的机械型人工肝进步至生物型人工肝,再到更为合理的混合型(机械+生物型)生物人工肝。生物反应器作为生物人工肝的核心部分[4-7],主要包括:中空纤维型,结合微载体培养型,细胞球悬浮培养型等多种,但对于临床治疗所要求的1010数量级细胞量,极少有生物反应器能达到上述要求[8,9]。本课题组经过多年的努力,研发了基于NASA微重力培养反应器[6,10]的微载体贴附悬浮式生物生物反应器的人工肝支持系统原理机和商品化机器,并在大动物(西藏小型猪及食蟹猴)上进行验证[11,12]。为了进一步向临床需求靠拢,我们在以上的基础上研制纤维支架循环灌注式生物反应器,以期达到临床治疗要求数量级的肝细胞,并在后续实验中尝试对108细胞进行培养,扩增至109-1010的肝细胞进行长达15d的培养,并初步取得成功,其主要由纤维支架反应罐,外置灌流系统,氧气检测系统,二氧化碳检测系统,温度控制系统,悬浮搅拌系统等构成;其具有独特的特点:①.逆重力式灌流,降低流体剪切力;②.外置循环灌注培养模式,增加液体交换效率;③.全自动氧气、二氧化碳、酸碱度检测调整系统;④.体型变小,重量变轻,易于运输。第一章纤维支架载体肝细胞生物相容性评价目的:对纤维支架材料的构成,理化性质加以分析,完成对纤维支架循环灌注式生物反应器的设计及组装,检测纤维支架材料与肝细胞之间的生物相容性。方法:采用电镜,X射线光电子能谱分析(X-ray photoelectron spectroscopy,XPS)等技术分析纤维支架材料特征,元素构成,表面电荷及氨基酸分析纤维支架材料表面粘附氨基酸分子;通过高温高压(121℃,45min),强酸(10%盐酸,浸泡24h)强碱(10%碳酸氢钠,浸泡24h)处理后通过电镜观察其表面性质及破损情况分析其稳定性。在纤维支架载体上接种C3A细胞,并随后采用Live/dead试剂盒细胞生长状况进行评价。结果:纤维支架载体,此载体长10cm,宽1.13cm,为细胞提供接近11.3cm2贴附面积,电镜下观察纤维直径均一,多层交叉,形成多种空隙,面积体积比大,可以提供较大细胞生长表面积。此支架主要成分为C,N,O分别74.32%,2.84%,22.84%,密度为p=0.2254mmol/g。支架表面携带赖氨酸,携带正电荷,增强细胞粘附。在高温,高压,强酸,强碱处理后,纤维支架材料均呈现出稳定状态,电镜下未观察到纤维破裂、变形、融化等不良现象。细胞对纤维支架载体贴附良好,5d内细胞活力维持大于90%,与对照组比较无显著差异(P0.05)。结论:纤维支架材料面积体积比大,高温高压强酸强碱处理后理化性质稳定,细胞帖附紧密生长旺盛,肝细胞与支架细胞相容性良好。第二章纤维支架循环灌注式生物反应器大规模培养肝细胞目的:使用纤维支架循环灌注式生物反应器大规模培养肝细胞,达到1010临床治疗要求水平,为新型生物人工肝系统提供可靠的生物反应器部件。方法:按照说明调试机器,校准PH、温度溶氧探头,并提前预湿载体(600-700ml全培,37℃C,2cm/s空转24h;将108的C3A细胞量,并消化离心成50ml悬浊液,接种入反器,4H后取样检测细胞悬浮情况,随后每日取样进行台盼蓝染色、结晶紫染色、细胞计数,Q-PCR、电镜观察;每日取培养基上清送检葡萄糖、白蛋白、ALT、AST、乳酸。结果:细胞生长良好,在15d的培养中,细胞从1.07×108增长至约90-100×108个,结晶紫染色,染色面积逐渐增加,至15d载体面积贴附约为70%-80%,细胞活力维持在75%以上,ALT、AST漏出水平低,细胞损伤小,细胞基因表达水平较平板对照组显著增高,电镜观察发现细胞-细胞间连接增多。结论:纤维支架循环灌注式生物反应器可大规模培养肝细胞至109数量级,细胞活力维持良好,细胞功能进一步增强是优良的肝细胞大规模培养反应器。
[Abstract]:Acute liver failure ( ALF ) refers to acute liver failure ( ALF ) , which refers to a patient with no basic liver disease . It is urgent for liver function damage or failure after various trauma ( physical , chemical , biological , etc . ) in the liver . There is no special effect treatment method . The mortality rate of the patients is still up to over 50 % . The most effective treatment method is liver transplantation , but due to the shortage of donor liver , the widespread use of liver transplantation is limited . Most of the patients with acute liver failure can not wait until the transplant liver source has died . Therefore , there is a need for a machine which can replace the liver function temporarily , so that the patients with acute liver failure can be given sufficient time to transition to the state of liver transplantation or liver cell regeneration , and the artificial liver is an effective way for treating liver failure patients , and the purpose of partial or total replacement of liver function can be achieved in a short time , and the precious time for patients with acute liver failure can be achieved . In recent ten years , we have made great progress in artificial liver research , from simple mechanical artificial liver to biological artificial liver , and then to a more reasonable hybrid ( mechanical + biological ) bioartificial liver . The biological reactor is composed of fiber support reaction tank , external perfusion system , oxygen detection system , carbon dioxide detection system , temperature control system , suspension stirring system , etc . The biological reactor is mainly composed of fiber support reaction tank , external perfusion system , oxygen detection system , carbon dioxide detection system , temperature control system , suspension stirring system and so on . The results showed that the cell viability was higher than that of the control group . The results showed that the cell viability was higher than that of the control group . The results showed that the cell viability was higher than that of the control group . The results showed that the cell viability was increased from 1.07 脳 108 to about 90 - 100 脳 108 , the cell viability was maintained at over 75 % , the level of ALT and AST was lower , the cell damage was small , the expression level of the cells was higher than that of the control group . Conclusion : The fibrous scaffold circulating perfusion bioreactor can be used for large - scale culture of hepatocytes to 109 orders of magnitude , the viability of the cells is maintained well , and the cell function is further enhanced to be an excellent large - scale culture reactor for hepatocytes .
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575.3

【参考文献】