一种新型非生物人工肝系统的构建及其疗效评估

发布时间：2018-06-24 11:22

本文选题：人工肝 + 急性肝衰竭　；参考：《浙江大学》2015年博士论文

【摘要】：背景肝衰竭是由多种因素引起肝细胞严重损害,导致其合成、解毒和生物转化等功能发生严重障碍,出现以黄疸、凝血功能障碍、肝性脑病和腹水等为主要表现的一种临床综合征[1]。肝功能衰竭患者往往病情危重,进展迅速,预后极差[2]。尽管随着医疗技术的进步,近年来在肝衰竭的诊治上取得了较大的进展,但其病死率仍然居高不下。根据病理组织学特征和病情的发展速度,肝衰竭通常分为急性肝衰竭、亚急性肝衰竭、慢加急性肝衰竭、慢性肝衰竭四类[3]。欧美国家,主要以药物诱发的急性肝衰竭为主,其中乙酰氨基酚(acetaminophen)诱导的急性肝衰竭占50%左右,而在亚太地区则以病毒性肝炎(主要为乙型病毒)诱发的慢加急性肝衰竭最为常见[4]。肝移植是肝衰竭患者最有效的治疗手段,但往往由于供体不足,即便在美国,每年也仅有不足三分之一的病人能够获得肝移植的机会,而更多患者在等待供体期间死去[5]。人工肝支持系统是一种有效的肝脏功能替代手段。它借助体外循环装置,采用物理、化学、生物的血液净化手段,可以暂时、部分替代肝脏功能,改善机体内环境,促进肝细胞再生[6],或者成为肝移植的“桥梁”,使患者能够赢得更多的时间等待合适的供肝,降低围手术期风险,减少并发症[7]。目前,根据人工肝系统中是否包含活性细胞成分,人工肝支持系统可分为非生物人工肝及生物型人工肝两大类。生物型人工肝是以人工培养具有活性的肝细胞为基础,与生物反应器相结合,构建的体外生物反应装置。目前在研究的生物人工肝系统主要有HepatAssist[8,9], ELAD[10-12], MELS[13,14], BLSS[15], AMC-BAL[16]等。生物人工肝支持系统是人工肝支持系统的主要研究方向,但现在技术发展仍不成熟,目前仍没有一种治疗系统被批准用于临床治疗。非生物型人工肝是目前广泛应用于临床的人工肝治疗手段。国内主要采用以血浆置换或者血浆置换联合血液透析滤过、血浆灌流等治疗方法为主,但往往因为血浆供应紧张,大量的患者得不到及时有效的救治。在欧美则常采用基于白蛋白透析的多种血液净化方法的联合系统,包括白蛋白透析吸附系统(包括单次白蛋白通过透析(single-pass albumin dialysis, SPAD)[17,18]、重复白蛋白透析(repeated albumin dialysis, RAD)、分子吸附再循环系统(molecular adsorbent recirculating system, MARS)[19-21]等。已有的研究表明该类方法缺乏补充蛋白质、凝血因子等有益物质,难以完成肝脏合成功能的替代,且治疗过程中消耗大量白蛋白,成本较高,疗效难以令人满意。如何高效整合各种基础的血液净化手段,构建新的有效的组合式非生物型人工肝系统,提高临床治疗的效果,目前是一个值得挑战的课题。最近,在李兰娟院士指导下,围绕着如何减少非生物人工肝治疗过程中的血浆用量,最大效率的清除不同分子量的各类毒素,更好的实现肝脏功能的替代这些关键问题,我们实验室团队结合了血浆置换联合血浆吸附和血液滤过的优势探索出了一套名为李氏人工肝系统(Li's artificial liver system, Li-ALS)的方法,。该方法借鉴了MARS的多净化循环设计,构建了一个多循环血液净化系统,通过多种净化手段的联合有效降低了血浆使用量,并且利用置换出的废弃血浆作为血浆吸附滤过循环的循环介质,有效的解决了复杂净化循环导致体外引血容量过大的问题。本研究中,我们利用D-氨基半乳糖建立猪肝衰竭模型,采用新型李氏人工肝系统对急性肝衰竭猪进行治疗,初步观察其治疗效果,评估该系统治疗的有效性、稳定性与安全性。目的：本研究构建了一套新型李氏人工肝系统,并采用猪急性肝衰竭模型,对新系统的安全性及有效性进行评价。方法：选取体重18-23千克的雄性巴马香猪40只作为模型动物,颈静脉置入6.5F双腔静脉导管,无麻醉条件下静脉注射D-氨基半乳糖(1.3g/kg)诱导猪急性肝衰竭。D-gal注射36h后,实验用猪随机抽签法分为5组,每组8只,开始持续6小时的干预：(1)急性肝衰竭对照组(n=8)：动物仅进行6h麻醉和基本生命体征监护,不接受任何治疗措施；(2)血浆循环吸附滤过治疗组(n=8)：动物先接受1h体外循环,随后进行5h血浆循环吸附滤过治疗；(3)低剂量血浆置换组(n=8)：动物先接受1h血浆置换治疗,血浆置换量按全血浆置换量的一半进行,置换结束后再进行5h体外循环；(4)李氏人工肝治疗组(n=8)：动物先接受1h低剂量血浆置换治疗,随后进行5h血浆循环吸附滤过治疗；(5)全血浆置换组(n=8)：动物接受2h全血浆置换,置换结束后再进行4h体外循环。观察及检测治疗过程前后实验动物临床表现、动物生存时问、凝血功能、生化指标、炎症因子以及肝组织病理改变。结果：所有动物均能很好的耐受6小时的麻醉及对应的治疗,5组实验动物生存时间依次为60±2h.74±2h、75±2h、90±3h和88±3h。Kaplan-Meier生存分析提示,相较于急性肝衰竭对照组,所有治疗组生存时间均显著延长(p0.001)；李氏人工肝治疗组生存时间显著长于低剂量血浆置换及血浆循环吸附滤过组(p0.001),其生存时间略高于全血浆置换组,但差异不具有统计学意义(p0.05)。李氏人工肝治疗之后,动物的凝血功能得到显著改善,凝血酶原时间显著下降,纤维蛋白原水平显著升高(p0.05),血清肝酶、胆红素、胆汁酸、血氨水平显著下降(p0.05),电解质水平保持稳定,炎症因子上升趋势得到明显缓解,死后肝组织病理提示肝脏增生明显,Ki-67标志指数显著较对照组显著升高(p0.05)。结论： 1.低剂量血浆置换和血浆循环吸附滤过治疗均能显著延长急性肝衰竭小猪生存时间,两者均为有效的方法； 2.李氏人工肝基于低剂量血浆置换并联合血浆循环吸附滤过,取得了较两者单独使用更加显著的治疗效果； 3.李氏人工肝仅用常规置换量一半的血浆,取得了与全量血浆置换相同的生存时间延长效果,但治疗过程中血浆用量显著减少。
[Abstract]:background
Liver failure is caused by a variety of factors causing serious damage to the liver cells, resulting in serious obstacles to its synthesis, detoxification and biotransformation, and a clinical syndrome characterized by jaundice, coagulation dysfunction, hepatic encephalopathy and ascites, such as the main manifestation of [1]. liver failure, is often in critical condition, rapid progress and poor prognosis, although the prognosis is extremely poor [2]. although With the progress of medical technology, great progress has been made in the diagnosis and treatment of liver failure in recent years, but its mortality is still high. According to the histopathological characteristics and the development speed of the disease, the liver failure is usually divided into four kinds of [3]., European and American countries, including acute liver failure, subacute liver failure, chronic liver failure, chronic liver failure, and chronic liver failure. Acute liver failure induced by drugs, of which acetaminophen (acetaminophen) induced acute liver failure is about 50%, while in the Asia Pacific region, the most common [4]. liver transplantation is the most effective treatment for patients with chronic liver failure induced by viral hepatitis (mainly B virus), but often due to insufficient donor, Even in the United States, only less than 1/3 of the patients can get a liver transplant every year, and more patients who are waiting for the donor to die of the [5]. artificial liver support system are an effective alternative to liver function. To replace the liver function, improve the body environment, promote the regeneration of [6], or become a "bridge" of liver transplantation, so that patients can win more time to wait for the appropriate donor liver, reduce the risk of perioperative period, reduce the complications of [7].
At present, artificial liver support system can be divided into two major categories, namely, abiotic artificial liver and biotype artificial liver, based on the inclusion of active cell components in artificial liver system. Biological artificial liver is based on artificial liver cells with active liver and bioreactor combined with bioreactor. Artificial liver system mainly includes HepatAssist[8,9], ELAD[10-12], MELS[13,14], BLSS[15], AMC-BAL[16] and so on. Biological artificial liver support system is the main research direction of artificial liver support system, but the development of the technology is still not mature now, and there is still not a kind of treatment system approved for clinical treatment. The main use of artificial liver treatment in clinical. The main use of plasma exchange or plasma exchange combined hemodialysis filtration, plasma perfusion and other treatment methods, but often because of tension in the plasma, a large number of patients can not get timely and effective treatment. In Europe and America, often use a variety of blood purification methods based on albumin dialysis. The combined system, including the albumin dialysate adsorption system (including single-pass albumin dialysis, SPAD) [17,18], repeated albumin dialysis (repeated albumin dialysis, RAD), molecular adsorption recirculation system (molecular adsorbent recirculating), etc. The method is lack of supplemental protein, coagulation factor and other beneficial substances, it is difficult to complete the replacement of liver synthesis function, and the cost of a large amount of albumin is consumed in the treatment process, the cost is high and the curative effect is difficult to be satisfactory. How to efficiently integrate various basic blood purification methods and construct a new and effective combined non biological artificial liver system and improve the clinical treatment. The effect is a challenging task at the moment.
Recently, under the guidance of academician Li Lanjuan, we have combined the advantages of plasma exchange and plasma adsorption and hemofiltration on how to reduce the amount of plasma in the process of abiotic artificial liver treatment, to maximize the efficiency of eliminating the various molecular weights of all kinds of toxins, and to better realize the replacement of liver function. A set of methods called Li's artificial liver system (Li-ALS) was given. The method used the multi purification cycle design of MARS to construct a multi circulation blood purification system. The plasma use was effectively reduced by a combination of various purification methods, and the discarded plasma was used as a plasma adsorption. The circulating medium of filtration circulation can effectively solve the problem of excessive blood volume in vitro caused by complex purification cycle.
In this study, we used D- amino galactose to establish a pig liver failure model and use a new Li's artificial liver system to treat acute liver failure pigs. The effect of the treatment was preliminarily observed and the effectiveness, stability and safety of the system were evaluated.
Objective:
In this study, we constructed a new Li's artificial liver system and evaluated the safety and efficacy of the new system by using the pig acute liver failure model.
Method:
A 18-23 kilogram male Bama pig was selected as a model animal. The 6.5F double caval catheter was inserted into the jugular vein in the jugular vein. After the intravenous injection of D- amino galactogram (1.3g/kg) under the anesthesia to induce.D-gal injection of 36h in swine acute liver failure, the experiment was divided into 5 groups with 8 pigs in each group, which began for 6 hours of intervention: (1) acute liver. Exhaustion control group (n=8): animals were only subjected to 6h anesthesia and basic vital signs monitoring, and no treatment was accepted; (2) plasma circulatory adsorption filtration therapy group (n=8): animals received 1H extracorporeal circulation, followed by 5h plasma circulatory adsorption filtration therapy; (3) low dose plasma exchange group (n=8): the animals received 1H plasma replacement therapy first. 5h extracorporeal circulation was carried out at the end of total plasma replacement, and (4) Li's artificial liver treatment group (n=8): the animals were first treated with 1H low dose plasma exchange, followed by 5h plasma circulatory adsorption filtration, and (5) total plasma replacement group (n=8): animals received 2H total plasma replacement, and then reentered after the replacement. 4h cardiopulmonary bypass was performed. The clinical manifestations of experimental animals, animal survival time, coagulation function, biochemical indexes, inflammatory factors and pathological changes of liver tissue were observed and detected before and after the treatment.
Result:
All the animals were well tolerated for 6 hours of anesthesia and corresponding treatment. The survival time of the 5 experimental animals was 60 + 2h.74 + 2h, 75 + 2h, 90 + 3H and 88 + 3h.Kaplan-Meier. The survival time of all the treatment groups was significantly longer than that in the control group of acute liver failure (p0.001); the survival time of the Li's artificial liver treatment group was significant. The survival time was slightly higher than the total plasma exchange group (p0.001), but the difference was not statistically significant (P0.05). After Li's artificial liver treatment, the blood coagulation function of the animal was significantly improved, the prothrombin time decreased significantly, the fibrinogen level increased significantly (P0.05), the serum level was significantly higher (P0.05). The level of liver enzyme, bilirubin, bile acid and blood ammonia decreased significantly (P0.05), the level of electrolyte remained stable, and the rising trend of inflammatory factors was obviously alleviated. The liver histopathology indicated that liver hyperplasia was obvious, and the index of Ki-67 marker was significantly higher than that of the control group (P0.05).
Conclusion:
1. low dose plasmapheresis and plasma circulating adsorption filtration treatment can significantly prolong the survival time of pigs with acute liver failure, both of which are effective methods.
2. Li artificial liver based on low dose plasma exchange combined with plasma circulating adsorption filtration has achieved a more significant therapeutic effect than the two alone.
3. Li's artificial liver was used only half of the conventional replacement plasma, and the same survival time was achieved with the total amount of plasma exchange, but the amount of plasma was significantly reduced during the treatment.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R318.14

【参考文献】