CD44反义寡核苷酸对肿瘤细胞表面抗原和对CD3AK杀伤敏感性的调节作用

目的：观察CD44反义寡核苷酸调节人低分化黏液腺胃癌MCC80-3细胞Fas分子和凋亡抵抗基因bcl-2的表达水平，提高免疫效应细胞杀伤敏感性的作用和机制。方法：RT-PCR法检测CD44、bcl-2mRNA的表达水平。MTT法检测CD3AK杀伤活性。流式细胞术检测细胞表面CD44及Fas、FasL蛋白表达水平。结果：CD44反义寡核苷酸（1.6μmol／L）处理后，明显地抑制MGC80-3细胞CD44mRNA和蛋白表达水平，在CD44配体低分子量透明质酸（HA）存在下，使MCC80-3表面下调的Fas分子显著增高，表达率从6．69％提高到16．81％（P〈0．01）。CD3AK对反义寡核苷酸处理的MGC80-3细胞杀伤活性显著增高，并呈效靶比依赖效应（P〈0．01）。MGC80-3细胞bcl-2mRNA的相对表达定量值由1．06降至0．32。结论：CD44反义寡核苷酸可通过抑制MGC80-3细胞CD44mRNA和蛋白表达，上调Fas分子的表达，下调凋亡抵抗基因bcl-2的表达，并提高其对免疫效应细胞的杀伤敏感性。     

ICAM-1反义寡核苷酸对小鼠肾小管上皮细胞ICAM-1表达的影响

目的:探讨ICAM1反义寡核苷酸对小鼠肾小管上皮细胞表达ICAM1的影响,为利用ICAM1反义寡核苷酸类药物治疗肾小管间质病变奠定基础。方法:小鼠ICAM1反义寡核苷酸及其对照物参照文献合成并行全硫代磷酸化修饰。部分ICAM1反义寡核苷酸在其5′端作FITC荧光标记。将ICAM1反义寡核苷酸单独或脂质体转染试剂DOTAP混合后转染至培养的肾小管上皮细胞之中,转染成功后加入IL1β诱导细胞产生ICAM1。采用对照寡核苷酸及不含寡核苷酸的细胞转染液作为实验对照及空白对照。利用免疫组化染色的方法检测细胞ICAM1表达变化并提取细胞总RNA行逆转录PCR及Northern杂交观察ICAM1mRNA的表达的变化。结果:两个不同浓度的ICAM1反义寡核苷酸均可明显阻断IL1β诱导的肾小管上皮细胞ICAM1及ICAM1mRNA的表达;对照的寡核苷酸不能减少IL1β诱导的肾小管上皮细胞ICAM1及ICAM1mRNA的表达。结论:小鼠ICAM1反义寡核苷酸可明显抑制IL1β诱导的小鼠肾小管上皮细胞ICAM1及ICAM1mRNA的表达,提示ICAM1的反义寡核苷酸有可能应用于肾小管间质病变的实验治疗之中。     

IL-6表达增高及TNFα下调参与bcl-2反义寡脱氧核苷酸诱导的细胞凋亡的调控

目的 探讨bcl-2反义硫代磷酸寡脱氧核苷酸（antisense phosphorothioate oligodeoxynucleotide,AS-PS-ODN)诱导细胞凋亡的调控机理。方法 合成bcl-2 AS－PS－ODN作用于小细胞肺癌细胞株NCI－H446，流式细胞仪DNA倍体分析和脱氧核苷酸末端转移酶介导的缺口末端标记法检测细胞凋亡；多重半定量逆转录－聚合酶链反应检测bcl-2 AS－PS－ODN处理前后bcl-2，c-myc,survivin,bax,s100A2,TNFα，TGFβ1及IL－6等基因mRNA表达的变化。结果 bcl-2 AS－PS－ODN能够诱导NCI－H446细胞凋亡，随着bcl-2 mRNA水平的下降，IL－6表达增高72．71％（P＜0．05），TNFα表达下调65．90％（P＜0．05），而c-myc,survivin,bax,s100A2,TGFβ1等基因的表达无明显变化。结论 IL－6表达增高及TNFα下调参与bcl-2反义寡脱氧核苷酸所诱导的细胞凋亡的调控。     

LMP-1 mRNA反义寡核苷酸抑制大鼠成骨细胞的分化

为深入探讨LIM矿化蛋白1（LMP－1）在成骨细胞分化中的作用，体外培养大鼠成骨细胞，在培养基中加入LMP－1反义寡核苷酸（LMP－1antisense终浓度为0．8mol／L），以阻断大鼠成骨细胞中LMP－1mRNA的表达。对照组加nonsense（终浓度为0．8mol／L）。测定细胞的碱性磷酸酶（ALP）活性、培养基中骨钙素（OC）的含量，免疫组化分析Ⅰ型胶原蛋白的表达，Northern blotting检测I型胶原mRAN的表达。结果显示：LMP－1mRNA被LMP－lantisense阻断后，ALP活性降低、OC分泌减少、I型胶原蛋白和mRNA的表达下降。上述结果表明：LMP－1mRNA被LMP－1反义寡核酸阻断后，成骨细胞分化受到明显的抑制，提示LMP－1是成骨细胞分化必不可少的正调节因子。     

反义核酸对气道平滑肌细胞内皮素转换酶—1基因表达的影响

目的：探讨肿瘤坏死因子（TN－α）对人气道平滑肌细胞内皮素转换酶－1（ECE－1）基因表达的影响及体外反义内皮素转换酶－1寡聚核苷酸（ECE—1oDNS）是否可拮抗TNF－α诱导人气道平滑肌细胞ECE－1表达分泌．方法：高体培养人气过平滑肌细胞共导入由阳离子脂质体Lipofectin携载的不同浓度反义ECEoDNS，用RTPCR观察其对前炎症因子TNF-α诱导人气道平滑肌细胞ECE基因表达的影响．结果：TNF－α可刺激ECE-1mRNA表达．ECE－1mRNA由0．1318±0．0318升至0．2277±0．0150（t=4．724，P＜0．01）；导入反义ECEoDNS后，ECE－1mRNA表达受到抑制，抑制率分别为34．74％±11．6％及29．24％±4.96％．结论：反义ECE－1oDNS能拮抗TNF－α诱导人气道平滑肌细胞的ECE—1mRNA表达，为从基因治疗角度探讨控制气道局反应提供新线索．     

阻断内源性IL-15的表达对人胚胎横纹肌肉瘤细胞的影响

目的　观察应用反义RNA阻断胚胎横纹肌肉瘤细胞内源性IL 15表达后 ,肿瘤细胞对NK细胞杀伤敏感性及其体内、外增殖的变化。方法　应用流式细胞技术检测RD细胞及转染IL 15反义RNA的RD 10细胞表面MHCⅠ类 (包括HLA ABC重链及β2M链 )分子的表达 ;应用LDH释放法检测NK细胞对RD和RD 10细胞的细胞毒活性 ;通过对转染细胞的细胞周期分析以及接种到裸鼠体内来评价IL 15对肿瘤细胞增殖的影响。结果　①阻断IL 15的表达可使HLA ABC分子阳性细胞百分率从 19.5 %升至 95 .5 % ;β2M分子阳性细胞百分率从 31%升至 90 .6 %。由于MHCⅠ类分子表达的增高 ,RD 10细胞对NK细胞的杀伤敏感性低于RD细胞 (P <0 .0 1)。②RD 10细胞的增殖减慢 ,阻滞在G0 /G1期。动物实验的结果也表明RD 10细胞在体内的致瘤性低于RD细胞。结论　阻断RD细胞内源性IL 15的表达可抑制该细胞的增殖 ,从而提示IL 15在肿瘤发生及发展过程中作用的多样性     

HLA-G反义寡核苷酸对绒癌细胞免疫逃逸的影响

采用非经典MHCI类免疫耐受分子HLA G反义基因 ,封闭和阻断肿瘤细胞上HLA G分子的释放 ,逆转肿瘤细胞的免疫抑制作用 ,为肿瘤生物治疗提供新的理论和实验依据。利用反义核酸技术 ,合成HLA G反义寡核苷酸 (ASODN ) ,硫代化修饰与脂质体形成复合物 ,转导入表达HLA G的绒毛膜癌细胞系JEG 3。采用RT PCR方法检测HLA GmRNA表达水平的变化。流式细胞术检测细胞表面HLA G蛋白表达水平的变化 ;ASODN处理后 ,对JEG 3诱导外周血单个核细胞凋亡的影响 ;MTT法检测ASODN作用后 ,JEG 3作为第 3种抑制细胞对同种异体混合淋巴细胞反应的影响。结果表明 ,HLA GASODN可显著抑制JEG 3细胞HLA GmRNA和蛋白水平的表达 ,逆转HLA G分子诱导的免疫效应细胞的凋亡作用 ,逆转HLA G分子对同种异体混合淋巴细胞反应的抑制作用     

反义锁核酸封闭c-myc第二外显子翻译起始区对肝癌细胞凋亡的影响

目的:探讨针对c-myc第二外显子翻译起始区的反义锁核酸对肝癌细胞活力和凋亡的影响。方法:设计合成能特异性封闭c-myc基因第二外显子翻译起始区的反义寡核苷酸、硫代寡核苷酸和锁核酸,分别以阳离子脂质体介导转染HepG2细胞,采用RT-PCR检测细胞内c-Myc的mRNA表达变化;Western blot检测细胞内cMyc的蛋白表达变化;流式细胞技术检测细胞凋亡情况;MTT法检测反义锁核酸的细胞毒性作用。结果:转染第5天后,反义锁核酸组c-Myc的mRNA相对表达量为0.335±0.016,明显低于对照组(P0.05);c-Myc蛋白相对表达量为0.448±0.037,也明显低于对照组(P0.01);凋亡细胞比例为32%±6%,显著高于对照组(P0.05)。结论:针对c-myc第二外显子翻译起始区的反义锁核酸能有效促进肝癌细胞的凋亡。     

ICA、PJA逆转人高转移肺癌细胞Fas/FasL途径免疫逃逸机制的研究

目的：研究ICA、PJA对人高转移肺癌细胞PG细胞免疫逃逸的逆转作用。方法：MTT法检测ICA、PJh对PG细胞增殖的影响以及对CD3AK杀伤敏感性的影响。流式细胞仪检测细胞表面分子Fas、FasL表达水平和细胞凋亡。应用PG细胞与Jurkat T细胞其培养的方法体外研究FasL诱导T淋巴细胞凋亡的作用。结果：PG细胞高表达FasL，低表达Fas，对CD3AK细胞杀伤敏感性较低，并在与Jurkat T细胞共培养中诱导高表达Fas的Jurkat T细胞凋亡。：ICA、PJA对PG细胞有明显的增殖抑制作用。ICA可明显提高PG细胞Fas的表达率。ICA、PJA可明显降低PG细胞FasL的表达率。ICA、PJA可使PG细胞与Jurkat T细胞共培养中，降低Jurkat T细胞的凋亡率。ICA、PJA可提高CD3AK细胞对PG细胞的杀伤活性。结论：ICA、PJA可逆转人高转移肺癌细胞PG通过Fas／FasL途径逃避机体免疫活性细胞的攻击。     

Langerhans cells - dendritic cells of the epidermis

Langerhans cells (LC) are dendritic cells of the epidermis. They are highly specialized leukocytes that serve immunogenic and tolerogenic purposes. Here, we review some aspects of LC biology, emphasizing those areas where LC are or may turn out to be special.     

Langerhans cells: antigen presenting cells of the epidermis

While epidermis in the skin provides an excellent barrier to the environment, it is an incomplete one. Some antigenic material can penetrate through the stratum corneum (or be introduced pathologically) where strategically placed epidermal Langerhans cells reside. In this review, we have assembled relevant data concerning the antigen presenting potential of epidermal Langerhans cells. Strong circumstantial evidence derived from in vitro studies of epidermal cell suspensions enriched for Langerhans cells indicates that Langerhans cells possess this capability. In vivo studies with intact skin indicate that critical numbers of functioning Langerhans cells are essential for successful induction of contact hypersensitivity by epicutaneously applied haptens. And within the past several months, experiments with purified preparations of epidermal Langerhans cells have proven that these cells, and perhaps they alone among epidermal cells, possess the capacity of processing and presenting haptenic determinants to the immune system. The challenge for the future is to determine the extent to which this unique property of Langerhans cells affords physiologic protection to the skin and under what pathologic circumstances altered Langerhans cell function leads to disease.     

Mast cells: the forgotten cells of renal fibrosis

BACKGROUND/AIMS: Mast cells, when activated, secrete a large number of fibrogenic factors and have been implicated in the development of fibrotic conditions of the liver, lung, and skin. There is evidence that renal fibrosis is closely linked with a chronic inflammatory cell infiltrate within the interstitium, but a potential role for mast cells in this process has yet to be defined. Therefore, the numbers of mast cells in normal and fibrotic kidneys with various pathologies were investigated. METHODS: Mast cells were quantified in renal transplants showing acute and chronic rejection and cyclosporin toxicity, kidneys removed for chronic pyelonephritis, and renal biopsies from patients with IgA nephropathy, membranous nephropathy, and diabetic nephropathy. Mast cells were stained using two methods: acid toluidine blue detected less than 30% of the mast cells revealed by immunohistochemistry for mast cell tryptase. RESULTS: Mast cells were scarce or absent in normal kidney (median, 1.6 mast cells/mm2) but numerous throughout the cortex and medulla in all specimens that showed fibrosis. They were almost entirely confined to the renal interstitium. Mast cells were present in large numbers in biopsies from patients with membranous nephropathy (median, 21.7 mast cells/mm2) and diabetic nephropathy (median, 29.2 mast cells/mm2), which were selected on the basis of showing chronic injury. In 24 unselected IgA nephropathy biopsies there was a close correlation between numbers of mast cells and the extent of interstitial fibrosis (r = 0.771; p < 0.0001). In renal transplant biopsies, mast cells were associated with allograft fibrosis in chronic rejection (median, 27.1 mast cells/mm2) and chronic cyclosporin toxicity (median, 10.6 mast cells/mm2) but not acute rejection (median, 2.7 mast cells/mm2) or acute cyclosporin toxicity (median, 2.0 mast cells/mm2). There was no detectable increase in mast cell numbers during acute rejection in those transplants that subsequently progressed to chronic rejection. In some biopsies the mast cells were largely intact, but in most cases some or all were degranulated. CONCLUSIONS: An increased number of mast cells is a consistent feature of renal fibrosis, whatever the underlying pathology, and the number of mast cells correlates with the extent of interstitial fibrosis. This suggests that mast cells might play a pathogenetic role in the fibrotic process.     

从G-CSF动员的外周血细胞悬液培养的成纤维细胞样细胞的特性分析

目的： 研究从G-CSF动员的外周血细胞（PBC）悬液培养的成纤维细胞样（F-L）细胞的特性。 方法： 取PBC中贴壁细胞分4组培养：①RPMI-1640组；②L-DMEM组；③粒细胞集落刺激因子（G-CSF）组；④白细胞介素-3（IL-3）组。流式细胞仪分析各组培养的F-L细胞特性。 结果： 培养2-3周后，4组均能收获到F-L细胞，细胞贴壁生长，但不融合，不能连续传代培养，③④组细胞数量明显多于①②组，4组F-L细胞表型相似：CD33+、CD11c+、CD64+、CD14+、CD45+、HLA-DR+、CD86+、CD34-、CD38-、CD3-、CD19-、CD56-、CD29-、CD44-、CD105-；与单核细胞（PB-M）表型差异仅CD38表达不同而与间质干细胞（MSC）或树突状细胞（DC）表型明显不同。 结论： 从PBC培养的F-L细胞为巨噬细胞，不是MSC或DC；G-CSF、IL-3能提高F-L细胞培养数量，不改变PB-M向巨噬细胞分化的分化方向。     

Hepatoma cells inhibit the differentiation and maturation of dendritic cells and increase the production of regulatory T cells

Tumor cells may escape from the immune responses because of defective differentiation of dendritic cells (DC). Recent studies have found an increased number of regulatory T cells (Treg) in both peripheral blood and tissues from patients with hepatocellular carcinoma. In the present study, we used tumor culture supernatants (TSN) from hepatoma-derived cell lines to investigate whether TSN interfere with the differentiation of human monocyte-derived DC and/or their ability to increase Treg. The results showed that exposure to TSN significantly inhibited the differentiation of monocytes into DC with retained CD14 molecule and reduced expression of CD1a. These TSN-exposed immature DC also produced significant amount of immunosuppressive cytokine IL-10 and displayed an increased expression of co-stimulatory molecules. Upon stimulation with LPS, however, the TSN-exposed DC failed to undergo full maturation, with a blockage of the upregulation of co-stimulatory molecules on their surface and a switch to an IL-10(high)IL-12(low)TNF-alpha(low) phenotype. Moreover, exposure of DC to TSN selectively inhibited their capacity to stimulate the proliferation of allogeneic CD8(+) T cells, but promoted the generation of CD4(+)CD25(hi)Foxp3(+) Treg cells. These findings, together with previous clinical studies showing that CD4(+)CD25(hi) Treg cells are concentrated within hepatocellular carcinoma tissue, suggest that the local tumor microenvironment may favor the induction of Treg cells through inhibiting the differentiation and maturation of DC.     

Human plasmacytoid-derived dendritic cells and the induction of T-regulatory cells

Suppression by T-regulatory (Tr) cells is essential for the induction of T-cell tolerance and the prevention of autoimmune diseases, organ rejection, and graft-versus-host disease. Increasing attention has been devoted to understand the role of dendritic cells (DC) in the control of Tr-cell differentiation. Here we review the recent evidence that cluster designation (CD)40-ligand activated plasmacytoid-derived DCs (DC2) have the ability to induce primary Tr-cell differentiation. We propose that in addition to the regulatory functions of immature myeloid DC, Tr-cell induction by DC2 represents a nonredundant mechanism for the safeguard of peripheral T-cell tolerance. DC2 can be used as tool to drive potent antigen specific Tr-cell differentiation and expansion in vitro and in vivo.     

Endocrine cells and parietal cells in the stomach of the developing rat

Gastrin-immunoreactive cells were fairly numerous in the pancreas and upper duodenum of the rat at about the time of birth. A minor population of these cells stained with antibodies directed against the N-terminal region of gastrin-34 as well as with antibodies directed against the C-terminal region. The remainder of the cells stained with the C-terminally directed antibodies only. Within a fortnight after birth all gastrin-immunoreactive cells disappeared from the pancreas and were greatly reduced in number in the duodenum; those that remained were probably CCK cells. Gastrin cells were rare in the antrum at birth and remained rare during the first days after birth. They increased in number, slowly until after weaning (15-20 days of age) and then more rapidly, until 25-30 days of age when the gastrin cell density reached that in adult rats. At the time of birth the gastrin concentration in serum was low; the subsequent increase during the first 2 weeks paralleled the development of the antral gastrin cell system. Adult postprandial serum gastrin concentrations were reached 12 days after birth. Somatostatin cells were rare in both the antral and oxyntic mucosa at birth. They increased gradually in number until about a month after birth when the cell density reached that seen in adult rats. In the oxyntic mucosa the ECL and A-like cells are the predominant endocrine (argyrophil) cell types. They were not detected until about 4 days after birth. Their number increased slowly until about 30 days of age. They did not stain argyrophil until about 2-4 weeks after birth. Parietal cells were few at birth; ultrastructurally they appeared to be in an active state and histochemically they were shown to contain carbonic anhydrase. The pH of the gastric content of newborn rats was close to 5; 15-17 days after birth the pH was about 4 in freely fed rats. In fasted rats shortly after birth the pH was about 4. Two weeks later it was around 2, which is the pH measured in older rats. Hence, the full capacity for acid secretion is probably not established until weaning. Fasting greatly lowers the serum gastrin concentration and the histidine decarboxylase activity of the ECL cells in adult rats. Before weaning, fasting produced these effects only to a minor degree.(ABSTRACT TRUNCATED AT 400 WORDS)     

TCRγδ cells: Mysterious cells of the immune system

γδ cells participate in pathogenic infections and autoimmune conditions, yet, almost a decade after their discovery, little is known regarding their TCR repertoire or effector functions. Unlike MHC-restricted antigen recognition employed by TCRαβ cells, TCRγδ cells can recognize whole unprocessed antigens in an MHC-independent manner. The nature of positive and negative selection used to shape the repertoire of TCRγδ cells is unclear, especially in the nonlymphoid tissues where these cells predominate. While TCRγδ cells express an activated phenotype and are present in pathological conditions, their roles in immunological protection is unknown. This review will focus on our efforts to study these issues of TCRγδ biology.