新型混合性生物人工肝体外功能和安全性研究
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摘要
急性肝衰蝎,病情危笃,进展迅速,预后凶险,是临床亟待解决的治疗难题。尽管近年来内科综合治疗措施取得了较大进展,患者病死率依然高达50-80%。肝移植可以显著降低肝衰竭患者病死率,但由于供肝严重短缺,事实上仅有不足1／3的患者接受了肝移植手术,而大多数患者在等待供肝的过程中死亡。基于此种前提下,国内外学者设计、构建了不同类型的人工肝系统,希冀借助一种体外的机械、理化或者生物的装置,清除患者体内蓄积的各种有害物质,补充必需物质,改善内环境,暂时替代衰竭肝脏的部分功能,为肝衰竭患者肝细胞再生及肝功能恢复创造条件,或等待供肝进行肝移植,从而降低患者病亡率。
经过50余年的发展,目前人工肝支持系统(artificial liver support system,ALSS)治疗技术你逐渐成熟,基本形成三大类十几种方法,国外多数学者按照人工肝的组成和性质分为以下三类：(1)非生物型人工肝(2)生物型人工肝(bioartificial liver, BAL) (3)混合型生物人工肝(hybrid bioartificial liver, HAL)。其中,HAL代表着人工肝的发展方向.
无论是BAL还是HAL,肝细胞都是其核心原材料,对肝功能衰竭患者的肝支持作用几乎完全依赖于所用肝细胞的生物学功能。BAL和HAL细胞来源种类较多,但目前尚未有一种可以完全满足临床的需要,如人肝细胞来源的匾乏、胎肝细胞的伦理问题、肝细胞株的瘤源性等原因、猪肝细胞潜在的异种细胞免疫反应、合成的蛋白质种属差异和猪体内普遍存在着内源性反转录病毒(procine endogenous retroviral, PERV)。我们的前期研究发现,中国人肝细胞系CL-1细胞分化程度高且生物代谢功能良好,并且CL-1细胞组织学上来源于正常肝组织,较其他来源于肿瘤源性的肝细胞系更为安全,但是目前尚未对CL-1细胞作为种子细胞的BAL和HAL进行研究。
迄今已有4种BAL或HAL系统业已开始进行Ⅱ—Ⅲ期临床验证,其中大多采用中空纤维管型生物反应器,氧合的血浆流经中空纤维内腔以供应肝细胞氧气和营养物质。初步结果证明这些治疗是安全、有效的,没有明显的不良事件出现。但是,由于血浆携氧能力不足全血的10%,因此,这些系统的氧供远不能满足肝细胞的需要,很大部分的肝细胞可能处于缺氧状态,进而导致肝细胞活性降低和肝细胞功能不良,最终导致这些人工肝系统的疗效降低,并且中空纤维反应器中肝细胞分布不均匀,细胞粘附能力差,膜的污染和堵塞,细胞生长或过量气体产生会破坏纤维,既影响细胞功能的发挥又影响对生物反应器内细胞功能状态的观察。
为解决生物反应器中肝细胞缺氧问题,我们与中科院共同研制了一种全接触灌流型生物反应器。该反应器采用双进口和双出口的透明有机塑料制成,并且这种反应器在治疗时做180度的往返旋转运动,生物反应器中的肝细胞与血浆直接接触,可以充分的发挥肝细胞的代谢和生物转化功能,并且HAL系统的生物部分有独立的共给氧气的装置,氧气通过膜式氧合器进入血清中,供给反应器中的肝细胞,以保证生物反应器中的肝细胞得到充分的氧气供应。
本研究中,我们以这种全接触灌流型生物反应器接种微载体微重力肝细胞共培养至第五天的CL-1细胞,结合非生物部分的血浆灌流器,生物反应箱、蠕动泵、膜式氧合器等构建了一种简单、新颖的HAL,并用模拟肝衰竭血清(专利公开号：CN101556275)对其进行疗效和安全性评价。具体包括以下三部分研究内容：
一；配置一种可以在常温下稳定保存的、能够反映部分肝衰竭患者血清成分变化的组合物；
二：混合型生物人工肝的构建和评价；
三：CL-1细胞作为种子细胞安全性的研究。
第一部分模拟肝衰竭血清的配置及其稳定性研究
目的：依据临床上肝功能衰竭时血清成分变化的主要特点,研制一种可用于体外检测混合型生物人工肝支持功能的组合物方法：(1)以未结合胆红素,鹅去氧胆酸,胆酸和氯化铵为组合物中的主要成分,将其先后溶解胎牛血清浓度为10％DMEM细胞培养基中,并使未结合胆红素,鹅去氧胆酸,胆酸和氯化铵的浓度分别在171-342μmol／L、80-160μmol／L、20-40μmol／L和240-400μmol／L范围内；(2)先将未结合胆红素制备成胆红素二钠后,再将其与鹅去氧胆酸,胆酸、氯化铵按照上述同样浓度范围先后溶解在DMEM细胞培养基中。上述两种不同方法配制的组合物放置在37.5℃的环境中,分别检测其在0,24和48小时组合物中各物质的浓度。
结果：在37.5℃时,按上述两种方法配置的组合物中各物质在0、24和48h性质稳定,浓度变化无显著性差异(P>0.05)。
结论：所配置的组合物性质稳定,能够在同一基线水平检测生物人工肝的体外功能。
第二部分
目的：本研究旨在设计一种新型混合型生物人工肝,并通过对模拟肝衰竭血清的净化作用评价其疗效,探讨其临床应用的可行性。
材料和方法：整个HAL的设计采用血液灌流+生物人工肝模式,非生物部分通过血浆分离后进入血液灌流器中,经过灌流后的血液在通过蠕动泵部分进入生物反应器中,生物反应器部分置于37.5℃的恒温环境中,生物部分通过膜式氧合器供给反应器中肝细胞,氧气和二氧化碳气体时间比约为110：10(即2分钟内通氧气110s,通二氧化碳10s),模拟肝衰竭血清经过肝细胞代谢后,在通过二次分浆回流至非生物部分,整个系统的预冲量约为800m1.其中灌流型生物反应器的预冲量约为300m1.,整个系统通过两套聚乙烯胶管构成一个封闭的环路。
选用中国人肝细胞系1(CL-1)作为肝细胞供体,采用微载体微重力肝细胞共培养方法培养至第五天,灌入反应器中,制成生物部分(细胞总量约为4.0×109,细胞密度约为4.0×107／ml)。
观察整个治疗过程中有无液体渗漏,动态监测模拟肝衰竭血清中未结合胆红素、鹅去氧胆酸,胆酸和血氨浓度的变化以及肝细胞对利多卡因代谢产物的检测,此外每过24h取样计数反应器中肝细胞数量和肝细胞的状态,MTT法检测肝细胞活性。
结果：整个治疗过程中未发现管路中有液体渗漏；循环前24h内,模拟肝衰竭血清中未结合胆红素、鹅去氧胆酸,胆酸和血氨的浓度呈直线型下降,与Oh相比有统计学意义(P<0.05),24h后浓度下降较平稳；循环48h后,CL-1细胞功能发生变化,生物反应器中模拟肝衰竭血清中的ALT、AST和LDH水平明显升高,与0h相比有统计学意义(P<0.05)；并且整个反应器中肝细胞的数量和活力在循环48h后也呈现显著性下降(P<0.05)。
结论：1、构建了一种新型灌流型生物反应器。该生物反应器中的CL-1细胞能够保持较高的活性和良好的功能。
2、构建了一种新型混合生物型人工肝。该型人工肝可以显著降低模拟肝衰竭血清中的毒性物质的浓度,提示其具有明显的肝支持作用。
第三部分CL-1细胞及其细胞碎片致瘤性研究
目的：探讨CL-1细胞的安全性
方法：取培养至第五天的CL-1细胞,以2.5g／L胰蛋白酶消化后离心,加DMEM调至细胞密度为1.0×1010／L,-70℃反复冻溶三次,使细胞裂解,将0.2ml含细胞碎片的溶液分别接种到5只裸鼠的颈部、后背部皮下(n=10))。以0.2ml,密度为1.0×1010／L的CL-1细胞接种到5只裸鼠的颈部、后背部皮下(n=10),苔盘蓝拒染试验计数活细胞率为97%,作对照,观察皮下细胞致瘤情况。4周后切取注射部位瘤组织及肝、肺、脑组织标本,苏木精-伊红(HE)染色镜检。
结果：接种后4周后,实验组未见接种部位出现种植瘤,对照组共有10个接种部位出现肿瘤,致瘤率为100%.切下裸鼠皮下的种植瘤,大体标本显示：瘤体呈光滑的圆球型,界限清楚,有完整的包膜。肝、肺、脑组织切片染色未发现有转移.
结论：CL-1细胞可以安全的作为HAL细胞材料。
Backgroud:
Acute liver failure is severe illness, the progress is rapid and the prognosis is poor.. How to improve survival rate of patients with acute liver failure are urgent need to solve.Despite medicine has made great progress in the comprehensive treatment in recent years, the mortality rate is still high. Liver transplantation can significantly reduce mortality of patients with liver failure, but because of severe shortage of donor liver,, in fact, less than 1/3 of the patients received liver transplantion, the majority of patients died in the process.of awaiting for liver. Based on this premise, domestic and foreign scholars designed different types of artificial liver support system and hope that use a mechanical, physical and chemical or biological device to clear the accumulation in patients with a variety of harmful substances to supplement the required material in vitro, to improve the the environment of patients, and to replace some functions of liver. Creating conditions for liver regeneration and recovery, or waiting for liver to transplantation, thereby reducing the death rate in patients with liver failure.
After more than 50 years of development, the technology of artificial liver support system has gradually development At abroad,most scholars, according to the composition and nature of the artificial liver.ALSS is divided into the following three categories:(1) non-bioartificial liver (2) bioartificial liver (bioartificial liver, BAL) (3) hybrid bioartificial liver (hybrid bioartificial liver, HAL).. HAL represents the direction of development of ALSS.
Whether BAL or HAL, liver cells are the core, the function of BAL or HAL almost entirely dependent on liver cells functions used in the bioreactor.there are some categories cell can be used as seed cell for BAL or HBAL,but there is no one can fully meet the needs of clinical. For example, as the source of human liver cells are limited, the ethical aspects of fetal liver cells, liver tumor cell line-derived and other reasons, procine liver cells, a potential xenograft cellular immune response, species differences of protein and widespread in pigs endogenous retrovirus (procine endogenous retroviral, PERV). Our preliminary study found that Chinese human liver cell line(CL-1)is high differentiation and biological metabolic functions well, and the CL-1 cells derived from normal liver tissue, compared with other liver from tumor-derived cell lines is more safety, but it is not yet that use the CL-1 cells as seed cells for BAL or the HAL.
So far four kinds of BAL or the HBAL system has been startedⅡ-Ⅲa clinical validation, most of which use hollow fiber bioreactor, Oxygen dissol ved in plasma, with the plasma flow to supply oxygen and nutrients liver cells Preliminary study proved that hollow fiber bioreactor, is safe and effective, no significant adverse events occurred. However, due to lack of oxygen carrying capacity of blood plasma and, therefore, these systems are far from the oxygen supply can not meet the needs of liver cells in bioreactor,, the majority of liver cells may be in a hypoxic state, which led to reduced activity of liver cells and liver cell dysfunction, eventually leading to reduced efficacy of these artificial liver support system. And hollow fiber bioreactor in the uneven distribution of liver cells, poor cell adhesion, membrane pollution and congestion, cell growth or excessive gas production would undermine the fiber, not only affect cell function,.but also affect observation cell function in bioreactor.
To address problems of bioreactor supply oxygen for liver cells, We are in co-operation with the Chinese Academy of Sciences has developed a perfusion bioreactor. The reactor is made of transparent organic plastic with two imports and two exports and this reactor take round-trip time to do a 180-degree rotation when in the treatment, liver cells in direct contact with plasma in the bio-reactor, liver cells can be full metabolism and bio-transformation function, and the biological part of the HAL system, has an independent of the device to oxygen, oxygen into the serum through the membrane oxygenator, the supply of reactor in the liver cells to ensure that bio-reactor in the liver cells, adequate oxygen supply.
In this study, we use CL-1 cell co-cultured with microcarrier (cytodex 3). In the fifth day, put the cells into reactor, combined with the plasma perfusion devices, biological reaction tank, peristaltic pump, membrane oxygenator, etc. to build a simple, novel HAL,. Specific research contents include the following three parts:
1:Configuration of a combination can reflect some changes in the surem of patients with liver failure, and can be stable stored at 37.5℃;
2:Construction hybrid bioartificial liver and evaluation its function:in vitro;
3:Study.the safety of CL-1 cell as seed cells for the HAL
Part I Configuration of a combination can reflect some changes in the surem of patients with liver failure
Object To provide a composition, on the basis of the serum component changed in patients with hepatic failure, for detecting the function of bioartificial liver in vitro and its preparation.
Method (1)The major components of the composition:non-conjugated bilirubin, chenodeoxycholic acid, cholic acid and ammonium chloride are dissolved in cell medium:DMEM and make the composition contains 10%Serum of Fetal bovine, the level of non-conjugated bilirubin, chenodeoxycholic acid, cholic acid and ammonium chloride was 171-342μmol/L、80-160μmol/L、20-40μmol/L and 240-40μmol/L, respectively. (2)We convert non-conjugated bilirubin into non-conjugated bilirubin disodium, then, follow the concentration of above, non-conjugated bilirubin disodium, chenodeoxycholic acid, bile acid and ammonium chloride dissolved in serum-free cell culture medium. Then,at37℃, we detect the concentration of various substances of composition at 0,24 and 48-hour, respectively.
Result The composition is stable at 37.5℃and the concentration of various substances of the composition were no significant difference, at 0,24 and 48-hour(p>0.05).
Conclusion At 37.5℃, within the scope of the concentration:171-342μmol/L of non-conjugated bilirubin、80-160μmol/L of chenodeoxycholic acid、20-40μmol/L of cholic acid and 240-400μmol/L of ammonium chloride, the composition is stable. We could use it to test the function of bioartificial liver in vitro in baseline.
PartⅡConstruct a new hybrid bioartificial liver and evaluate its function:in vitro
objective
This study aimed to design a new type of hybrid bioartificial liver and evaluated its efficacy through purify the simulation serum of liver failure, to explore the feasibility of its clinical application
Method
CL-1 cells and microcarrier are co-culture in three-dimensional with microgravity,in the fifth day, CL-1 cells and microcarrier are poured into in the bioreactor in sterile environment, the new hybrid BAL system construction: bio-reactor plus hemoperfusion device,the volume of bio-reactor is about 300 ml (cell volume 1.0×109 cell density 4.0×108/ml), oxygen and carbon dioxide are intermittent injected in bio-reactor of, the time ratio is 110:10 (ie, in 2 minutes, inject oxygen 110s, inject carbon dioxide 10s). Simulated liver failure serum is 20ml/min, and 6ml/min of them are separated through the plasma separator and inflow into the blood perfusion device, after purified by perfusion device,4ml/min inflow into the bioreactor, while bio-reactor parts,100ml/min from the loop, and through the second sub-paste to 4ml/min flow from the bioreactor, the bio-reactor maintain at 37.5℃. Detection the compositions of simulated liver failure serum before the cycles,1,12, 24,48,72,96 and 120 h of cycles,such as unconjugated bilirubin, chenodeoxycholic acid, cholic acid and ammonium chloride concentrations,we also measured AL T, AST, LDH level in the bio-reactor. MTT way to determine the rate of liver cells alive.
Result
There was no liquid leaks from the pipeline and no liver cell and micro-carrier leak from the reactor throughout the course of treatment; the total number of liver cells and the viability was no significant change within 24 h, the new hybrid BAL system has a strong ability to clear toxins in patients with liver failure and lidocaine metabolism.
Conclusions
We therefore conclude that (1)In the newly designed HAL system, CL-1 cells can keep their viability and function in vitro, and (2)the HAL appears to be effective in purified the simulation serum of liver failure, with improvement non-conjugated bilirubin, chenodeoxycholic acid, cholic acid and ammonium chloride. The safety and efficacy of this HAL system seems to be promising for animal experimental.
PartⅢStudy the safety of CL-1 cells and its debris tumorigenicity
Objective
Evaluate the safety of CL-1 cells as the seed cell for HAL
Method
Culture the CL-1 cells to the fifth dayuse the 2.5g/L trypsin digestion and centrifugation, DMEM transferred cell density to 1.0×1010/L, under the-70℃, CL-1 cells repeated freezing and melting three times, so that cell lysised,0.2ml cell lysis solution were inoculated to the five nude mice,the neck and the back (n= 10)). In control group,0.2ml CL-1 cells solution with the same density s were inoculated into the the other five nude mice (n= 10),typan blue exclusion rate of living cells, observation subcutaneous tumorigenicity.4 weeks after, the tumor tissues were cut from the injection site and hematoxylin-eosin (HE) staine. liver, lungs and brain tissue of nude mouse.
Result:
After inoculation 4 weeks, there was no cultivation of tumor in the inoculation site in the experimental group; in the control group,10 inoculation sites appear tumors, tumorigenic rate was 100%.Cut the cultivation of subcutaneous tumors in nude mice, gross specimen shows:tumor was smooth, the ball type, clear boundaries, there is a complete capsule. There was no tumor founed in Liver, lung and brain tissue.
Conclusion
CL-1 cell line could be safety as seed cell for HAL
经过50余年的发展,目前人工肝支持系统(artificial liver support system,ALSS)治疗技术你逐渐成熟,基本形成三大类十几种方法,国外多数学者按照人工肝的组成和性质分为以下三类：(1)非生物型人工肝(2)生物型人工肝(bioartificial liver, BAL) (3)混合型生物人工肝(hybrid bioartificial liver, HAL)。其中,HAL代表着人工肝的发展方向.
无论是BAL还是HAL,肝细胞都是其核心原材料,对肝功能衰竭患者的肝支持作用几乎完全依赖于所用肝细胞的生物学功能。BAL和HAL细胞来源种类较多,但目前尚未有一种可以完全满足临床的需要,如人肝细胞来源的匾乏、胎肝细胞的伦理问题、肝细胞株的瘤源性等原因、猪肝细胞潜在的异种细胞免疫反应、合成的蛋白质种属差异和猪体内普遍存在着内源性反转录病毒(procine endogenous retroviral, PERV)。我们的前期研究发现,中国人肝细胞系CL-1细胞分化程度高且生物代谢功能良好,并且CL-1细胞组织学上来源于正常肝组织,较其他来源于肿瘤源性的肝细胞系更为安全,但是目前尚未对CL-1细胞作为种子细胞的BAL和HAL进行研究。
迄今已有4种BAL或HAL系统业已开始进行Ⅱ—Ⅲ期临床验证,其中大多采用中空纤维管型生物反应器,氧合的血浆流经中空纤维内腔以供应肝细胞氧气和营养物质。初步结果证明这些治疗是安全、有效的,没有明显的不良事件出现。但是,由于血浆携氧能力不足全血的10%,因此,这些系统的氧供远不能满足肝细胞的需要,很大部分的肝细胞可能处于缺氧状态,进而导致肝细胞活性降低和肝细胞功能不良,最终导致这些人工肝系统的疗效降低,并且中空纤维反应器中肝细胞分布不均匀,细胞粘附能力差,膜的污染和堵塞,细胞生长或过量气体产生会破坏纤维,既影响细胞功能的发挥又影响对生物反应器内细胞功能状态的观察。
为解决生物反应器中肝细胞缺氧问题,我们与中科院共同研制了一种全接触灌流型生物反应器。该反应器采用双进口和双出口的透明有机塑料制成,并且这种反应器在治疗时做180度的往返旋转运动,生物反应器中的肝细胞与血浆直接接触,可以充分的发挥肝细胞的代谢和生物转化功能,并且HAL系统的生物部分有独立的共给氧气的装置,氧气通过膜式氧合器进入血清中,供给反应器中的肝细胞,以保证生物反应器中的肝细胞得到充分的氧气供应。
本研究中,我们以这种全接触灌流型生物反应器接种微载体微重力肝细胞共培养至第五天的CL-1细胞,结合非生物部分的血浆灌流器,生物反应箱、蠕动泵、膜式氧合器等构建了一种简单、新颖的HAL,并用模拟肝衰竭血清(专利公开号：CN101556275)对其进行疗效和安全性评价。具体包括以下三部分研究内容：
一；配置一种可以在常温下稳定保存的、能够反映部分肝衰竭患者血清成分变化的组合物；
二：混合型生物人工肝的构建和评价；
三：CL-1细胞作为种子细胞安全性的研究。
第一部分模拟肝衰竭血清的配置及其稳定性研究
目的：依据临床上肝功能衰竭时血清成分变化的主要特点,研制一种可用于体外检测混合型生物人工肝支持功能的组合物方法：(1)以未结合胆红素,鹅去氧胆酸,胆酸和氯化铵为组合物中的主要成分,将其先后溶解胎牛血清浓度为10％DMEM细胞培养基中,并使未结合胆红素,鹅去氧胆酸,胆酸和氯化铵的浓度分别在171-342μmol／L、80-160μmol／L、20-40μmol／L和240-400μmol／L范围内；(2)先将未结合胆红素制备成胆红素二钠后,再将其与鹅去氧胆酸,胆酸、氯化铵按照上述同样浓度范围先后溶解在DMEM细胞培养基中。上述两种不同方法配制的组合物放置在37.5℃的环境中,分别检测其在0,24和48小时组合物中各物质的浓度。
结果：在37.5℃时,按上述两种方法配置的组合物中各物质在0、24和48h性质稳定,浓度变化无显著性差异(P>0.05)。
结论：所配置的组合物性质稳定,能够在同一基线水平检测生物人工肝的体外功能。
第二部分
目的：本研究旨在设计一种新型混合型生物人工肝,并通过对模拟肝衰竭血清的净化作用评价其疗效,探讨其临床应用的可行性。
材料和方法：整个HAL的设计采用血液灌流+生物人工肝模式,非生物部分通过血浆分离后进入血液灌流器中,经过灌流后的血液在通过蠕动泵部分进入生物反应器中,生物反应器部分置于37.5℃的恒温环境中,生物部分通过膜式氧合器供给反应器中肝细胞,氧气和二氧化碳气体时间比约为110：10(即2分钟内通氧气110s,通二氧化碳10s),模拟肝衰竭血清经过肝细胞代谢后,在通过二次分浆回流至非生物部分,整个系统的预冲量约为800m1.其中灌流型生物反应器的预冲量约为300m1.,整个系统通过两套聚乙烯胶管构成一个封闭的环路。
选用中国人肝细胞系1(CL-1)作为肝细胞供体,采用微载体微重力肝细胞共培养方法培养至第五天,灌入反应器中,制成生物部分(细胞总量约为4.0×109,细胞密度约为4.0×107／ml)。
观察整个治疗过程中有无液体渗漏,动态监测模拟肝衰竭血清中未结合胆红素、鹅去氧胆酸,胆酸和血氨浓度的变化以及肝细胞对利多卡因代谢产物的检测,此外每过24h取样计数反应器中肝细胞数量和肝细胞的状态,MTT法检测肝细胞活性。
结果：整个治疗过程中未发现管路中有液体渗漏；循环前24h内,模拟肝衰竭血清中未结合胆红素、鹅去氧胆酸,胆酸和血氨的浓度呈直线型下降,与Oh相比有统计学意义(P<0.05),24h后浓度下降较平稳；循环48h后,CL-1细胞功能发生变化,生物反应器中模拟肝衰竭血清中的ALT、AST和LDH水平明显升高,与0h相比有统计学意义(P<0.05)；并且整个反应器中肝细胞的数量和活力在循环48h后也呈现显著性下降(P<0.05)。
结论：1、构建了一种新型灌流型生物反应器。该生物反应器中的CL-1细胞能够保持较高的活性和良好的功能。
2、构建了一种新型混合生物型人工肝。该型人工肝可以显著降低模拟肝衰竭血清中的毒性物质的浓度,提示其具有明显的肝支持作用。
第三部分CL-1细胞及其细胞碎片致瘤性研究
目的：探讨CL-1细胞的安全性
方法：取培养至第五天的CL-1细胞,以2.5g／L胰蛋白酶消化后离心,加DMEM调至细胞密度为1.0×1010／L,-70℃反复冻溶三次,使细胞裂解,将0.2ml含细胞碎片的溶液分别接种到5只裸鼠的颈部、后背部皮下(n=10))。以0.2ml,密度为1.0×1010／L的CL-1细胞接种到5只裸鼠的颈部、后背部皮下(n=10),苔盘蓝拒染试验计数活细胞率为97%,作对照,观察皮下细胞致瘤情况。4周后切取注射部位瘤组织及肝、肺、脑组织标本,苏木精-伊红(HE)染色镜检。
结果：接种后4周后,实验组未见接种部位出现种植瘤,对照组共有10个接种部位出现肿瘤,致瘤率为100%.切下裸鼠皮下的种植瘤,大体标本显示：瘤体呈光滑的圆球型,界限清楚,有完整的包膜。肝、肺、脑组织切片染色未发现有转移.
结论：CL-1细胞可以安全的作为HAL细胞材料。
Backgroud:
Acute liver failure is severe illness, the progress is rapid and the prognosis is poor.. How to improve survival rate of patients with acute liver failure are urgent need to solve.Despite medicine has made great progress in the comprehensive treatment in recent years, the mortality rate is still high. Liver transplantation can significantly reduce mortality of patients with liver failure, but because of severe shortage of donor liver,, in fact, less than 1/3 of the patients received liver transplantion, the majority of patients died in the process.of awaiting for liver. Based on this premise, domestic and foreign scholars designed different types of artificial liver support system and hope that use a mechanical, physical and chemical or biological device to clear the accumulation in patients with a variety of harmful substances to supplement the required material in vitro, to improve the the environment of patients, and to replace some functions of liver. Creating conditions for liver regeneration and recovery, or waiting for liver to transplantation, thereby reducing the death rate in patients with liver failure.
After more than 50 years of development, the technology of artificial liver support system has gradually development At abroad,most scholars, according to the composition and nature of the artificial liver.ALSS is divided into the following three categories:(1) non-bioartificial liver (2) bioartificial liver (bioartificial liver, BAL) (3) hybrid bioartificial liver (hybrid bioartificial liver, HAL).. HAL represents the direction of development of ALSS.
Whether BAL or HAL, liver cells are the core, the function of BAL or HAL almost entirely dependent on liver cells functions used in the bioreactor.there are some categories cell can be used as seed cell for BAL or HBAL,but there is no one can fully meet the needs of clinical. For example, as the source of human liver cells are limited, the ethical aspects of fetal liver cells, liver tumor cell line-derived and other reasons, procine liver cells, a potential xenograft cellular immune response, species differences of protein and widespread in pigs endogenous retrovirus (procine endogenous retroviral, PERV). Our preliminary study found that Chinese human liver cell line(CL-1)is high differentiation and biological metabolic functions well, and the CL-1 cells derived from normal liver tissue, compared with other liver from tumor-derived cell lines is more safety, but it is not yet that use the CL-1 cells as seed cells for BAL or the HAL.
So far four kinds of BAL or the HBAL system has been startedⅡ-Ⅲa clinical validation, most of which use hollow fiber bioreactor, Oxygen dissol ved in plasma, with the plasma flow to supply oxygen and nutrients liver cells Preliminary study proved that hollow fiber bioreactor, is safe and effective, no significant adverse events occurred. However, due to lack of oxygen carrying capacity of blood plasma and, therefore, these systems are far from the oxygen supply can not meet the needs of liver cells in bioreactor,, the majority of liver cells may be in a hypoxic state, which led to reduced activity of liver cells and liver cell dysfunction, eventually leading to reduced efficacy of these artificial liver support system. And hollow fiber bioreactor in the uneven distribution of liver cells, poor cell adhesion, membrane pollution and congestion, cell growth or excessive gas production would undermine the fiber, not only affect cell function,.but also affect observation cell function in bioreactor.
To address problems of bioreactor supply oxygen for liver cells, We are in co-operation with the Chinese Academy of Sciences has developed a perfusion bioreactor. The reactor is made of transparent organic plastic with two imports and two exports and this reactor take round-trip time to do a 180-degree rotation when in the treatment, liver cells in direct contact with plasma in the bio-reactor, liver cells can be full metabolism and bio-transformation function, and the biological part of the HAL system, has an independent of the device to oxygen, oxygen into the serum through the membrane oxygenator, the supply of reactor in the liver cells to ensure that bio-reactor in the liver cells, adequate oxygen supply.
In this study, we use CL-1 cell co-cultured with microcarrier (cytodex 3). In the fifth day, put the cells into reactor, combined with the plasma perfusion devices, biological reaction tank, peristaltic pump, membrane oxygenator, etc. to build a simple, novel HAL,. Specific research contents include the following three parts:
1:Configuration of a combination can reflect some changes in the surem of patients with liver failure, and can be stable stored at 37.5℃;
2:Construction hybrid bioartificial liver and evaluation its function:in vitro;
3:Study.the safety of CL-1 cell as seed cells for the HAL
Part I Configuration of a combination can reflect some changes in the surem of patients with liver failure
Object To provide a composition, on the basis of the serum component changed in patients with hepatic failure, for detecting the function of bioartificial liver in vitro and its preparation.
Method (1)The major components of the composition:non-conjugated bilirubin, chenodeoxycholic acid, cholic acid and ammonium chloride are dissolved in cell medium:DMEM and make the composition contains 10%Serum of Fetal bovine, the level of non-conjugated bilirubin, chenodeoxycholic acid, cholic acid and ammonium chloride was 171-342μmol/L、80-160μmol/L、20-40μmol/L and 240-40μmol/L, respectively. (2)We convert non-conjugated bilirubin into non-conjugated bilirubin disodium, then, follow the concentration of above, non-conjugated bilirubin disodium, chenodeoxycholic acid, bile acid and ammonium chloride dissolved in serum-free cell culture medium. Then,at37℃, we detect the concentration of various substances of composition at 0,24 and 48-hour, respectively.
Result The composition is stable at 37.5℃and the concentration of various substances of the composition were no significant difference, at 0,24 and 48-hour(p>0.05).
Conclusion At 37.5℃, within the scope of the concentration:171-342μmol/L of non-conjugated bilirubin、80-160μmol/L of chenodeoxycholic acid、20-40μmol/L of cholic acid and 240-400μmol/L of ammonium chloride, the composition is stable. We could use it to test the function of bioartificial liver in vitro in baseline.
PartⅡConstruct a new hybrid bioartificial liver and evaluate its function:in vitro
objective
This study aimed to design a new type of hybrid bioartificial liver and evaluated its efficacy through purify the simulation serum of liver failure, to explore the feasibility of its clinical application
Method
CL-1 cells and microcarrier are co-culture in three-dimensional with microgravity,in the fifth day, CL-1 cells and microcarrier are poured into in the bioreactor in sterile environment, the new hybrid BAL system construction: bio-reactor plus hemoperfusion device,the volume of bio-reactor is about 300 ml (cell volume 1.0×109 cell density 4.0×108/ml), oxygen and carbon dioxide are intermittent injected in bio-reactor of, the time ratio is 110:10 (ie, in 2 minutes, inject oxygen 110s, inject carbon dioxide 10s). Simulated liver failure serum is 20ml/min, and 6ml/min of them are separated through the plasma separator and inflow into the blood perfusion device, after purified by perfusion device,4ml/min inflow into the bioreactor, while bio-reactor parts,100ml/min from the loop, and through the second sub-paste to 4ml/min flow from the bioreactor, the bio-reactor maintain at 37.5℃. Detection the compositions of simulated liver failure serum before the cycles,1,12, 24,48,72,96 and 120 h of cycles,such as unconjugated bilirubin, chenodeoxycholic acid, cholic acid and ammonium chloride concentrations,we also measured AL T, AST, LDH level in the bio-reactor. MTT way to determine the rate of liver cells alive.
Result
There was no liquid leaks from the pipeline and no liver cell and micro-carrier leak from the reactor throughout the course of treatment; the total number of liver cells and the viability was no significant change within 24 h, the new hybrid BAL system has a strong ability to clear toxins in patients with liver failure and lidocaine metabolism.
Conclusions
We therefore conclude that (1)In the newly designed HAL system, CL-1 cells can keep their viability and function in vitro, and (2)the HAL appears to be effective in purified the simulation serum of liver failure, with improvement non-conjugated bilirubin, chenodeoxycholic acid, cholic acid and ammonium chloride. The safety and efficacy of this HAL system seems to be promising for animal experimental.
PartⅢStudy the safety of CL-1 cells and its debris tumorigenicity
Objective
Evaluate the safety of CL-1 cells as the seed cell for HAL
Method
Culture the CL-1 cells to the fifth dayuse the 2.5g/L trypsin digestion and centrifugation, DMEM transferred cell density to 1.0×1010/L, under the-70℃, CL-1 cells repeated freezing and melting three times, so that cell lysised,0.2ml cell lysis solution were inoculated to the five nude mice,the neck and the back (n= 10)). In control group,0.2ml CL-1 cells solution with the same density s were inoculated into the the other five nude mice (n= 10),typan blue exclusion rate of living cells, observation subcutaneous tumorigenicity.4 weeks after, the tumor tissues were cut from the injection site and hematoxylin-eosin (HE) staine. liver, lungs and brain tissue of nude mouse.
Result:
After inoculation 4 weeks, there was no cultivation of tumor in the inoculation site in the experimental group; in the control group,10 inoculation sites appear tumors, tumorigenic rate was 100%.Cut the cultivation of subcutaneous tumors in nude mice, gross specimen shows:tumor was smooth, the ball type, clear boundaries, there is a complete capsule. There was no tumor founed in Liver, lung and brain tissue.
Conclusion
CL-1 cell line could be safety as seed cell for HAL
引文
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[9]Kamath P S, Wiesner R H, Malinchoc M, et al. A model to predict survival in patients with end-stage liver disease.[J]. Hepatology,2001,33(2):464-470.
[10]刘青,刘铮,王泰龄等.应用MELD系统预测慢性重型肝炎的预后及其治疗效果[J].中华医学杂志,2005,85(25)：1773-1777.
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[17]戴朝六.肝脏储备功能的评估[J].中国实用外科杂志,2005,25(12).708-710
[18]卢新兆,黄晓华.血清总胆汁酸测定在肝胆疾病临床诊断中的价值[J].现代中西医结合杂志,2004,13(08)：1058-1059.
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[20]Saxena A, Sun W, Luo C, et al. Human serum butyrylcholinesterase:in vitro and in vivo stability, pharmacokinetics, and safety in mice.[J]. Chem Biol Interact,2005,157-158:199-203.
[21]朱新功.血清胆碱脂酶和甲胎蛋白对慢性乙型重型肝炎预后判断的价值[J].右江医学,2007,35(05)：530-531.
[22]Izumi S, Langley P G, Wendon J, et al. Coagulation factor V levels as a prognostic indicator in fulminant hepatic failure.[J]. Hepatology,1996,23(6): 1507-1511.
[23]Anand A C, Nightingale P, Neuberger J M. Early indicators of prognosis in fulminant hepatic failure:an assessment of the King's criteria.[J]. J Hepatol, 1997,26(1):62-68.
[24]Yoshiba M, Sekiyama K, Inoue K, et al. Accurate prediction of fulminant hepatic failure in severe acute viral hepatitis:multicenter study.[J]. J Gastroenterol,2002,37(11):916-921.
[25]Venturini I, Corsi L, Avallone R, et al. Ammonia and Endogenous Benzodiazepine-like Compounds in the Pathogenesis of Hepatic Encephalopathy [J]. Scandinavian Journal of Gastroenterology,2001,36(4): 423-425.
[26]Carraro P, Burighel D, De S G, et al. Early prognostic biochemical indicators of fulminant hepatic failure [J]. International Journal of Clinical& Laboratory Research,1998,28(3):196-199.
[27]Murray-lyon I M, Orr A H, Gazzard B, et al. Prognostic value of serum alpha-fetoprotein in fulminant hepatic failure including patients treated by charcoal haemoperfusion[J]. Gut,1976,17(8):576-580.
[28]李庭赞,孙士其,孙丹莉等.血清甲状腺激素改变对重型肝炎预后的判断价值[J].中华传染病杂志,2000,18(02).：118-119
[29]Poddar U, Thapa B R, Prasad A, et al. Natural history and risk factors in fulminant hepatic failure.[J]. Arch Dis Child,2002,87(1):54-56.
[30]向德栋,张盛,王宇明等.477例重型病毒性肝炎预后影响因素分析[J].第三军医大学学报,2001.23(6)：716-717.
[31]Acharya S K, Dasarathy S, Kumer T L, et al. Fulminant hepatitis in a tropical population:clinical course, cause, and early predictors of outcome.[J]. Hepatology,1996,23(6):1448-1455.
[32]黎一鸣,吕凡,吉鸿等.肝脏体积变化与病肝储备功能的关系研究[J].中华普通外科杂志,2003,18(02).79-81.