丝氨酸蛋白酶抑制剂PI-9的研究进展

丝氨酸蛋白酶抑制剂-9(serine proteinase inhibitor9,PI-9)是丝氨酸蛋白酶抑制剂的重要成员,也是目前发现的颗粒蛋白酶B(granzyme B,GrB)唯一的内源性丝氨酸蛋白酶抑制剂.近年研究发现,PI-9能抑制GrB所致的靶细胞DNA断裂而阻断其诱导的细胞凋亡.在生理状况下,PI-9能阻止细胞毒淋巴细胞之间的相互攻击,维持机体免疫豁免部位的功能.近年研究发现PI-9还涉及-多种病理过程,诸如调节炎性介质反应,移植免疫应答及介导肿瘤免疫耐受等多种功能.     

丝氨酸蛋白酶抑制剂PI-9的研究进展

丝氨酸蛋白酶抑制剂-9(serine proteinase inhibitor9,PI-9)是丝氨酸蛋白酶抑制剂的重要成员,也是目前发现的颗粒蛋白酶B(granzyme B,GrB)唯一的内源性丝氨酸蛋白酶抑制剂.近年研究发现,PI-9能抑制GrB所致的靶细胞DNA断裂而阻断其诱导的细胞凋亡.在生理状况下,PI-9能阻止细胞毒淋巴细胞之间的相互攻击,维持机体免疫豁免部位的功能.近年研究发现PI-9还涉及-多种病理过程,诸如调节炎性介质反应,移植免疫应答及介导肿瘤免疫耐受等多种功能.     

PI-9和GrB在肿瘤发生发展中作用的研究进展

丝氨酸蛋白酶抑制剂-9（PI-9）是目前发现的惟一具有抑制颗粒酶B（GrB）功能的丝氨酸蛋白酶抑制剂，能阻断GrB所致的靶细胞凋亡，从而维持机体内环境的自稳。GrB存在于活化的细胞毒T淋巴细胞（CTL）和自然杀伤（NK）细胞中，是细胞免疫应答中最主要的效应分子。近年发现在一些肿瘤细胞中有GrB和/或PI-9的表达，并认为它们可能与肿瘤的发生、发展，免疫耐受等有关，笔者就PI-9和GrB的分子结构和功能特点以及它们在肿瘤细胞中表达和意义作一综述。     

基因干扰对人肾小球系膜细胞megsin基因表达及α平滑肌肌动蛋白和转化生长因子β1生成的影响

megsin,即SERPINB7,是丝氨酸蛋白酶抑制剂进化单位B的第7位成员[1],为近年发现的特异性表达于肾脏的基因.     

分泌颗粒酶B的调节性B细胞在器官移植术后免疫调节作用的研究进展

器官移植术后的排斥反应以及长期使用免疫抑制所致的代谢性疾病、肾功能不全、感染、肿瘤复发等并发症,严重影响着受者的长期生存和生活质量。因此,诱导器官移植术后免疫耐受是移植科医师所追求的最终目标。目前研究发现,调节性B细胞(Breg)在移植术后免疫耐受的形成中发挥了重要作用。本文主要从分泌颗粒酶B(GrB)的Breg(GrB+Breg)的发现和特点、GrB发挥免疫调节作用的方式、GrB+Breg在器官移植术后的免疫调节作用以及常用免疫抑制剂对GrB+Breg的作用进行文献综述。     

NF-κB在急性胰腺炎病程进展中的作用

细胞因子的级联释放是急性胰腺(acute pancreatitis,AP)病情加重的重要因素,最终导致多器官功能障碍和死亡.核因子-κB(nuclear factor-kappa B,NF-κB)作为一种多项性调节作用的核转录因子,能与多种基因启动子部位kB位点特异性结合,引起炎症反应和凋亡等多种病理生理反应.研究发现选用NF-kB抑制剂对其活性进行抑制,能抑制机体的炎症反应,明显改善急性胰腺炎的预后,可以作为一个潜在的新靶点治疗急性胰腺炎.然而,亦有研究发现抑制NF-κB能明显加重急性胰腺炎病情,因此,研究NF-κB在炎症反应中占有重要的研究地位.     

颅脑损伤标志物研究进展

颅脑损伤是一类临床常见的疾病和疾病的并发症.近年来,脑损伤的特异性生化标志物的研究得到了临床医生和研究人员越来越多的关注.这些指标对于及时诊断、早期判断病情、评估预后及调整治疗方案等方面有着重要临床意义.其中星形胶质源性蛋白(S100B)、神经元特异性烯醇化酶(NSE)、基质金属蛋白酶-9(MMP-9)和组织基质金属蛋白酶抑制剂-1 (TIMP-1)是目前研究较多的几种.     

甲磺酸加贝脂抑制人肝癌HepG2细胞增殖及相关机制

甲磺酸加贝脂(gabexate mesilate,GM)是一种人工合成的丝氨酸蛋白酶抑制剂,近年来有文献报道将GM用于直肠癌、胰腺癌等肿瘤治疗的探索性研究,初步显示GM可能是一种新型的基质金属蛋白酶抑制剂[1-2].我们探索GM在体外对肝癌细胞HepG2增殖和凋亡的影响,探讨其可能的作用机制.     

甲磺酸加贝脂抑制人肝癌HepG2细胞增殖及相关机制

甲磺酸加贝脂(gabexate mesilate,GM)是一种人工合成的丝氨酸蛋白酶抑制剂,近年来有文献报道将GM用于直肠癌、胰腺癌等肿瘤治疗的探索性研究,初步显示GM可能是一种新型的基质金属蛋白酶抑制剂[1-2].我们探索GM在体外对肝癌细胞HepG2增殖和凋亡的影响,探讨其可能的作用机制.     

甲磺酸加贝脂抑制人肝癌HepG2细胞增殖及相关机制

甲磺酸加贝脂(gabexate mesilate,GM)是一种人工合成的丝氨酸蛋白酶抑制剂,近年来有文献报道将GM用于直肠癌、胰腺癌等肿瘤治疗的探索性研究,初步显示GM可能是一种新型的基质金属蛋白酶抑制剂[1-2].我们探索GM在体外对肝癌细胞HepG2增殖和凋亡的影响,探讨其可能的作用机制.     

Serine proteinase inhibitor-9, an endogenous blocker of granzyme B/perforin lytic pathway,is hyperexpressed during acute rejection of renal allografts

BACKGROUND: Serine proteinase inhibitor (PI)-9 with a reactive center P1 (Glu)-P1' is a natural antagonist of granzyme B and is expressed in high levels in cytotoxic T lymphocytes (CTL). In view of the role of CTL in acute rejection, we explored the hypothesis that PI-9 would be hyperexpressed during acute rejection. Because PI-9 can protect CTL from its own fatal arsenal and potentially enhance the vitality of CTL, we examined whether PI-9 levels correlate with the severity of rejection as well as predict subsequent graft function. METHODS: We obtained 95 urine specimens from 87 renal allograft recipients. RNA was isolated from the urinary cells and mRNA encoding PI-9, granzyme B, or perforin and a constitutively expressed 18S rRNA was measured with the use of real-time quantitative polymerase chain reaction assay, and the level of expression was correlated with allograft status. RESULTS: The levels of PI-9 (P=0.001), granzyme B (P<0.0001), and perforin mRNAs (P<0.0001), but not the levels of 18S rRNA (P=0.54), were higher in the urinary cells from the 29 patients with a biopsy-confirmed acute rejection than in the 58 recipients without acute rejection. PI-9 levels were significantly higher in patients with type II or higher acute rejection changes compared with those with less than type II changes (P=0.01). Furthermore, PI-9 levels predicted subsequent graft function (r=0.43, P=0.01). CONCLUSIONS: PI-9 mRNA levels in urinary cells are diagnostic of acute rejection, predict renal allograft histology grade, and predict functional outcome following an acute rejection episode.     

Cloning of pig serine proteinase inhibitor 9 and its use in protecting against apoptosis

The activity of granzyme B, a main effector molecule of natural killer (NK) cells and cytotoxic T lymphocytes, is regulated by the intracellular serine proteinase inhibitor 9 (PI-9). Pig PI-9 was first cloned, and the sequences that encode pig PI-9, including the start codon and stop codon, were identified. The cDNA was inserted into the cloning site of pCXN2 (chicken beta actin promoter and cytomegalovirus enhancer), transfected into pig endothelial cells (PEC), and several stable PEC clones were established. An NK cell-mediated cytolysis test was next applied to the PEC clones, using YT cells (an NK-like cell line). The PEC transfectants with pig PI-9 had a significant inhibitory effect on NK cell-mediated PEC lysis. The overexpression of the anti-apoptotic molecule, pig PI-9, has the potential for use in protecting graft cells from human NK cells.     

Hyperexpression of the granzyme B inhibitor PI-9 in human renal allografts: a potential mechanism for stable renal function in patients with subclinical rejection

BACKGROUND: Granzyme B-positive T lymphocytes infiltrate renal allografts during acute cellular rejection and cause graft injury by inducing apoptosis of tubular cells. Protease inhibitor 9 (PI-9), an intracellular serpin that inhibits granzyme B, is known to protect cells from the action of cytotoxic T lymphocytes. METHODS: Expression of granzyme B and PI-9 in transplant biopsies from patients with acute cellular rejection (N= 18), subclinical rejection showing a mononuclear cell infiltrate without deterioration of renal function (N= 15), or stable transplant function (N= 13) were studied. Immunohistochemical stainings were analyzed and scored semiquantitatively by two independent observers who were not aware of clinical results. RESULTS: Granzyme B was expressed by mononuclear cells in all biopsies with cellular infiltrates. PI-9 was diffusely expressed by tubular cells in the allografts of all patients with subclinical rejection. In contrast, PI-9 expression was only focally in the patients with clinical rejection or without rejection. Although no difference was observed in granzyme B levels between acute and subclinical rejection, in subclinical rejection tubular epithelial cells showed significantly stronger expression of PI-9 than in acute rejection (P= 0.011). CONCLUSION: These data suggest that a high expression of PI-9 by tubular epithelial cells can serve as one of the factors protecting renal allografts from rejection in spite of the presence of inflammatory cell infiltrates.     

Insulin-like growth factor-1 improves postischemic recovery in hypertrophied hearts.

BACKGROUND: Severe myocardial hypertrophy is associated with decreased tolerance to ischemia compared with normal hearts. We hypothesized that treatment with insulin-like growth factor-1 (IGF-1) improves postischemic myocardial recovery by increasing glucose uptake during ischemia and early reperfusion. METHODS: Banding of the thoracic aorta in 10-day-old rabbits created pressure-overload hypertrophy. At 5 weeks of age (severe hypertrophy), aortic banded and sham-operated isolated hearts underwent 30 minutes of normothermic ischemia with or without IGF-1 in the preischemic perfusate and cardioplegia followed by 30 minutes of reperfusion. RESULTS: 2-Deoxyglucose uptake (31P-NMR) and phosphatidylinositol-3-kinase (PI-3-kinase) activity were significantly lower in hypertrophied hearts. Insulin-like growth factor-1 restored glucose uptake and PI-3-kinase activity to control levels in the hypertrophied hearts and both effects were blocked by wortmannin (a PI-3-kinase inhibitor). Postischemic developed pressure was significantly improved in IGF-1-treated hearts compared with untreated or IGF-1+wortmannin-treated hypertrophied hearts. CONCLUSIONS: These data indicate that IGF-1 improves glucose uptake and tolerance to ischemia in hypertrophied hearts. Myocardial IGF-1 effects are likely mediated through a PI-3-kinase-dependent pathway.     

A synthetic heparanase inhibitor reduces proteinuria in passive Heymann nephritis

The beta-D-endoglycosidase heparanase has been proposed to be important in the pathogenesis of proteinuria by acting to selectively degrade the negatively charged side chains of heparan sulfate proteoglycans (HSPG) within the glomerular basement membrane (GBM). A loss of the negatively charged HSPG may result in alteration of the permselective properties of the GBM, loss of glomerular epithelial and endothelial cell anchor points, and liberation of growth factors. This study examined the effect of PI-88, a sulfated oligosaccharide heparanase inhibitor, on renal function, glomerular ultrastructure, and proteinuria. Continuous PI-88 infusion at 25 mg/kg per d did not adversely affect animal behavior, growth, or GFR. Cortical tubular vacuolation, however, was observed by light microscopy, and GBM thickness was significantly reduced in these animals (P < 0.0002). Tissue distribution studies using [(35)S]-labeled PI-88 revealed high levels of radioactivity in the kidney after a single subcutaneous injection of 25 mg/kg, suggesting protracted accumulation; moreover, active PI-88 was detected in urine. In passive Heymann nephritis, PI-88 delivered as a continuous infusion at 25 mg/kg per d significantly reduced autologous-phase proteinuria, at day 14 (P < 0.009), in the absence of altered sheep antibody deposition, C5b-9 deposition, and circulating rat anti-sheep antibody titers. Glomerular vascular endothelial growth factor and fibroblast growth factor expression was unaffected by PI-88 administration. However, PI-88 administration significantly prevented glomerular HSPG loss as demonstrated by quantitative immunofluorescence studies (P < 0.0001) in the absence of altered agrin distribution. These data therefore confirm the importance of heparanase in the development of proteinuria.     

Retrovirally transferred genes inhibit apoptosis in an insulin-secreting cell line: implications for islet transplantation

The transplantation of pancreatic islets for the treatment of type I diabetes is hindered by the enormous loss of cells due to early apoptotic events. Genetic engineering of islets with cytoprotective genes is an important strategy aimed to enhance the survival of these cells in the transplant setting. The present study was designed to evaluate and compare the effects of five genes on a cell line derived from insulin-producing beta-cells, NIT-1. Cells were transduced using a Maloney murine leukemia virus (MLV) vector coding for yellow fluorescent protein (YFP) and for one of the following antiapoptotic genes: cFLIP, FADD-DN, BcL-2, PI-9, and ICAM-2. These genes were able to protect NIT-1 cells from cytokine-induced apoptosis to varying degrees ranging from no protection to significant protection equivalent to an optimal dose of a chemical caspase inhibitor. The data demonstrate that cFLIP, FADD-DN, and PI-9 are significantly more effective in protecting NIT-1 cells than BcL-2 and ICAM-2. Additionally, the data show that despite its weak in vitro inhibition of caspase-3, PI-9 affords significant protection against TNF-alpha-induced apoptosis in these cells. These genes may be ideal candidates to augment islet survival following transplantation.     

硫酸寡聚糖复合物对实验性腹主动脉瘤的抑制作用

目的 观察乙酰肝素酶抑制剂PI-88对腹主动脉瘤形成的抑制作用。方法 利用豚鼠-SD大鼠异种移植腹主动脉瘤模型，于移植术后4周内连续PI-88治疗；术后4周利用Northem blot杂交和免疫组化技术，检测移植腹主动脉直径、乙酰肝素酶表达、微血管增生及炎性细胞浸润程度。结果 与非治疗组相比，治疗组腹主动脉乙酰肝素酶表达、移植血管直径、微血管增生及炎性细胞浸润程度均显著降低，但仍高于阴性对照组。结论 PI-88可通过抑制乙酰肝素酶作用，控制腹主动脉瘤形成。     

Protein kinase Czeta activation mediates glucagon-like peptide-1-induced pancreatic beta-cell proliferation

Glucagon-like peptide-1 (GLP-1), an insulinotropic and glucoincretin hormone, is a potentially important therapeutic agent in the treatment of diabetes. We previously provided evidence that GLP-1 induces pancreatic beta-cell growth nonadditively with glucose in a phosphatidylinositol-3 kinase (PI-3K)-dependent manner. In the present study, we investigated the downstream effectors of PI-3K to determine the precise signal transduction pathways that mediate the action of GLP-1 on beta-cell proliferation. GLP-1 increased extracellular signal-related kinase 1/2, p38 mitogen-activated protein kinase (MAPK), and protein kinase B activities nonadditively with glucose in pancreatic beta(INS 832/13) cells. GLP-1 also caused nuclear translocation of the atypical protein kinase C (aPKC) zeta isoform in INS as well as in dissociated normal rat beta-cells as shown by immunolocalization and Western immunoblotting analysis. Tritiated thymidine incorporation measurements showed that the p38 MAPK inhibitor SB203580 suppressed GLP-1-induced beta-cell proliferation. Further investigation was performed using isoform-specific pseudosubstrates of classical (alpha, beta, and gamma) or zeta aPKC isoforms. The PKCzeta pseudosubstrate suppressed the proliferative action of GLP-1, whereas the inhibitor of classical PKC isoforms had no effect. Overexpression of a kinase-dead PKCzeta acting as a dominant negative protein suppressed GLP-1-induced proliferation. In addition, ectopic expression of a constitutively active PKCzeta mutant stimulated tritiated thymidine incorporation to the same extent as GLP-1, and the glucoincretin had no growth-promoting action under this condition. The data indicate that GLP-1-induced activation of PKCzeta is implicated in the beta-cell proliferative signal of the insulinotropic hormone. The results are consistent with a model in which GLP-1-induced PI-3K activation results in PKCzeta translocation to the nucleus, which may play a role in the pleiotropic effects (DNA synthesis, metabolic enzymes, and insulin gene expression) of the glucoincretin.