膜截流分子量在新型生物人工肝免疫安全性中的作用研究

目的 探讨膜截流分子量对新型生物人工肝(bioartificial liver,BAL)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肝功能衰竭模型.急性肝功能衰竭犬根据BAL隔离膜截流分子量的大小分为两组:A组:200 kD组;B组:1200 kD组.两组均接受新型2次BAL治疗,每次6h.观察各组体内IgG、IgM、50％补体溶血单位(CH50)变化及反应器内抗体漏过情况.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.在第2次治疗后第7天,1200 kD组的IgG和IgM水平明显升高,200 kD组IgG和IgM水平仍未出现明显上升或下降.在第一次BAL治疗后,两组CH50都出现暂时性的下降,在治疗后1h达到最低值,而后缓步上升,在7d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,1200 kD组在治疗结束后IgG、IgM及CH50含量显著高于200 kD组.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.     

新型多层平板型生物人工肝体外支持系统治疗肝功能衰竭的临床研究

目的 评价新型多层平板型生物人工肝(BAL)体外支持系统治疗肝功能衰竭的临床效果及安全性.方法 2010年12月至2011年12月,有38例肝功能衰竭患者接受了新型多层平板型BAL治疗共计48例次,每次治疗时间为4h,于治疗前,治疗中,以及治疗后1、3、7和14d,抽取患者外周血监测肝功能和凝血功能等生化指标,并观察患者临床症状及体征的变化,以判定BAL的治疗效果.采用酶联免疫吸附试验法检测患者血浆及反应器中IgG、IgM及补体CH50的水平,采用聚合酶链反应检测外周血单个核细胞(PBMC)中猪内源性逆转录病毒(PERV) DNA、猪特异性细胞色素B(SsCytB)基因序列及逆转录酶的活性等,以评估BAL治疗的安全性.结果 大部分患者接受BAL治疗后感觉良好,精神状态、临床症状、各项指标较治疗前均有所缓解或明显改善,未发生严重不良反应.38例患者中,临床治愈9例,好转25例,治愈好转率为89.5％(34/38),治疗无效4例;有7例患者经BAL治疗后病情好转,顺利等到供肝接受了肝移植.治疗期间,患者血浆IgG、IgM和CH50水平均未出现明显变化,仅补体CH50在治疗1h时出现一过性下降,随后很快恢复正常水平;反应器内未检测出IgG,仅治疗4h时检出极少量IgG,治疗4h内均未检测出IgM.各时间点患者PBMC均未检测到PERV DNA,且患者血浆中均未检测到猪特异性SsCytB基因序列和逆转录酶活性.结论 新型多层平板型BAL体外支持系统对肝功能衰竭患者具有良好的临床治疗效果和安全性,其在患者等待供肝期间也可作为良好的过渡治疗手段.     

腹腔镜与开腹直肠癌根治术对机体C反应蛋白及体液免疫影响的比较

目的探讨腹腔镜直肠癌手术中C反应蛋白（CRP）及免疫功能的变化特点。方法将54例行直肠癌根治术的患者按患者意愿分成腹腔镜组（26例）和开腹组（28例）,分别于术前1d和术后1、2、3、7d清晨检测患者静脉血CRP、免疫球蛋白（Ig）：IgG、IgA、IgM及补体C3、C4、CH50水平,比较两组患者手术前后CRP、Ig及补体的变化。结果两组患者CRP术后3d均较术前显著增高（P〈0．01）,术后2d达峰值,腹腔镜组术后3d内CRP显著低于开腹组（P〈0．01）。术后两组患者Ig均较术前明显下降,腹腔镜组高于开腹组,但IgG和IgM两组差异无统计学意义（P〉0．05）;而IgA两组差异有统计学意义（P〈0．05）。两组患者术后补体C3、C4及CH50与术前比较差异均有统计学意义（P〈0．05）：腹腔镜组血清补体在术后48～72h已恢复到术前水平,而开腹组在术后72h尚未恢复到术前水平。结论腹腔镜直肠癌根治术和传统的开腹手术均可导致术后机体C反应蛋白浓度增加和免疫水平降低。但腹腔镜手术较开腹手术程度轻。     

猪—猴异种血管移植免疫反应初探

目的 探讨猪－猴异种血管移植超急性排斥反应（HAR）的机理。方法 猪股静脉原位异种移植于恒河猴,发生HAR后通过免疫组化检测移植血管IgG、IgG、C3及C4的沉积。结果 大量IgM、C3和C4沉积于移植静脉内皮, 未发现IgG沉积于移植血管内皮。结论 猪－猴异种移植HAR是由异种自然抗体IgM与异抗原特异结合启动,进而以经典途径激活补体系统而发生。     

腹腔镜胃大部切除术对机体免疫功能的影响

目的:比较腹腔镜与开腹胃大部切除术对机体免疫功能的影响。方法:选择40例有胃大部切除指征的患者,分为腹腔镜组和开腹组,各20例,测定免疫球蛋白IgG、IgM、IgA,补体C3、C4水平及测定、组间比较CD3+(T细胞总数)、CD4+(T辅助/诱导细胞)、CD8+(T抑制/杀伤细胞)的数量。结果:两组IgM、IgA、C4手术前后均无明显变化,组间无统计学差异。腹腔镜组术后1d IgG、C3较术前有所下降,术后3d恢复至术前水平。开腹组IgG、C3术后1d明显低于术前水平,术后5d恢复至术前水平。腹腔镜组淋巴细胞亚群手术前后均无统计学差异,开腹组术后1d CD3+、CD4+、CD8+与术前比较均明显降低,术后5d恢复至术前水平。结论:腹腔镜对机体免疫功能的影响小,术后恢复较快。     

膜截流分子量对新型生物人工肝支持系统内细胞材料影响的实验研究

目的 探索膜截流分子量对新型生物人工肝(bioartificial liver,BAL)支持系统内细胞材料功能的影响.方法 选用200×103和1200×103两种不同膜截流分子量的半透膜作为BAL隔离膜.正常比格犬接受新型BAL治疗6h,定时收集BAL系统中培养液.观察各组细胞活力及功能变化.结果 200×103组细胞活力保持在90％左右,明显高于1200×103组的22％.细胞功能检测显示,200×103组细胞白蛋白分泌水平及尿素合成分别为53.3 μg/106细胞和3.6 μg/106细胞,显著高于1200×103组的5.6 μg/106细胞和0.3 μg/106细胞.1200×103组反应器内免疫球蛋白分子浓度显著高于200×103组,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.     

腹腔镜胆囊切除术对机体免疫功能的影响

目的：比较腹腔镜与开腹胆囊切除术对机体免疫功能的影响。方法：随机将有胆囊切除手术指征的80例患者分为2组,腹腔镜胆囊切除组（laparoscopic cholecystectomy,LC组）和开腹胆囊切除组（open cholecystectomy,OC组）各40例,测定并比较手术前后IgG、IgM、IgA,补体C3、C4水平及CD3^＋（T细胞总数）、CD4^＋（T辅助/诱导细胞）和CD8^＋的数量。结果：两组IgM、IgA、C4手术前后均无明显变化,两组间差异无统计学意义。LC组术后1d IgG、C3较术前有所下降,术后3d恢复至术前水平;OC组术后1d IgG、C3明显低于术前水平,术后5d恢复至术前水平;组间比较,OC组术后IgG、C3下降明显。LC组T淋巴细胞亚群手术前后差异无统计学意义,OC组术后1d CD3^＋、CD4^＋、CD8^＋与术前比较明显降低,术后5d恢复至术前水平;组间比较,术后1d、3d OC组CD3^＋、CD4^＋、CD8^＋均明显低于LC组。结论：腹腔镜手术对机体的免疫功能影响小,术后恢复快。     

猪到猕猴异种心脏移植超急性排斥反应的免疫学及病理学变化

目的观察猪到猕猴异种心脏移植超急性排斥反应时的免疫学及病理学变化。方法采用猪到猕猴腹腔内异位心脏移植模型,检测发生超急性排斥反应者的血液中补体、天然抗体及T淋巴细胞亚群的变化,并对移植心脏进行免疫组化(测定C3、C4、C5b9、IgG及IgM的沉积)及病理学分析。结果发生超急性排斥反应时,血清补体C3、C4的含量、总补体活性及抗猪内皮细胞天然抗体均有一定程度的下降;CD4 /CD8 T淋巴细胞的比率也有所下降;移植心脏中均有补体C3、C4、C5b9的沉积,IgG及IgM也均有沉积,但IgG和IgM沉积强度的差异无统计学意义;病理学改变主要为心肌间质弥漫性出血、水肿,毛细血管内普遍淤血。结论补体通过经典途径激活参与猪到猕猴异种心脏移植超急性排斥反应;超急性排斥反应时受者血中天然抗体水平明显下降;CD4 T淋巴细胞可能参与异种移植超急性排斥反应过程并有所消耗;发生超急性排斥反应的移植物突出病理表现为间质出血。     

抗炎灵对腹部外科SIRS/MODS患者免疫功能的影响

目的：动态研究观察抗炎灵对腹部外科SIRS／MODS患者免疫功能的影响。方法：以腹部外科手术SIRS／MODS52例患者为研究对象,按入院时间分为对照组21例和抗炎灵治疗组31例,分别于术后1、3、7d观察血清I gA,IgG,IgM,C3,C4水平。结果：血清IgG,IgM,IgA和补体C3、C4后逐渐上升,于第3d时IgG,IgM,IgA两组有显著差别（P＜0．05）,于第7d时,C3、C4两组有差异（P＜0．05）,治疗组上升较快,结论：抗炎灵能提高机体免疫能力,对SIRS／MODS有一定的治疗作用。     

中药对手术后免疫功能的影响

目的:观察中等以上手术病人应用中药免疫调理后机体免疫功能的变化.方法:80例病人随机分为中药调理组和对照组各40例,分别于术前、术后第1 d及第7 d检测IgG、IgA、IgM、C3、T细胞亚群.结果:术后第1 d,IgG、IgA、IgM、C3、T细胞亚群,中药调理组与术前比较IgG、IgA、IgM、C3无显著变化,CD3 、CD4 、CD4 /CD8 显著下降(P＜0.05,或P＜0.01),对照组均显著下降(P＜0.01),术后第7 d中药调理组恢复快,达到或超过术前水平,对照组恢复慢,与术前比较仍有显著差异(P＜0.01).手术前后中药调理组IgG、IgA、IgM、C3、CD3 、CD4 、CD4 /CD8 的差值显著高于对照组(P＜0.01).结论:中等以上手术的病人,术后早期免疫功能受抑制,在围手术期应用中药调理可以有效地改善免疫功能.     

The influence of membrane molecular weight cutoff on a novel bioartificial liver

Given the xenogeneic immune reaction relevant to the molecular weight cutoff of the membrane of a bioartificial liver (BAL) system, we investigated the influence of membrane molecular weight cutoff in our BAL system in this study. Acute liver failure in beagles was induced by d-galactosamine administration. Eight beagles were divided into two groups by the membrane molecular weight cutoff of the plasma component separator. Group 1 beagles were treated with BAL containing 200 kDa retention rating membrane. Group 2 beagles were treated with BAL containing 1200 kDa retention rating membrane. Each group underwent two 6-h BAL treatments that were performed on day 1 and day 21. The hemodynamic and hematologic response, humoral immune responses, and cytotoxic immune response to BAL therapy were studied before and after treatments. All beagles remained hemodynamically and hematologically stable during BAL treatments. BAL treatment was associated with a significant decline in levels of complement; however, a longer time of level maintenance was observed in Group 2. Group 2 beagles experienced a significant increase in levels of IgG and IgM after two BAL treatments. Significant levels of canine proteins were detected in BAL medium from Group 2; only trace levels of canine proteins were detected in BAL medium from Group 1. The posttreatment viability of co-culture cells in Group 2 was lower compared with Group 1, and the viability of co-culture cells after treatments was associated with deposition of canine proteins on the cells. Xenogeneic immune response was influenced by membrane molecular weight cutoff in the BAL.     

Effects of membrane molecular weight cutoff on performance of a novel bioartificial liver

Immunoisolation using semipermeable membranes has been incorporated into bioartificial liver (BAL) devices to separate cellular components of the recipient's immune system from the cells within the BAL device. This study was designed to explore the influence of membrane molecular weight cutoff on performance of the multilayer radial‐flow BAL using porcine hepatocytes cocultured with mesenchymal stem cells. In this study, healthy beagles underwent 6‐h treatment with a BAL containing membrane with 200 kDa retention rating or 1200 kDa retention rating. Functional markers of BAL performance were monitored before and after treatment, as well as cytotoxic immune response to BAL therapy. The results showed that hepatocyte performance levels such as albumin secretion, urea synthesis, and viability were all significantly higher in 200 kDa retention rating group compared with the 1200 kDa retention rating group after treatment (P < 0.05). Significant levels of canine proteins were detected in BAL medium from the 1200 kDa retention rating group. Fluorescence microscopy further verified that heavy deposition of canine IgG, IgM, and complement (C3) on coculture cells was obtained after BAL treatment in the 1200 kDa retention rating group. However, only trace deposits of canine immunoproteins were observed on coculture cells obtained from BAL in the 200 kDa retention rating group. Small membrane molecular weight cutoff of the BAL could reduce the transfer of xenoreactive antibodies into the BAL medium and improve the performance of the BAL.     

生物人工肝的细胞培养及反应器进展

生物人工肝支持系统(BALSS)由肝细胞、专用反应器和体外循环系统三大要素共同构建。其中生物活性部分的体外培养技术与反应器设计的优劣是影响BALSS性能的重要因素。为使培养肝细胞的数量、活性和功能更理想,近年各种培养技术及其相应反应器均从各个方面改进以期实现"拟肝化培养"。现就目前常用于BAL的肝细胞培养方法及其相应反应器的现状及进展进行综述。     

Effect of immune response during cardiopulmonary bypass on post-operative infections

To study the effects of extracorporeal circulation on the post operative infections, the relationship between the influences of the immunologic change during cardiopulmonary bypass (CPB) and post operative change of neutrophilia was evaluated in 52 patients. These patients were divided into two groups: group A (non-infected), 19 patients whose neutrophil count increased within 15,000/microliters in peripheral blood after operation. Group B (infected), 33 patients whose neutrophil count increased 15,000/microliters or more within one week after operation. Immunoglobulins (IgG, IgA, IgM) and complement fraction (C3, C4, CH50) levels decreased significantly during CPB, but there was no significant difference between the two groups. The amount of lymphocyte subsets of helper/inducer T cell and suppressor/cytotoxic T cell showed similar change in both groups. On the contrary the amount of lymphocyte subset of Natural Killer cell showed significant increase in A group during CPB when compared with B group, suggesting immunological activation, however, in the group which showed less than 40% increase of NK cell during CPB, there was significant increase of neutrophils after operation compared with the other group which showed high increase of NK cell. These findings suggested the increased activation of the Natural Killer cell during CPB is a favorable response, especially for preventing infections after operation.     

The influence of extracorporeal circulation on erythrocytes and flow properties of blood

The influence of extracorporeal circulation on red blood cells and flow properties of blood was studied in 10 patients undergoing aorta-coronary bypass grafting. Blood samples were drawn on admission, under general anesthesia before the operation, during extracorporeal circulation, immediately after extracorporeal circulation, and 24 hours after extracorporeal circulation. Echinocytes were found during and shortly after extracorporeal circulation, but disappeared within 24 hours. Washing the cells in buffer restored the normal discocytic shape, which indicated that a plasma factor was responsible. Red cell membrane lipids were not affected. Analysis of the membrane proteins revealed a decrease of ankyrin after extracorporeal circulation, which was prevented by protease inhibitors during preparation. This suggests an increased proteolytic activity of the plasma after extracorporeal circulation. Red cell deformability was not altered. Plasma viscosity and hematocrit were markedly reduced by hemodilution with the priming solution. Their low levels resulted in a low blood viscosity during extracorporeal circulation, which was even lower at 26 degrees C than before or after the operation at 37 degrees C. We conclude that the red cell is affected by extracorporeal circulation. The flow properties of blood, however, are not impaired, but are improved by hemodilution.     

IgM and IgG alloantibody production by splenocytes and deposition in rat renal allografts are decreased by donor-specific blood transfusion

Untreated PVG (RT1c) rats reject ACI (RT1a) renal grafts in 6-8 days. Autologous (PVG) blood transfusions (ABT) do not alter ACI allograft rejection, but donor-specific blood transfusions (DSBT) 7 or 11 days prior to transplantation usually results in indefinite graft survival. We have reported previously that DSBT is associated with the development of antiidiotypic antibody and reduced circulating cytotoxic alloantibodies in this model. To further define the effects of DSBT on the alloantibody responses to renal allografts, we examined PVG rats that received DSBT or ABT prior to ACI renal transplantation. Antibody production by cells in the spleen was investigated by tissue culture techniques; circulating antibody titers were measured by antibody binding to target ACI lymphoblasts with flow cytometry; and antibodies bound to the ACI allograft were recovered by hypertonic acid elution and quantitated by flow cytometry and ELISA. Seven days after DSBT alone, circulating IgM alloantibodies to ACI reached peak titers. After renal allografting, serum IgM alloantibody titers decreased in DSBT-pretreated rats and little IgG could be detected. In contrast, renal allografts in ABT-pretreated rats elicited high titers of IgM and moderate titers of IgG in the circulation by 5-7 days posttransplantation. Spleens harvested one week posttransplantation from ABT-pretreated rats produced high titers (16-32) of IgM and IgG antibodies to ACI antigens, but no such antibody production was detected in spleens cultured from DSBT-pretreated rats. In addition, 4-32-fold more IgM and IgG was eluted from kidneys removed 6-7 days after grafting to ABT-treated rats than from allografts in DSBT-treated rats. IgG2a was the predominant subclass of IgG that bound to target ACI cells. No IgA was detected in graft eluates from any rat. Polyacrylamide gel electrophoresis demonstrated that the eluates contained predominantly IgM and IgG without significant contamination by other serum proteins. These data suggest that DSBT decreases the levels of IgM and IgG normally produced in the spleen and deposited in the graft following renal transplantation. Because IgM fixes complement and IgG (especially IgG2a) triggers ADCC, the reduced deposition of IgM and IgG in the graft may be of particular importance in DSBT enhancement.     

Cytotoxic immune response to a xenogeneic bioartificial liver

Prior studies have suggested the possibility of immune-mediated death of xenogeneic hepatocytes in a bioartificial liver (BAL) during hemoperfusion. This study was designed to elucidate how immunity may cause death of xenogeneic hepatocytes in the BAL. Healthy dogs were treated with a BAL containing hollow fiber membranes with large pores (200 nm) or small pores (400 kDa). The immune response of recipient dogs to BAL therapy was monitored over 3 h of treatment. We observed significantly greater loss of viability of hepatocytes in the 200 nm group compared with the 400 kDa group (p < 0.001). Low viability after treatment with the large pore membrane was associated with positive staining for dog IgG, dog IgM, and dog complement on dead hepatocytes. Significant levels of dog antibody were detected in samples of BAL medium from the 200 nm group. These canine antibodies were cytotoxic to porcine hepatocytes. In contrast, medium from the 400 kDa group contained only trace levels of dog IgG and were noncytotoxic. We conclude that antibody-mediated cytotoxicity contributed to the death of hepatocytes during treatment with a xenogeneic BAL. Immune-mediated death of hepatocytes was reduced by increasing selectivity of the BAL membrane.     

生物人工肝支持系统生物反应器的建立及体外转流试验

目的 探讨由微载体培养的L－02人肝细胞和中空纤维舱构成的生物反应器的生物效能。方法 采用微载体培养高浓度L－02人肝细胞，同时使用中空纤维型生物反应器和血泵等共同构成生物人工肝系统。在体外转流试验中观察循环液中游离胆红素、葡萄糖、白蛋白、谷草转氨酶浓度的变化以及实验对肝细胞的影响等。结果 L－02肝细胞能很好地粘附于cytodex 3微载体上形成微载体诱导的肝细胞聚集体；移入生物反应器内进行体外循环，4h后可见循环液中游离胆红素和葡萄糖浓度显著降低，分别为27.1μmmol/L和1.75mmol/L；白蛋白含量明显增加，为15．21mg/L；肝细胞仍有较高活力达75％。结论 本实验所建立的生物反应器作为生物人工肝系统的核心组件具备一定的生物合成和解毒代谢功能，为进一步建立混合型生物人工肝支持系统奠定了基础。     

Acute humoral rejection of renal transplants in alloimmunized pigs

BACKGROUND: Despite progress in immunosuppression, acute humoral rejection (AHR) remains an unsolved issue in transplantation, with a possible reversibility in only about 50% of cases. AHR is characterized by antidonor antibodies in the recipient circulation and characteristic histological lesions within the graft itself, as well as complement degradation products and immunoglobulins (IgM and IgG) deposition in vascular zones. The lack of a large animal models of AHR in experimental allotransplantation led us to establish such a model in the pig. MATERIALS AND METHODS: Pigs were immunized with peripheral blood mononuclear cells from allogeneic donors and subsequently received a kidney from the same donor, once antidonor antibodies (Ab) had reached a plateau. The efficiency of the alloimmunization, the nature of the induced antibodies and rejection were studied. RESULTS: Six out of seven recipients developed specific antidonor Ab. Three days post-transplantation, characteristic lesions of AHR were observed together with intragraft IgG, IgM, and complement deposition. The AlloAb were found to be cytotoxic and directed against donor MHC class I molecules. CONCLUSIONS: We described, for the first time, a large animal model of AHR, which will enable us to more extensively study phenomena implicated in AHR and to test new strategies aimed at its prevention or cure, as well as improve transplant protocols in the case of presensitized or hyperimmunized patients.