微囊化人肝细胞移植对小鼠急性肝衰竭的治疗作用

Therapeutic effect of transplantation of microencapsulated human immortalized hepatocytes on acute liver failure in mice

目的:评价微囊化人源性肝细胞腹腔内移植对四氯化碳诱导的小鼠急性肝功能衰竭的治疗作用,为微囊化异种肝细胞在肝细胞移植的临床应用提供实验依据。方法:实验于2005-02/2006-01在中国科学院大连化学物理研究所,生物技术部生物医学材料工程实验室完成。①采用自制静电液滴发生器制备海藻酸钠-聚赖氨酸-海藻酸钠胶囊,应用四氯化碳腹腔注射建立急性肝功能衰竭动物模型,以谷草转氨酶、谷丙转氨酶高出正常值10倍以上作为判定肝功能衰竭的具体标准。②取造模成功的急性肝衰竭小鼠90只,随机分为空囊组、微囊化细胞组、游离细胞组,30只/组。各组均于注射四氯化碳24h后进行移植实验:空囊组腹腔注射含有空囊的生理盐水,微囊化细胞组腹腔注射微囊化肝细胞悬液,游离肝细胞组腹腔注射游离肝细胞悬液,均1mL/只。③每组随机取出10只用于观察8d存活率,其余小鼠分别于移植后1,2,4,8d经眼球后静脉丛采血,进行肝功能检测谷丙转氨酶和谷草转氨酶水平,每组5只/次。并回收微胶囊观察其形态,对回收的微囊化细胞进行组织切片细胞活性观察。结果:成功建立急性肝衰竭模型的90只小鼠全部进入结果分析。①移植后不同时间点各组小鼠血清谷丙转氨酶和谷草转氨酶水平的变化:移植后第1,2天,微囊化细胞组的两种转氨酶水平均显著低于游离细胞组和空囊组(P<0.05)。到移植后第8天各组间转氨酶水平基本相似(P>0.05)。②各组小鼠移植8d存活率的比较:微囊化细胞组小鼠的存活率显著高于空囊组、游离细胞组(90.0%,50.0%,60.0%,P<0.05)。③腹腔移植微胶囊的形态及囊内细胞活性观察结果:回收的微胶囊形态完整光滑,未见明显的纤维包裹,囊内细胞呈圆形,细胞膜完整光滑,胞浆饱满,仍有90%以上肝细胞存活。结论:微囊化人源性肝细胞腹腔内移植可以提高药物诱导急性肝衰竭小鼠的存活率,改善急性肝衰小鼠的肝脏功能。提示生物微胶囊可以有效阻断免疫排斥作用,保护移植的肝细胞,有利于其发挥生物功能。

AIM： To investigate the therapeutic effect of transplantation of microencapsulated human hepatocytes on acute liver failure in mice induced by carbon tetrachlofide （CCl4）, so as to provide evidence for clinical application of microencapsulated heterogenous hepatocytes in transplantation. METHODS： The experiment was conducted in the laboratory of Biomedial Material Engineering, Department of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Science between February 2005 and January 2006. ①Human hepatocytes were encapsulated in 400 μm of alginate-poly-L-lysine-alginate microcapsules with the static eleetrleity drip generator to establish the acute liver failure models, which had 10 times glutamic-oxalacetic transaminase （GOT） and glutamic pyruvic transaminase （GPT） than the normal values, by intraperitoneal injection of CC14. ②Ninety successful mice models were selected and randomly divided into 3 groups： Empty capsule group injected with saline without cells; microencapsulated cell group intraperitoneally injected with mierocapsules cell suspension and free cell group injected with free cell suspension, 1 mL per rat. ③Ten mice in each group were randomly selected to observe the survival rate on the 8^th day, and the rest mice were taken the/etrobulbar venous plexus blood to measure the level of GOT and GPT on the 1^st, 2^rd, 4^th and 8^th days after transplantation, once 5 mice in each group. The microcapsules were retrieved for observation of their appearance, and the cells were performed tissue slice cytoactive observation. RESULTS： The 90 mice models with acute liver failure were all involved in the result analysis. ①The changes of GPT and GOT level at different time points after transplantation： On the 1^st and 2^rd days after transplantation, the level of two kinds of transaminase in the mieroeneapsulated cells group was obviously lower than the other two groups （P 〈 0.05）. On the 8^th day, the level in each group was nearly the same （P 〉 0.05）. ②Comparison of survival rate of each group on the 8^th day after transplantation： It was significantly higher in the microencapsulated cells group than the other two groups （90.0%, 50.0% and 60.0%, P 〈 0.05）. ③ The observed results on appearance and cytoactive of the microcapsules： The retrieved microcapsules had complete and smooth appearance, without obvious fibrous encapsulation, and the cells were round, the cell membrane was silky and complete with full cytoplasm, more than 90% of the hepatocytes remained survival. CONCLUSION： Microencapsulated hepatocytes transplantation can increase the survival rate of mice with CCl4-induced acute liver failure, and improve the liver biochemical function, indicating that the microcapsule can effectively inhibit immunologic rejection and protect the transplanted hepatocytes to display its biological function.