| 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 (ALSS) has gradually development. In its developing process experienced the person - animal hemodialysis, in vitro exsomatize liver irrigation, the person - person cross circulation, the dissociation liver cell to transplant successively to the liver cell hangs the fluid dialysis, hybrid bioartificial liver (HBAL) and so on.HBAL represents the direction of development of ALSS.Liver cell as HBAL its core raw material, the function of HBAL 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 the HBALHBAL another core is the bioreactor. The ideal bioreactor ought to be able to provide the good growth metabolism environment for the liver cell, and serves the good material interchange purpose. 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).Put the cells into reactor, combined with the plasma perfusion devices, biological reaction tank, peristaltic pump, membrane oxygenator, etc. to build a simple, novel HBAL,. Specific research contents include the following two parts:1. Establishment of Cynomolgus acute liver failure Model2. The safety and effectiveness evaluation of new human hepatocytes hybrid bioartificial liverPart I Establishment of Cynomolgus acute liver failure ModelObjective:To establish a large animal ALF model for study in artificial liver support system with stable reversibility and reproducibility.In our study, Cynomolgus. ALF model was set by intravenous injection of different doses of D-gal under ketamine (0.1 ml/kg) anesthesia condition. Clinical, biochemistry and histology changes were observed to explore suitable D-gal doses for Cynomolgus. ALF model and settle a solid foundation for further study and remedy for acute liver failure.Methods:Male Cynomolgus were selected as model animals.15 Cynomolgus were randomly divided into 3 groups after preliminary experiment:the first one is large dose group (n=5, medicine dose:0.45g/kg), the second moderate dose group (n=5, medicine dose:0.3g/kg), the third low dose group (n=5, medicine dose:0.15g/kg). Blood ammonia, PT, AST and TBL were under dynamic monitoring. D-gal were injected into Cynomolgus through vena jugularis externa in 10 minutes under anesthesia state induced by combined intramuscular injection of sumianxinⅡ(0.1 mL/kg) and ketamine (0.1 ml/kg). ICP was monitored by the encephalic Codman micro sensor which was placed through the hole in the calvarium. Blood was drawn daily before and after surgery to monitor hepatic function, kidney function, blood sugar, blood ammonia, coagulation test, BCAA/AAA through laboratory dept. in Nanfang hospital while postoperation survival condition was under dynamic monitoring. Clinical manifestations as well as disease progression were also observed and the survival time was recorded. Thorough Autopsy was immediately done after the death to take liver, kidney, spleen, pancreas, heart, lung, gastric and brain. Specimens prepared for light microscope were fixed by 10% formaldehyde to be made into paraffin sections dyed by HE. Specimens prepared for electron microscope were fixed by glutaraldehyde and osmic acid to be made into resin sections dyed by uranyl acetate and lead citrate. Pathological changes were observed under light and electron microscope.Results:The mean survival time of the first group was (56.1±8.1) h with the occurrence of hepatic coma in 80% Cynomolgus before death. The mean survival time of the second group was (109.8±11.2)h with the occurrence of hepatic coma in 60% Cynomolgus before death. All is survival except one was death after 98h in third group after injection D-gal, they take a better turn after 4 days, all index return normal gradually. Among the 5 Cynomolgus induced by 0.3g/ kg D-gal,2 Cynomolgus was 105h and 120h separately. Blood pressure of all Cynomolgus in the third group remained stable after being given medicine with ICP more than doubled. Serum aminotransferase of Cynomolgus increased 12h after being given medicine and became higher and higher as the disease progressed with the continuous rise of bilirubin, cholic acid, ammonia and lactic acid, the decrease of platelet, the prolonging of activated clotting time and normal blood gas analysis results. There was significant difference between the survival time of different groups. Linear correlation analysis showed that there was negative correlation between the D-gal dose and survival time. K-M survival analysis showed that survival time of the Cynomolgus receiving 0.45g/kg D-gal was significant shorter than the other two groups (x2=21.933,P=0.000). Autopsy showed that Cynomolgus receiving D-gal had smaller liver with sharp margin, mottled surface and scattered from pin-point like to rice-like bleeding points. There was kermesinus congestion on the cut surface of liver with yellow chyliform necrotic tissues. Intestinal gas could also been seen in the intestinal tract with congested and swelling wall and scattered bleeding points. The brain tissue had sever edema with the most obvious pathological change around stellate cells and perivascular space.The shape and size of gall bladder was normal. There was no abnormality of spleen, lung and kidney under naked eyes.The structure of hepatic lobule in the control group was normal with no hepatic necrosis under light microscope. However, there were hepatic patchy necrosis with significant bleeding and swelling hepatic cells as well as narrow hepatic sinus in Cynomolgus receiving D-gal. Cytoplasmic puffing and vacuolar degeneration could also be seen in hepatic cells. Some hepatic cells had fatvacuole and some had karyoclasis. Cellular infiltration of neutrophil and lymphocyte could also be seen in some portal area. Large patchy necrosis with obscure hepatic lobules and severe bleeding appeared in the hepatic tissue of Cynomolgus receiving 1.0 g/kg. The hepatic ultrastructure of Cynomolgus without receiving D-gal under electron microscope showed rich glycogenosome in cytoplasma, abundant normal endoplasmic reticulum, plentiful normal mitochondria with clear mitochondrialcrista and membrane and nucleus with even chromatin and clear membrane as well as nucleolus. After given D-gal, the hepatic ultrastructure showed homogenized glycogenosome in cytoplasma, endoplasmic reticulum degranulation, swelling mitochondria with obscure mitochondrialcrista, some dissolved mitochondria,abnormal nucleus with congested chromatin and intranuclear pseudoinclusions and significant decrease of microvilli of intracellular bile canaliculi.Conclusion:1. Successful establishment of ALF induced by D-gal was done with the finding of optimum dose.2. Cynomolgus ALF model induced by D-gal has the similar clinical, biochemistry and pathological characteristic with human being.3. Cynomolgus ALF model, which is suitable for the assessment of artificial liver support system, has nice reversibility and reproductivity, appropriate time window with all animals dying from ALF.PartⅡThe safety and effectiveness evaluation of novel human hepatocytes hybrid bioartificial liverObjective:This study is to design a nonel type of hybrid bioartificial liver and evaluate its safety and effectiveness of the treatment of machin acute liver failure,also discusses the feasibility of its clinical application.Materials and methods:The whole HBAL design uses plasma perfusion and bioartificial liver, the biological parts, after the plasma separating, enter into the blood perfusion, and then into biological reactor, which is being in 37.5℃environment, e suppling oxygenator to reactor liver cell by the membrane typ, oxygen and carbon dioxide gas is approx 110:10 (in two minutes, the carbon dioxide 110s general oxygen 10s), analog liver failure serum after liver cell metabolism in through the second points after grouting backflow to the biological parts, the whole system is about 800ml,while the perfusion type bioreactor is about 300ml and the entire system constitute a closed through polyethylene.Choosing Chinese liver cell line 1 (CL-1) as liver cell donors and adopting micro carrier of microgravity liver cells to the fifth day, cultivating cultivation method into the reactor, made into biological part (cells is about 10 total x 109, cell density 4. 0x 108/ml).While selecting male machin (mass 6.5-7 kg) as animal model, inject D - gal 0.3 g/kg to induce ALF model through external carotid intravenous. After injection about 48h,15 machines were divided into two groups at random, (1) ALF control group (n=5):only give water and food, do not give any treatment measures; (2) HBAL treatment group (n= 10):animals,HBAL 6h, accept treatment within a bioreactor vaccination 10 x 109 CL-1 cells.Observe the leakproofness during treatment, as well as the vital signs ,adverse reaction of mixed bioartificial liver before treatment, treatment and after treatment; Record animal survival time, dynamic monitoring aspartate transaminase, albumin, total bilirubin, total bile acid, urea nitrogen, serum creatinine, blood ammonia and calculation Fischer index, etc.; take pathological examination of liver, kidneys, spleen, pancreas, heart, lungs, stomach, and brain tissue specimen under the microscope and electron microscopy after the death of mcahin immediately, then exam the pathological changes of the organization. In addition, before and after HBAL treatment,use MTT method to detect liver cell activity.After treatment, take second points pulp to microcopy, and the CL-1 cells, with 2.5 g/L trypsin digestion centrifugal, moves to a cell density of 1.0x 1010/L, 70℃repeated frozen soluble three times,0.2 ml cracking and cellular debris will be injected into the neck, back subcutaneous of 5 nude mouse, respectively (n 10).As a control,0.2 ml CL - 1, density of 1.0 x 1010/L,are injected into the neck, back subcutaneous of 5 nude mouse (n=10), living cells rate is 97% by Placenta blue, observing oncogenesis. Four weeks after, take the injection site tumor tissue and the liver, lungs and brain tissue specimens to observe cell's form, arrangement, the nucleus and whether dyeing or infringing upon the peripheral tissues, etc by and hematoxylin-eosin fine - Iraqi red (HE).Results:10 for treatment of acute liver failure in Cynomolgus, none died during the treatment. None of The circulation line of liquid leakage, obvious bleeding, allergies, fever nor other serious adverse reactions have be found in the course of treatment, and is well tolerated. Monitoring vital signs, except when the blood began to lead a transient heart rate and blood pressure fluctuation, the heart rate, oxygen saturation, breathing and mean arterial pressure can remain stable during the whole treatment.After treatment, HBAL treatment group stay awake since five cynomolgus gradually appeared fatigue, reduced activity, unresponsive, drowsiness, coma, and death happened after injection with D-gal 108-123h. However, compared with the ALF group, HBAL treated cynomolgus without vomiting, convulsions and other serious gastrointestinal and neurological symptoms.In the HBAL treatment group, five Cynomolgus survived, and the remaining five died, survival time was 128±3; ALF control group of cynomolgus monkeys all died, survival time was 112±2. Kaplan-Meier (KM) survival analysis methods suggest HBAL treatment can significantly prolong the survival time of animals tested (p＜0.01). 48 hours after injection of D-gal, following changes ocurred to all the cynomolgus monkeys,serum aspartate aminotransferase, total bilirubin, total bile acid, urea nitrogen, creatinine, blood ammonia concentration increased rapidly;albumin,Fischer index was significantly reduced. After HBAL reatment, serum aspartate aminotransferase, total bilirubin, total bile acid, urea nitrogen, creatinine, blood ammonia concentration of the treatment group was significantly lower than those of the ALF control group. The activity of CL-1 cells was 97-99% before the start of HBAL treatment, after the end of treatment, the activity of CL-1 cells can be maintained to 85-90%.CL - 1 cells are not seen in the nutrient-containing medium after Second points. nude mouse' Neck, Back subcutaneous which CL - 1 cells inject,after 4 weeks, no tumou occurrence, vaccinations control group were 10 vaccination site appear tumor, the rate is 100%. The tumor is smooth round, Circumscribed, enveloped,and liver, lungs, brain tissue organ is not found metastases.Conclusion:1. Building a new type bioreactor-perfusion. The biological CL - 1 cells can maintain higher activity and good function in the bioreactor, and CL - 1 cells can be safely as HBAL cell materials.2. Construct a nonel hybrid bioartificial liver.This type of artificial liver can significantly reduce aspartic acid transaminase, total bilirubin, total bile acid, urea nitrogen, serum creatinine, ammonia concentration of biooxyrate animals, improve blood Fischer index, and extend animal survival time, suggesting that its has obvious liver support role. |
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