丝氨酸蛋白酶HtrA2/Omi与细胞存亡

HtrA2/Omi是一种寡聚丝氨酸蛋白酶,存在于大部分的正常组织器官中。在最初的研究中发现[1],当细胞受到凋亡刺激时,HtrA2/Omi可由线粒体膜间隙(mitochondrial intermembrane space)释放进入细胞质中,接着出现大量的细胞凋亡性死亡,这些现象表明HtrA2/Omi与细胞死亡之间有着密切的关系[2]。随着近年来研究的不断深入,人们又惊奇地发现,它不仅出现在细胞凋亡的进程中,当细胞面对各种应激环境时,HtrA2/Omi活性和浓度也会上调,以增加细胞对刺激的耐受能力,使细胞得以生存下来。这是一对看似互相矛盾的事实,却更体现出HtrA2/Omi在细胞存亡调…     

A novel serine protease of the mammalian HtrA family is up-regulated in mouse uterus coinciding with placentation

This paper characterizes a novel gene, previously identified as uniquely regulated at implantation in mouse uterus. We cloned its full mRNA sequence encoding a serine protease possessing an IGF-binding domain and named it pregnancy-related serine protease (PRSP). PRSP is structurally similar to mammalian HtrA1 (56% amino acid similarity). Northern analysis revealed that the expression of PRSP mRNA was low before pregnancy, but it was increased at implantation and markedly up-regulated post-implantation. In-situ hybridization localized low levels of mRNA expression to the epithelium and stroma during very early pregnancy, but high expression to the decidual cells on day 8.5, primarily at the mesometrial pole where the placenta was forming. By day 10.5, PRSP mRNA was detected in the placenta. We also cloned an alternatively spliced PRSP mRNA that is expressed at a very low level. We located PRSP gene on chromosome 5 and established its intron/exon structure, which unambiguously explains how the two mRNA variants are produced through alternative splicing. Based on PRSP protein domain structure and its unique expression during pregnancy, we propose that PRSP plays an important role in the formation/function of the placenta.     

The expression levels and prognostic value of high temperature required A2 (HtrA2) in NSCLC

High temperature required A2 (HtrA2) is a serine kinase that is released from mitochondria into the cytosol upon apoptotic stimuli, inducing apoptosis in various cancers. Thus, analysis of the expression of HtrA2 in non-small-cell lung cancer (NSCLC) tissues is needed for the understanding of this malignancy. In this study we firstly analyzed the apoptosis effect of HtrA2 in A549 cells by RNA interference and cisplatin with Western blot and flow cytometry. Then HtrA2 expression was evaluated by Western blot and immunohistochemistry in NSCLC tissues. Western blot and flow cytometry analyses indicated that deletion of HtrA2 was negatively correlated with apoptosis-induced protein in A549 cells. HtrA2 was lowly expressed in NSCLC and significantly associated with histological differentiation and clinical stage. Besides, low expression of HtrA2 was a prognostic factor for NSCLC patients’ inferior survival. In conclusion, HtrA2 might promote the apoptosis of NSCLC cells, and serve as a target for NSCLC's treatment.     

HtrA 对乳牙高致龋性变异链球菌 GTFs 表达及活性的影响

目的：研究乳牙高致龋性变异链球菌（S．mutans）高温需要 A 蛋白（HtrA）基因缺陷株和 HtrA 高毒力株在体外非应激环境中的葡萄糖基转移酶（GTFs）表达能力及其生物学活性的差异。方法：选用前期已获得的乳牙高致龋性 S．mutans HtrA 基因缺陷株和高毒力株（HtrA 高毒力株和 HtrA 基因缺陷株）,在 BHI 培养基培养至指数期第10 h 后。以实时逆转录聚合酶链反应（real-time RT-PCR）方法检测2菌株 gtfB,gtfC,gtfD 的表达情况。提取蛋白,以蒽酮硫酸法检测其生物学活性,以蛋白免疫印迹（Western Blot）检测其表达量。结果：GTFs 蛋白和基因在乳牙 S．mutans HtrA 基因缺陷株中的表达高于 HtrA 高毒力株,但其生物学活性低于高毒力株。结论：HtrA 基因在乳牙 S．mutans GTFs 的分泌过程中起重要的调控作用。     

Omi/HtrA2在窒息后血清诱导人近曲肾小管皮细胞凋亡中的作用

Objective To investigate the mechanism of inducing apoptosis of Omi/HtrA2 in renal tubular cells with postasphyxial serum of neonate. Methods Human renal proximal tubular cell line HK-2 cell was used as target cell. They were divided into three groups: control group, asphyxia group and Ucf-101 (Omi/HtrA2 special inhibitor) treated group. The challenge concentration of serum obtained from neonates 24 hours after asphyxia was 20%,and the treatment concentration of Ucf-101 was 10 μmol/L.The Omi/HtrA2 translocation in renal tubular cells was observed with confocal microscopy, and the rate of apoptosis was detected with flow cytometer. Results It was found that Omi/HtrA2 was translocated into cytoplasm in asphyxia group, and the rate of Omi/HtrA2 translocation in HK-2 cells of asphyxia group was significantly increased [(28. 1 % vs. (9.4±2.1)%, P<0. 01]. Compared with the control group, after being treated with postasphyxial serum, the rate of apoptosis of HK-2 cells in asphyxia group wa ssignificantly increased [(36. 3±4. 4)% vs. (12. 4±2. 9) %, P<0. 01]. Compared with asphyxia group, the rate of apoptosis in HK-2 cells in Ucf-101 treated group was significantly decreased [(27. 0± 3. 9)% vs. (36.3±4.4) %, P<0. 01]. Conclusion These experimental data demonstrates that postasphyxial serum of neonate can induce apoptosis of HK-2 cells, and translocation of Omi/HtrA2 from mitochondria into cyto-plasm may play an important role in its intracellular signal transduetion mechanism in induction of apoptosis.     

胃癌组织中Omi/HtrA2的表达及与预后的关系

目的 探讨丝氨酸蛋白酶Omi/HtrA2在胃癌组织中的表达及其与胃癌临床病理特征及预后的关系.方法 采用免疫组化法检测68例胃癌组织、15例癌旁组织及15例正常胃黏膜组织中Omi/HtrA2的表达,并分析其表达与胃癌临床病理特征及预后的关系.结果 Omi/HtrA2在胃癌组织中的阳性表达率为73.5%（50/68）,高于癌旁组织（13.3%,2/15）和正常胃黏膜（6.7%,1/15）,差异有统计学意义（P＜0.05）.胃癌组织中Omi/HtrA2的表达与患者的性别、年龄、肿瘤大小及浸润深度无关（P＞0.05） 与肿瘤的分化程度、淋巴结转移和临床分期有关（P＜0.05）.本组胃癌患者5年总体生存率为63.3%,其中Omi/HtrA2表达阳性组和阴性组分别为72.0%和61.1%,两组比较,差异并无统计学意义（P＞0.05）.结论 Omi/HtrA2在胃癌组织中高表达,其表达与胃癌分化程度、淋巴结转移及TNM分期有关,但并不影响胃癌患者的预后.     

Omi/HtrA2短发夹RNA在大鼠肾小管上皮细胞凋亡中的作用及机制

目的探讨Omi/HtrA2短发夹RNA(shRNA)对缺氧/复氧诱导大鼠肾小管上皮细胞(NRK-52E)凋亡的作用及机制。方法细胞分为5组:正常组(常规培养),模型组(缺氧/复氧组)、HK组(转染重组质粒Pgenesil-1/HK)、shRNA1组(转染Pgenesil-1/Omi/HtrA2shRNA1)、shRNA2组(转染Pgenesil-1/Omi/HtrA2shRNA2)。用荧光显微镜观察荧光蛋白的表达,Westernblot检测各组Omi/HtrA2、半胱氨酰天冬氨酸特异性蛋白酶(caspase)-3/-9蛋白表达,比色法测定caspase-3/-9的活性。结果在荧光显微镜下,转染组细胞均可见绿色荧光,未转染组未见绿色荧光。与模型组相比,shRNA1组和shRNA2组Omi/HtrA2、caspase-3/-9蛋白表达明显减少(P<0.01)。模型组和HK组、shRNA1组和shRNA2组之间Omi/HtrA2、caspase-3/-9蛋白表达差异无统计学意义。与正常组相比,模型组Omi/HtrA2、caspase-3/-9蛋白表达明显增强(P<0.01)。结论Pgenesil-1/Omi/HtrA2shRNA1和Pgenesil-1/Omi/HtrA2shRNA2能明显减少缺氧/复氧诱导的NRK-52E中Omi/HtrA2蛋白的表达。通过抑制procaspase-9的活化,进而减少了下游procaspase-3的激活,最终减轻了缺氧/复氧诱导的NRK-52E的凋亡程度。     

慢性细菌性前列腺炎患者精子中促凋亡蛋白Omi/HtrA2的表达

目的:检测慢性细菌性前列腺炎患者精子中促凋亡蛋白Omi/HtrA2的表达,探讨该疾患影响男性生育的可能机制。方法:采集41例慢性细菌性前列腺炎患者(A组)和12例健康生育男性(B组)的精液后,分别检测精液中炎症细胞因子(TNF-α、IL-1β)的浓度和精子密度、活动力及形态。精液经47.5%、57%、76%和95%Percoll梯度分离法分离后,分别用RT-PCR和Western印迹法检测精子促凋亡蛋白Omi/HtrA2的表达。结果:A组与B组炎症因子TNF-α、IL-1β的浓度分别为:(53.62±27.60)pg/ml、(60.75±32.42)pg/ml和(25.03±5.02)pg/ml、(18.70±7.06)pg/ml,两组相比差异有显著性(P<0.05)。A组中促凋亡蛋白Omi/HtrA2的转录和表达水平亦显著升高(P<0.05)。结论:慢性细菌性前列腺炎患者精子中促凋亡蛋白Omi/HtrA2的表达上调。     

Omi／HtrA2对人肝癌细胞系凋亡的调控作用研究

目的探讨丝氨酸蛋白激酶Omi／HtrA2在肝癌细胞中在促凋亡基因p53与抑凋亡基因XIAP之间调控机制的研究,并探讨其今后用于靶向治疗的可能性。方法用Western blotting的方法半定量测定4种不同的肝细胞株（L02、HepG2、Hep3B、Huh7）中p53、XIAP、Omi／HtrA2蛋白的表达量,以及使用Omi／HtrA2特异性抑制剂ucf-101后各自的表达量。结果在p53表达不同的细胞株中XIAP蛋白和Omi／HtrA2蛋白的表达也是不同的,p53可上调Omi／HtrA2蛋白表达,下调XIAP蛋白表达。使用ucf-101后,肝癌细胞中XIAP蛋白表达明显上调。结论Omi／HtrA2是p53与XIAP之间凋亡调控作用的信号因子之一,参与细胞凋亡的调控,有可能作为肿瘤细胞凋亡的治疗靶点。