【摘要】：目的：探討膜截流分子量對(duì)新型生物人工肝支持系統(tǒng)免疫安全性的影響 方法：應(yīng)用D氨基半乳糖靜脈滴注比格犬建立急性肝功能衰竭模型。采用豬肝細(xì)胞-骨髓間充質(zhì)干細(xì)胞（mesenchymal stem cells, MSCs）共培養(yǎng)體系作為種子細(xì)胞，以基于乳糖�；鶜ぞ厶羌{米纖維支架的多層平板作為反應(yīng)器，構(gòu)建新型生物人工肝（bioartificial liver, BAL）。急性肝功能衰竭犬根據(jù)BAL中血漿成分分離柱內(nèi)半透膜截流分子量的大小分為兩組：A組：200KD組；B組：1200KD組，各組均接受新型2次BAL治療，，時(shí)間點(diǎn)為第1天和第21天，每次6小時(shí)。觀察各組體內(nèi)IgG、IgM、CH50變化及反應(yīng)器內(nèi)抗體漏過情況。免疫組化檢測(cè)各組動(dòng)物模型心臟、肝臟、脾臟、肺臟和腎臟組織中IgG、IgM及補(bǔ)體C3沉積情況。 結(jié)果：在第一次BAL治療后兩組的IgG和IgM水平均沒有發(fā)生明顯的變化；而在第2次治療后的第7天，1200KD組的IgG和IgM水平出現(xiàn)明顯的升高，200KD組的IgG和IgM水平仍未出現(xiàn)明顯上升或下降。CH50的結(jié)果顯示，在第一次BAL治療后，兩組的CH50都出現(xiàn)了暫時(shí)性的下降，在治療后1小時(shí)達(dá)到最低值，而后緩步上升，在7天后恢復(fù)至治療前水平。反應(yīng)器內(nèi)培養(yǎng)液中IgG、IgM及CH50檢測(cè)結(jié)果顯示，1200KD組在治療結(jié)束后IgG、IgM及CH50含量顯著高于200KD組。采用免疫組化技術(shù)對(duì)所有犬心臟、肝臟、脾臟、腎臟、肺臟等重要器官檢測(cè)抗體沉積情況后發(fā)現(xiàn)，各重要臟器均未檢測(cè)有IgG、IgM及補(bǔ)體C3的沉積 結(jié)論：膜截流分子量可能是影響B(tài)AL異種免疫排斥的重要因素之一。 目的：探索膜截流分子量對(duì)新型生物人工肝支持系統(tǒng)內(nèi)細(xì)胞材料功能的影響 方法：以基于乳糖�；鶜ぞ厶羌{米纖維支架的多層平板作為生物反應(yīng)器，豬肝細(xì)胞-骨髓間充質(zhì)干細(xì)胞共培養(yǎng)體系作為核心細(xì)胞材料，構(gòu)建新型生物人工肝。首先采用免疫熒光染色及逆轉(zhuǎn)錄聚合酶鏈?zhǔn)椒磻?yīng)（RT-PCR）觀察離體豬肝細(xì)胞、骨髓間充質(zhì)干細(xì)胞表面是否表達(dá)異種抗原Galα(1,3) Gal；然后依據(jù)新型BAL中血漿成分分離柱內(nèi)半透膜截流分子量的大小分為兩組：200KD組；1200KD組。動(dòng)物模型采用正常比格犬，各組均接受新型BAL治療6小時(shí)，定時(shí)收集BAL系統(tǒng)中培養(yǎng)液。檢測(cè)各組反應(yīng)器內(nèi)細(xì)胞材料清蛋白、尿素表達(dá)水平，同時(shí)觀察各組反應(yīng)器內(nèi)細(xì)胞活力及細(xì)胞損傷情況。 結(jié)果：熒光顯微鏡觀察發(fā)現(xiàn)豬肝細(xì)胞和骨髓MSCs在體外培養(yǎng)過程中仍然能穩(wěn)定表達(dá)Galα(1,3) Gal。RT-PCR結(jié)果顯示離體豬肝細(xì)胞、骨髓MSCs和共培養(yǎng)細(xì)胞內(nèi)均存在ggta-1mRNA序列。細(xì)胞功能檢測(cè)結(jié)果顯示，200KD組細(xì)胞清蛋白分泌水平及尿素合成分別為53.3ug/10~6細(xì)胞和3.6ug/10~6細(xì)胞，顯著高于1200KD組的5.6ug/10~6細(xì)胞和0.3ug/10~6細(xì)胞。細(xì)胞活力觀測(cè)結(jié)果顯示200KD組細(xì)胞活力保持在90%左右，明顯高于1200KD組的22％；細(xì)胞損傷結(jié)果提示1200KD組反應(yīng)器內(nèi)AST含量從13.3U/L上升至60.2U/L，200KD組濃度則在13.2-14.8U/L之間波動(dòng)；LDH結(jié)果與之類似。反應(yīng)器內(nèi)免疫球蛋白分子含量檢測(cè)結(jié)果發(fā)現(xiàn)200KD組在循環(huán)開始3小時(shí)后才檢測(cè)出微量的IgG，直至實(shí)驗(yàn)結(jié)束其含量穩(wěn)定在0.004-0.005mg/ml之間；而1200KD組從循環(huán)開始后30分鐘的0.4mg/ml上升至2.7mg/ml。兩組結(jié)果具有統(tǒng)計(jì)學(xué)差異（P0.05）。CH50的檢測(cè)結(jié)果表現(xiàn)出類似的趨勢(shì)。200KD組在6小時(shí)的體外循環(huán)灌注過程中未能檢測(cè)出IgM，1200KD組則從開始后30分鐘的0.7mg/ml上升至結(jié)束時(shí)的2.3mg/ml。免疫熒光試驗(yàn)進(jìn)一步證實(shí)了上述結(jié)果。 結(jié)論：降低BAL膜截流分子量可以有效減少免疫球蛋白分子的透過，從而維持BAL中細(xì)胞材料的功能。 第三部分新型生物人工肝治療應(yīng)用的免疫安全性研究 目的：探討新型生物人工肝系統(tǒng)治療急性肝功能衰竭犬模型的免疫安全性。 方法：采用基于乳糖�；鶜ぞ厶羌{米纖維支架的多層平板生物反應(yīng)器與豬肝細(xì)胞-骨髓間充質(zhì)干細(xì)胞構(gòu)建新型的BAL系統(tǒng)。其中血漿成分分離柱內(nèi)半透膜截流分子量為200KD。D氨基半乳糖誘導(dǎo)建立犬急性肝功能衰竭模型。根據(jù)治療次數(shù)的不同，實(shí)驗(yàn)犬被分成兩組，1組：接受1次BAL治療；2組：接受3次BAL治療，每次治療時(shí)間6小時(shí)。治療期間對(duì)動(dòng)物血流動(dòng)力學(xué)及血液動(dòng)力學(xué)進(jìn)行監(jiān)測(cè)。ELISA法檢測(cè)血漿及培養(yǎng)液中IgG，IgM及CH50水平。免疫熒光片檢測(cè)心、肝、脾、肺、腎等器官及反應(yīng)器內(nèi)細(xì)胞材料表面免疫蛋白的沉積情況。 結(jié)果：治療期間，每條實(shí)驗(yàn)犬的心率、血壓及呼吸頻率平穩(wěn)，血常規(guī)檢測(cè)顯示，白細(xì)胞、血小板及淋巴細(xì)胞未出現(xiàn)明顯的升高或降低。犬在接受1次BAL治療后，體內(nèi)IgG，IgM免疫抗體水平未出現(xiàn)明顯的升高或下降，而補(bǔ)體CH50水平出現(xiàn)一過性下降，但很快恢復(fù)至正常水平，且并未發(fā)生嚴(yán)重的過敏反應(yīng)及排斥反應(yīng)。接受3次BAL治療的實(shí)驗(yàn)犬體內(nèi)IgG、IgM和CH50水平的變化規(guī)律亦表現(xiàn)出類似的現(xiàn)象。反應(yīng)器內(nèi)培養(yǎng)液中IgG，IgM及CH50檢測(cè)結(jié)果顯示，前3h內(nèi)未能檢測(cè)出IgG與CH50，3h及6h兩個(gè)時(shí)間點(diǎn)檢出極其少量的IgG與CH50。6h內(nèi)均未檢測(cè)出IgM。免疫熒光的結(jié)果與ELISA檢測(cè)結(jié)果類似；循環(huán)結(jié)束后反應(yīng)器內(nèi)細(xì)胞材料表面免疫復(fù)合物檢測(cè)有微量的IgG與補(bǔ)體C3沉積，但未能檢測(cè)出IgM各重要臟器均未檢測(cè)到IgG，IgM及補(bǔ)體C3的沉積。 結(jié)論：新型BAL行實(shí)驗(yàn)性治療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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R318.14

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