新型生物人工肝免疫安全性的实验研究

发布时间：2018-08-02 17:22

【摘要】：目的：探讨膜截流分子量对新型生物人工肝支持系统免疫安全性的影响方法：应用D氨基半乳糖静脉滴注比格犬建立急性肝功能衰竭模型。采用猪肝细胞-骨髓间充质干细胞（mesenchymal stem cells, MSCs）共培养体系作为种子细胞，以基于乳糖酰基壳聚糖纳米纤维支架的多层平板作为反应器，构建新型生物人工肝（bioartificial liver, BAL）。急性肝功能衰竭犬根据BAL中血浆成分分离柱内半透膜截流分子量的大小分为两组：A组：200KD组；B组：1200KD组，各组均接受新型2次BAL治疗，，时间点为第1天和第21天，每次6小时。观察各组体内IgG、IgM、CH50变化及反应器内抗体漏过情况。免疫组化检测各组动物模型心脏、肝脏、脾脏、肺脏和肾脏组织中IgG、IgM及补体C3沉积情况。结果：在第一次BAL治疗后两组的IgG和IgM水平均没有发生明显的变化；而在第2次治疗后的第7天，1200KD组的IgG和IgM水平出现明显的升高，200KD组的IgG和IgM水平仍未出现明显上升或下降。CH50的结果显示，在第一次BAL治疗后，两组的CH50都出现了暂时性的下降，在治疗后1小时达到最低值，而后缓步上升，在7天后恢复至治疗前水平。反应器内培养液中IgG、IgM及CH50检测结果显示，1200KD组在治疗结束后IgG、IgM及CH50含量显著高于200KD组。采用免疫组化技术对所有犬心脏、肝脏、脾脏、肾脏、肺脏等重要器官检测抗体沉积情况后发现，各重要脏器均未检测有IgG、IgM及补体C3的沉积结论：膜截流分子量可能是影响BAL异种免疫排斥的重要因素之一。目的：探索膜截流分子量对新型生物人工肝支持系统内细胞材料功能的影响方法：以基于乳糖酰基壳聚糖纳米纤维支架的多层平板作为生物反应器，猪肝细胞-骨髓间充质干细胞共培养体系作为核心细胞材料，构建新型生物人工肝。首先采用免疫荧光染色及逆转录聚合酶链式反应（RT-PCR）观察离体猪肝细胞、骨髓间充质干细胞表面是否表达异种抗原Galα(1,3) Gal；然后依据新型BAL中血浆成分分离柱内半透膜截流分子量的大小分为两组：200KD组；1200KD组。动物模型采用正常比格犬，各组均接受新型BAL治疗6小时，定时收集BAL系统中培养液。检测各组反应器内细胞材料清蛋白、尿素表达水平，同时观察各组反应器内细胞活力及细胞损伤情况。结果：荧光显微镜观察发现猪肝细胞和骨髓MSCs在体外培养过程中仍然能稳定表达Galα(1,3) Gal。RT-PCR结果显示离体猪肝细胞、骨髓MSCs和共培养细胞内均存在ggta-1mRNA序列。细胞功能检测结果显示，200KD组细胞清蛋白分泌水平及尿素合成分别为53.3ug/10~6细胞和3.6ug/10~6细胞，显著高于1200KD组的5.6ug/10~6细胞和0.3ug/10~6细胞。细胞活力观测结果显示200KD组细胞活力保持在90%左右，明显高于1200KD组的22％；细胞损伤结果提示1200KD组反应器内AST含量从13.3U/L上升至60.2U/L，200KD组浓度则在13.2-14.8U/L之间波动；LDH结果与之类似。反应器内免疫球蛋白分子含量检测结果发现200KD组在循环开始3小时后才检测出微量的IgG，直至实验结束其含量稳定在0.004-0.005mg/ml之间；而1200KD组从循环开始后30分钟的0.4mg/ml上升至2.7mg/ml。两组结果具有统计学差异（P0.05）。CH50的检测结果表现出类似的趋势。200KD组在6小时的体外循环灌注过程中未能检测出IgM，1200KD组则从开始后30分钟的0.7mg/ml上升至结束时的2.3mg/ml。免疫荧光试验进一步证实了上述结果。结论：降低BAL膜截流分子量可以有效减少免疫球蛋白分子的透过，从而维持BAL中细胞材料的功能。第三部分新型生物人工肝治疗应用的免疫安全性研究目的：探讨新型生物人工肝系统治疗急性肝功能衰竭犬模型的免疫安全性。方法：采用基于乳糖酰基壳聚糖纳米纤维支架的多层平板生物反应器与猪肝细胞-骨髓间充质干细胞构建新型的BAL系统。其中血浆成分分离柱内半透膜截流分子量为200KD。D氨基半乳糖诱导建立犬急性肝功能衰竭模型。根据治疗次数的不同，实验犬被分成两组，1组：接受1次BAL治疗；2组：接受3次BAL治疗，每次治疗时间6小时。治疗期间对动物血流动力学及血液动力学进行监测。ELISA法检测血浆及培养液中IgG，IgM及CH50水平。免疫荧光片检测心、肝、脾、肺、肾等器官及反应器内细胞材料表面免疫蛋白的沉积情况。结果：治疗期间，每条实验犬的心率、血压及呼吸频率平稳，血常规检测显示，白细胞、血小板及淋巴细胞未出现明显的升高或降低。犬在接受1次BAL治疗后，体内IgG，IgM免疫抗体水平未出现明显的升高或下降，而补体CH50水平出现一过性下降，但很快恢复至正常水平，且并未发生严重的过敏反应及排斥反应。接受3次BAL治疗的实验犬体内IgG、IgM和CH50水平的变化规律亦表现出类似的现象。反应器内培养液中IgG，IgM及CH50检测结果显示，前3h内未能检测出IgG与CH50，3h及6h两个时间点检出极其少量的IgG与CH50。6h内均未检测出IgM。免疫荧光的结果与ELISA检测结果类似；循环结束后反应器内细胞材料表面免疫复合物检测有微量的IgG与补体C3沉积，但未能检测出IgM各重要脏器均未检测到IgG，IgM及补体C3的沉积。结论：新型BAL行实验性治疗具有良好的免疫安全性。
[Abstract]:Objective: To investigate the effect of molecular weight of membrane closure on immune safety of a new bioartificial liver support system.
Methods: the model of acute liver failure was established by intravenous infusion of D amino galactose. The co culture system of pig hepatocyte - bone marrow mesenchymal stem cells (mesenchymal stem cells, MSCs) was used as seed cell, and a new type of bioartificial liver was constructed with a multi layer plate based on lactose acyl chitosan nanofiber scaffold as a reactor. (bioartificial liver, BAL). The dogs of acute liver failure were divided into two groups according to the size of interceptor interceptor in BAL plasma, A group: group 200KD, group B: 1200KD group, each group received a new 2 times BAL treatment, the time point was first days and twenty-first days, each time of 6 hours. The IgG, IgM, CH50 changes and reactor in each group were observed. Immunohistochemistry was used to detect the deposition of IgG, IgM and complement C3 in heart, liver, spleen, lung and kidney tissues.
Results: there was no significant change in the level of IgG and IgM in the two groups after the first BAL treatment, while the level of IgG and IgM in the 1200KD group increased significantly on the seventh day after the second treatment, and the IgG and IgM levels in the 200KD group were still not significantly increased or decreased in the.CH50. The two groups of CH50 appeared after the first BAL treatment. IgG, IgM and CH50 in the culture fluid of the reactor showed that the IgG, IgM and CH50 content in the 1200KD group was significantly higher than that of the 200KD group at the end of the treatment. Immunohistochemistry technique was used for the heart, liver, spleen, and kidney of all dogs. After detection of antibody deposition in lung and other important organs, no deposition of IgG, IgM and complement C3 was detected in all important organs.
Conclusion: molecular weight of membrane closure may be one of the important factors affecting BAL xenograft rejection.
Objective: To explore the effect of molecular weight of membrane closure on the function of cell material in a new bioartificial liver support system.
Methods: the multi-layer plate based on lactose acyl chitosan nanofiber scaffold was used as a bioreactor, pig hepatocytes and bone marrow mesenchymal stem cells co culture system was used as the core cell material to construct a new type of biological artificial liver. First, immunofluorescence staining and reverse transcriptase polymerase chain reaction (RT-PCR) were used to observe the liver cells in vitro, bone and bone. Whether the surface of medullary mesenchymal stem cells (MSCs) expressed the heterologous antigen Gal alpha (1,3) Gal, and then divided into two groups according to the size of the intramedullary semi permeable membrane intercepted by the plasma components in the new BAL: 200KD group and 1200KD group. The animal model was used in normal beagle dogs, each group received a new BAL treatment for 6 hours, and the culture solution in BAL system was collected regularly. The expression levels of albumin and urea, cell viability and cell damage were observed.
Results: the fluorescence microscope showed that the pig liver cells and bone marrow MSCs could still express Gal alpha (1,3) Gal.RT-PCR in vitro, and the results showed that there were ggta-1mRNA sequences in the bone marrow MSCs and co culture cells. The cell function detection results showed that the secretion level of albumin and the urea synthesis score of the cell of the 200KD group were in the 200KD group. Not as 53.3ug/10~6 cells and 3.6ug/10~6 cells, significantly higher than the 5.6ug/10~6 and 0.3ug/10~6 cells in group 1200KD. Cell viability observation showed that cell viability in group 200KD remained about 90%, obviously higher than that of group 1200KD, 22%. The results of cell damage suggested that AST content in 1200KD group increased from 13.3U/L to 60.2U/L, 200KD group concentration. The results were similar between the 13.2-14.8U/L and the LDH results. The results of the immunoglobulin content detection in the reactor found that the 200KD group detected a trace IgG after 3 hours of circulation until the end of the experiment, and the content of the 1200KD was stable between 0.004-0.005mg/ml; and the 1200KD group increased from the 0.4mg/ml to 2.7mg/ml. from the beginning of the cycle. The results of the two groups were statistically different (P0.05).CH50 detection results showed a similar trend in the.200KD group failed to detect IgM during the 6 hour cardiopulmonary bypass, and the 1200KD group confirmed the above results by the 2.3mg/ml. immunofluorescence test at the end of the 0.7mg/ml 30 minutes after the beginning.
CONCLUSION: Reducing the molecular weight of BAL membrane can effectively reduce the permeation of immunoglobulin molecules and maintain the function of cell materials in BAL.
The third part is about the immune safety of new bioartificial liver.
Objective: To explore the immune safety of a new bioartificial liver system in the treatment of acute liver failure dogs.
Methods: a new type of BAL system was constructed by the multi-layer bioreactor based on lactose acyl chitosan nanofiber scaffold and pig liver cells bone marrow mesenchymal stem cells, in which the molecular weight of the plasma component isolated column interceptor was induced by 200KD.D amino galactose to establish the model of acute liver failure in dogs. The experimental dogs were divided into two groups, 1 groups, 1 BAL treatments, 2 groups, 3 times of BAL, and 6 hours each time. During the treatment, the hemodynamics and hemodynamics were monitored by.ELISA method for the detection of IgG, IgM and CH50 levels in the plasma and culture fluid. The immune fluorescein was used to detect the heart, liver, spleen, lung, kidney and other organs and reactors. The deposition of immunoglobulin on the surface of the internal cell material.
Results: during the treatment, the heart rate, blood pressure and respiratory frequency of each experimental dog were stable. The blood routine test showed that white blood cells, platelets and lymphocytes did not rise or decrease obviously. After 1 BAL treatment, the level of IgG, IgM immunization antibody in the body was not significantly elevated or decreased, and the level of complement CH50 had an excessive decrease. But it quickly recovered to the normal level and did not have serious anaphylaxis and rejection. The changes in the levels of IgG, IgM and CH50 in the experimental dogs receiving 3 BAL treatments were also similar. The results of IgG, IgM and CH50 in the culture medium of the reactor showed that the two time points of IgG, CH50,3h and 6h were not detected in the former 3H. The results of undetected IgM. immunofluorescence in a very small number of IgG and CH50.6h were similar to that of ELISA, and there were trace IgG and complement C3 deposition in the cell material surface immune complex in the reactor after the end of the cycle, but no IgG, IgM and the deposition of complement C3 were not detected in all the important organs of IgM.
Conclusion: the new BAL has good immunological safety.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R318.14

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