自体移植脾巨噬细胞C3b、Fc受体及蛋白质表达的实验研究

目的 探讨自体脾组织移植后的功能状况。方法 采用小鼠进行自体脾组织网膜内移植，术后6个月切取移植脾组织，检测巨噬细胞的Fc、C3b受体及蛋白表达。结果 自体移植脾巨噬细胞Fc受体的含量与原位脾相近，C3b受体的功能正常；蛋白质的表达与原位睥相同。结论 大网膜内自体移植脾组织的功能在细胞水平是正常的，移植脾组织具有原位脾的功能。     

大剂量免疫球蛋白对抑制性受体的免疫调控作用

目的通过观察大剂量免疫球蛋白(IVIG)作用前后某些免疫细胞表面抗体活化性受体(FcγRⅡa)和抑制性受体(FcγRⅡb)的表达，探讨IVIG免疫调节作用的机理。方法运用RTPCR、Western blot和FACS的方法来检测某些免疫效应细胞表面的FcγRⅡa和FcγRⅡb的表达，并观察IVIG作用前后两者的表达差异。结果FcγRⅡa主要表达于单核巨噬细胞，在B细胞和T细胞中没有表达。FcγRⅡb在检测的细胞中无表达。在组织淋巴瘤U937细胞中FcγRⅡa的表达量比较高。U937细胞在经过IVIG作用后，抑制性FcγRⅡb的表达逐渐升高，24h时表达达到最高。在IVIG作用后，FcγRⅡb的表达在蛋白水平也逐渐增高，与RT-PCR的结果一致；U937细胞表面的FcγRⅡb表达显著增高，表现为荧光强度峰值的增强和右移。用IVIG治疗的患者，单核巨噬细胞表面的FcγRⅡb的表达比正常人外周血单核巨噬细胞明显增加。结论IVIG的免疫调节作用可部分归因于增加某些免疫效应细胞表面抗体抑制性受体FcγRⅡb的表达。     

高迁移率族蛋白B1对小鼠巨噬细胞免疫功能影响的受体机制

目的探讨高迁移率族蛋白B1(HMGB1)对巨噬细胞免疫功能影响的可能受体机制。方法分离培养小鼠腹腔巨噬细胞,HMGB1刺激后,采用激光扫描共聚焦显微镜和流式细胞术检测细胞表面晚期糖基化终末产物受体(RAGE)的表达。实验分为正常对照组、HMGB1组、HMGB1 抗RAGE抗体组、HMGB1 rmRAGE/Fc组,分别观察巨噬细胞摄取中性红能力、对L1210细胞的杀伤作用(MTT法)、趋化活性(采用Transwell小室趋化装置)、细胞表面I-Ak抗原表达(流式细胞术)的变化。结果100μg/L的HMGB1作用于巨噬细胞48 h后,巨噬细胞表面受体RAGE表达明显上调(P<0.01);HMGB1组巨噬细胞吞噬功能、杀伤活性、趋化活性及I-Ak抗原表达均显著高于对照组、HMGB1 抗RAGE组与HMGB1 rmRAGE/Fc组(P<0.01)。结论HMGB1可诱导RAGE表达增强,RAGE是HMGB1作用于巨噬细胞免疫功能的主要受体之一。     

自体移植脾组织VEGF、KDR表达与血管再生的实验研究

目的 研究自体移植脾组织血管再生及VEGF、KDR表达规律，阐明VEGF、KDR对移植脾组织血管再生的调控作用，为脾脏外科临床及实验研究提供理论依据。方法 健康Wistar大鼠70只，体重100—120g，随机分为7组，每组10只中又设脾切除自体脾移植组5只，假手术组5只，分别于术后7，14，30，60，90，120，180d进行：(1)自体移植脾组织病理学检测；(2)大鼠行主动脉插管灌注墨汁，光镜观测再生血管并采用图像分析测定其密度；(3)免疫组化抗VEGF、KDR抗体染色，图像分析定量，阐明其表达规律及与血管再生的关系。结果 (1)自体脾组织移植术后7d即有血管从大网膜向脾组织内伸展，移植脾组织内血管密度逐渐增大，至术后180d血管再生接近正常；(2)自体脾组织移植术后7d、14d，VEGF、KDR阳性染色细胞密度迅速升高，术后60d达高峰，以后逐渐降低，至术后180d VEGF、KDR阳性染色细胞密度趋向正常。结论 自体脾组织大网膜内移植术是简便有效的脾移植方法；移植脾组织新生血管由大网膜再生而来；术后移植脾组织内VEGF、KDR表达量升高，促进血管形成，血管再生完成后恢复正常水平。     

I型IP_3受体在自体中厚移植皮片中的表达

目的：检测I型1,4,5--三磷酸肌醇受体（type I inositol 1,4,5-t riphosphate receptor,IP3R1）在人自体中厚移植皮片中的表达,初步认识由IP3受体介导的钙信号转导在自体中厚移植皮片过度色素沉着发生机制中的作用。方法：利用免疫组化SP方法,检测IP3R1在自体移植中厚皮片、受区周围与供区正常皮肤中的表达并进行统计学分析。结果：IP3R1的表达定位于表皮基底部黑素细胞、角质形成细胞胞浆,在大部分自体中厚移植皮片中呈强阳性表达,与自体正常对照皮肤中的表达差异有显著性意义（P〈0.05）;IP3R1在受区周围和供区正常对照皮肤中的表达差异无显著性意义。结论：IP3R1在自体中厚移植皮片中的表达比自体正常对照皮肤中显著提高,推测由IP3R1介导的胞内钙库钙离子释放参与了钙信号转导在自体移植皮片过度色素沉着中的调控作用。     

脾损伤自体脾组织移植的临床应用

目的：探讨自体脾组织移植在临床中的应用。方法：总结32例脾外伤行全脾切除自体脾组织移植手术，其中采用大网膜囊内移植18例，去粘膜游离空肠段内移植12例，腹直肌鞘内移植2例。结果：术后随访均显示脾功能满意，尤以去粘膜游离空肠段内移植效果最好。结论：自体脾组织移植可作为严重脾外伤、全脾切除术后保留脾功能的一个重要有效手段，移植脾的功能恢复与血供有密切的关系。     

自体移植脾组织的血流量及形态学的变化

对自体移植脾的组织量和血流量以及形态学进行动态观察，实验用大鼠分别于术后３、５、７、１２月通过生物微球法对和人动物保留１／４原位脾和地脾进行检测。结果得出，自体移植各时相点的单位重量血流量、血容积和吞噬指数与原位保留的相差不显著。原位保留的残脾重量代偿性增加，但移植脾组织量明显减轻，因而移植脾的绝对血流量也相应减少，揭示移植脾组织应尽量增多。镜检移植脾组织随着时间推移显示正常结构。     

自体脾组织移植后病理学变化的实验研究

目的　研究自体脾组织移植后不同时相点病理形态学变化规律。　方法　健康Wistar大鼠 5 6只 ,雌雄不限 ,体重 10 0～ 12 0 g ,随机分为 7组 ,每组 8只中又设脾切除自体组织大网膜内脾移植组 5只 ,假手术组 3只 ,分别于术后 7、14、3 0、60、90、12 0、180d取脾组织 ,光镜、透射电镜观察。　结果　移植脾组织之重量在术后 7d最轻 ,为 0 .0 72 g ,其后逐渐增加 ,180d时为 0 .5 11g ,各时相点之间有明显差异 (P <0 .0 5 )。病理检查提示移植脾组织经历急性期、缓解期、修复期三个病理时期 ,逐步恢复并接近正常的组织结构。　结论　自体脾组织大网膜内移植术是简便有效的脾移植方法。自体移植脾组织再生过程可分为 :急性期、缓解期与修复期     

体外转染可溶性IL-1RI-Ig基因延长糖尿病小鼠移植胰岛细胞存活时间

目的 探讨糖尿病小鼠移植经可溶性白细胞介素1(IL-1)受体胞外段Fc融合蛋白(sIL-1RI-Ig)基因修饰的胰岛细胞对延长移植物存活时间的作用及机制.方法 将Ad-sIL-1RI-Ig体外转染Balb/c小鼠的胰岛细胞,并将其移植给糖尿病C57BL/6小鼠,观察移植物的存活时间,并检测移植局部组织炎症细胞的浸润以及炎症细胞因子的表达.结果 糖尿病小鼠移植转染sIL-1RI-Ig基因的胰岛细胞后,血糖水平很快下降至正常范围,胰岛移植物存活时间达(39±3)d,而移植正常胰岛细胞的小鼠胰岛移植物存活时间为(9±2)d(P<0.01).糖尿病小鼠移植转染sIL-1RI-Ig基因的胰岛细胞后,胰岛素分泌量随糖负荷增加而升高,并与胰岛素分泌功能正常的小鼠呈相似变化趋势(P>0.05).糖尿病小鼠移植转染sIL-1RI-Ig基因的胰岛细胞后,移植局部组织表达肿瘤坏死因子α(TNF-α)、γ干扰素(IFN-γ)和RANTES等水平明显下调;病理学观察发现移植局部组织浸润的炎症细胞明显少于移植正常胰岛细胞的小鼠.结论 sIL-1RI-Ig基因转染胰岛细胞后,可通过表达sIL-1RI-Ig,阻断IL-1的作用,降低TNF-α、IFN-γ、RANTES等细胞因子的表达,从而减轻排斥反应,延长胰岛细胞移植物的存活时间和提高胰岛素分泌功能.     

脾组织移植保留脾损伤患者脾功能的实验研究

自体脾组织移植已成为保留严重脾损伤患者脾功能的措施之一,但移植牌组织的功能如何,对机体究竟有多大影响,尚未定论。提高自体移植脾组织的功能已成为人们普遍关心的问题。因此,本文研究了动物自体移植脾组织的结构与功能;初步探索评价了促进自体移植脾组织生长的措施。     

Immunoarchitecture and specific functions of splenic autotransplants at different implantation sites.

The present study deals with the morphological and functional development of intraomentally and subcutaneously implanted splenic tissue. Spleens and splenic transplants from 138 Lewis rats were investigated with immunohistological, immunological and molecular biological methods at different times after operation (up to 200 days postoperatively). The analysis of the development revealed a nonsignificant reduction concerning the weight of subcutaneous replants and a nonsignificant decrease of the weight of female transplants of both groups at different phases after operation. The cell composition of cell suspensions from spleen and both transplant types showed a deficiency of T, B, MHC-I+ cells and a certain macrophage subset (ED-3+ cells) in transplants. In a quantitative immunohistological analysis of compartments (red pulp, periarteriolar lymphoid sheaths, marginal zone and follicles) the T cell reduction was related to the Tsupp/cyt cells and T cell receptor bearing cells in the periarteriolar lymphoid sheaths, whereas the density of T helper cells was normal. In addition, a different homing of kappa-light chain positive and leukocyte common antigen (B cell type)-positive B cells in follicles and marginal zone was detected. The amount of two macrophage subsets (ED-1+ and ED-2+ cells) was increased in the red pulp. Only minor differences in the immunoarchitecture of transplants at different implantation sites were measured. A functional analysis of spleen compared to both transplant groups elicited a B cell defect after LPS stimulation in subcutaneous transplants and a reduced allogeneic response of both transplant types but a normal proliferation of T cells after ConA stimulation and a correct IgM antibody response against sheep red blood cells. The in vivo mRNA expression and the expression kinetics of interferon-gamma and granulocyte-macrophage colony-stimulating factor after antigen stimulation differed in both transplant groups with a remarkable permanent expression of both mediators in subcutaneous transplants. It can be summarized that the results clearly indicate a development of spleen-like immunoarchitecture of intraomental replants with subtle cellular, functional and molecular alterations. In contrast, despite a comparable development, some severe functional defects occurred in subcutaneous implants pointing out the important role of interactions between the regenerating splenic tissue and the target tissue on a functional and molecular level.     

Alteration of proliferation and subtle changes of protein synthesis in autologous transplanted spleens.

In the case of massive splenic rupture, heterotopic autologous transplantation of splenic tissue into the omentum majus may be used to restore splenic function. Yet little is known about specific functions of the transplants compared to the normal spleen. The goal of this study was to get more information about immunologic functions and protein expression in splenic transplants. As an animal model we used the pig, whose splenic morphology and immunoarchitecture is similar to that of the human spleen. Histologic examination of transplants revealed structures that were comparable to normal spleens (consisting of red and white pulp and including germinal centers). Immunologic tests such as the hemolytic plaque assay and mitogen stimulation revealed that the number of plaque-forming cells was not changed significantly, but the stimulation index for T cells was drastically increased in the autotransplants. Electrophoresis and immunochemical methods showed differences in the protein patterns between both tissues. Several proteins were found to be produced only in the spleen, or were produced in much higher amounts in the spleen than in the splenic transplants. More information about these differences between spleen and splenic transplants is needed before we can recommend a general clinical application of autologous spleen transplantation.     

Influence of donor and host age on the regeneration and blood flow of splenic transplants

After splenectomy there is an increased risk of fatal overwhelming postsplenectomy sepsis, especially in children. If all alternatives to splenectomy fail, autotransplantation of splenic fragments is indicated. These fragments regenerate after a necrotic phase to small splenic nodules. Regulatory factors governing the regeneration process are largely unknown. Inbred rats were used as a model to define the influence of recipient and donor age on the regenerated mass and the blood flow of transplanted splenic fragments. These are both important factors for the protective function of the spleen. Fetal, newborn, weanling, or adult spleens were implanted into the greater omentum of newborn, weanling, or adult rats. The younger the recipient and donor, the better the regeneration and perfusion of transplants. However, these did not reach more than 40% of the normal splenic mass. In addition, no experimental group achieved more than one third of the normal splenic blood flow. There is an obvious age dependency in splenic regeneration and blood flow, but the transplants are far from attaining a normal splenic mass and perfusion.     

Regeneration and function of autologous splenic grafts in pigs

The growth kinetics, perfusion and immune response of autotransplanted splenic tissue were studied in pigs. Splenic fragments were transplanted subfascially or in the greater omentum and regenerated to small splenules with a normal histologic structure 6 months later. The grafts showed a normal function of the white pulp after antigenic stimulation, and of the red pulp as demonstrated scintigraphically. The blood flow was reduced. The growth kinetic was less rapid than in rodents and resulted in a relatively lower mass of splenic tissue, but these results are closer to findings in human splenosis.     

Experimental study on function of regenerated splenic tissue after splenic autotransplantation

To prevent postsplenectomy overwhelming sepsis, splenic autotransplantation has been clinically attempted. However, function of regenerated splenic tissue after splenic autotransplantation has not been completely understood. Changes in weigh of regenerated splenic tissue, splenic blood flow, splenic immune responses and phagocytic function were studied for one year after splenic autotransplantation using Sprague-Dawley rats. At one year after autotransplantation, the weight of regenerated splenic tissue was increased to 80% of the originally implanted spleen and the blood flow was increased to 80% of the control spleen. The counts of lymphocytes and macrophages in the regenerated splenic tissue were significantly low at eight weeks after transplantation, however lymphocytes was increased to 58.8% and macrophages was increased to 29.5% of the control spleen at 16 weeks after transplantation. The blast formation of splenic lymphocytes was lower at the early stage after transplantation, thereafter, it was increased at the later time after transplantation. Microangiography of the regenerated spleen showed new capillaries around the implanted tissue 2 weeks after transplantation. These results suggested that the transplanted splenic tissue was regenerated to the similar structure to normal spleen and immunological function was recovered close to the normal splenic tissue.     

Does survival depend on the amount of autotransplanted splenic tissue?

Susceptibility to Streptococcus pneumoniae infection was studied in 11 groups of rats allocated to sham operation, splenectomy, or splenic autotransplantation of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the removed spleen. Three months later, all rats were exposed intravenously to type 1 Streptococcus pneumoniae (median lethal dose, LD50, for control group). Survivors were killed 13 days after the bacterial challenge. Autopsy showed that more splenic tissue was recovered in rats that received less than 50% splenic tissue compared with those that received 50% or more. More survivors were found among sham-operated rats (47.5%; 95% confidence intervals, 32 to 68) and rats that had 40% splenic tissue implanted (35%; confidence interval, 20 to 54) or those that were found to have regenerated 40% splenic tissue. We conclude that 40% of the spleen should be autotransplanted to protect the rat optimally against infection after splenectomy.     

Splenic tissue autotransplantation in rabbits: no restoration of host defense

BACKGROUND: The loss of spleen may increase the incidence of overwhelming sepsis. To prevent this, splenic autotransplantation has been performed in humans and experimental animals. However, there is still controversy about the effectiveness of regenerated splenic tissue in preventing infection. This study explored the effectiveness of splenic tissue autotransplantation in restoring host defense. MATERIALS AND METHODS: Rabbits were divided into three groups: splenic autotransplantation, sham operation, and total splenectomy. Histomorphology, T-lymphocyte count, serum lysozyme levels, hemolysin titers, and pneumococcal clearance were observed as read-out parameters over 24 weeks. RESULTS: Histological study showed that the white pulp was poorly developed and central arterioles were missing in the regenerated splenic tissue of the autotransplanted rabbits. The weight of regenerated spleens recovered 6 months later in the splenic autotransplantation group was 11% of that in the sham operation group and was significantly less than the weight at implantation. There was no significant difference in the number of T lymphocytes or level of serum lysozyme between the three groups. A poor antibody response by the rabbits in the splenic autotransplantation and total splenectomy groups was noted after the primary intravenous administration of sheep red blood cells compared to those of sham operation group. After the challenge with type 3 pneumococci intravenously, pneumococcal clearance from the bloodstream in the splenic autotransplantation group did not differ significantly from that in the total splenectomy group, but was markedly delayed compared with that in the sham operation group. CONCLUSIONS: The low quantity and poor quality of the regenerated splenic tissue contribute to the inferior immunoprotective ability of animals autotransplanted with one-third of the original spleen. This suggests that the regenerated spleen cannot compensate for the immunological function of the original one, especially host resistance to infection.     

Serum antibody responses to pneumococcal vaccine after splenic autotransplantation

Immunization with pneumococcal capsular polysaccharide vaccines is advocated after splenectomy; however, experimental and clinical data suggest an impaired antibody response in splenectomized individuals. This study examined the value of splenic autotransplantation at various sites in augmenting the antibody response to Type III pneumococcal capsular polysaccharide in mice immunized 3 months after operation. Splenectomy resulted in impaired antibody responses compared to sham-operated mice (p less than 0.001) using an enzyme-linked immunosorbent assay. Mice with intraperitoneal splenic autotransplants, but not mice with subcutaneous or intramuscular transplants, had greater antibody responses compared to splenectomized mice (p less than 0.05). Antibody responses were elevated only in mice autotransplanted with 50% or more of the original splenic mass. Since autotransplantation of splenic tissue augments the antibody response to pneumococcal capsular polysaccharides, the combination of splenic autotransplantation and pneumococcal vaccination may confer more protection than either modality alone in individuals who must undergo splenectomy.