【作者】 曹红翠；

【导师】 李兰娟；

【作者基本信息】 浙江大学 ， 内科学， 2007， 硕士

【摘要】 研究背景：肝细胞属于高度分化的多功能、实质性脏器细胞。肝细胞离体后，在不适宜环境中很快出现低功能、去分化甚至死亡，如缺少肝细胞生长因子（HGF）、表皮生长因子（EGF）等引起细胞衰老和失功能，pH值不适和无激素支持导致细胞死亡，基因的激活造成细胞凋亡等。随着近代细胞工程学的进展，细胞生物学、组织培养技术、生物学技术的最新成就，肝细胞作为构建生物人工肝的生物部分，作为细胞移植治疗急慢性肝衰竭的种子细胞以及作为药物筛选的靶细胞，成为目前研究的热点，而制备和培养大量的、高质量的肝细胞是这些研究的最基本环节。肝细胞分离方法有多种：机械分离法、螯合法和酶消化法等。最初的机械分离法始于半个多世纪前，1956年Sorrentino第一次用机械的方法把新鲜肝组织制成匀浆，并在体外进行药物解毒试验，证实了它有代谢水杨酸、巴比妥钠、酮体的能力，同时还证实了它能够利用氨合成尿素。但机械分离法获得的肝细胞，细胞数量少、活性差，现已很少被使用。1967年Howard创建了胶原酶灌流法，后经Berry和Friend等人的改良，采用无钙的平衡盐、胶原酶和透明质酸酶配制成的消化液进行灌流，获得的肝细胞的数量和活性均优于机械分离法。1972年Seglen进一步将这种方法发展为两步灌流法，即首先灌注预热的无钙平衡盐溶液，以去除肝血窦内的血细胞及部分非实质细胞，然后再灌注含有胶原酶的平衡盐溶液，进行体外消化后，分离提纯而获得肝细胞。用此法获得的肝细胞，细胞数量和活性都得到了提高，而且减少了肝细胞悬液中的杂质。肝细胞的二步灌流法也成为目前实验室常用的方法。二步灌流法适合中小型动物如小鼠、大鼠、兔及犬等动物肝脏的分离。猪的肝脏因含有更多的胶原及纤维组织，人的肝脏体积较大，用二步分离法分离效果不佳。而生物人工肝和细胞移植等研究需要大规模制备高质量肝细胞。因此建立从猪等较大动物脏器和人肝脏分离肝细胞的细胞分离技术成为必需。本研究以二步灌流法为对照，建立四步灌流的新方法，为获得高质量的肝细胞提供新途径。材料和方法：中性蛋白酶（Dispase）、DNA酶（DNase）和牛血清白蛋白（BSA）购自美国罗氏公司；Ⅳ型胶原酶（typeⅣcollagenase）、谷胺酰胺（Glutamine）购自美国Sigma公司；Walliams’ E购自美国Gibco公司；葡萄糖（Glucose）、羟乙基哌嗪乙磺酸（Hepes）、碳酸氢钠（NaHCO₃）、氯化钠（NaCl）、氯化钾（KCl）、磷酸二氢钾（KH₂PO₃）、磷酸氢二钠（Na₂HPO₄·7H₂O）、氯化钙（CaCl₂·2H₂O）、台盼蓝、氯化镁（MgCl₂·6H₂O）、硫酸镁（MgSO₄·7H₂O）、胰蛋白酶（Trypsin）和乙二胺四乙酸（EDTA·4Na）购自上海生物工程技术公司公司；胎牛血清（FBS）购自美国Hyclone公司；肝素购自上海第一生化药业有限公司；培养瓶和培养板为丹麦Nunc公司产品；0.22μm过滤器为德国Millipore公司产品。中国小型猪购自北京农业大学实验猪场。实验仪器包括Olympus倒置相差显微镜、日本HITACHI H-600A透射电镜、美国Forma二氧化碳培养箱、美国Thermo液氮储存罐、德国Sartorius酸度计和电子天平、德国Millipore超纯水仪、德国Scotsman制冰机、日本HITACHI立式大容量低温离心机、日本Tomy蒸汽消毒锅、德国Eppendorf蠕动泵、上海精宏实验设备有限公司电热恒温水浴箱、杭州仪表电机厂磁力加热搅拌器等，80目和150目不锈钢网筛购自华东医药股份有限公司，体外灌流装置自行设计。无菌手术取中国小型猪肝脏，用含EDTA、胶原酶等灌流液进行常规的体外二步灌流法分离猪肝细胞。同样方法取肝后，将肝脏放在不锈钢盆中，轻轻挤压肝脏，用4℃预冷的灌流缓冲液（PB）500ml尽可能多地冲洗去肝脏外部血液和大血管中残留的血液。第一步经蠕动泵向肝脏中连续灌注常温的含EDTA的灌流缓冲液（PBE）300ml、预热至39℃的PBE液700ml；第二步灌注预热至39℃的含Hepes的PB液500ml；弃去上述灌流液，将肝脏转于新的无菌不锈钢盆中，第三步灌注预热39℃的含Dispase的灌流缓冲液（PBD）500ml，第四步弃去其中约200ml灌流液，继续用预热至39℃的含胶原酶的灌流缓冲液（PBC）500ml灌流，这500mlPBC液和约300ml的PBD液重复循环灌注，直至肝包膜分离；摘除胆囊，锐性撕裂肝包膜，去除纤维结缔组织，在冰浴条件下用80目和150目不锈钢网筛过滤肝细胞，收集肝细胞悬液。洗涤两种灌流法收集的肝细胞，台盼蓝染色，计算肝细胞的活率和得率。用William’s Medium E培养肝细胞，计算肝细胞24h贴壁率。结果：采用自行设计的体外灌流系统，对中国小型猪肝脏进行常规的胶原酶两步灌流和我们创建的四步灌流法分离肝细胞。二步法分离肝细胞的活率是88％±5％，后者为95％±4％；二步法分离得到的肝细胞总数是（3.1±0.7）×10⁷／克肝组织，四步灌流法所得的分离细胞总数为（5.3±1.1）×10⁷／克肝组织；二步灌流法分离的肝细胞贴壁率为80％，四步灌流法获得的肝细胞贴壁率达90％以上。四步分离法分离得到肝细胞的活率、得率和24h贴壁率显著高于二步灌流法（P＜0.05），且细胞碎片和其它非实质细胞污染极少，镜下观察非实质细胞污染率低。结论：四步灌流法分离肝细胞，可获得更高的细胞活率和得率，对于培养后的细胞贴壁也有明显促进作用，且细胞碎片和其它非实质细胞污染极少。该法可广泛应用于生物人工肝用肝细胞的分离和其他大规模分离肝细胞的研究。为生物人工肝、细胞移植和药物筛选等研究提供高质量的肝细胞。更多还原

【Abstract】 Background:Hepatocyte belongs to a sort of high-differentiated parenchymal cell with multiple functions. It may change gradually to be of hypofunction, dedifferentiation and even death rapidly under the inappropriate environment in vitro, for instance, absence of hepatocyte growth factor （HGF） and epidermal growth factor （EGF） can cause cell senescence and nonfunction, inappropriate pH value and lack of hormone can cause cell death, and gene activation can cause apoptosis.With the development of modern cell engineering and the latest achievements in cytobiology, tissue culture technique and biology, nowadays hepatocyte has become the hotspot of multiple studies because it can be used as the biological section in the construction of bioartificial liver, the seed cell in cellular transplantation to cure acute and chronic liver failure and the target cell in the drug screening. Thus, the preparation and culture of a large number of high-active hepatocytes have become the most basic link among all these studies.There are many kinds of hepatocyte isolation method, such as mechanical separation, chelation, enzyme digestion, and so on. The mechanical separation began more than half a century ago. In 1956, for the first time, Sorrentino prepared fresh hepatic tissue homogenate using mechanical method and many in vivo drug detoxification trials were performed. It was demonstrated that the homogenate could digest salicylic acid, barbital sodium and ketone body, and induce the urea synthesis of ammonia. Gradually, this method was abandoned because of a little number of hepatocytes with poor motility. In 1967, Howard established collagenase perfusion method that was then improved by Berry and Friend. Using the perfusion of digestive liquid that was prepared through balanced saline without calcium, collagenase and hyaluronidase, the method could yield a larger number of hepatocytes with better motility than the mechanical separation. In 1972, Seglen further developed the method into two-step perfusion, during which the preheated balanced saline without calciumwas first infused to remove blood cells within hepatic sinusoid and some nonparenchymal cells, and then balanced saline containing collagenase was infused to perform in vitro digestion. Following these procedures, hepatocyte was separated, purified and obtained. By this method, the number of hepatocyte and its motility were both improved and foreign matters in hepatocyte suspension were reduced. Nowadays, the two-step perfusion has become one common method used in the laboratory.However, two-step perfusion method is appropriate in liver separation of small animals such as mice, rats, rabbits and dogs, and the effectiveness of separation is poor in pigs because of the more collagens and fibrous tissue in livers. Nevertheless, a large number of high-quality hepatocytes are needed in bioartificial liver and cellular transplantation studies. Therefore, it is necessary to establish novel cell separation techniques from large animal organs （i.e. pig） and human livers.In the current study, with two-step perfusion as control, we established a novel four-step perfusion method in order to provide new approaches to obtaining high quality hepatocytes.Materials and Methods:The following materials were obtained commercially:Dispase, DNase and bovine serum albumin （BSA） were purchased from Roche Holding AG, USA; type Ⅳ collagenase and glutamine were from Sigma corporation of USA; Walliams’ E was from Gibco corporation of USA; glucose, Hepes, NaHCO₃, NaCl, KCl, KH₂PO₃, Na₂HPO₄·H₂O, CaCl₂·2H₂O, Trypan Blue, MgCl₂·6H₂O, MgSO₄·7H₂O, Trypsin and EDTA·4Na were from Shanghai Bio-engineering Co., Ltd; fetal bovine serum （FBS） was from Hyclone corporation of USA and heparin was from Shanghai No.1 Biochemical & Pharmaceutical Co., Ltd. Culture flask and culture plate were from Nunc corporation of Denmark. Filtrator（0.22μm） was from Millipore ultrapure water system （Germany）. Chinese Experimental Miniature Pigs were purchased from the Experimental Hogpen of China Agricultural University.Experimental instruments include Olympus inverted phase contrast microscope, H-600A transmission electron microscope （Hitachi, Japan）, CO₂ incubator （FormaScientific, USA）, liquid nitrogen tank （Thermo Scientific, USA）, acidometer and electronic balance （Sartorius, Germany）, Millipore ultrapure water system （Germany）, ice machine （Scotsman, Germany）, large capacity refrigerated centrifuge （Hitachi, Japan）, vapor sterilization machine （Tomy, Japan）, peristaltic pump （Eppendorf, Germany）, electrothermal thermostatic waterbath box （Shanghai Jinghong Laboratory Instrument Co., Ltd） and heating magnetic stirrer （Hangzhou Instrument and Electrical Machinery Factory）. Stainless steel wire mesh （#80 and #150） were purchased from Huadong Medicine Co., Ltd and the extracorporeal perfusion apparatus were self-designed.Porcine hepatocyte separation was performed using perfusate containing EDTA and collagenase according to the routine two-step perfusion method in vitro.Liver was obtained by the same method and put in stainless steel pot, and then was crushed gently. Blood outside the liver and residual blood in great vessels were washed as much as possible using 500 ml Perfusion Buffer （PB） precooled at 4℃. First, liver was consecutively infused with 300 ml Perfusion Buffer with EDTA（PBE） with normal temperature and 700 ml PBE liquid preheated at 39 °C through peristaltic pump. Second, 500 ml PB liquid preheated at 39 °C was infused into liver and then was removed. Then liver was transferred into a new sterile stainless steel pot. Third, 500 ml Perfusion Buffer with Dispase （PBD） preheated at 39 °C was infused. Fourth, about 200 ml perfusate was removed and then 500 ml Perfusion Buffer with Collagenase （PBC） preheated at 39℃ is infused. The 500 ml PBC liquid and the residual 300 ml PBD liquid were infused in the recycling way until liver capsule was separated. Cholecyst was removed and liver capsule was sharply torn. Fibrous connective tissue was removed and hepatocyte was obtained under the ice bath through #80 and #150 stainless steel wire mesh, and then hepatocyte suspension was collected.Hepatocyte was collected via the two different perfusion methods and was stained using trypan blue. Motility and recovery rates were calculated. Hepatocyte was cultured using William’s Medium E and 24 h adherent rate of hepatocytes was calculated.Results:Using self-designed extracorporeal perfusion system, hepatocyte was separated from miniature pig via the routine two-step perfusion and the four-step perfusion method we had established, respectively. The hepatocyte motility rate was 88 %±5 % and 95%±4% in two- and four-step methods, respectively. The total number of separated hepatocytes was 3:1±0.7×10⁷/g hepatic tissue and 5.3±1.1×10⁷/g hepatic tissue, respectively. The adherent rate of hepatocyte was 80% and higher than 90%, respectively. There were significantly higher hepatocyte motility and recovery rates and 24 h adherent rate in the four-step method as compared with the two-step method （P<0.05 ）, with extremely little cellular fractions and nonparenchymal cell contamination. Under the microscope, the contamination rate of nonparenchymal cells was lower.Conclusion:Four-step perfusion method can result in high hepatocyte motility and recovery rates, and significantly improve the adherence ability of cultured cells. Furthermore, there are extremely little cellular fractions and nonparenchymal cell contamination. This method may be extensively used during separating hepatocyte for bioartificial liver and in other large studies on hepatocyte separation. It can provide high quality hepatocytes in bioartificial liver and cellular transplantation studies.更多还原