Granzyme B基因联合姜黄素对肝癌细胞影响的初步研究

原发性肝细胞癌（PHCC）是临床上较为常见的一种恶性肿瘤,我国肝癌发病率在全世界是最高的。笔者将对Granzyme B基因联合姜黄素对肝癌细胞的影响进行初步的探讨。

Participation of CD45,NKR-P1A and ANK61 antigen in rat hepatic NK cell(pit cell)-mediated target cell cytotoxicity

AIM Several triggering receptors have beendescribed to be involved in natural killer（NK）cell-mediated target cytotoxicity.In these studies,NKcells derived from blood or spleen were used.Pitcells are liver-specific NK cells that possess ahigher level of natural cytotoxicity and a differentmorphology when compared to blood NK cells.The aim of this study was to characterize the roleof the NK-triggering molecules NKR-P1A,ANK61antigen,and CD45 in pit cell-mediated killing oftarget cells.METHODS 51Cr-release and DNA fragmentationwere used to quantify target cell lysis andapoptosis,respectively.RESULTS Flow cytometric analysis showed thatpit cells expressed CD45,NKR-P1A,and ANK61antigen.Treatment of pit cells with monoclonalantibody（mAb）to CD45（ANK74）not onlyinhibited CC531s or YAC-1 target lysis but alsoapoptosis induced by pit cells.The mAbs to NKR-P1A（3.2.3）and ANK61 antigen（ANK61）had no effect on pit cell-mediated CC531s or YAC-1 targetcytolysis or apoptosis,while they did increase theFcγ receptor positive（FcγR+）P815 cytolysis andapoptosis.This enhanced cytotoxicity could beinhibited by 3,4-dichloroisocoumarin,an inhibitorof granzymes.CONCLUSION These results indicate that CD45participates in pit cell-mediated CC531s and YAC-1target cytolysis and apoptosis.NKR-P1A andANK61 antigen on pit cells function as activationstructures against FcγR+ P815 cells,which wasmediated by the perforin/granzyme pathway.

Role for urinary biomarkers in diagnosis of acute rejection in the transplanted kidney

Despite the introduction of potent immunosuppressive medications within recent decades, acute rejection still accounts for up to 12% of all graft losses, and is generally associated with an increased risk of late graft failure. Current detection of acute rejection relies on frequent monitoring of the serum creatinine followed by a diagnostic renal biopsy. This strategy is flawed since an alteration in the serum creatinine is a late clinical event and significant irreversible histologic damage has often already occurred. Furthermore, biopsies are invasive procedures that carry their own inherent risk. The discovery of non-invasive urinary biomarkers to help diagnose acute rejection has been the subject of a significant amount of investigation. We review the literature on urinary biomarkers here, focusing on specific markers perforin and granzyme B m RNAs, FOXP3 m RNA, CXCL9/CXCL10 and mi RNAs. These and other biomarkers are not yet widely used in clinical settings, but our review of the literature suggests that biomarkers may correlate with biopsy findings and provide an important early indicator of rejection, allowing more rapid treatment and better graft survival.

Intracellular protease activation in apoptosis and cell- mediated cytotoxicity characterized by cell-permeable fluorogenic protease substrates

Over the past decade the importance of signaling from reporter molecules inside live cells and tissues has been clearly established. Biochemical events related to inflammation, tumor metastasis and proliferation, and viral infectivity and replication are examples of processes being further defined as more molecular tools for live cell measurements become available. Moreover, in addition to quantitating parameters related to physiologic processes, real-time imaging of molecular interactions that compose basic cellular activities are providing insights into understanding disease mechanisms as well as extending clini- cal efficacy of therapeutic regimens. In this review the use of highly cell-permeable fluorogenic substrates that report protease activities inside live cells is described; applications to defining the molecular events of two cellular processes, i.e., apoptosis and cell-mediated cytotoxicity, are then illustrated.

Recombinant GrB and PFP Co-expression in Hep-2 Cells

Objective： To achieve the co-expression of GrB and PFP in Hep-2 cells and analyze the growth inhibiting effects on Hep-2 cells. Methods： Lymphocytes were separated from human laryngeal carcinoma tissue, complete Exon fragments of GrB and PFP were amplified by RT-PCR via extracting lymphocytes total RNA, and they were recombined to the downstream of T7 promoter in the vector pVAX1. The recombinant plasmid pVAX1-PIG was transfected into Hep-2 cells with Lipofectamine 2000. The expression of proteins was identified by RT-PCR, MTT and western blot assay. Results： The gene sequence of the RT-PCR products of GrB and PFP were consistent with the data of GenBank by DNA sequencing analysis. The GrB and PFP cDNA fragment were cloned into the vector of pVAX1 in the right direction and the open reading fragment of GrB and PFP were maintained. The target proteins were detected in the transfected Hep-2 cells, and the inhibitive effect of PFP and GrB on Hep-2 cells growth were studied by thiazolyl blue （MTT） test. Conclusion： The pVAX1-PFP-IRES-GrB plasmid was successfully constructed and expressed, and the expression of PFP and GrB could inhibit the growth of Hep-2 cells.

Enhanced Gene Expression Following Vaccination in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis

Vaccines have been shown to cause differential expression of genes and increase antibody titers against antigens. Influenza vaccines may have an effect on unexplained disorders such as Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME). Immunological changes have been identified following immunization with trivalent influenza vaccine (TIV). The objective of this pilot study was to examine the consequences of TIV on cytokine and cytotoxic genes in CFS/ME. Peripheral blood mononuclear cells were preferentially isolated from whole blood of 7 CFS/ME patients and 8 controls. Following total RNA extraction and synthesis of cDNA, reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) was used to determine the expression levels of mRNAs for cytotoxic genes (perforin (PRF1), granzyme A (GZMA), granzyme B (GZMB) and cytokine genes. GZMB was significantly increased overall in the CFS/ME patients compared to the controls. GZMA was significantly increased 28 days after vaccination while PRF1 was reduced prevaccination but increased 14 days post-vaccination in the CFS/ME patients. There were no significant changes in cytokine genes pre or post vaccination. Administration of TIV may increase the expression of lytic genes in CFS/ME and this may contribute to the increase in cytotoxic activity we observed in these patients post vaccination.

Immunogenic cell death effects induced by doxorubicin improved chemo-immunotherapy via restoration of granzyme B activity

Chemotherapy remains one of the irreplaceable treatments for cancer therapy.The use of immunogenic cell death(ICD)-inducing chemotherapeutic drugs offers a practical strategy for killing cancer cells,simultaneously eliciting an antitumor immune response by promoting the recruitment of cytotoxic immune cells and production of granzyme B(GrB).However,numerous malignant cancers adaptively acquired the capacity of secreting serpinb9(Sb9),a physiological inhibitor of GrB,which can reversibly inhibit the biological activity of GrB.To circumvent this dilemma,in this study,an integrated tailor-made nanomedicine composed of tumor-targeting peptide(Arg-Gly-Asp,RGD)decorated liposome,doxorubicin(DOX,an effective ICD inducer),and the compound 3034(an inhibitor of Sb9),is developed(termed as D3RL)for breast cancer chemo-immunotherapy.In vitro and in vivo studies show that D3RL can directly kill tumor cells and trigger the host immune response by inducing ICD.Meanwhile,D3RL can competitively relieve the inhibition of Sb9 to GrB.The restored GrB can not only effectively induce tumor immunotherapy,but also degrade matrix components in the tumor microenvironment,consequently improving the infiltration of immune cells and the penetration of nanomedicines,which in return enhance the combined antitumor effect.Taken together,this work develops an integrated therapeutic solution for targeted production and restoration of GrB to achieve a combined chemo-immunotherapy for breast cancer.

High Expression of NKG2A/CD94 and Low Expression of Granzyme B Are Associated with Reduced Cord Blood NK Cell Activity

Human umbilical cord blood （CB） has recently been used as a source of stem cells in transplantation. NK cells derived from CB are the key effector cells involved in graft-versus-host disease （GVHD） and graft-versus-leukemia （GVL）. It was reported that the activity of CB NK cells was lower than that of adult peripheral blood （PB） NK cells. In this study, we analyzed the expression of some NK cell receptors and cytotoxicity-related molecules in CB and PB NK cells. The expressions of activating NK receptors, CD16, NKG2D and NKp46, did not show significant difference between CB and PB NK cells. But the expression of inhibitory receptor NKG2A/CD94 was significantly higher on CB NK cells. As to the effector function molecules, granzyme B was expressed significantly lower in CB NK cells, but the expressions of intracellular perforin, IFN-γ, TNF-α and cell surface FasL and TRAIL did not show difference between CB and PB NK cells. The results indicated that the high expression of NKG2A/CD94 and low expression of granzyme B may be related with the reduced activity of CB NK cells.

Ⅱ型糖尿病人来源胰岛细胞抗原2特异性T细胞克隆的建立及其细胞毒性研究

目的:为了研究I型糖尿病中细胞介导的自身免疫损伤因子。方法:采用有限稀释法建立糖尿病病人自身抗原IA2抗原特异性的T细胞克隆,51Cr释放细胞毒性试验测定T细胞激活的HLA限制性和IA2肽段中最小的抗原表位,并用流式细胞仪分析其表面凋亡相关的肿瘤坏死因子（Tumor Necrosis Factor,TNF）家族细胞因子Fas配体（Fas Ligand,FasL）、TNF凋亡诱导配体（Tumor Necrosis Factor Apoptosis-Inducing Ligand,TRAIL）、TNFα表达和细胞内的Perforin和Granzyme B的表达。结果:建立了IA2抗原特异性的T细胞克隆JDM IA2A2,其为HLA DR5限制,IA2最小抗原表位在IA2（838-846）,其表面没有 FasL表达,39.3％细胞表达TRAIL,29.23％细胞表达TNFα,细胞内没有Perforin,但59.16％细胞有Granzyme B。结论:TRAIL、TNFα、GranzymeB可能参与T细胞介导的胰岛β细胞损伤。

运用组织芯片检测淋巴瘤中活化的细胞毒性细胞的意义(英文)

目的运用组织芯片及常规技术检测活化的细胞毒性细胞在各型淋巴瘤中的表达分布情况,为临床治疗和判断预后提供依据。方法运用免疫组织化学方法检测以60例淋巴瘤标本制成的淋巴瘤组织芯片中穿孔素、颗粒酶B的表达和分布情况;同时选择10例鼻NK/T细胞淋巴瘤常规标本与组织芯片进行比较研究,10例淋巴组织反应性增生作为对照。结果在60例淋巴瘤制成的组织芯片中,发生在淋巴结内者48例,淋巴结外者12例。B细胞淋巴瘤42例,T细胞淋巴瘤16例,2例霍奇金淋巴瘤。42例B细胞淋巴瘤的瘤细胞均不表达穿孔素和颗粒酶B。10例外周T细胞淋巴瘤中8例表达穿孔素和9例表达颗粒酶B,阳性表达细胞均为非肿瘤细胞。芯片中的2例和常规10例鼻NK/T细胞淋巴瘤中均见穿孔素和颗粒酶B过度表达。T、B细胞淋巴瘤与NK/T细胞淋巴瘤比较有显著性差异(P<0.01)。结论穿孔素和颗粒酶B是鉴定活化的细胞毒性细胞的免疫标志物,可作为NK/T细胞淋巴瘤的诊断性标志物;其在T细胞淋巴瘤中的表达反映了机体存在抗肿瘤的免疫反应机制。组织芯片技术具有高信息量、简捷、高效、实验误差小、重复性好等优点,可作为研究淋巴瘤的有用工具。

活动性结核病患者自然杀伤(NK)细胞CD160的表达及其与细胞功能的关系

目的分析外周血单个核细胞(PBMC)中CD160 mRNA和蛋白的表达与结核病免疫的关系。方法使用荧光定量PCR检测PBMC中CD160 mRNA的表达,流式细胞术分析PBMC中主要细胞群(T细胞、B细胞、自然杀伤(NK)细胞和单核细胞)表面CD160蛋白表达情况,使用流式细胞术分析NK细胞中CD160与穿孔素(perforin)、颗粒酶B(granzyme B)、颗粒溶素(granulysin)、CD69、CD107和γ干扰素(IFN-γ)的关系。结果活动性肺结核患者PBMC中CD160 mRNA表达显著性下调,且结核分枝杆菌(MTB)阳性患者显著低于MTB阴性患者;B细胞和单核细胞表面CD160表达比例低;CD3^(+)T细胞表面CD160表达在活动性肺结核患者和正常对照者之间差异不明显;活动性肺结核患者NK细胞表面CD160表达显著低于正常对照者;NK细胞与MTB抗原体外培养可下调NK细胞表面CD160的表达;活动性肺结核患者NK细胞表面活化标志CD69表达显著低于正常对照者;CD160^(+)NK细胞的perforin、granzyme B、granulysin、CD69和CD107表达都显著高于CD160^(-)NK细胞;但是CD160^(+)NK细胞的IFN-γ表达显著低于CD160^(-)NK细胞。结论活动性肺结核患者CD160 mRNA和蛋白表达显著下调,CD160促进NK细胞与结核抗原相关的活化和脱颗粒,抑制NK细胞的IFN-γ的表达,CD160可能成为结核病诊治的新靶标。

Biochemical analysis of granzyme H and elucidation of its structurally important residues involved in substrate and inhibitor binding

Apoptotic cell death plays an important role in the maintenance of the normal physiological state and in the pathogenesis of diseases.Granule exocytosis is the main pathway for the immune elimination of virus-infected cells and tumor cells by cytotoxic T lymphocytes and natural killer cells.In recent study,we have investigated the level of granzyme H in patients with breast cancer and in control subjects using enzymatic method.Our study also included the prediction of different sites of granzyme H that play a role in substrate and inhibitor recognition in apoptosis process by using 3D structural model of the enzyme.The research described the possible post-translational modification sites that may help the enzyme in immune elimination of tumor cells.Our study shows that the level of granzyme H was reduced in patients when compared to normal control subjects.There are a number of amino acids that function as substrate recognition sites in granzyme H.However,inhibitors may inhibit their activity and affect the process of autolysis.

Up-regulated intragraft gene expression, ICAM-1 and IL-2R molecules, and apoptotic epithelial cells during rejection of rat small intestine allografts

Objective To investigate the kinetics and the magnitude of intragraft gene expression of interleukin-2(IL-2), interferon-gamma (IFN-y), perforin and granzyme B, and intragraft expression of interieukin-2receptor (IL-2R) and intercellular adhesion molecule-1 ( ICAM-1 ) during acute rejection episodes, and to analyze the changes in apoptosis in small intestinal allograft rejection.Methods Heterotopic small intestine transplantation was performed with inbred rats F344/N (RT11) and Wistar/A (RT1-Ak, RT1-Ed). All recipients were divided into four groups: group 1 : Wistar, native control;group 2: Wistar→Wistar; group 3: F344→Wistar and group 4: F344→Wistar + cyclosporine A (6 mg·kg-1 ·d-1 I.M. ). The grafts were harvested on postoperative days (PODs) 3, 5 and 7. All samples were examined pathologically. Intragraft mRNA expression of IL-2, IFN-γ, perforin and granzyme B were detected with reverse transcriptase polymerase chain reaction (RT-PCR) and intragraft expression of IL-2R and ICAM-1 were stained using immunohistochemistry. We also analyzed the change in apoptosis rejection with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL).Results Mild acute rejection occurred on POD 3 in the ailograft group, moderate acute rejection on POD 5, and severe acute rejection on POD 7, while none of the isografts had histological evidence of acute rejection. Cyclosporine A could effectively control rejection. Gene expression was virtually negative in the native control. Only on POD 5 was IL-2 mRNA expression of ailografts significantly higher than that of isografts ( P ＜ 0.05). IFN-γ mRNA expression was significantly higher than that of the control groups ( P ＜0.01 ) on PODs 3, 5 and 7, and the level of perforin and granzyme B mRNA expression reached significantly higher levels than in the other two control groups on POD 5 and POD 7. Intragraft IL-2Rexpression of the allograft was significantly higher than that of the other three control groups. Only on POD 3 was intragraft ICAM-1 expression of allografts significantly higher than isografts. The number of apoptotic cells per crypt of allografts was significantly higher than that of the other three control groups on POD 3 and POD 5 ( P ＜ 0.01 ).Conclusion Transcription of IL-2, IFN-γ, perforin and granzyme B, and expression of IL-2R and ICAM-1as well as apoptosis of epithelial cells of the grafts play an important role in small intestine allograft rejection.Intragraft gene expression of IFN-γ and intragraft expression of IL-2R as well as apoptotic epithelial cells may become a specific and sensitive diagnostic method of clinical value. Furthermore, therapeutic strategies to alter these molecules in small intestine transplantation may improve the outcome of current antirejection therapy.

Suppressed expression of miR-378 targeting gzmb in N cells is required to control dengue virus infection

Dengue virus （DENV） remains a major public health threat because no vaccine or drugs are available for the prevention and treatment of DENV infection, and the immunopathogenesis mechanisms of DENV infection are not fully understood. Cytotoxic molecules, such as granzyme B （GrzB）, may be necessary to control viral infections. However, the exact role of GrzB during DENV infection and the mechanisms regulating GrzB expression during DENV infection are not clear. This study found that miR-27a~, miR-3Oe, and miR-378 were down-regulated in DENV-infected patients, and DENV infection in humans induced a significant up-regulation of GrzB in natural killer （NK） cells and CD8＋ T cells. Further investigation indicated that NK cells, but not CD8＋ T cells, were the major sources of GrzB, and miR-378, but not miR-27a~ or miR-3Oe, suppressed GrzB expression in NK cells. Notably, we found that overexpression of miR-378 using a miR-378 agomir in DENV-infected mice inhibited GrzB expression and promoted DENV replication. These results suggest the critical importance of miR-378 in the regulation of GrzB expression and a protective role for GrzB in controlling DENV replication in vivo. Therefore, this study provides a new insight into the immunopathogenesis mechanism of DENV infection and a biological basis for the development of new therapeutic strategies to control DENV infection.

Monomethyl fumarate augments NK cell lysis of tumor cells through degranulation and the upregulation of NKp46 and CDIO7a

Dimethyl fumarate （DMF） is a new drug used to treat multiple sclerosis （MS） patients. Here, we examined the effects of DMF and the DMF metabolite monomethyl fumarate （MMF） on various activities of natural killer （NK） cells. We demonstrated that MMF augments the primary CD56^＋, but not CD56^-, NK cell lysis of K562 and RAJI tumor cells. MMF induced NKp46 expression on the surface of CD56^＋, but not CD56^-, NK cells after incubation for 24 h. This effect was closely correlated with the upregulation of CD107a expression on the surface of CD56＋ NK cells and the induction of Granzyme B release from these cells through this metabolite. An anti-NKp46 antibody inhibited the MMF-induced upregulation of CD107a and the lysis of tumor cells through CD56^＋ NK cells. Thus, these results are the first to show that MMF augments CD56^＋ NK cell lysis of tumor target cells, an effect mediated through NKp46. This novel effect suggests the use of MMF for therapeutic and/or preventive protocols in cancer.

The Mechanism of Organophosphorus Pesticide-Induced Inhihition of Cytolytic Activity of Killer Cells

The main toxicity of organophosphorus pesticides （OPs） is neurotoxicity, which is caused by the inhibition of acetylcholinesterase. OPs also affect immune responses including effects on antibody production, IL-2 production, T cell proliferation, decreasement of CD5 cells, and increasement of CD26 cells and autoantibodies. However, there have been few papers investigating the mechanism of OP-induced inhibition of cytolytic activity of killer cells. This study reviews the new mechanism of OP-induced inhibition of activities of natural killer （NK）, lymphokine-activated killer （LAK） and cytotoxic T lymphocytes （CTL）. NK, LAK and CTL induce cell death in tumor or virus-infected target cells by two main mechanisms. The first mechanism is direct release of cytolytic granules that contain perforin, granzymes, and granulysin by exocytosis to kill target cells, which is called the granule exocytosis pathway. The second mechanism is mediated by the Fas ligand （Fas-L）/Fas pathway. To date, it has been reported that OPs inhibit NK, LAK and CTL activities by at least the following three mechanisms： 1） OPs impair the granule exocytosis pathway of NK, LAK and CTL cells by inhibiting the activity of granzymes, and by decreasing the intracellular level of perforin, granzyme A and grannlysin, which was mediated by inducing degranulation of NK cells and by inhibiting the transcript of mRNA of perforin, granzyme A and granulysin; 2） OPs impair the FasL/Fas pathway of NK, LAK and CTL cells, as investigated by using perforin-knockout mice, in which the granule exocytosis pathway of NK cells does not function and only the FasL/Fas pathway remains functional; 3） OPs induce apoptosis of immune cells. Cellular ＆ Molecular Immunology. 2006;3（3）：171-178.