1.异种胰岛移植中巨噬细胞免疫排斥及调节的研究 2.GLUT1及Ang1与糖尿病视网膜病变关系研究
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摘要
研究背景
细胞移植是治疗很多疾病的有效治疗手段，异种细胞移植为供体短缺提供了良好的解决办法，但是，免疫排斥问题仍然是阻碍它走上临床的最大难题。与实体器官移植不同的是，针对细胞异种移植物的免疫排斥反应主要由细胞介导的排斥机制参与，CD4+T细胞在其中起效应细胞的作用。已有实验通过间接手段证实，巨噬细胞在异种移植的免疫反应中起效应细胞的作用，破坏移植物。早已有人提出，巨噬细胞可以浸润异种移植物，发挥特异性排斥的功能，但是缺乏直接的证据。我们以前的实验将胎猪胰岛细胞移植入NODSCID小鼠肾被膜下，然后用CD4+细胞重建，巨噬细胞被分离并转入未经过CD4+T细胞重建的、已接受猪胰岛异种移植的NODSCID受体身上，在无需CD4+T细胞再次活化的情况下，后者的异种移植物被特异性的排斥，为CD4+T细胞活化的巨噬细胞作为效应细胞排斥异种移植物提供了证据。
目的
本实验中，我们建立了混合移植的糖尿病小鼠模型，以寻找支持活化的巨噬细胞能够特异性的识别并排斥猪胰岛细胞的更直接的证据。
方法
1胰岛移植与巨噬细胞重建：STZ诱导的糖尿病NODSCID小鼠分别接受：Balb／c小鼠的胰岛移植(AlloTx组)，混合移植(MixTx组)(左侧接受小鼠胰岛与NICC的混合移植物，右肾接受NICC异种重建后移植物)和NICC异种移植。用CD4+T细胞活化的巨噬细胞重建接受同种移植及混合移植的NODSCID，重建后第5周取出混合移植物，第8周处死所有小鼠。实验过程中检测血糖。
2．对组织块进行形态学观察及胰岛素、F4／80巨噬细胞免疫组化染色，观察移植物。
结果
1．血糖变化AlloTx组和MixTx组，在胰岛移植后血糖明显下降，巨噬细胞重建后血糖仍然维持正常重建后第5周，MixTx组小鼠混合移植物被取出，血糖上升，明显高于AlloTx组小鼠。
2．组织学观察H&E染色显示，AlloTx组小鼠肾被膜下可见完整的小鼠胰岛。在MixTx组小鼠的左肾组织，还可以见到完整的胰岛组织，但是在右肾，未见胰岛结构，可见大量的细胞浸润。在NICC对照组异源性胰岛组织清晰可见。
3．免疫组化在AlloTx组，胰岛素染色可见棕色的阳性颗粒。在MixTx组，左肾可见清晰的胰岛素染色阳性，而在右肾，胰岛素几乎没有阳性颗粒，在NICC对照组可见大量的胰岛素阳性染色。巨噬细胞染色，在AlloTx组，仅可见少量阳性染色，在胰岛内部，看不到任何阳性颗粒，在MixTx组，左肾可见巨噬细胞浸润，但是没有侵蚀到胰岛内部。巨噬细胞在右肾被膜下大量浸润。
结论
1．本实验直接证明在猪异种胰岛抗原存在的情况下，用CD4+T细胞活化的巨噬细胞可以特异性的识别并排斥猪异种胰岛移植物。
2．活化的巨噬细胞可以从外周自动聚集至移植物局部。
3．这种特异性识别的具体作用机制需要进一步研究。
研究背景
异种胰岛移植是治疗1型糖尿病的有效手段，但移植物排斥仍是它的主要副作用。细胞异基因移植物易受细胞介导的免疫破坏，在早期的排斥反应中，非特异性免疫系统中的抗体和补体成分是介导排斥的主要因素，在急性和慢性细胞免疫排斥中，T细胞则是重要的反应细胞，它可以通过募集巨噬细胞发挥排斥作用。巨噬细胞既是早期非特异性反应细胞，在被CD4+T细胞活化后，又是主要的效应细胞。人们利用药物治疗以及基因调控供体动物的方法，有限抑制了超急性排斥甚至迟发性血管性排斥，延长了猪异种器官在非人类灵长类的存活时间，于是由特异性免疫系统介导的细胞排斥反应成为克服移植物排斥和增加移植物存活时间的亟待解决的问题。上世纪90年代，研究者们发现，占外周淋巴细胞总数5％—10％的CD4+CD25+调节性细胞(CD4+CD25+Regulatory T Cells，Tregs)可以制止器官特异性自身免疫性疾病的发病，维持同种异体移植的免疫耐受。在异种移植中，Porter等报道人Tregs可以抑制CD4+CD25—T细胞的增殖和细胞因子的产生，还可以明显抑制CD4+CD25—T细胞介导的对猪靶细胞的溶细胞作用。亦有证据显示人Tregs可以抑制树突状细胞的成熟和抗原递呈功能，可以激发直接抑制单核／巨噬细胞的作用进而影响非特异性和特异性免疫。然而，目前Tregs能否对异基因刺激的巨噬细胞产生抑制作用尚未见研究。
目的
我们以前的研究显示，体外扩增的Tregs可以抑制T细胞介导的针对猪异种抗原的免疫反应，在本实验中，我们利用扩增的Tregs，研究其对异种细胞激活的巨噬细胞是否具有抑制作用以及可能的机制
方法
新鲜Tregs、CD14+单核细胞、去CD4+细胞的PBMC细胞，均来自健康志愿者外周血单个核细胞(Peripheral blood mononuclear cells，PBMC)，用含10％FCS，Anti-CD3／CD28 beads，IL2及Rapamycin的RPMI1640培养液体外扩增，培养2至3周的细胞用于下游实验。经射线照射的猪PBMC(PIG)作为异种抗原。
实验中有3个培养体系：1．PBMC体系分PBMC、PBMC+PIG、PBMC+PIG+Tregs三组，Transwell用于第四组，将Tregs与PBMC+Pig细胞隔开培养。2．CD14+细胞体系分CD14+细胞、CD14+细胞+PIG、CD14+细胞+PIG+Tregs三组。3．去CD4+PBMC体系分去CD4+PBMC、去CD4+PBMC+PIG、去CD4+PBMC+PIG+Tregs三组。每组细胞的共培养比例均为1∶1∶1。
48小时之后，收集细胞做标志分子及凋亡染色，做流式细胞术分析。
结果
1．在PBMC培养系统中，CD14+细胞的CD40、IL12及TNF，加入猪异种抗原之后，较PBMC单独培养组的明显增加，在与Tregs共培养组，表达下调，组间差别有统计学意义。在CD14+培养体系及在去CD4+PBMC体系中这些分子的表达在三组间没有差别。
2．Transwell与PBMC+PIG组相比，CD40的表达在Transwell组没有下降，IL12和TNF-α在Transwell组有下降的趋势，但是统计分析显示没有明显的差异。
3．细胞凋亡本实验比较了PBMC+PIG组和PBMC+PIG+Treg组的细胞凋亡情况，统计学显示两组间没有显著性差异。
结论
1．通过减少巨噬细胞表达活化标志分子而不是诱导巨噬细胞死亡，人Tregs可以抑制异种抗原引起的单核／巨噬细胞激活。
2．在巨噬细胞激活的过程中，需要CD4+T细胞的参与，Tregs或许通过对CD4+T细胞的抑制来实现对巨噬细胞的调节。
3．Tregs需要通过细胞接触实现对巨噬细胞的抑制。
4．Tregs是否能够抑制巨噬细胞在猪异种移植免疫反应中的功能以及作用机制尚待阐明。
研究背景
糖代谢紊乱在糖尿病视网膜病变发生发展中起着重要的作用，而葡萄糖转运蛋白-1(GLUT1)作为调节葡萄糖进入细胞的主要载体，其基因表达及功能状态，有可能是决定视网膜是否受累的因素之一。关于GLUT1在糖尿病病程中的表达变化，目前尚有争议。高糖、缺氧、氧化应激、AngiotensinⅡ、VEGF等均参与调节GLUT1的表达。最近有研究表明，Akt(丝氨酸／苏氨酸蛋白激酶，又称PKB)的激活可调节GLUT1的表达。Angiopoietin(Ang)是最近新发现的血管调节因子，其中Ang1的作用有：参与新生血管生成；抑制炎症及渗出；抑制粘附与凋亡等。在糖尿病病程中，Ang1在视网膜有所表达，可能参与了糖尿病视网膜病变的发展。已有研究证实Ang1与其受体Tie2结合后可激活Akt。
目的
已知Ang1可通过Tie2受体调节Akt的活性，而Akt又可调节GLUT1活性，那么，GLUT1与Ang1在糖尿病视网膜中表达部位及数量究竟如何；在糖尿病视网膜病变中是否有一定的作用；Ang1能否通过Akt来调节GLUT1的表达，进而影响视网膜的糖代谢过程?本研究将检测糖尿病大鼠视网膜中Ang1与GLUT1的含量，为临床诊断治疗提供理论依据。
材料与方法：
健康雄性Wistar大鼠40只，体重180-220g。随机选出10只作为正常对照组(NC组)，另外30只采用链脲佐菌素(STZ)溶液腹腔一次性注射建立糖尿病模型(成模20只)，确定成模后随机分为2组：糖尿病血糖未控制组(DM1组)和糖尿病血糖控制组(DM2组)，每组10只。利用腹腔内注射鱼精蛋白锌胰岛素(PZI)将2个糖尿病组大鼠血糖分别控制在＞16.7mmol／L、和＜14mmol／L两个水平。标准饲养3个月后取血测定糖化血红蛋白(HbAlc)，并分离大鼠视网膜，利用免疫组化方法进行GLUT1蛋白的检测，用逆转录—聚合酶链式反应(RT-PCR)方法检测mRNA含量。统计学分析：所有数据均以均数±标准差((?)±SD)表示，采用SPSS11.0软件处理。样本间均数的比较采用t检验，变量间的关系采用直线相关分析。P＜0.05表示有统计学意义。
结果：
1、实验期间DM1、DM2两组糖尿病大鼠体重明显低于NC组大鼠同期体重，而血糖和糖化血红蛋白(HbAlc)则显著高于NC组大鼠。
2、免疫组化染色显示NC组大鼠视网膜中可见GLUT1阳性染色颗粒，DM1组、DM2组大鼠视网膜中GLUT1阳性染色颗粒明显增加，且DM1组比DM2组增加更明显。
3、RT-PCR结果显示三组大鼠视网膜中均含Ang1及GLUT1 mRNA。与NC组相比，DM1组大鼠视网膜内Ang1及GLUT1 mRNA表达增加，DM2组大鼠较DM1组下降，差别均具有统计学意义(p＜0.01)。
结论：
1、糖尿病高血糖状态下，大鼠视网膜中GLUT1蛋白表达较正常对照组增加，尤以血糖未控制组增加明显。糖尿病病程中，视网膜中GLUT1表达并未降低。
2、糖尿病高血糖状态下，Wistar大鼠视网膜中Ang1及GLUT1mRNA含量较正常对照组增加。用胰岛素控制血糖后含量有所下降。
Background
Cellular xenografts have been proposed as having therapeutic potential in several disease states.. However, rejection remains the major impediment to their use. Cellular xenografts are rejected predominantly by cell-mediated mechanisms. This response is initiated by CD4~+ T cells. Macrophages have been proposed as an important effector cell in this response, and several studies have provided indirect evidence supporting its role. Macrophages have been identified as the predominant cell within rejecting xenografts and were shown to be capable of destroying xenografts. Early studies proposed that xenografts were IFNiltrated by a macrophage specificly. But the direct evidence is lacking. Our previous study has shown that porcine antigen-primed and CD4+ T cell- activated macrophages (MO) are capable of specific recognition and rejection of porcine xenografts but not mouse allografts.
Aims:
In this experiment, we aim to find direct evidence to support the specificity of the macrophage response.
Methods
NODSCID mice were induced diabetic by STZ. AlloTx group of mice received moue islet allotransplantation from normal Balb/c mice. Mice from MixTx group were transplanted with mouse islets and NICC admix grafts in the left kidney and NICC grafts in the right kidney. Only normoglycemic mice were undergoing the subsequent macrophage challenge. Normal NODSCID mice, transplanted with NICC, were reconstituted with CD4+ T cells isolated from Balb/c mice and macrophages were separated and injected into normoglycemic NODSCID. 5 weeks after macrophage transfer, left nephrectomy was performed on MixTx mice. Diabetic NODSCID transplanted with NICC alone without macrophage transfer were used as parallel controls. All mice were sacrificed 8 weeks post macrophage transfer
H&E staining and immunohistochemistry were done to analyze graft survival.
Results
The blood glucose went down to normal after transplantation and kept controlled after macrophage transfer. At week 5, in MixTx group, after left nephrectomy the BG increased obviously compared with AlloTx group, which indicated xenografts have been rejected specifically and it was allografts which kept secreting insulin controlling BG. Histology shows intact mouse islets on kidneys from mouse-islet transplanted mice and the left kidy from MixTx mice. But there was little islet left in the right kidney. Immunohistochemistry of Insulin shows insulin-secreting from mouse islet, admix graft and NICC alone graft but little from the right kidney of mixTx mice. Anti-F4/80 staining shows a dense macrophage IFNiltrate surrounding but not IFNiltrating the mouse islets, and the NICC xenografts in right kidneys were completely destroyed.
Conclusions
1. In this study, we showed porcine antigen-primed and CD4+ T cell-activated MO are capable of specifically rejecting porcine islet xenografts after adoptive transfer.
2. Macrophages recruit from peripheral to local grafts automatically.
3. The involved mechanism remains to be investigated. Background
Islet xenotransplantation is considered to have therapeutic potential in type 1 diabetes, but rejection remains the major obstacle to clinical use. Cellular xenogeneic tissue is subject to vigorous cell mediated destruction. While antibody and components of the innate immune system are primary mediators of earlier rejection responses, T cells have been identified as important mediators of acute and chronic cellular xenorejection, which appears to lead to rejection via the recruitment of macrophages. Macrophages are both the dominant early innate responding cells and the major effector cells after activation by CD4+ T cells. A number of approaches including treatment of recipients and genetic manipulations of source animals, have extended survival of porcine organs in non-human primates beyond hyperacute and even delayed vascular rejection, with limited success, and cellular rejection by the adaptive immune system is likely to emerge as a significant immunological barrier as the more rapid types of graft rejection are overcome and graft survival time increases. In the late 1990s, Sakaguchi et al. and other investigators showed that CD4+CD25+ T cells(5%-10% of peripheral lymphocytes) are able to prevent organ-specific autoimmune disease and maintain allotransplantation tolerance. In xenotransplantation, Porter et al reported that human CD4+CD25+ T cells can suppress the proliferation and cytokine production by CD4+CD25- T cells induced in responses to porcine cells. They can also dramatically inhibit the CD4+CD25- T cell-mediated cytolytic response against porcine target cells. There were also evidence to show that human CD4+CD25+ T cells can restrain the maturation and antigen-presenting function of dendritic cells and exert direct suppressive effects on monocytes/macrophages, thereby affecting subsequent innate and adaptive immune responses. However whether Tregs are capable of suppressing macrophages in response to xenogeneic antigen still need to be addressed.
Aims
Our previous study has shown that expanded Tregs are capable of suppressing in vitro T cell-mediated anti-porcine xenoresponses. In this study we aimed to investigate the suppressive effect of expanded Tregs on macrophage activation in the porcine xenoimmune response.
Methods
Peripheral blood mononuclear cells (PBMC) were separated from healthy donors by density gradiant centrifugation and CD4+CD25+ cells were isolated from PBMC using MACS separation system. Fresh Tregs were expanded in vitro in RPMI 1640, plus 10%FCS, Anti-CD3/CD28 beads, IL-2 and Rapamycin. Cells in week 2 and week 3 were used in subsequent co-culture experiment. CD14+ monocytes were isolated by anti-human CD14 antibody-coated microbeads. Non-CD4 PBMC were obtained by depleting CD4+ cells from PBMC. Porcine PBMC (PIG) were used in our experiment as xenoantigen. They were in-activited by irradiation before added into culture system.
Generally, three kinds of co-culture system were used in this experiment. There were four groups in PBMC system: PBMCalone, PBMC+PIG, PBMC+PIG+Tregs at 1:1:1 ratio. In the fourth group, cell culture inserts were used to separate Tregs from PBMC and pig cells. CD14+ cells or Non-CD4 PBMC were co-cultured alone, with pig cells, or with Pig cells and Tregs.
After 48 hrs co-culture, all the cells were collected and the variety of markers and apoptosis/death on macrophages was analyzed by flow cytometry.
Results
1. The expression of CD40, IL12 and TNF in PBMC+PIG group on CD14+ cells increased compared with PBMC. Tregs could down-regulate their expression. There was no difference of their expression among groups in CD14 and non-CD4 PBMC couture systems.
2. The exppression of CD40 in transwell group was similar with that of PBMC+PIG group. The expression of IL12 and TNF slightly decreased in transwell group but there was no significant difference between them.
3. The proportion of macrophages undergoing apoptosis didn' t increase compared with other groups.
Conclusions
1. Human Tregs are able to suppress monocyte/macrophage activation in xenoimmune response in vitro via reducing proportion of macrophages co-expressing their activation markers but not killing them.
2. CD4+ T cells are required for macrophage activation in response to porcine-antigen stimulation, and modulation of this macrophage activation by Tregs may be via their suppressive effect on CD4+ T cells.
3. Cell-to-cell contact pathway is involved in the suppression of Tregs on macrophage activation.
4. Whether human Tregs are able to inhibit macrophage function in anti-porcine xenoimmune response and the mechanism involved remains to be investigated. Background
Glucose is an essential metabolic substrate for retinal metabolism, and its
transport from blood to retina is tightly coupled with retinal glucose utilization. The disorder of glucose metabolism plays an essential role in the occurance and development of diabetic retinopathy. Glucose entry into the endothelial cells of the inner BRB is mediated by a saturable, facilitated transport process involving GLUT1, a member of a family of sodium-independent glucose transporter proteins. The expression of GLUT1 is regulated by many factors. It is reported that Akt mediates a step in the activation of GLUT1 gene expression. Ang1 is an angiogenic growth factor , playing an essential role in retinal vascularization. Studies have suggested that Ang1 could regulate Akt pathway through phosphorylation of Ser473 and Thr308 in Akt. But whether Ang1 could regulate the expression of GLUT1 in the retina of diabetic rats via the regulation of Akt is still unknown.
Aims
Our study aimed to use diabetic rats to explore expression of GLUT1 and the content of Ang1 and Glut1 mRNA in diabetic retina.
Methods
A total of 40 healthy male Wistar rats weighting 180-220g were used, 10 rats were randomly chosen to be normal control group (group NC), the rest 30 rats were induced to be diabetes mellitus by STZ which was injected into the abdomens and only 20 rats became diabetes finally. After the diabetic model was successfully established, the 20 rats were randomly divided into 2 groups: diabetic group without blood glucose control (group DM1), diabetic rats treated with insulin (group DM2). The DM2 group were treated with protamine zinc insulin (PZI)) to control their blood glucose levels to<14mmol/L. After three months the HbA1c of three groups were measured, then all rats were decapitated and their retinas were dissected. The immunhistochemistry method was used to measure the expression GLUT1 and reverse transcription-polymerase chain reaction (RT-PCR) was used to measure the content of mRNA.
Datas were analysed using the Statistical Package of the SPSS 11.0. Discriptive data were given as means±SD. Continuous variables were tested by analysis of t-test and the relations between the variables were tested by linear correlation. The p-values are two-tailed and a p-value of less than 0.05 being considered to be significant.
Results
1. During the experiment the weights of DM1 and DM2 groups were lower than the control group, but the blood glucose and HbA1c were much higher than the control group.
2. Immunohistochemistry method showed that the positive stained granules of GLUT1 protein in BRB were found in NC group. In DM1 and DM2 group the positive stained granules of GLUT1 protein increased markedly, especially in DM1 group. The differences were significant (P<0.05).
3. Compared with NC group, the Ang1 and Glut1 mRNA content in DM1 group increased and decreased in DM2 group. The differences were significant.
Conclusions
1. GLUT1 protein expression was increased in the diabetic rat retina. After we controlled the blood glucose level using insulin, the expressions of GLUT1 protein was reduced. The upregulation of GLUT1 expression was probably the absence of defensive reaction of the retina, which could avoid excessive glucose entering the cell and leading to the damnification of the cell.
2. The mRNA expression of Ang1 and Glut1 was increased in the diabetic rat retina. Insulin treatment could down-regulate the expression.
细胞移植是治疗很多疾病的有效治疗手段，异种细胞移植为供体短缺提供了良好的解决办法，但是，免疫排斥问题仍然是阻碍它走上临床的最大难题。与实体器官移植不同的是，针对细胞异种移植物的免疫排斥反应主要由细胞介导的排斥机制参与，CD4+T细胞在其中起效应细胞的作用。已有实验通过间接手段证实，巨噬细胞在异种移植的免疫反应中起效应细胞的作用，破坏移植物。早已有人提出，巨噬细胞可以浸润异种移植物，发挥特异性排斥的功能，但是缺乏直接的证据。我们以前的实验将胎猪胰岛细胞移植入NODSCID小鼠肾被膜下，然后用CD4+细胞重建，巨噬细胞被分离并转入未经过CD4+T细胞重建的、已接受猪胰岛异种移植的NODSCID受体身上，在无需CD4+T细胞再次活化的情况下，后者的异种移植物被特异性的排斥，为CD4+T细胞活化的巨噬细胞作为效应细胞排斥异种移植物提供了证据。
目的
本实验中，我们建立了混合移植的糖尿病小鼠模型，以寻找支持活化的巨噬细胞能够特异性的识别并排斥猪胰岛细胞的更直接的证据。
方法
1胰岛移植与巨噬细胞重建：STZ诱导的糖尿病NODSCID小鼠分别接受：Balb／c小鼠的胰岛移植(AlloTx组)，混合移植(MixTx组)(左侧接受小鼠胰岛与NICC的混合移植物，右肾接受NICC异种重建后移植物)和NICC异种移植。用CD4+T细胞活化的巨噬细胞重建接受同种移植及混合移植的NODSCID，重建后第5周取出混合移植物，第8周处死所有小鼠。实验过程中检测血糖。
2．对组织块进行形态学观察及胰岛素、F4／80巨噬细胞免疫组化染色，观察移植物。
结果
1．血糖变化AlloTx组和MixTx组，在胰岛移植后血糖明显下降，巨噬细胞重建后血糖仍然维持正常重建后第5周，MixTx组小鼠混合移植物被取出，血糖上升，明显高于AlloTx组小鼠。
2．组织学观察H&E染色显示，AlloTx组小鼠肾被膜下可见完整的小鼠胰岛。在MixTx组小鼠的左肾组织，还可以见到完整的胰岛组织，但是在右肾，未见胰岛结构，可见大量的细胞浸润。在NICC对照组异源性胰岛组织清晰可见。
3．免疫组化在AlloTx组，胰岛素染色可见棕色的阳性颗粒。在MixTx组，左肾可见清晰的胰岛素染色阳性，而在右肾，胰岛素几乎没有阳性颗粒，在NICC对照组可见大量的胰岛素阳性染色。巨噬细胞染色，在AlloTx组，仅可见少量阳性染色，在胰岛内部，看不到任何阳性颗粒，在MixTx组，左肾可见巨噬细胞浸润，但是没有侵蚀到胰岛内部。巨噬细胞在右肾被膜下大量浸润。
结论
1．本实验直接证明在猪异种胰岛抗原存在的情况下，用CD4+T细胞活化的巨噬细胞可以特异性的识别并排斥猪异种胰岛移植物。
2．活化的巨噬细胞可以从外周自动聚集至移植物局部。
3．这种特异性识别的具体作用机制需要进一步研究。
研究背景
异种胰岛移植是治疗1型糖尿病的有效手段，但移植物排斥仍是它的主要副作用。细胞异基因移植物易受细胞介导的免疫破坏，在早期的排斥反应中，非特异性免疫系统中的抗体和补体成分是介导排斥的主要因素，在急性和慢性细胞免疫排斥中，T细胞则是重要的反应细胞，它可以通过募集巨噬细胞发挥排斥作用。巨噬细胞既是早期非特异性反应细胞，在被CD4+T细胞活化后，又是主要的效应细胞。人们利用药物治疗以及基因调控供体动物的方法，有限抑制了超急性排斥甚至迟发性血管性排斥，延长了猪异种器官在非人类灵长类的存活时间，于是由特异性免疫系统介导的细胞排斥反应成为克服移植物排斥和增加移植物存活时间的亟待解决的问题。上世纪90年代，研究者们发现，占外周淋巴细胞总数5％—10％的CD4+CD25+调节性细胞(CD4+CD25+Regulatory T Cells，Tregs)可以制止器官特异性自身免疫性疾病的发病，维持同种异体移植的免疫耐受。在异种移植中，Porter等报道人Tregs可以抑制CD4+CD25—T细胞的增殖和细胞因子的产生，还可以明显抑制CD4+CD25—T细胞介导的对猪靶细胞的溶细胞作用。亦有证据显示人Tregs可以抑制树突状细胞的成熟和抗原递呈功能，可以激发直接抑制单核／巨噬细胞的作用进而影响非特异性和特异性免疫。然而，目前Tregs能否对异基因刺激的巨噬细胞产生抑制作用尚未见研究。
目的
我们以前的研究显示，体外扩增的Tregs可以抑制T细胞介导的针对猪异种抗原的免疫反应，在本实验中，我们利用扩增的Tregs，研究其对异种细胞激活的巨噬细胞是否具有抑制作用以及可能的机制
方法
新鲜Tregs、CD14+单核细胞、去CD4+细胞的PBMC细胞，均来自健康志愿者外周血单个核细胞(Peripheral blood mononuclear cells，PBMC)，用含10％FCS，Anti-CD3／CD28 beads，IL2及Rapamycin的RPMI1640培养液体外扩增，培养2至3周的细胞用于下游实验。经射线照射的猪PBMC(PIG)作为异种抗原。
实验中有3个培养体系：1．PBMC体系分PBMC、PBMC+PIG、PBMC+PIG+Tregs三组，Transwell用于第四组，将Tregs与PBMC+Pig细胞隔开培养。2．CD14+细胞体系分CD14+细胞、CD14+细胞+PIG、CD14+细胞+PIG+Tregs三组。3．去CD4+PBMC体系分去CD4+PBMC、去CD4+PBMC+PIG、去CD4+PBMC+PIG+Tregs三组。每组细胞的共培养比例均为1∶1∶1。
48小时之后，收集细胞做标志分子及凋亡染色，做流式细胞术分析。
结果
1．在PBMC培养系统中，CD14+细胞的CD40、IL12及TNF，加入猪异种抗原之后，较PBMC单独培养组的明显增加，在与Tregs共培养组，表达下调，组间差别有统计学意义。在CD14+培养体系及在去CD4+PBMC体系中这些分子的表达在三组间没有差别。
2．Transwell与PBMC+PIG组相比，CD40的表达在Transwell组没有下降，IL12和TNF-α在Transwell组有下降的趋势，但是统计分析显示没有明显的差异。
3．细胞凋亡本实验比较了PBMC+PIG组和PBMC+PIG+Treg组的细胞凋亡情况，统计学显示两组间没有显著性差异。
结论
1．通过减少巨噬细胞表达活化标志分子而不是诱导巨噬细胞死亡，人Tregs可以抑制异种抗原引起的单核／巨噬细胞激活。
2．在巨噬细胞激活的过程中，需要CD4+T细胞的参与，Tregs或许通过对CD4+T细胞的抑制来实现对巨噬细胞的调节。
3．Tregs需要通过细胞接触实现对巨噬细胞的抑制。
4．Tregs是否能够抑制巨噬细胞在猪异种移植免疫反应中的功能以及作用机制尚待阐明。
研究背景
糖代谢紊乱在糖尿病视网膜病变发生发展中起着重要的作用，而葡萄糖转运蛋白-1(GLUT1)作为调节葡萄糖进入细胞的主要载体，其基因表达及功能状态，有可能是决定视网膜是否受累的因素之一。关于GLUT1在糖尿病病程中的表达变化，目前尚有争议。高糖、缺氧、氧化应激、AngiotensinⅡ、VEGF等均参与调节GLUT1的表达。最近有研究表明，Akt(丝氨酸／苏氨酸蛋白激酶，又称PKB)的激活可调节GLUT1的表达。Angiopoietin(Ang)是最近新发现的血管调节因子，其中Ang1的作用有：参与新生血管生成；抑制炎症及渗出；抑制粘附与凋亡等。在糖尿病病程中，Ang1在视网膜有所表达，可能参与了糖尿病视网膜病变的发展。已有研究证实Ang1与其受体Tie2结合后可激活Akt。
目的
已知Ang1可通过Tie2受体调节Akt的活性，而Akt又可调节GLUT1活性，那么，GLUT1与Ang1在糖尿病视网膜中表达部位及数量究竟如何；在糖尿病视网膜病变中是否有一定的作用；Ang1能否通过Akt来调节GLUT1的表达，进而影响视网膜的糖代谢过程?本研究将检测糖尿病大鼠视网膜中Ang1与GLUT1的含量，为临床诊断治疗提供理论依据。
材料与方法：
健康雄性Wistar大鼠40只，体重180-220g。随机选出10只作为正常对照组(NC组)，另外30只采用链脲佐菌素(STZ)溶液腹腔一次性注射建立糖尿病模型(成模20只)，确定成模后随机分为2组：糖尿病血糖未控制组(DM1组)和糖尿病血糖控制组(DM2组)，每组10只。利用腹腔内注射鱼精蛋白锌胰岛素(PZI)将2个糖尿病组大鼠血糖分别控制在＞16.7mmol／L、和＜14mmol／L两个水平。标准饲养3个月后取血测定糖化血红蛋白(HbAlc)，并分离大鼠视网膜，利用免疫组化方法进行GLUT1蛋白的检测，用逆转录—聚合酶链式反应(RT-PCR)方法检测mRNA含量。统计学分析：所有数据均以均数±标准差((?)±SD)表示，采用SPSS11.0软件处理。样本间均数的比较采用t检验，变量间的关系采用直线相关分析。P＜0.05表示有统计学意义。
结果：
1、实验期间DM1、DM2两组糖尿病大鼠体重明显低于NC组大鼠同期体重，而血糖和糖化血红蛋白(HbAlc)则显著高于NC组大鼠。
2、免疫组化染色显示NC组大鼠视网膜中可见GLUT1阳性染色颗粒，DM1组、DM2组大鼠视网膜中GLUT1阳性染色颗粒明显增加，且DM1组比DM2组增加更明显。
3、RT-PCR结果显示三组大鼠视网膜中均含Ang1及GLUT1 mRNA。与NC组相比，DM1组大鼠视网膜内Ang1及GLUT1 mRNA表达增加，DM2组大鼠较DM1组下降，差别均具有统计学意义(p＜0.01)。
结论：
1、糖尿病高血糖状态下，大鼠视网膜中GLUT1蛋白表达较正常对照组增加，尤以血糖未控制组增加明显。糖尿病病程中，视网膜中GLUT1表达并未降低。
2、糖尿病高血糖状态下，Wistar大鼠视网膜中Ang1及GLUT1mRNA含量较正常对照组增加。用胰岛素控制血糖后含量有所下降。
Background
Cellular xenografts have been proposed as having therapeutic potential in several disease states.. However, rejection remains the major impediment to their use. Cellular xenografts are rejected predominantly by cell-mediated mechanisms. This response is initiated by CD4~+ T cells. Macrophages have been proposed as an important effector cell in this response, and several studies have provided indirect evidence supporting its role. Macrophages have been identified as the predominant cell within rejecting xenografts and were shown to be capable of destroying xenografts. Early studies proposed that xenografts were IFNiltrated by a macrophage specificly. But the direct evidence is lacking. Our previous study has shown that porcine antigen-primed and CD4+ T cell- activated macrophages (MO) are capable of specific recognition and rejection of porcine xenografts but not mouse allografts.
Aims:
In this experiment, we aim to find direct evidence to support the specificity of the macrophage response.
Methods
NODSCID mice were induced diabetic by STZ. AlloTx group of mice received moue islet allotransplantation from normal Balb/c mice. Mice from MixTx group were transplanted with mouse islets and NICC admix grafts in the left kidney and NICC grafts in the right kidney. Only normoglycemic mice were undergoing the subsequent macrophage challenge. Normal NODSCID mice, transplanted with NICC, were reconstituted with CD4+ T cells isolated from Balb/c mice and macrophages were separated and injected into normoglycemic NODSCID. 5 weeks after macrophage transfer, left nephrectomy was performed on MixTx mice. Diabetic NODSCID transplanted with NICC alone without macrophage transfer were used as parallel controls. All mice were sacrificed 8 weeks post macrophage transfer
H&E staining and immunohistochemistry were done to analyze graft survival.
Results
The blood glucose went down to normal after transplantation and kept controlled after macrophage transfer. At week 5, in MixTx group, after left nephrectomy the BG increased obviously compared with AlloTx group, which indicated xenografts have been rejected specifically and it was allografts which kept secreting insulin controlling BG. Histology shows intact mouse islets on kidneys from mouse-islet transplanted mice and the left kidy from MixTx mice. But there was little islet left in the right kidney. Immunohistochemistry of Insulin shows insulin-secreting from mouse islet, admix graft and NICC alone graft but little from the right kidney of mixTx mice. Anti-F4/80 staining shows a dense macrophage IFNiltrate surrounding but not IFNiltrating the mouse islets, and the NICC xenografts in right kidneys were completely destroyed.
Conclusions
1. In this study, we showed porcine antigen-primed and CD4+ T cell-activated MO are capable of specifically rejecting porcine islet xenografts after adoptive transfer.
2. Macrophages recruit from peripheral to local grafts automatically.
3. The involved mechanism remains to be investigated. Background
Islet xenotransplantation is considered to have therapeutic potential in type 1 diabetes, but rejection remains the major obstacle to clinical use. Cellular xenogeneic tissue is subject to vigorous cell mediated destruction. While antibody and components of the innate immune system are primary mediators of earlier rejection responses, T cells have been identified as important mediators of acute and chronic cellular xenorejection, which appears to lead to rejection via the recruitment of macrophages. Macrophages are both the dominant early innate responding cells and the major effector cells after activation by CD4+ T cells. A number of approaches including treatment of recipients and genetic manipulations of source animals, have extended survival of porcine organs in non-human primates beyond hyperacute and even delayed vascular rejection, with limited success, and cellular rejection by the adaptive immune system is likely to emerge as a significant immunological barrier as the more rapid types of graft rejection are overcome and graft survival time increases. In the late 1990s, Sakaguchi et al. and other investigators showed that CD4+CD25+ T cells(5%-10% of peripheral lymphocytes) are able to prevent organ-specific autoimmune disease and maintain allotransplantation tolerance. In xenotransplantation, Porter et al reported that human CD4+CD25+ T cells can suppress the proliferation and cytokine production by CD4+CD25- T cells induced in responses to porcine cells. They can also dramatically inhibit the CD4+CD25- T cell-mediated cytolytic response against porcine target cells. There were also evidence to show that human CD4+CD25+ T cells can restrain the maturation and antigen-presenting function of dendritic cells and exert direct suppressive effects on monocytes/macrophages, thereby affecting subsequent innate and adaptive immune responses. However whether Tregs are capable of suppressing macrophages in response to xenogeneic antigen still need to be addressed.
Aims
Our previous study has shown that expanded Tregs are capable of suppressing in vitro T cell-mediated anti-porcine xenoresponses. In this study we aimed to investigate the suppressive effect of expanded Tregs on macrophage activation in the porcine xenoimmune response.
Methods
Peripheral blood mononuclear cells (PBMC) were separated from healthy donors by density gradiant centrifugation and CD4+CD25+ cells were isolated from PBMC using MACS separation system. Fresh Tregs were expanded in vitro in RPMI 1640, plus 10%FCS, Anti-CD3/CD28 beads, IL-2 and Rapamycin. Cells in week 2 and week 3 were used in subsequent co-culture experiment. CD14+ monocytes were isolated by anti-human CD14 antibody-coated microbeads. Non-CD4 PBMC were obtained by depleting CD4+ cells from PBMC. Porcine PBMC (PIG) were used in our experiment as xenoantigen. They were in-activited by irradiation before added into culture system.
Generally, three kinds of co-culture system were used in this experiment. There were four groups in PBMC system: PBMCalone, PBMC+PIG, PBMC+PIG+Tregs at 1:1:1 ratio. In the fourth group, cell culture inserts were used to separate Tregs from PBMC and pig cells. CD14+ cells or Non-CD4 PBMC were co-cultured alone, with pig cells, or with Pig cells and Tregs.
After 48 hrs co-culture, all the cells were collected and the variety of markers and apoptosis/death on macrophages was analyzed by flow cytometry.
Results
1. The expression of CD40, IL12 and TNF in PBMC+PIG group on CD14+ cells increased compared with PBMC. Tregs could down-regulate their expression. There was no difference of their expression among groups in CD14 and non-CD4 PBMC couture systems.
2. The exppression of CD40 in transwell group was similar with that of PBMC+PIG group. The expression of IL12 and TNF slightly decreased in transwell group but there was no significant difference between them.
3. The proportion of macrophages undergoing apoptosis didn' t increase compared with other groups.
Conclusions
1. Human Tregs are able to suppress monocyte/macrophage activation in xenoimmune response in vitro via reducing proportion of macrophages co-expressing their activation markers but not killing them.
2. CD4+ T cells are required for macrophage activation in response to porcine-antigen stimulation, and modulation of this macrophage activation by Tregs may be via their suppressive effect on CD4+ T cells.
3. Cell-to-cell contact pathway is involved in the suppression of Tregs on macrophage activation.
4. Whether human Tregs are able to inhibit macrophage function in anti-porcine xenoimmune response and the mechanism involved remains to be investigated. Background
Glucose is an essential metabolic substrate for retinal metabolism, and its
transport from blood to retina is tightly coupled with retinal glucose utilization. The disorder of glucose metabolism plays an essential role in the occurance and development of diabetic retinopathy. Glucose entry into the endothelial cells of the inner BRB is mediated by a saturable, facilitated transport process involving GLUT1, a member of a family of sodium-independent glucose transporter proteins. The expression of GLUT1 is regulated by many factors. It is reported that Akt mediates a step in the activation of GLUT1 gene expression. Ang1 is an angiogenic growth factor , playing an essential role in retinal vascularization. Studies have suggested that Ang1 could regulate Akt pathway through phosphorylation of Ser473 and Thr308 in Akt. But whether Ang1 could regulate the expression of GLUT1 in the retina of diabetic rats via the regulation of Akt is still unknown.
Aims
Our study aimed to use diabetic rats to explore expression of GLUT1 and the content of Ang1 and Glut1 mRNA in diabetic retina.
Methods
A total of 40 healthy male Wistar rats weighting 180-220g were used, 10 rats were randomly chosen to be normal control group (group NC), the rest 30 rats were induced to be diabetes mellitus by STZ which was injected into the abdomens and only 20 rats became diabetes finally. After the diabetic model was successfully established, the 20 rats were randomly divided into 2 groups: diabetic group without blood glucose control (group DM1), diabetic rats treated with insulin (group DM2). The DM2 group were treated with protamine zinc insulin (PZI)) to control their blood glucose levels to<14mmol/L. After three months the HbA1c of three groups were measured, then all rats were decapitated and their retinas were dissected. The immunhistochemistry method was used to measure the expression GLUT1 and reverse transcription-polymerase chain reaction (RT-PCR) was used to measure the content of mRNA.
Datas were analysed using the Statistical Package of the SPSS 11.0. Discriptive data were given as means±SD. Continuous variables were tested by analysis of t-test and the relations between the variables were tested by linear correlation. The p-values are two-tailed and a p-value of less than 0.05 being considered to be significant.
Results
1. During the experiment the weights of DM1 and DM2 groups were lower than the control group, but the blood glucose and HbA1c were much higher than the control group.
2. Immunohistochemistry method showed that the positive stained granules of GLUT1 protein in BRB were found in NC group. In DM1 and DM2 group the positive stained granules of GLUT1 protein increased markedly, especially in DM1 group. The differences were significant (P<0.05).
3. Compared with NC group, the Ang1 and Glut1 mRNA content in DM1 group increased and decreased in DM2 group. The differences were significant.
Conclusions
1. GLUT1 protein expression was increased in the diabetic rat retina. After we controlled the blood glucose level using insulin, the expressions of GLUT1 protein was reduced. The upregulation of GLUT1 expression was probably the absence of defensive reaction of the retina, which could avoid excessive glucose entering the cell and leading to the damnification of the cell.
2. The mRNA expression of Ang1 and Glut1 was increased in the diabetic rat retina. Insulin treatment could down-regulate the expression.
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2. Nakajima M , Cooney M J , Alexander H T , et al. Normalization of retinal vascular permeability in experimental diabetes with genistein. Invest Ophthalmol Vis Sci ,2001, 42: 2110-2114
3. Davis S, Aldrich TH, Jones PF, et al. Isolation of angiopoietin -1, a ligand for the tie2 receptor by secretion - trap expression cloning. Cell, 1996, 87(7) :1161 - 1169.
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5. Kumagai AK, Vinores SA, Pardridge WM,et al. Pathological upregulation of inner-retinal barrier GLUT1 glucose transporter expression in diabetes mellitus. Brain Res, 1996,706:313-7
6. Giovanni E. Mann, David L. Yudilevich, and Luis Sobrevia. Regulation of Amino Acid and Glucose Transporters in Endothelial and Smooth Muscle. Cells Physiol Rev, Jan 2003; 83: 183-252.
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(Please refer to the text for tables and figures.)