The role of polymorphic cytochrome P450 gene(CYP2B6)in B-chronic lymphocytic leukemia(B-CLL)incidence and outcome among Egyptian patients

Cytochromes P450(CYPs)play a prominent role in catalyzing phase I xenobiotic biotransformation and account for about 75%of the total metabolism of commercially available drugs,including chemotherapeutics.The gene expression and enzyme activity of CYPs are variable between individuals,which subsequently leads to different patterns of susceptibility to carcinogenesis by genotoxic xenobiotics,as well as differences in the efficacy and toxicity of clinically used drugs.This research aimed to examine the presence of the CYP2B6*9 polymorphism and its possible association with the incidence of B-CLL in Egyptian patients,as well as the clinical outcome after receiving cyclophosphamide chemotherapy.DNA was isolated from whole blood samples of 100 de novo B-CLL cases and also from 100 sex-and age-matched healthy individuals.The presence of the CYP2B6*9(G516T)polymorphism was examined by PCR-based allele specific amplification(ASA).Patients were further indicated for receiving chemotherapy,and then they were followed up.The CYP2B6*9 variant indicated a statistically significant higher risk of B-CLL under different genetic models,comprising allelic(T-allele vs.G-allele,OR=4.8,p<0.001)and dominant(GT+TT vs.GG,OR=5.4,p<0.001)models.Following cyclophosphamide chemotherapy,we found that the patients with variant genotypes(GT+TT)were less likely to achieve remission compared to those with the wild-type genotype(GG),with a response percentage of(37.5%vs.83%,respectively).In conclusion,our findings showed that the CYP2B6*9(G516T)polymorphism is associated with B-CLL susceptibility among Egyptian patients.This variant greatly affected the clinical outcome and can serve as a good therapeutic marker in predicting response to cyclophosphamide treatment.

Identification of Cytochrome P450 (CYP) Genes in Zhikong Scallop (Chlamys farreri)

Cytochrome P450 （CYP） superfamily is one of the membership largest and function most diverse protein superfamily recogniozed among living beings. Members of this superfamily were further assigned to different families and subfamilies based on their amino acid similarities. According to their phylogenetic relationships, the CYP genes which likely diverged from common ancestor gene and may share common functions were grouped into one clan. Widely distributing scallops are a group of the most conspicuous bivalve; however the studies on their CYP is acarce. In this study, we searched the genome and expressed sequence tags of Zhikong scallop （Chlamysfarreri） for CYP genes. In total, 88 non-redundant CIfP were identified, which were homed in 13 CYPs gene families. Phylogenetic analysis divided these genes into 4 CYP clans. As in deuterostomes, Clan 2 was the largest, which contained 33 genes belonging to CYP1, CYP2, CYP17 and CYP356 families. Clan 3 contgained 19 genes belonging to CYP3, CYP5 and CYP30 families. Clan 4 contained 23 genes, all belonging to CYP4 family. The mitochondrial CYP clan contained 9 genes belonging to CYPIO and CYP24 families. In comparison, protostomes （C. farreri, D. pluex, D. melanogaster） contained more CYP genes than deuterostomes （S. purpuratus and vertebrates） in Clan 2 but less genes in Clan 3 and Clan 4. Our findings will aid to deciphering CYP function and evolution in scallops and bivalves.

Cytochrome P450 2E1 genetic polymorphism and gastric cancer in Changle,Fujian Province

AIM: Genetic polymorphism in enzymes of carcinogen metabolism has been found to have the influence on the susceptibility to cancer. Cytochrome P450 2E1 (CYP2E1) is considered to play an important role in the metabolic activation of procarcinogens such as N-nitrosoamines and low molecular weight organic compounds. The purpose of this study is to determine whether CYP450 2E1 polymorphisms are associated with risks of gastric cancer. METHODS: We conducted a population based case-control study in Changle county, Fujian Province, a high-risk region of gastric cancer in China. Ninety-one incident gastric cancer patients and ninety-four healthy controls were included in our study. Datas including demographic characteristics, diet intake, and alcohol and tobacco consumption of individuals in our study were completed by a standardized questionnaire.PCR-RFLP revealed three genotypes:heterozygote (C1/C2) and two homozygotes (C1/C1 and C2/C2) in CYP2E1. RESULTS: The frequency of variant genotypes (C1/C2 and C2/C2) in gastric cancer cases and controls was 36.3% and 24.5%, respectively. The rare homozygous C2/C2 genotype was found in 6 individuals in gastric cancer group(6.6%), whereas there was only one in the control group (1.1%). However, there was no statistically significant difference between the two groups (two-tailed Fisher's exact test P=0.066). Individuals in gastric cancer group were more likely to carry genotype C1/C2 (odds ratio, OR=1.50) and C2/C2 (OR=7.34) than individuals in control group (chi(2) =4.597, for trend P=0.032). The frequencies of genotypes with the C2 allele (C1/C2 and C2/C2 genotypes) were compared with those of genotypes without C2 allele (C1/C1 genotype) among individuals in gastric cancer group and control group according to the pattern of gastric cancer risk factors. The results show that individuals who exposed to these gastric cancer risk factors and carry the C2 allele seemed to have a higher risk of developing gastric cancer. CONCLUSION: Polymorphism of CYP2E1 gene may have some effect in the development of gastric cancer in Changle county, Fujian Province.

Identification of the metabolites of polybrominated diphenyl ether 99 and its related cytochrome P450s

Objective：To investigate the metabolites of polybrominated diphenyl ether 99（BDE-99）and its related cytochrome P450s in an in vitro system.Methods：Rat primary hepatocytes were isolated and treated with BDE-99 for 24-72 h.Metabolites were then extracted from the hepatocytes and media,and detected by GC/MS.Several mRNAs of metabolic enzymes were also extracted from the same cells and the gene expression levels were determined using quantitative real-time PCR.In addition,selected recombinant cytochrome P450s（CYPs） were expressed in a bacurovirus/sf9 system,and these were further used to explore the metabolism of BDE-99 in vitro.The parent depletion approach was used for screening the ability of CYPs to eliminate BDE-99.Results： A reductively debrominated metabolite,BDE-47,and three oxidative metabolites,2,4,5-tribromophenol,5-OH-BDE-47,and 5＇-OH-BDE-99,were identified from the BDE-99-treated rat hepatocytes,whereas no MeO metabolite was detected in the system.RT-PCR analysis showed that CYP 3A23/3A1,1A2,and 2B1/2 were induced by BDE-99.Furthermore,using the heterological expressed CYP proteins in in vitro BDE-99 metabolism experiments we found that CYP1A2 and CYP3A4 showed the highest metabolic efficiency for BDE-99,with the metabolic clearance rates of CYP1A2 and CYP3A4 being 30.3%and 27.7%,respectively.CYP1A1 and CYP2A6 displayed relatively low clearance rates,while CYP2E1 seemed not to be associated with the BDE-99 metabolism.Conclusions：In our in vitro rat primary hepatocyte metabolism system,four metabolites of BDE-99 were identified,and CYP3A4 and CYP1A2 were demonstrated to be involved in the BDE-99 metabolism.

Three new alternative splicing variants of human cytochrome P450 2D6 mRNA in human extratumoral liver tissue

AIM: To identify the new alternative splicing variants of human CYP2D6 in human extratumoral liver tissue with RT-PCR and sequencing. METHODS: Full length of human CYP2D6 cDNAs was amplificated by reverse transcription-polymerase chain reaction (RT-PCR) from a human extratumoral liver tissue and cloned into pGEM-T vector. The cDNA was sequenced. Exons from 1 to 4 of human CYP2D6 cDNAs were also amplificated by RT-PCR from extratumoral liver tissues of 17 human hepatocellular carcinomas. Some RT-PCR products were sequenced. Exons 1 to 4 of CYP2D6 gene were amplified by PCR from extratumoral liver tissue DNA. Two PCR products from extratumoral liver tissues expressing skipped mRNA were partially sequenced. RESULTS: One of the CYP2D6 cDNAs had 470 nucleotides from 79 to 548 (3' portion of exons 1 to 5' portion of exon 4), and was skipped. Exons 1 to 4 of CYP2D6 cDNA were assayed with RT-PCR in 17 extratumoral liver tissues. Both wild type and skipped mRNAs were expressed in 4 samples, only wild type mRNA was expressed in 5 samples, and only skipped mRNA was expressed in 8 samples. Two more variants were identified by sequencing the RT-PCR products of exons 1 to 4 of CYP2D6 cDNA. The second variant skipped 411 nucleotides from 175 to 585. This variant was identified in 4 different liver tissues by sequencing the RT-PCR products. We sequenced partially 2 of the PCR products amplified of CYP2D6 exon 1 to exon 4 from extratumoral liver tissue genomic DNA that only expressed skipped mRNA by RT-PCR. No point mutations around exon 1, intron 1, and exon 4, and no deletion in CYP2D6 gene were detected. The third variant was the skipped exon 3, and 153 bp was lost. CONCLUSION: Three new alternative splicing variants of CYP2D6 mRNA have been identified. They may not be caused by gene mutation and may lose CYP2D6 activity and act as a down-regulator of CYP2D6.

Self-sufficient Cytochrome P450s and their potential applications in biotechnology

Cytochrome P450s(CYPs)are ubiquitously found in all kingdoms of life,playing important role in various biosynthetic pathways as well as degradative pathways;accordingly find applications in a vast variety of areas from organic synthesis and drug metabolite production to modification of biomaterials and bioremediation.Significantly,CYPs catalyze chemically challenging CAH and CAC activation reactions using a reactive high-valent iron-oxo intermediate generated upon dioxygen activation at their heme center,while the other oxygen atom is reduced to the level of water by electrons provided through a reductase partner protein.Self-sufficient CYPs,encoding their heme domain and reductase protein in a single polypeptide,facilitate increased catalytic efficiency and render a less complicated system to work with.The self-sufficient CYP enzyme from CYP102A family(CYP102A1,BM3)is among the earliest and most-investigated model enzymes for mechanistic and structural studies as well as for biotechnological applications.An increasing number of self-sufficient CYPs from the same CYP102 family and from other families have also been reported in last decade.In this review,we introduce chemistry and biology of CYPs,followed by an overview of the characteristics of self-sufficient CYPs and representative reactions.Enzyme engineering efforts leading to novel self-sufficient CYP variants that can catalyze synthetically useful natural and non-natural(nature-mimicking)reactions are highlighted.Lastly,the strategy and efforts that aim to circumvent the challenges for improved thermostability,regio-and enantioselectivity,and total turnover number;associated with practical use of self-sufficient CYPs are reviewed.

In vitro and in vivo cytochrome P450 3A enzyme inhibition by Aframomum melengueta and Dennettia tripetala extracts

Objective: To evaluate the in vitro and in vivo inhibitory effects of two commonly used herbs, Aframomum melengueta(A. melengueta) and Dennettia tripetala(D. tripetala) on CYP 3A enzymes. Methods: In vitro inhibition of the enzymes were assessed with microsomes extracted from female albino rats using erythromycin-N-demethylation assay(EMND) method while their in vivo effects were measured by estimating simvastatin plasma concentrations in rats. Pharmacokinetic parameters were determined using non-compartmental anaysis as implemented in Win Nonlin pharmacokinetic program. Results: EMND assay with intestinal microsomes indicated that aqueous extracts of D. tripetala and A. melengueta significantly(P < 0.05) inhibited intestinal CYP 3A activity at both 50 μg and 100 μg concentrations. Petroleum ether extract of D. tripetala and ethanol extracts of A. melengueta inhibited intestinal CYP3 A activity at 100 μg but not at 50 μg concentrations. All the extracts showed an in vitrodose dependent CYP 3A inhibition with liver microsomes. In vivo analysis showed that pretreatment with the extracts enhanced systemic absorption of simvastatin with reductions in metabolizing enzymes activity as indicated in significant increases in maximal concentration, area under curve, area under moment curve and mean resident time of simvastatin(P < 0.05). Conclusions: Herbal preparations containing these plants' extracts should be used with caution especially in patients on CYP450 3A substrate medications.

光伏支架专用连续镀锌钢带S450GD+Z的开发

结合唐钢镀锌生产线的设备特点,按照光伏支架专用镀锌钢带高强度和高延展性的特点,重新设计了高强结构级镀锌产品S450GD+Z的生产工艺,制定了全流程工艺制度,并成功试制。生产结果显示:无论热基还是冷基光伏支架的屈服强度与抗拉强度均达到了设计值;热基光伏支架金相组织为细小的铁素体+珠光体,冷基光伏支架的晶粒没有长大,仍为拉长状态。表明热基光伏支架可通过细化晶粒与改变晶体结构来提高产品的强度与延伸率,冷基光伏支架则是通过退火过程中晶粒发生回复再结晶来提高产品的强度与延伸率。

基于文献报道的中药化学成分与CYP450s相互作用研究

目的:使用复杂网络技术研究中药化学成分与CYP 4 50s的关系,为中药分子机理研究和新药开发提供参考。方法:本文基于120篇文献报道,通过人工摘录的方式收集中药化学成分和CYP450s的相关信息,使用Cytoscape 2.8.2作为分析工具,构建化学成分与CYP450s的复杂网络图,并计算了点度中心度、接近中心度、中间中心度以及中药化学成分节点的相似度。结果:本文采集了169个中药化学成分和25种CYP450s的相关信息。CYP2C19、CYP1A2、CYP3A4等CYP450s和槲皮素、银杏内酯A、黄芩素等化学成分为目前研究的热点酶和热点中药化学成分。相似度较高的欧前胡素和银杏内酯B,槲皮素和黄芩素等化学成分可能存在协同相互作用。结论:复杂网络计算可以很好地辅助中药协同作用机理研究和新药开发工作。

Identification and analysis of a processed cytochrome P450 pseudogene of the disease vector Aedes aegypti

Objective: To clone cytochrome P450 from Aedes aegypti(Ae. aegypti) and determine the characteristics using bioinformatics tools. Methods: Cytochrome P450 of Ae. aegypti was amplified using polymerase chain reaction, cloned and sequenced. Evolutionary relationship of the sequence was inferred and bioinformatics tools were used to predict subcellular localisation, signal peptide, transmembrane helix, phosphorylation, O-glycosylation, secondary and tertiary structures of the deduced protein. Results: Polymerase chain reaction rather amplified a cytochrome P450 pseudogene which was named CYP4H44P(Gen Bank accession number KF779932). The pseudogene has 1537 nucleotides and an open reading frame of 335 amino acids containing cytochrome P450 motifs except the Wxxx R motif. It is highly homologous to CYP4H28 and CYP4H28v2. Phylogenetic analysis and evolutionary divergence showed strong clustering with CYP4H28 alleles and least divergence from the alleles respectively. The deduced protein was predicted to be found in the cytoplasm and likely to be phosphorylated but devoid of signal peptide, transmembrane helix and O-glycosylated sites. The secondary and tertiary structures were also generated. Conclusions: A cytochrome P450 pseudogene, CYP4H44 P was cloned from Ae. aegypti. The pseudogene is homologous with CYP4H28 alleles and seems to have recently diverged from this group. Isolating this pseudogene is an important step for evaluating its biological role in the mosquito and for the evolutionary analysis of Ae. aegypti CYPs.

Expression of cytochrome P450 2A13 in human non-small cell lung cancer and its clinical significance

Lung cancer is one of the most important causes of cancer-related mortality worldwide. Human cytochrome P450 2A13 enzyme （CYP2A13） is predominantly expressed in the respiratory tract and could catalyze various carcinogens. In this study, we quantified CYP2A13 expression in non-small cell lung cancer （NSCLC） tissues and examined the relation between CYP2A13 and clinicopathologic factors. Thirty-five paired lung cancer and normal tissues were studied for the expression of the CYP2A13 gene by using real-time PCR and Western blot- ting assays. We also investigated the relationship between CYP2A13 expression and clinicopathologic factors such as age, gender, histology and lymph node status in tumor tissues. SPSS （17.0） statistical software was applied for data analysis. The real-time PCR results showed that there was no significant difference in the CYP2A13 mRNA transcript levels between tumor and paired normal tissues in the 35 samples and in 12 paired squamous cell car- cinomas. In adenocarcinoma, the expression of CYP2A13 mRNA in tumor tissues was 12.5% of that in adjacent tissues （P 〈 0.05） and it was not associated with age, gender, histology and lymph node status of the patients. The amounts of CYP2A13 proteins detected by Western blotting assays correlated well with those of the correspond- ing mRNAs. In conclusion, the expression of CYP2A13 was downregulated in lung adenocarcinoma. CYP2A13 may be involved in the development and progression of lung adenocarcinoma.

糖尿病状态下CYP450s酶活性改变及其在糖尿病病情发展中的作用

细胞色素P450酶(CYP450s)是肝脏、肠道中参与大多数临床药物I相代谢的最重要的代谢酶家族。近年来有临床报道显示,糖尿病状态下,肝脏CYP450s酶的活性与表达发生改变。本文对CYP3A、CYP2E1、CYP1A2等多种CYP450s酶亚型在糖尿病状态下功能与表达发生的改变进行了评述,分析了种属差异、糖尿病类型以及病程对CYP450s酶活性的影响,并总结了糖尿病状态下因CYP450s酶活性改变造成的药物代谢的变化及药物不良反应。此外,CYP450s酶活性的改变可进一步引发内源性物质代谢改变,加剧胰岛素抵抗等,本文进一步阐述了CYP450s酶活性改变对糖尿病病情发展产生的影响。

Cytochrome P450 CitCYP97B modulates carotenoid accumulation diversity by hydroxylating β-cryptoxanthin in Citrus

Carotenoids in plant foods provide health benefits by functioning as provitamin A.One ofthe vital provitamin A carotenoids,β-cryptoxanthin,is typically plentiful in citrus fruit.However,little is known about the genetic basis of β-cryptoxanthin accumulation in citrus.Here,we performed a widely targeted metabolomic analysis of 65 major carotenoids and carotenoid derivatives to characterize carotenoid accumulation in Citrus and determine the taxonomic profile of b-cryptoxanthin.We used data from 81 newly sequenced representative accessions and 69 previously sequenced Citrus cultivars to reveal the genetic basis of β-cryptoxanthin accumulation through a genome-wide association study.We identified a causal gene,CitCYP97B,which encodes a cytochrome P450 protein whose substrate and metabolic pathways in land plants were undetermined.We subsequently demonstrated that CitCYP97B functions as a novel monooxygenase that specifically hydroxylates the β-ring of β-cryptoxanthin in a heterologous expression system.In planta experiments provided further evidence that CitCYP97B negatively regulates b-cryptoxanthin content.Using the sequenced Citrus accessions,we found that two critical structural cis-element variations contribute to increased expression of CitCYP97B,thereby altering β-cryptoxanthin accumulation in fruit.Hybridization/introgression appear to have contributed to the prevalence of two cis-element variations in different Citrus types during citrus evolution.Overall,these findings extend our understanding of the regulation and diversity of carotenoid metabolism in fruit crops and provide a genetic target for production of β-cryptoxanthin-biofortified products.

黄芩苷对中国对虾细胞色素P450酶及谷光甘肽-S-转移酶活性的影响

用紫外分光光度法及荧光分光光度法测定中国对虾肝胰腺及鳃氨基比林-N-脱甲基酶(APND)、乙氧基香豆素-O-脱乙基酶(ECOD)、谷光甘肽-S-转移酶(GST)几种药物代谢酶的活性,探讨黄芩苷对上述几种酶活性的影响。实验结果表明,中国对虾肝胰腺和鳃丝APND活性分别为3.172和2.242 nmol/(min.mgprotein-1)、ECOD活性分别为0.325 0和0.357 9 pmol/(min.mgprotein-1)、GST活性分别为70.59和13.97 nmol/(min.mgprotein-1)。中国对虾连续7 d投喂黄芩苷药饵(100 mg/kg)后,黄芩苷对几种酶均有不同程度的诱导作用,酶的活性均呈现先升高后降低的趋势。肝胰腺和鳃丝APND活性均在3 h左右达到最高,ECOD活性均在1 h左右达到最高,而GST活性迟于CYP450酶被诱导,2种组织GST活性分别在6,12 h达到最高水平。在临床用药中应考虑到黄芩苷对药物代谢酶的诱导作用,适当用药,合理配伍。

植物细胞色素P450s及其在植物新陈代谢中的作用

对目前已知功能的CYP450s进行了概述,综述了CYP450s在植物基础代谢和非基础代谢中的重要功能和作用,为进一步研究该基因家族及各成员间的功能提供参考。

人类细胞色素P450s研究概况及其在新药安全性评价中的应用

人类细胞色素P45 0s(CYP45 0s)对许多内源、外源性化合物在体内的I相生物转化有重要作用。人类编码CYP45 0s的基因已基本明确 ,为 5 5个基因 ,2 9个假基因。目前欧美各国已经把CYP45 0s及其同工酶测定用于新药的筛选及安全性评价。本实验室建立了不同种属动物肝细胞色素P45 0s同工酶的测定方法 ,并选择来氟米特、重组人白细胞介素 11(rhIL 11)及重组人表皮生长因子 (rh EGF) ,检测其对不同动物CYP45 0s的影响。实验结果表明 :①来氟米特对Beagle犬肝微粒体蛋白含量、CYP45 0s总量、7 乙氧基试卤灵脱乙基酶 (CYP1A1)、香豆素 7 羟化酶 (CYP2A6 /CYP2A2 )、甲苯磺丁脲羟化酶 (CYP2C8/9)、氯唑沙宗羟化酶 (CYP2E1)的比活性均无明显影响。②大鼠皮下注射rhIL 11后 ,肝微粒体中CYP1A1及CYP2A2比活性明显低于对照组 ,而肝微粒体蛋白含量、CYP45 0总量及CYP2E1比活性无明显变化。③rh EGF对猕猴肝微粒体蛋白含量、CYP45 0s总量、CYP1A1、CYP2A6、CYP2C8/9、S (+) 美芬妥英羟化酶 (CYP2C19)、丁呋洛尔 1 羟化酶 (CYP2D6 )、CYP2E1比活性均无明显影响。本实验建立的犬、大鼠及猴肝CYP45 0s测定方法具有快速、简单、重复性好等特点 ,可广泛用于药物筛选及临床前安全性评价。除合成药物外 。

S(+)盐酸氯胺酮对大鼠肝微粒体细胞色素P450同工酶的影响

目的研究S(+)盐酸氯胺酮对大鼠肝微粒体细胞色素P450(CYP450)6种主要同工酶的诱导或抑制作用。方法应用HPLC-UV测定CYP450 6种主要同工酶的特异性探针底物右美沙芬(CYP2D)、双氯酚酸钠(CYP2C6)、奥美拉唑(CYP2C)、氨苯砜(CYP3A)、非那西丁(CYP1A2)和氯唑沙宗(CYP2E1)的代谢速率。大鼠iv给予S(+)盐酸氯胺酮25 mg.kg-1.d-1,连续给药7 d,评价药物对大鼠肝微粒体CYP450主要同工酶的诱导或抑制作用。结果S(+)盐酸氯胺酮50μmol.L-1时可明显抑制大鼠肝脏CYP2C,2C6和3A,分别为对照组的61.4%,45.8%和31.3%,其中以对CYP2C的抑制作用最为明显。S(+)盐酸氯胺酮1和10μmol.L-1对CYP450同工酶无明显影响,仅10μmol.L-1S(+)盐酸氯胺酮对CYP2C有轻度抑制作用。S(+)盐酸氯胺酮iv给药7 d后,对大鼠肝微粒体CYP1A2和CYP2C有不同程度的诱导作用,分别诱导137.5%和117.6%,对CYP2E1,3A,2D,2C6无明显影响。结论S(+)盐酸氯胺酮50μmol.L-1时可明显抑制肝脏CYP2C,2C6和3A,连续给药7 d后可诱导CYP1A2和CYP2C,提示S(+)盐酸氯胺酮与经上述同工酶代谢的药物联合应用时,应注意药物-药物相互作用的可能性。

细胞色素P450s酶系催化除草剂代谢的研究进展

综述了细胞色素P450s酶系催化的单加氧反应机理,细胞色素P450s酶系在酰胺类、三氮苯类、磺酰脲类、脲类、苯氧羧酸类等除草剂的活性或降解代谢中的催化反应。讨论了研究细胞色素P450s酶系代谢作用在除草剂选择性、抗药性机理,抗除草剂作物的培育以及除草剂安全剂的解毒机理等方面的意义。

ZG230-450铸钢的重构疲劳可靠性S-N曲线

给定若干概率水平的常规疲劳可靠性曲线只能做相应概率水平的疲劳设计与寿命预测,要实现任意可靠性水平的疲劳设计、寿命预测和可靠性评定,需要重构全概率疲劳可靠性S-N曲线。首先应用Monte Carlo模拟技术在可接受的误差范围重构了铁道车辆ZG230-450车轮铸钢的疲劳极限和成组法S-N数据。然后应用广义极大似然法测定了其中短寿命范围的可靠性S-N曲线。最后应用概率疲劳极限外推法获得了该材料中短和长寿命范围的合理疲劳概率S-N曲线。并进一步发展了适于车辆结构用万公里单位表征的可靠性曲线。应用该曲线可实现任意可靠性水平的疲劳可靠性设计、寿命预测和可靠性评价。