ERCC1基因多态性与食管癌的研究进展

核苷酸切除修复交叉互补基因1(excision repair cross complementation 1,ERCC1)是核苷酸切除修复(NER)系统的关键酶,其表达情况与食管癌的发生发展及铂类耐药有关.检测ERCC1基因多态性有助于制定食管癌个体化治疗.     

非小细胞肺癌RAD51、ERCC2和BAG-1基因多态性的表达

目的:研究DNA修复基因RAD51和着色性干皮病基因(ERCC2/XPD)以及Bcl-2结合抗凋亡基因1(Bcl-2 associated athanogene 1,BAG-1)基因多态性与非小细胞肺癌(non-small cell lung cancer,NSCLC)遗传易感性的关系。方法:采用病例对照研究设计,选取100例非小细胞肺癌病例和80例正常对照。以ERCC2/XPD Lys751Gln和RAD51 codon 135以及BAG-1codon 324基因多态性为研究位点,聚合酶链反应-限制性片段长度多态性(PCR-RFLP)方法对多态性进行检测。应用Logistic回归计算OR值及95%CI,比较不同基因型与NSCLC发病风险的关系。结果:ERCC2/XPD 751基因型在病例组的分布频率为C/C型69例(69%)、C/A型26例(30%)和A/A型5例(5%)。与野生基因型C/C型相比,携带ERCC2/XPD C/A基因型和A/A基因型者患NSCLC的危险度比值比(odds ratio,OR)分别是1.53(95%CI:1.15-3.32)和0.58(95%CI:0.15-2.39)。BAG-1 codon 324基因型的分布频率为C/C型81%(81/100)、C/T型19%(19/100)以及T/T型0%(0例)。与野生基因型C/C型相比,携带BAG-1 C/T基因型者患NSCLC的OR是1.28(95%CI:1.08-2.74)。RAD51 codon 135基因型的分布频率为G/G型67%(67/100)、G/C型33%(33/100)、未现C/C型。与野生基因型G/G型相比,RAD51 G/C基因型者患NSCLC的OR是1.03(95%CI:1.06-2.29)。分析结果提示吸烟、环境危险因素与XPD Lys751Gln基因多态存在交互作用,交互效应OR值分别为2.24(95%CI:1.18-2.87)和2.53(95%CI:1.71-3.46),携带XPD Ly s751Gln突变基因者若同时暴露于吸烟、环境危险因素下,则患NSCLC的危险显著增加,相较未暴露于上述因素者,OR值均增大。结论:BAG-1和ERCC2/XPD以及RAD51基因多态性可能与当地居民NSCLC遗传易感性有关,ERCC2/XPD与吸烟、饮酒、环境危险因素存在交互作用。     

ERCC1与XPD基因多态性对NSCLC化疗敏感性影响的研究进展

临床III-IV期NSCLC占肺癌的大多数,以铂类药物为基础联合第三代新药是晚期NSCLC的一线化疗方案,近年来多项研究表明,个体间化疗敏感性的不同可能与基因修复能力有关,本文主要对核苷酸切除修复系统的关键基因ERCC1、XPD的多态性与铂类药物化疗敏感性关系的最新研究成果做简要综述。     

四川北部地区 AGT、XRCC1基因多态性与食管癌风险的关系

目的：：探讨 DNA 损伤修复酶基因 XRCC1Arg194Trp 多态性和 AGT 基因第三外显子 Leu84Phe 多态性与食管癌易感性的关系。方法：采用病例-对照研究方法分析四川北部地区食管癌病例（n ＝155）和年龄、性别分布无差异的对照组（n ＝127）XRCC1基因 Arg194Trp 多态性和 AGT 基因第三外显子 Leu84Phe 多态性,两个基因多态的交互作用和基因多态与吸烟、饮酒之间的交互作用对食管癌易感性的影响。结果：XRCC1基因194位点各基因型在两组间的分布无统计学差异（χ2＝0.614;P ＝0.736）。AGT 基因第三外显子第84位密码子各基因型在两组间的分布无统计学差异（χ2＝1.826;P ＝0.177）,吸烟、饮酒与 AGT 基因第三外显子第84位密码子突变基因型 TT存在正交互作用,交互作用指数（the synergy index S,S）分别为7.375、17.67。结论：四川北部地区 XRCC1基因194位点基因多态性与 AGT 基因第三外显子 Leu84Phe 多态性可能与食管癌的易感性无关,但 AGT 基因第三外显子 Leu84Phe 多态性可能与吸烟、饮酒与食管癌的易感性之间存在协同作用。     

ERCC1、XRCC1单核苷酸多态性与鼻咽癌放化疗敏感性的相关性研究

摘 要：［目的］ 初步探讨ERCC1、XRCC1单核苷酸多态性与鼻咽癌患者放疗敏感性的关系。［方法］ 经病理确诊且未经放疗、化疗或手术治疗的无远处转移鼻咽癌患者100例,接受顺铂单药同步放化疗,采用TaqMan 探针实时荧光定量聚合酶链反应（polymerase chain reaction,PCR）法和直接测序法对患者外周血ERCC1、XRCC1进行多态性分析,患者同步放化疗结束后及治疗后3个月复查鼻咽部磁共振成像(MRI)测量肿瘤大小,进而分析ERCC1、XRCC1基因型与鼻咽癌同步放化疗近期治疗敏感性及远期生存率的关系。［结果］ ERCC1、XRCC1基因多态性与鼻咽癌放化疗的短期疗效无关(均P>0.05),携带ERCC1C/C、C/T+T/T基因型患者放化疗后1年、2年、3年生存率分别为97.7%、94.3%、86.3%和98.2%、87.4%、73.8%,两组间比较差异有统计学意义（P<0.05);携带XRCC1G/G、A/A+G/A基因型患者放化疗后1年、2年、3年生存率分别为97.7%、91%、86.4%和86.9%、82.3%、69.6%,两组间比较差异有统计学意义（P<0.05)。［结论］ ERCC1、XRCC1基因多态性与鼻咽癌同步放化疗的短期疗效无相关性,但与生存期有相关性,因此可以作为鼻咽癌放化疗生存期的预测指标。     

XRCC1和XRCC2的基因多态性在乳腺癌治疗中的临床意义

目的:乳腺癌是女性常见的恶性肿瘤之一,其发生发展与遗传因素十分密切,寻找与乳腺癌发生、病理、治疗及预后相关的分子标志物对于该病的治疗具有重要的指导意义。XRCC是参与DNA损伤修复的重要基因。XRCC1及XRCC2基因的多态性已被研究与多种肿瘤易感性及生物学行为有关。本实验研究XRCC1 和XRCC2 多态性与中国女性乳腺癌发病风险、临床病理（腋窝淋巴结转移状态）及预后(复发和转移)的相关性,以探讨其在乳腺癌临床治疗中的潜在价值。方法:采用PCR-RFLP方法检测60例原发性乳腺癌患者新鲜血标本中XRCC1 Arg399Gln、XRCC2 C41657T、XRCC2 G4234C多态,采用SAS 9.1.3统计软件分析基因型与乳腺癌发病风险、临床病理及预后的相关性。结果:XRCC1 Arg399Gln 不同基因型与乳腺癌的发病风险不存在统计学相关性(P>0.05); XRCC1 Arg399Gln与乳腺癌的腋窝淋巴结转移情况及乳腺癌的远处转移、局部复发不存在相关性(P>0.05);XRCC2 C41657T、XRCC2 G4234C 基因型与乳腺癌的发病风险均无显著相关(P>0.05);XRCC2 C41657T C/T和T/T基因型的多态性可能与乳腺癌的腋窝淋巴结转移状态、乳腺癌的远处转移及局部复发具有相关性(P<0.05)。结论:XRCC1 Arg399Gln G/G、XRCC2 C41657T、XRCC2 G4234C 基因型可能均与乳腺癌的发病风险无关。XRCC2 C41657T C/T和T/T基因型的多态性可能与乳腺癌的腋窝淋巴结转移状态、乳腺癌的远处转移及局部复发具有相关性。     

ERCC1和XRCC3基因多态性在接受含铂方案化疗NSCLC中的疗效预测作用

[目的]探讨DNA修复基因ERCC1 118和XRCC3 241多态性对于接受一线含铂化疗方案的非小细胞肺癌（NSCLC）患者的疗效预测作用。[方法]130例晚期接受含铂化疗的NSCLC患者进入研究．应用Taqman探针结合实时荧光PCR方法分析其外周血基因多态性．分析ERCC1 118和XRCC3 241多态性与疗效、疾病进展时间和总体生存期之间的关系。[结果]130例患者的总体有效率为20％．中位生存时间为15个月．ERCC1 118和XRCC3 241多态性与疗效无显著相关性。ERCC1 118基因型C/T或T/T患者的生存时间显著延长（P=0.003）。Cox多因素分析显示,ERCC1 118基因型C/T或T/T以及化疗有效患者的生存期显著延长。[结论]DNA修复基因ERCC1 118基因型C／T或T/T多态性可以延长NSCLC患者铂类治疗后的生存时间．     

晚期食管癌患者血清XRCC1基因多态性与铂类化疗疗效相关性的研究

目的：探讨XRCC1Arg399Gln（G＋A）基因多态性与晚期食管癌对以奥沙利铂为主的化疗敏感性的关系。方法：晚期食管癌患者87例（Ⅲ期41例,Ⅳ期46例）,采用改良FOLFOX方案化疗,46例Ⅳ期患者3个周期后进行临床疗效评价,87例患者统计至疾病进展时间（TTP）。应用TaqMan-MGB探针等位基因分型技术进行基因分型。结果：54.2%为A/A,34.5%为A/G,11.5%为G/G。Ⅳ期46例患者化疗后临床获益率（CR＋PR＋SD）为52.17%,A/A、G/A＋G/G在化疗敏感组与不敏感组中的分布具有统计学意义（χ2=6.213,P=0.023）。87例患者中位TTP 8个月,A/A基因型11个月,G/A＋G/G基因型4个月,两者比较差异有统计学意义（χ2=27.781,P〈0.01）。结论：XRCCl Arg399Gln基因多态性与晚期食管癌患者对奥沙利铂化疗的敏感性与生存时间相关。     

晚期食管癌患者血清XRCC1基因多态性与铂类化疗疗效相关性的研究

目的:探讨XRCC1Arg399Gln(G+A)基因多态性与晚期食管癌对以奥沙利铂为主的化疗敏感性的关系。方法:晚期食管癌患者87例(Ⅲ期41例,Ⅳ期46例),采用改良FOLFOX方案化疗,46例Ⅳ期患者3个周期后进行临床疗效评价,87例患者统计至疾病进展时间(TTP)。应用TaqMan-MGB探针等位基因分型技术进行基因分型。结果:54.2%为A/A,34.5%为A/G,11.5%为G/G。Ⅳ期46例患者化疗后临床获益率(CR+PR+SD)为52.17%,A/A、G/A+G/G在化疗敏感组与不敏感组中的分布具有统计学意义(χ2=6.213,P=0.023)。87例患者中位TTP 8个月,A/A基因型11个月,G/A+G/G基因型4个月,两者比较差异有统计学意义(χ2=27.781,P<0.01)。结论:XRCCl Arg399Gln基因多态性与晚期食管癌患者对奥沙利铂化疗的敏感性与生存时间相关。     

ERCC1和XRCC1基因多态性与接受奥沙利铂化疗晚期大肠癌患者生存期的关系

目的: 探讨DNA损伤修复基因ERCC1 Asn118Asn和XRCC1 Arg399Gln多态性与接受奥沙利铂一线化疗的中国晚期大肠癌患者生存期的关系。 方法: 99例晚期大肠癌患者化疗前抽取静脉血并提取DNA,以real-time PCR法对ERCC1、XRCC1基因进行SNP分型。患者接受奥沙利铂为主的化疗方案化疗,比较不同基因型与患者生存期的关系。 结果: ERCC1 Asn118Asn基因位点在所研究的中国大肠癌患者中的突变频率为:C/C50.51%、C/T41.41%、T/T8.08%;XRCC1 Arg399Gln基因突变频率为:G/G52.53%、G/A38.38%、A/A9.09%。99例晚期大肠癌患者中位TTP为7个月。ERCC1C/C基因型患者中位TTP10个月,C/T+T/T型患者中位TTP5个月,二者有显著统计学差异(P<0.01);XRCC1G/G基因型中位TTP10个月,G/A+A/A基因型中位TTP5个月,二者比较差异有显著性(P<0.01)。两个基因联合多态性分析发现,同时携带ERCC1C/C和XRCC1G/G基因型、ERCC1C/C和XRCC1G/A+A/A基因型、ERCC1C/T+T/T和XRCC1G/G基因型、以及ERCC1C/T+T/T和XRCC1G/A+A/A基因型的患者中位TTP分别为11个月、6个月、5个月和5个月,4组比较差异有显著性(P<0.01)。 结论: ERCC1 Asn118Asn、XRCC1 Arg399Gln基因多态性与中国晚期大肠癌患者接受奥沙利铂一线化疗后的生存期有关。     

Polymorphisms of DNA repair genes XRCC1 and XPD and their associations with risk of esophageal squamous cell carcinoma in a Chinese population

Esophageal squamous cell carcinoma (ESCC), which is prevalent in China, is believed to be induced by environmental carcinogens. Accumulating evidence has shown that individual variation in DNA repair capacity resulting from genetic polymorphism influences risk of environmental carcinogenesis. We therefore investigated the associations between genetic polymorphisms in the DNA repair genes XRCC1 (Arg194Trp and Arg399Gln) and XPD (Asp312Asn and Lys751Gln) and risk of ESCC in an at-risk Chinese population. Genotypes were determined by a PCR-based approach in 433 patients with ESCC and 524 frequency-matched normal controls. We found that individuals with Trp/Trp genotype at XRCC1 Arg194Trp site had a 2-fold increased risk of this disease compared to Arg/Arg genotype (adjusted OR = 1.98; 95% CI 1.26-3.12). Furthermore, when compared to Arg/Arg and Arg/Trp genotype combined, homozygote for Trp/Trp genotype significantly increased the risk of developing ESCC, with the adjusted OR being 2.07 (95% CI 1.34-3.20). However, the XRCC1 Arg399Gln polymorphism was not significantly associated with risk of ESCC, with the adjusted OR being 0.87 (95% CI 0.55-1.37). Neither Asp312Asn nor Lys751Gln polymorphisms in the XPD gene influenced risk of ESCC in our study. These findings suggest that DNA repair gene XRCC1 but not XPD might play a role in esophageal carcinogenesis and might represent a genetic determinant in the development of the cancer.     

DNA repair gene ERCC1 and XPD polymorphisms predict glioma susceptibility and prognosis

Aims: We conducted a case-control study in a Chinese population to clarify the association betweenpolymorphisms in ERCC1 and XPD and susceptibility and survival of glioma. Methods: A total of 393 cases and410 controls were selected from March 2007 to December 2011. Genotyping of ERCC1 and XPD was conductedby TaqMan assays using the ABI Prism 7911HT Sequence Detection System. All analyses were performed usingthe STATA statistical package. Results: Polymorphisms in ERCC1 118C/T, ERCC1 8092C/A and XPD Asp312Asnshowed no statistically significant difference between glioma cases and controls. However, individuals with theXPD 751Gln/Gln genotype had an increased risk of developing glioma compared with those with the Lys/Lysgenotype (adjusted OR=1.64, 95% CI: 1.06-2.89). The ERCC1 118T/T genotype was associated with significantlyhigher median survival than the ERCC1 C/C genotype (HR=0.67, 95%CI=0.35-0.96). In addition, individualswith XPD 751Gln/Gln had a lower median survival time than XPD Lys/Lys carriers (HR=0.54, 95%CI=0.37-0.93). Conclusion: In conclusion, we observed that the XPD 751Gln/Gln genotype is associated with gliomasusceptibility, and ERCC1 118 T/T and XPD 751Gln/Gln genotypes confer a significantly better prognosis.     

ERCC2、RAD51和BAG-1基因多态性与晚期非小细胞肺癌铂类化疗敏感性的研究

目的:研究着色性干皮病基因(ERCC2/XPD)、DNA修复基因RAD51 codon 135以及Bcl-2结合抗凋亡基因1(Bcl-2 associated athanogene 1,BAG-1)codon 324多态性与晚期非小细胞肺癌(non-small cell lung cancer,NSCLC)以铂类药物为基础的化疗敏感性的关联。方法:选取100例晚期NSCLC患者,使用PCR-RFLP方法检测ERCC2/XPD Lys 751 Gln、RAD51 codon 135以及BAG-1 codon 324基因多态性,应用非条件Logistic回归计算OR值及95%CI,分析不同基因型与化疗敏感性的关系。结果:100例晚期NSCLC患者中,ERCC2/XPD 751基因Lys/Lys、Lys/Gln、Gln/Gln基因型的分布频率分别为69例(69%)、26例(26%)和5例(5%);RAD51 codon 135的基因型频率分布为G/G型占67%(67/100)、G/C型33%(33/100)、未发现C/C型;BAG-1 codon 324基因型频率分布C/C型占81%(81/100)、C/T型占19%(19/100),未发现T/T型。100例NSCLC患者化疗后,其中完全缓解(CR)、部分缓解(PR)、稳定(SD)和进展(PD)患者分别为0、31、41和28例。ERCC2/XPD C/A基因型多态性与化疗敏感性具有相关性(OR=3.53,95%CI=1.58-11.46,P<0.01);BAG-1 codon 324 C/C型患者与C/T型患者的化疗有效率差异有统计学意义(P=0.036),携带C/C基因型患者比C/T基因型患者化疗敏感(OR=2.67,95%CI=1.16-8.23,P<0.05);RAD51 codon 135G/C基因型患者是G/G基因型患者化疗敏感性的2.12倍(OR=2.12,95%CI=1.08-7.41,P<0.05)。结论:ERCC2/XPD、BAG-1 codon 324以及RAD 51 codon 135基因多态性可能与晚期NSCLC铂类药物化疗敏感性有关。     

XRCC1 and XPD Gene Polymorphisms in a South Indian Population

DNA repair systems play an important role in maintaining the integrity of the human genome. Deficiency inthe repair capacity due to either mutations or inherited polymorphisms in DNA repair genes may contribute tovariations in the DNA repair capacity and subsequently susceptibility to cancer. The interindividual variabilityas well as ethnic differences in DNA repair polymorphisms, stress the importance to establish genotype profilesunique to a population. Hence the present study aimed to determine the frequencies of XRCC1 and XPD genepolymorphisms in 255 healthy random unrelated individuals from South India. DNA was isolated from theperipheral blood sample of these individuals and the XRCC1 and XPD genotypes were determined by PCRRFLPwith Msp1 and Pst1 enzymes respectively. The XRCC1 genotype frequencies revealed 36% Arg/Arg,47% Arg/Gln and 17% Gln/Gln with Gln allele frequency of 0.41. Analysis of XPD genotypes revealed 51% Lys/Lys, 41% Lys/Gln and 8% Gln/Gln with Gln allele frequency of 0.29. No significant difference in the distributionof genotypes was seen based on gender. Comparison of the frequencies of XRCC1 and XPD polymorphismsobserved in the present study with other populations revealed a distinctive nature of the South Indian population.An understanding of DNA repair gene polymorphisms might not only enable risk assessment of humans exposedto environmental carcinogens but also response to therapy, which target the DNA repair pathway.     

Polymorphisms of ERCC1, XPD, XRCC1 and XPG predict clinical outcome in advanced gastric cancer patients receiving oxaliplatin-based chemotherapy in Chinese population

OBJECTIVE To investigate whether polymorphisms in ERCC1, XPD, XPG, XRCC1 genes are associated with clinical outcomes in advanced gastric cancer （AGC） patients treated with oxaliplatinbased chemotherapy. METHODS The genetic polymorphisms in ERCCI, XPD, XPG, XRCC1 were determined in 94 advanced gastric cancer patients treated with oxaliplatin-based chemotherapy, using TaqMan-MGB probes. The clinical response of 60 patients with stage IV disease, time to progression （TTP） and overall survival （OS） of 94 patients were evaluated. RESULTS The overall disease control rate （CR ＋ PR ＋ SD） of the 60 patients in stage IV was 70% （42/60）. Patients with XRCC1 399 G/G, XPG 46 C/C genotypes showed enhanced response to the oxaliplatin-based chemotherapy compared to those with other genotypes （P 〈 0.05）. The median OS and TTP of the patients were 5.5 months and 9.0 months, respectively. Among the 4 types of polymorphisms in the study, XRCC1 399 G/A ＋ A/A, XPG 46 C/T ＋ T/T genotypes were regarded to be associated with chemoresistance and poor survival （P 〈 0.05）. Combination analysis of the 2 polymorphisms using the Kaplan-Meier method revealed that the TTP and OS of the patients with a number of risk genotypes were significantly shortened （P 〈 0.05）. No significant association was found between the genotypes of the XPD codon 751, the ERCC1 codon 118 and the clinical outcome （P 〉 0.05）. CONCLUSION Testing for XRCC1 399, XPG 46 polymorphisms may allow identification of the gastric cancer patients who will benefit from oxaliplatin-based chemotherapy. Specific polymorphisms may influence clinical outcomes of AGC patients. Selecting specific chemotherapy based on pretreatment genotyping represents an innovative strategy that warrants prospective studies.     

Polymorphisms at XPD and XRCC1 DNA repair loci and increased risk of oral leukoplakia and cancer among NAT2 slow acetylators

Polymorphisms at N-acetyl transferase 2 locus (NAT2) lead to slow, intermediate and rapid acetylation properties of the enzyme. Improper acetylation of heterocyclic and aromatic amines, present in tobacco, might cause DNA adduct formation. Generally, DNA repair enzymes remove these adduct to escape malignancy. But, tobacco users carrying susceptible NAT2 and DNA repair loci might be at risk of oral leukoplakia and cancer. In this study, 389 controls, 224 leukoplakia and 310 cancer patients were genotyped at 5 polymorphic sites on NAT2 and 3 polymorphic sites on each of XRCC1 and XPD loci by PCR-RFLP method to determine the risk of the diseases. None of the SNPs on these loci independently could modify the risk of the diseases in overall population but variant genotype (Gln/Gln) at codon 399 on XRCC1 and major genotype (Lys/Lys) at codon 751 on XPD were associated with increased risk of leukoplakia and cancer among slow acetylators, respectively (OR = 4.2, 95% CI = 1.2-15.0; OR = 1.6, 95% CI = 1.1-2.3, respectively). Variant genotype (Asn/Asn) at codon 312 on XPD was also associated with increased risk of cancer among rapid and intermediate acetylators (OR = 1.9, 95% CI = 1.2-2.9). Variant C-G-A haplotype at XRCC1 was associated with increased risk of leukoplakia (OR = 1.7, 95% CI = 1.2-2.4) but leukoplakia and cancer in mixed tobacco users (OR = 3.1, 95% CI = 1.4-7.1, OR = 2.4, 95% CI = 1.1-5.4, respectively) among slow acetylators. Although none of the 3 loci could modulate the risk of the diseases independently but 2 loci in combination, working in 2 different biochemical pathways, could do so in these patient populations.     

ERCC2/XPD基因312位点单核苷酸多态与肺癌发病关联的Meta分析

目的 核苷酸切除修复交叉互补基因2/人类着色性干皮病基因D(excision repair cross complementation group2/Dxeroderma pigmentosum group D,ERCC2/XPD)单核苷酸多态(single nucleotide polymorphism,SNP)与肺癌发病相关联.本研究通过对近15年间发表的相关文献进行Meta分析,评价ERCC2/XPD Asp312Asn位点SNP与肺癌发病的关联.方法 检索2000-01-2014-05年PubMed、中国知网(CNKI)、维普资讯网(cqVIP)和万方全文数据库中关于ERCC2/XPD基因312位点SNP与肺癌发病关联的病例对照研究.按拟定的入选标准和排除标准进行筛选,提取符合纳入标准文献的相关数据,采用RevMan 4.2软件计算合并相对危险度(OR值)及其95％可信区间(95％CI),同时绘制漏斗图,估计发表偏倚的影响.结果 共纳入国内外文献20篇,累计病例组7 525例,对照组8 570例.Asp/Asn versusAsp/Asp异质性检验结果显示,x2=19.97,P=0.40.采用固定效应模型分析,合并OR=1.02,95％CI为0.94～1.10,P=0.65.Asn/Asn versus Asp/Asp异质性检验结果显示,x2=20.99,P=0.28.采用固定效应模型分析,合并OR=1.22,95％CI为1.07～1.40,P=0.004.根据种族分组后,结果显示,无论在高加索人群,还是亚洲人群中携带Asn/Asn基因型个体与肺癌发病升高均有关,差异有统计学意义,P＜0.05.结论 ERCC2/XPD基因312位点多态与肺癌发病风险相关联,携带312位点Asn/Asn基因型的个体,其肺癌发病风险显著高于携带Asp/Asp基因型的个体,按种族进行分层分析后,此关联仍然存在.