生物人工肝的构建及其治疗急性肝衰的实验研究
摘要
急性肝衰（ALF）的治疗是当代临床医学的一大难题，常规治
疗病死率高达80％。虽然肝移植（LT）可将ALF的病死率降低到
30-50％，但是，LT存在急诊手术时供体来源困难、手术复杂、患
者需要终生接受免疫抑制治疗及费用昂贵等缺点。作为肝脏替代疗
法的另一个方向，人工肝的研究在近40年来取得了长足的进展。目
前，国外以培养的肝细胞与人工辅助装置相结合的生物人工肝
（BAL）所进行的动物实验及临床初步应用结果已表明，BAL不仅
可能代偿ALF患者肝脏解毒、生物合成功能及稳定内环境、阻断恶
性循环，而且为具有强大再生能力的肝细胞提供了再生的条件和时
间。经过逐渐成熟和完善后，BAL可望将不会仅停留为等待LT的
一种过渡治疗手段，而极有可能象肾透析曾给肾功能衰竭的治疗带
来革命性变化一样，为ALF的现代治疗提供最大的希望。结合本课
题的前期工作，本研究用微载体粘附悬浮培养的原代正常猪肝细胞
作为生物材料构建BAL，进行治疗猪ALF的实验，旨在为BAL早
期进入临床Ⅰ期试验提供依据。
第一部分：猪肝细胞的分离与微载体粘附悬浮培养
目的 获得一个具有丰富来源的肝细胞生产技术。
方法 取本地杂种小猪作为肝细胞供体。用Seglen改良二步胶
原酶原位循环灌注法分离获取肝细胞悬液，台盼蓝（TB）染色法计
算细胞数及细胞活率。将Cytodex-3微载体按5mg/mL的浓度加入
经5％甲基硅树脂乙酸乙酯溶液硅化的STUART搅拌培养瓶中。猪
肝细胞按5×10~6/mL的密度全部接种于培养瓶，随后加入培养液及
附加因子至1/3最终体积，搅拌培养6小时后加入培养液及附加因
子至最终体积200mL，然后持续搅拌培养。光镜及电镜下观察细胞
生长情况，动态检测培养上清中白蛋白及尿素浓度。
结果 每只猪肝平均获取肝细胞数为N．26土 0．75）XIO‘＼活
率为o91．22士0．83）％。猪肝细胞在接种后即呈明显的Cytodex刁
聚集趋势，在培养48－72小时后可见典型的多细胞聚球形成，该形
态特点及白蛋白、尿素合成能力可保持约4周左右。
结论 采用IV型胶原酶原位门腔循环灌注可分离获取大量。高
活性的原代正常猪肝细胞，经微载体粘附悬浮培养后可达到高密度。
高活性、长期离体培养的目的，可满足BAL对生物材料的要求。
The treatment of acute liver failure (ALF) is extremely difficult in
the current medicine. With a routine treatment, the mortality of ALF is
high about 80%. Although liver transplantation (LT) can reduce that to
30-50%, LT has the shortcomings such as the shortage of donor liver
so not to be able to meet the urgent need of treating ALF with
emergency LT, complex operation, the life-long immunosuppressant
treatment on sufferer and high expenses. As another way of
liver-substituting therapy, the study on artificial liver has made great
achievements in the past forty years. Nowadays the animal
experiments and primary clinical applications with bioartificial liver
(BAL) based on cultured hepatocytes and artificial assistant device at
abroad have shown that BAL can not only be possible to compenste
the functions of detoxification and biosynthesis of liver, stabilization
of inner condition and interdiction of vicious circle, but also provide
conditions and time for regeneration of hepatocytes which possess
great ability to regenerate. With gradually maturation and perfection
BAL can be promise to bring the lightest hope for the treatment of
ALF just as the revolutionary change of the treatment of kidney failure
with kidney dialysis instead of only being a bridging therapy for
waiting LT. Combining with the prophase work on this subject, we
cultured the normal primary porcine hepatocytes on microcarriers
suspendingly as biomaterial to develop BAL to experiment on treating
ALF pigs with aiming to provide evidences for BAL to enter phase I
clinical examination earlier.
??
Part I Seperation and culture on microcarriers suspendingly of
porcine hepatocytes
Aim To establish a technique of harvesting hepatocytes of
abundant sources.
Methods Hepatocytes were from the donors of native hybrid
pigs and isolated on the way of two-step orthotopic collagenase
circulating perfusing method as described by Seglen. The yield and
viability were assessed by trypan blue exclusion test. Microcarriers
Cytodex-3 were added at the concentration of Smg/L into the
STUART stirring culture vessel siliconized by 5% methyl silicon resin
ethyl acetate. Porcine hepatocytes were entirely inoculated into the
vessel at the concentration of 5 X 106/mL. Together with the
supplemental factors, the culture matrix was added in according to one
third of the final volume. After being stirred for 6 hours the culture
matrix and supplemental factors were added to the 200mL final
volume and from then on stirring were kept on. Cell growth was
observed on light and electron microscopes. Concentrations of
albumin and urea were examined dynamically in the supemation.
Results Each liver provided (4.26?.75) X 1010 hepatocytes
averagely and their vivid rate was (91.22 ?0.83)%. Most hepatocytes
tended to conglomerate obviously after being inoculated and 48-72
hours later typical multicellular aggregate spheroids could be observed.
Their morphological characteristics and abilities to synthesize albumin
and urea could be maintained for about 4 weeks.
Conclusion large-amount and high-activity normal primary
porcine hepatocytes could be isolated on the way of two-step
orthotopic tyep IV collagenase circulating perfusing method. When
being cultured on microcarriers they could be achieved to the objects
of high
疗病死率高达80％。虽然肝移植（LT）可将ALF的病死率降低到
30-50％，但是，LT存在急诊手术时供体来源困难、手术复杂、患
者需要终生接受免疫抑制治疗及费用昂贵等缺点。作为肝脏替代疗
法的另一个方向，人工肝的研究在近40年来取得了长足的进展。目
前，国外以培养的肝细胞与人工辅助装置相结合的生物人工肝
（BAL）所进行的动物实验及临床初步应用结果已表明，BAL不仅
可能代偿ALF患者肝脏解毒、生物合成功能及稳定内环境、阻断恶
性循环，而且为具有强大再生能力的肝细胞提供了再生的条件和时
间。经过逐渐成熟和完善后，BAL可望将不会仅停留为等待LT的
一种过渡治疗手段，而极有可能象肾透析曾给肾功能衰竭的治疗带
来革命性变化一样，为ALF的现代治疗提供最大的希望。结合本课
题的前期工作，本研究用微载体粘附悬浮培养的原代正常猪肝细胞
作为生物材料构建BAL，进行治疗猪ALF的实验，旨在为BAL早
期进入临床Ⅰ期试验提供依据。
第一部分：猪肝细胞的分离与微载体粘附悬浮培养
目的 获得一个具有丰富来源的肝细胞生产技术。
方法 取本地杂种小猪作为肝细胞供体。用Seglen改良二步胶
原酶原位循环灌注法分离获取肝细胞悬液，台盼蓝（TB）染色法计
算细胞数及细胞活率。将Cytodex-3微载体按5mg/mL的浓度加入
经5％甲基硅树脂乙酸乙酯溶液硅化的STUART搅拌培养瓶中。猪
肝细胞按5×10~6/mL的密度全部接种于培养瓶，随后加入培养液及
附加因子至1/3最终体积，搅拌培养6小时后加入培养液及附加因
子至最终体积200mL，然后持续搅拌培养。光镜及电镜下观察细胞
生长情况，动态检测培养上清中白蛋白及尿素浓度。
结果 每只猪肝平均获取肝细胞数为N．26土 0．75）XIO‘＼活
率为o91．22士0．83）％。猪肝细胞在接种后即呈明显的Cytodex刁
聚集趋势，在培养48－72小时后可见典型的多细胞聚球形成，该形
态特点及白蛋白、尿素合成能力可保持约4周左右。
结论 采用IV型胶原酶原位门腔循环灌注可分离获取大量。高
活性的原代正常猪肝细胞，经微载体粘附悬浮培养后可达到高密度。
高活性、长期离体培养的目的，可满足BAL对生物材料的要求。
The treatment of acute liver failure (ALF) is extremely difficult in
the current medicine. With a routine treatment, the mortality of ALF is
high about 80%. Although liver transplantation (LT) can reduce that to
30-50%, LT has the shortcomings such as the shortage of donor liver
so not to be able to meet the urgent need of treating ALF with
emergency LT, complex operation, the life-long immunosuppressant
treatment on sufferer and high expenses. As another way of
liver-substituting therapy, the study on artificial liver has made great
achievements in the past forty years. Nowadays the animal
experiments and primary clinical applications with bioartificial liver
(BAL) based on cultured hepatocytes and artificial assistant device at
abroad have shown that BAL can not only be possible to compenste
the functions of detoxification and biosynthesis of liver, stabilization
of inner condition and interdiction of vicious circle, but also provide
conditions and time for regeneration of hepatocytes which possess
great ability to regenerate. With gradually maturation and perfection
BAL can be promise to bring the lightest hope for the treatment of
ALF just as the revolutionary change of the treatment of kidney failure
with kidney dialysis instead of only being a bridging therapy for
waiting LT. Combining with the prophase work on this subject, we
cultured the normal primary porcine hepatocytes on microcarriers
suspendingly as biomaterial to develop BAL to experiment on treating
ALF pigs with aiming to provide evidences for BAL to enter phase I
clinical examination earlier.
??
Part I Seperation and culture on microcarriers suspendingly of
porcine hepatocytes
Aim To establish a technique of harvesting hepatocytes of
abundant sources.
Methods Hepatocytes were from the donors of native hybrid
pigs and isolated on the way of two-step orthotopic collagenase
circulating perfusing method as described by Seglen. The yield and
viability were assessed by trypan blue exclusion test. Microcarriers
Cytodex-3 were added at the concentration of Smg/L into the
STUART stirring culture vessel siliconized by 5% methyl silicon resin
ethyl acetate. Porcine hepatocytes were entirely inoculated into the
vessel at the concentration of 5 X 106/mL. Together with the
supplemental factors, the culture matrix was added in according to one
third of the final volume. After being stirred for 6 hours the culture
matrix and supplemental factors were added to the 200mL final
volume and from then on stirring were kept on. Cell growth was
observed on light and electron microscopes. Concentrations of
albumin and urea were examined dynamically in the supemation.
Results Each liver provided (4.26?.75) X 1010 hepatocytes
averagely and their vivid rate was (91.22 ?0.83)%. Most hepatocytes
tended to conglomerate obviously after being inoculated and 48-72
hours later typical multicellular aggregate spheroids could be observed.
Their morphological characteristics and abilities to synthesize albumin
and urea could be maintained for about 4 weeks.
Conclusion large-amount and high-activity normal primary
porcine hepatocytes could be isolated on the way of two-step
orthotopic tyep IV collagenase circulating perfusing method. When
being cultured on microcarriers they could be achieved to the objects
of high
引文
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84．上海市卫生局，中华医学会上海分会编著，实验室诊断与基础治疗常规，第一版，上海：上海科学技术出版社，1999；98-107
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87． Shnyra A, Bocharov A, Bochataova N, et al. Large-scale production and cultivation of hepatocytes on biosilon microcarriers. Artif Organs, 1990; 14: 421
88． Catapano G. Mass transfer limitations to the performance of memberane bioartificial liver support devices. Int J of Artif Organs, 1996; 19(1) : 18-31
89． Jauregui HO, Mullon CJ, Trenkler D, et al. In vivo evaluation of a hollow fiber liver assist device. Hepatology, 1995; 21: 460
90． Wolf CF, Munkelt BE. Bilirubin conjugation by an artificial liver composed of cultured cell and synthetic capillaries. ASAIO Trans, 1975; 21(1) : 16-27
91． Nyberg SL, Platt JL, Shirabe K, et al. Immunoprotection of xenocytes in a hollow fiber bioartificial liver. ASAIO J, 1992; 38: 463-7
92． Rozga J, Demetriou AA. Artificial liver evolution and future perspectives. ASAIO J, 1995; 41(6) : 831-6
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94． Hughes R, Williams R. Clinical experience with charcoal and resin hemoperfusion. Semin Liver Dis, 1986; 6: 164-73
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97． Chang TMS. Clinical experience with ACAC coated charcoal hemoperfusion in acute intoxication. Clin Toxicol, 1980; 7: 529-42
98． Mito M, Hepatic assist: present and future. Artif Organs, 1986; 10(3) : 214-8
99． Omori Y. New apparatus for artificial liver support: PAT resin-removal of protein-bound substance and its effect on hepatic failure in animals. Nippon Geka Gakkai Zasshi, 1985; 86(5) : 566-75
100． Sakagami K, Miyazaki M, Matsuoka J, et al. Artificial liver support for postoperative hepatic failure with anion exchange resin(BR-601) . Acta Med Okayama, 1986; 40(5) : 249-55
101． Filip K, Maly J, Horky J, et al. Bilirubin and bile acids removal by hemoperfusion through synthetic resin "Persorb" . Czech Med, 1990; 13(1) : 34-7
102． Gerlach J. Special issue:hybrid liver support. Int J of Artif Organs, 1996; 19(1) : 1