The prevalence and distribution of human papillomavirus genotypes in oral epithelial hyperplasia: proposal of a concept

Background:  Evidence is accumulating for the aetiological role of human papillomavirus (HPV) in the pathogenesis of potentially malignant oral mucosal lesions and squamous cell carcinomas.
Methods:  Paraffin tissue sections from 49 patients with 'white patches' of the oral mucosa were investigated histologically, by broad-spectrum PCR followed by genotyping and chromogenic in situ hybridisation (CISH).
Results:  Histologically, 33 flat hyperplasias and 16 papillary hyperplasias were diagnosed. Twenty-two of 28 samples studied (78.6%) were positive for HPV DNA by PCR and six were negative. The following HPV types were detected in decreasing order of prevalence: HPV 35, HPV 6, HPV16, HPV 53, HPV 18, HPV 51 and HPV 55. Seventeen samples (60.7%) contained high-risk HPV DNA. Using CISH, ≥ 1 HPV signals were detected at least in a few epithelial cells in 95% of cases studied. All but one case were positive with the high-risk HPV probe and all HPV infections contained low viral load. Concordant positive results both by PCR and CISH were detected in 14 of 19 cases (73.7%) analysed.
Conclusions:  The high prevalence of HPV infection in hyperplastic 'white patches' of the oral mucosa supports the putative role of HPV at an early stage of oral carcinogenesis. These results further indicate that the majority of white oral mucosal lesions – flat, exophytic, wart-like or papillary proliferations – could be considered as the clinical manifestations of oral HPV infection. This finding has clinical relevance regarding therapy and patient management and may help in elucidating the role of HPV infection in oral carcinogenesis.     

Variant haplotypes at XRCC1 and risk of oral leukoplakia in HPV non-infected samples

Background:  One of the mechanisms in human papillomavirus (HPV)-related carcinogenesis is inhibition of DNA repair by HPV oncoprotein. In this study, we investigated whether polymorphisms at XRCC1 , one of the DNA repair loci, could modulate the risk of tobacco-related leukoplakia and cancer in HPV-infected individuals.
Methods:  Tissue DNA from 83 oral cancer, 91 leukoplakia and 100 healthy controls were screened for HPV 16/18 infection and polymorphisms at XRCC1 by PCR–RFLP to estimate the risk of diseases independently and jointly.
Results:  Human papillomavirus infection was significantly associated with increased risk of leukoplakia and cancer (OR = 2.8, 95% CI = 1.2–6.5 and OR = 5.5, 95% CI = 1.6–19, respectively). Independently, genotypes at three polymorphic sites on XRCC1 did not modulate the risk of diseases but pooled variant haplotypes increased the risk of leukoplakia in overall and HPV non-infected (OR = 1.8, 95% CI = 1.2–2.8; OR = 2.2, 95% CI = 1.2–4.0, respectively) samples but not that of cancer.
Conclusion:  The association between variant haplotypes at XRCC1 and risk of leukoplakia is pronounced in non-infected individuals since HPV oncoprotein could inhibit directly the DNA repair activity of XRCC1. But more samples of leukoplakia and cancer are essential to validate these results.     

Expression of p16 in oral cancer and premalignant lesions

Background:  Expression of p16 has been proposed as a marker for malignant transformation. This study aimed to evaluate p16 expression in oral squamous cell carcinoma (OSCC) and premalignant lesions including oral leukoplakia (OL) with and without dysplasia.
Methods:  Expression of p16 was investigated in 56 samples including OSCC, OL with and without dysplasia, and normal oral mucosa. Expression of p16 was identified by immunohistochemistry, using the CINtecTM p16INK4a Histology Kit. Both nuclear and/or cytoplasmic staining of the keratinocytes were considered to be positive and the percentage of positive cells was calculated.
Results:  Expression of p16 was detected in 3/16 (18.75%) cases of OSCC, in 4/15 (26.7%) cases of OL without dysplasia, and in none of OL with dysplasia and normal mucosa. No significant differences in p16 expression prevalence were found among OSCC, OL with and without dysplasia and normal mucosa. The percentages of positive cells in OSCC and OL without dysplasia were 0.89 and 0.17, respectively. No significant difference in the percentage of positive keratinocytes was found.
Conclusion:  As a marker, p16 is not reliable for oral mucosal dysplasia and malignant transformation.     

人乳头瘤病毒和人巨细胞病毒与口腔癌的关系

目的：检测口腔白斑和口腔鳞状细胞癌中的人乳头瘤病毒（HPV16）DNA和人巨细胞病毒（HCMV）DNA,探讨HPV16和HCMV与口腔癌之间的关系。方法：应用多聚酶链反应（PCR）技术扩增HPV16DNA和HCMVDNA,采用2%琼脂糖凝胶电泳分析,检测口腔白斑和口腔鳞状细胞癌中的HPV16DNA和HCMVDNA。结果：6例口腔白斑中,HPV16DNA的阳性率为16.7%,HCMVDNA为16.7%,16例口腔鳞癌组织HPV16DNA的阳性率为31.3%,HCMVDNA为25%,例正常组织均未检测出。结论：4HCMV与HPV16在口腔癌的发生中具有协同致癌作用。     

Patients with oral cancer developing from pre‐existing oral leukoplakia: do they do better than those with de novo oral cancer?

Background:  It has been suggested that patients with squamous cell carcinomas derived from oral leukoplakia have a better prognosis than patients with carcinomas that are not associated with oral leukoplakia.
Aim:  To study the mortality rate of 19 patients with a squamous cell carcinoma derived from pre-existing oral leukoplakia.
Method:  The mortality rate of 19 patients with a proven oral squamous cell carcinoma derived from a pre-existing oral leukoplakia was compared with that of a similar size group of patients with oral carcinoma without a pre-existing oral leukoplakia, being matched for gender, age, smoking habits, use of alcohol, oral subsite and histopathologic grade. Treatment in all patients was primarily by surgical excision. The mortality rates up to 5 years have been computed according to the Kaplan–Meier method.
Result:  No significant difference of the mortality rates up to 5 years of follow-up was observed between the two groups of patients.
Conclusion:  Patients with oral cancer developing from pre-existing oral leukoplakia do not do better than those with de novo oral cancer.     

Progressive increase of human papillomavirus carriage rates in potentially malignant and malignant oral disorders with increasing malignant potential

Introduction:  We investigated the potential role of human papillomaviruses (HPVs) in potentially malignant oral disorders, oral leukoplakia (OL) and oral lichen planus (OLP), and in oral squamous cell cancer (OSCC) in an Eastern Hungarian population with a high incidence of OSCC.
Methods:  Excised tumor samples (65 OSCC patients) and exfoliated cells from potentially malignant lesions (from 44 and 119 patients with OL and OLP, respectively) as well as from healthy controls (72 individuals) were analysed. OLPs were classified based on clinical appearance, 61 patients had erosive–atrophic lesions (associated with higher malignancy risk, EA-OLP) and 58 had non-erosive non-atrophic lesions (with lower risk of becoming malignant, non-EA-OLP), respectively. Exfoliated cells collected from apparently healthy mucosa accompanied each lesion sample. HPV was detected by MY/GP polymerase chain reaction (PCR) and genotyped by restriction analysis of amplimers. Copy numbers in lesions were determined using real-time PCR. Prevalence rates, copy number distributions, and association with risk factors and diseases were analysed using chi-square test, t -test, and logistic regression, respectively.
Results:  We detected HPVs significantly more frequently in lesions than in controls ( P  ≤ 0.001 in all comparisons). HPV prevalence increased gradually with increasing severity of lesions (32.8, 40.9, and 47.7% in OLP, OL, and OSCC, respectively). Copy number distribution patterns roughly corresponded to prevalence rates, but OLP and OL were comparable. HPV prevalence differed significantly between EA-OLP and non-EA-OLP groups (42.6 vs. 22.4%); EA-OLP group showed a prevalence similar to that found in OL.
Conclusion:  HPVs may be involved in the development or progression of not only OSCC but also of potentially malignant oral lesions.     

Evaluation of myofibroblasts in oral epithelial dysplasia and squamous cell carcinoma

Background:  Carcinogenesis is accompanied by a number of changes in the adjacent stroma including the appearance of myofibroblasts. The purpose of this study was to evaluate and compare the presence of myofibroblasts in normal mucosa, oral epithelial dysplasia, and different grades of oral squamous cell carcinoma.
Methods:  The study sample consisted of three groups, including 40 oral squamous cell carcinomas, 15 dysplasias, and 15 sections of normal oral epithelium. Vimentin, desmin, and alpha-smooth muscle actin were used to identify myofibroblasts.
Results:  The percentage and intensity of alpha-smooth muscle actin were examined, and positive immunostaining was observed in the myofibroblasts of all squamous cell carcinomas; however these cells did not stain in the dysplasias or normal epithelium specimens. The presence of myofibroblasts was significantly higher in oral squamous cell carcinomas compared to both, dysplasias and normal mucosa cases ( P  < 0.001). A significant difference was not observed between the different grades of oral squamous cell carcinoma ( P  = 0.2).
Conclusions:  These findings show the presence of myofibroblasts in the stroma of oral squamous cell carcinoma but not dysplasia and normal mucosa, suggesting further investigation to clarify the role of myofibroblasts in the carcinogenesis of this tumor.     

Demonstration of ethanol-induced protein adducts in oral leukoplakia (pre-cancer) and cancer

Background:  Excessive alcohol consumption is a common cause for upper gastrointestinal tract cancers. However, the primary mechanisms of alcohol-induced carcinogenesis have remained poorly defined.
Method:  We examined the generation and subcellular distribution of protein adducts with acetaldehyde (AA), the first metabolite of ethanol, and end products of lipid peroxidation, malondialdehyde (MDA) and 4-hydroxynonenal (HNE), from oral biopsy specimens obtained from 36 subjects (11 British, 25 Japanese) reporting alcohol misuse. All patients had been diagnosed with oral pre-cancer (leukoplakia, n  = 7) or squamous cell carcinoma (SCC; n  = 29). Automated immunostaining for AA, MDA and HNE adducts was performed using monospecific antibodies.
Results:  Positive staining for AA, MDA and HNE adducts was observed in the dysplastic or malignant epithelial cells, HNE being relatively the most abundant adduct species. The subgroup of Japanese patients had higher levels of AA and MDA, although not HNE, than the British sample. When the material was divided to those with SCC or leukoplakia, MDA adducts but not the other antigens were more prominent in the former group. Significant correlations were found between the different adducts (AA vs. MDA, r  = 0.68, P  < 0.001; AA vs. HNE, r  = 0.47, P  < 0.01 and MDA vs. HNE, r  = 0.59, P  < 0.001). In addition, cytochrome P450 2E1 staining was found in these samples, correlating with both AA and MDA adducts.
Conclusion:  The data indicates that AA- and lipid peroxidation-derived adducts are formed in oral tissues of alcohol misusers with oral leukoplakia and cancer. The findings also support a pathogenic role of AA and excessive oxidative stress in carcinogenesis.     

Use of quantum dots to detect human papillomavirus in oral squamous cell carcinoma

Objective:  To examine the association of oral squamous cell carcinoma with human papillomavirus (HPV) using quantum dots (QD) in situ hybridization (ISH).
Methods:  Expression of HPV16/18 was analyzed in a representative collection of 21 oral squamous cell carcinomas by tissue microarrays. The presence of HPV16/18 high risk was detected by applying QDISH which is compared with conventional ISH.
Results:  Seven cases out of 21 (33.3%) were positive for QDISH while 1 out of 21 (4.8%) was positive for ISH, although all of HPV DNA were localized in the nuclei in the spinous and basal cell layer of the epithelium. The difference between these two methods was significant ( P  < 0.05).
Conclusions:  These results demonstrate that the QD might be an efficient method for determination of HPV infection and HPV-associated oral squamous cell carcinoma.     

Detection of human papillomavirus DNA in oral mucosal lesions using in situ DNA-hybridization applied on paraffin sections

The in situ DNA hybridization technique, carried out under stringent conditions, was used to detect human papillomavirus (HPV) DNA of types 6, 11, and 16 in paraffin sections of 32 surgically treated oral mucosal lesions. Expression of HPV structural proteins was analyzed by means of the immunoperoxidase (IP-PAP) method. A total of 10 (31.3%) of the 32 lesions proved to express HPV antigens, which were found in 4 of 7 squamous cell papillomas, in 2 of 2 classic condylomas, in 2 of 10 papillary hyperplasias, and in 2 of 3 leukoplakia lesions. Two of the squamous cell papillomas contained HPV 6 DNA, and 4 additional lesions were positive for HPV 11 DNA. In one of the condylomas, a double infection by HPV 6 and 11 was found, while the second was positive for HPV 11 alone. Both the HPV antigen-positive papillary hyperplasias contained HPV 6 DNA, as did the HPV antigen-positive leukoplakia lesions. Of the latter, one was infected by HPV 6 and 11. DNA of the "high-risk" HPV 16 was contained in two lesions: one lichen planus lesion and one of the two squamous cell carcinomas. The results confirm the previously reported evidence of HPV involvement in oral mucosal lesions. The implications of these findings are discussed in terms of the well-established premalignant character of oral leukoplakia and oral lichen planus, although far less commonly versus leukoplakia, with special emphasis on the discovery of the "high-risk" HPV 16 in the latter as well as in oral cancer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Association between proliferative verrucous leukoplakia and infection with human papillomavirus type 16

Proliferative verrucous leukoplakia (PVL) is a recently described clinical entity characterized by multifocal oral lesions that frequently progress to oral cancer despite abstinence from tobacco use by most patients. To determine if this condition is associated with human papillomavirus (HPV), a polymerase chain reaction (PCR) for HPV DNA was performed on 9 lesions from 7 patients with PVL, histologically diagnosed with focal keratosis (1), papilloma (1), epithelial dysplasia (5) and squamous cell cancer (2). Eight (89%) were HPV positive, 7 for HPV 16. For comparison, we studied 55 non-PVL-associated oral specimens, including 24 oral squamous cell cancers. Of the cancers, 8 (33%) were HPV positive, 4 for HPV 16. These data suggest that HPV 16 infection may play an important role in the pathogenesis of PVL-associated oral dysplasia and possibly cancer, but is found in only a small proportion of the more common, non-PVL associated-oral lesions.     

Human papillomavirus (HPV) DNA sequences in oral precancerous lesions and squamous cell carcinoma demonstrated by in situ hybridization

A series of routinely processed, paraffin-embedded biopsies from 73 surgically treated oral precancerous lesions (OPL) (22 cases), and oral squamous cell carcinomas (SCC) (51 cases), was first screened using an in situ DNA hybridization technique with a human papillomavirus (HPV) DNA probe cocktail containing the 35S-labelled DNA of HPV types 6, 11, 13, 16, 18 and 30. The specific HPV types in lesions shown to contain HPV DNA in this procedure were further analysed by using in situ hybridization and the 6 HPV DNA probes separately. A total of 12/73 (16.4%) of the lesions proved to contain HPV DNA; 6/51 (11.8%) carcinomas and 6/21 (28.6%) dysplasias. The most frequent sites of HPV DNA-positive lesions were palate (4/7; 57%), followed by the floor of the mouth (2/8; 25%), the tongue and gingiva (11.8%). HPV 13 or HPV 30 were not found in any of the lesions studied. HPV 11 DNA was demonstrated in 2 mild dysplasia lesions, but not in carcinomas. One additional mild dysplasia proved to contain HPV 6 DNA. HPV 16 DNA was present in 5 biopsies; 3 carcinomas and 2 dysplasias. In one of the HPV 16-positive carcinomas, HPV 18 DNA was simultaneously present. HPV 18 alone was found in 3 additional carcinomas and in one moderate dysplasia lesion. The results confirm the recently reported evidence on HPV involvement in OPL and oral cancer. The implications of these findings are discussed in terms of the possible HPV etiology of oral SCC.(ABSTRACT TRUNCATED AT 250 WORDS)     

High frequency of hypermethylation of p14, p15 and p16 in oral pre-cancerous lesions associated with betel-quid chewing in Sri Lanka

Background:  Oral squamous cell carcinoma and the most common oral pre-malignancies appear to be related to the habit of betel-quid chewing in Sri Lanka. Although hypermethylation of the tumour suppressor genes in oral cancer have been well documented, little information has been available concerning hypermethylation in oral pre-cancerous lesions. In the present study, we investigated the hypermethylation of p14, p15 and p16 in pre-cancerous lesions including epithelial dysplasia and submucous fibrosis.
Methods:  All samples were obtained from patients with a betel-quid chewing habit in Sri Lanka. Sixty-four patients were clinically diagnosed with leukoplakia, and histopathologically diagnosed with mild or severe dysplasia. Ten patients were diagnosed with submucous fibrosis without epithelial dysplasia. CpG island hypermethylation was assessed by a methylation-specific PCR method. Immunohistochemical staining was performed using anti-p53 antibodies.
Results:  A high frequency of hypermethylation of p14, p15 and p16 was detected in the pre-cancerous lesions, although no hypermethylation was found in normal epithelium. The frequency of hypermethylation was higher than that of positive staining for p53 mutation except in the case of p16 in mild dysplasia. No significant correlation was observed between p53-positive reactions and hypermethylation in any lesions. The hypermethylation was highly detectable even in p53-negative lesions, suggesting that hypermethylation of p14, p15 and p16 occur regardless of whether the lesions have p53 mutations or not.
Conclusions:  The present study indicates that hypermethylation may be involved in the pathogenesis of oral pre-cancerous lesions associated with betel-quid chewing in Sri Lanka.     

Human papillomavirus (HPV) DNA sequences in oral precancerous lesions and squamous cell carcinoma demonstrated by in situ hybridization

A series of routinely processed, paraffin-embedded biopsies from 73 surgically treated oral precancerous lesions (OPL) (22 cases), and oral squamous cell carcinomas (SCC) (51 cases), was first screened using an in situ DNA hybridization technique with a human papillomavirus (HPV) DNA probe cocktail containing the 35S-labelled DNA of HPV types 6, 11, 13, 16, 18 and 30. The specific HPV types in lesions shown to contain HPV DNA in this procedure were further analysed by using in situ hybridization and the 6 HPV DNA probes separately. A total of 12/73 (16.4%) of the lesions proved to contain HPV DNA; 6/51 (11.8%) carcinomas and 6/21 (28.6%) dysplasias. The most frequent sites of HPV DNA-positive lesions were palate (4/7; 57%), followed by the floor of the mouth (2/8; 25%), the tongue and gingiva (11.8%). HPV 13 or HPV 30 were not found in any of the lesions studied. HPV 11 DNA was demonstrated in 2 mild dysplasia lesions, but not in carcinomas. One additional mild dysplasia proved to contain HPV 6 DNA. HPV 16 DNA was present in 5 biopsies; 3 carcinomas and 2 dysplasias. In one of the HPV 16-positive carcinomas, HPV 18 DNA was simultaneously present. HPV 18 alone was found in 3 additional carcinomas and in one moderate dysplasia lesion. The results confirm the recently reported evidence on HPV involvement in OPL and oral cancer. The implications of these findings are discussed in terms of the possible HPV etiology of oral SCC. The use of the in situ DNA hybridization as a powerful tool (enabling the localization of specific HPV DNA sequences and the proper classification of the lesion at the same site) in the study of routinely processed oral biopsies is strongly advocated.     

整联蛋白连接激酶在口腔白斑及早期浸润癌中的表达

目的 研究整联蛋白连接激酶(integrin-linked kinase,ILK)在口腔自斑及早期浸润癌中的表达特点及演变规律,以期为口腔癌前病变的诊断、治疗及口腔癌的预防提供依据.方法 联合应用免疫组织化学及过碘酸希夫反应(periodic acid Schiff reaction,PAS)方法研究ILK在19例正常口腔黏膜(正常黏膜组)、43例白斑伴上皮单纯增生(单纯增生组)、84例白斑伴上皮异常增生[异常增生组,包括轻、中度异常增生44例(轻中度异常增生组),重度异常增生和原位癌40例(重度异常增牛组)]及54例早期浸润癌(浸润癌组)中的表达情况及分布规律.结果 ILK在正常口腔黏膜中呈阴性表达,在其他3组中的阳性表达率可高达90%(163/181),并且ILK在间质的表达随病变程度的加重而增加(χ2=41.585,P<0.001).白斑从单纯增生至癌变,ILK的表达呈现从上皮浅层向基底层下移的趋势,基底细胞由阴性转为阳性着色,并且伴随从胞质向胞膜的分布改变.重度异常增生组基底层和间质表达之间差异有统计学意义(P=0.029).ILK在早期浸润癌的癌巢表达较癌旁上皮浅,间质阳性的病例[76%(41/54)]较重度异常增生[45%(18/40)]增多.结论 ILK在口腔白斑的癌变中可能起莺要作用,确切机制需进一步研究.     

人类主要组织相容抗原Ⅰ类分子在口腔白斑中表达的临床意义

目的通过检测人类主要组织相容抗原Ⅰ类分子（MHC-Ⅰ）在伴有不同异常增生程度的口腔白斑中的表达,以探讨MHC-Ⅰ类抗原表达改变后其局部免疫状态的变化,以及与口腔白斑发生的关系。方法应用MHC-Ⅰ特异性单抗通过免疫组织化学方法从蛋白表达水平检测28例正常口腔黏膜、55例口腔白斑、31例口腔鳞癌内MHC-Ⅰ类抗原的表达。结果伴有重度异常增生的口腔白斑与口腔鳞癌内MHC-Ⅰ类抗原的表达显著低于正常口腔黏膜组（P<0.05）。不伴异常增生及伴有轻、中度异常增生的口腔白斑内MHC-Ⅰ类抗原的表达和正常黏膜未见统计学差异（P＞0.05）。结论MHC-Ⅰ在口腔白斑中存在有表达降低的现象,特别在伴有重度异常增生时,其降低与异常增生的程度有关,对判断口腔白斑的预后有一定价值。     

Promoter hypermethylation of mismatch repair genes, hMLH1 and hMSH2 in oral squamous cell carcinoma

Objectives:  Major risk factors of oral squamous cell carcinoma (OSCC) are environmental and can lead to DNA mutagenesis. Mismatch repair (MMR) system functions to repair small DNA lesions, which can be targeted for promoter hypermethylation. We therefore wanted to test whether hypermethylation of MMR genes ( hMLH1, hMSH2 ) could contribute to oral carcinogenesis by correlating the information to patient clinical data.
Methods:  Genomic DNA was extracted from 28 OSCC and six normal oral epithelium samples. The methylation status of the two MMR genes was assessed using Methylation Specific PCR after DNA modification with sodium bisulfite. Serial sections of the same tissues were immunostained with antibodies against hMLH1 and hMSH2 protein.
Results:  Promoter hypermethylation was observed in 14/28 OSCC cases. Remarkably, 100% of patients with multiple oral malignancies showed hypermethylation in hMLH1 or hMSH2 compared with 31.5% of single tumor patients. In 10 cancer cases, expression of the hMLH1 and hMSH2 genes by immunostaining showed reduced or absence of expression of one of the genes, although some did not reflect the methylation status.
Conclusions:  Hypermethylation of hMLH1 and hMSH2 might play a role in oral carcinogenesis and may be correlated with a tendency to develop multiple oral malignancies.     

口腔粘膜组织中HPV16DNA和空泡细胞关系的研究

本研究采用斑点杂交技术，检测８３例口腔扁平苔藓、口腔白斑、口腔鳞癌及正常口腔粘膜组织中的ＨＰＶ１６ ＤＮＡ，并分析ＨＰＶ１６ ＤＮＡ与空泡细胞出现的关系。口腔粘膜组织ＨＰＶ１６ ＤＮＡ的检出率为１０．８％，空泡细胞的检出率为３４．９％。两者均阳性的检出率为７．２％，结果提示空泡细胞不是ＨＰＶ感染所特有的，不能作为诊断ＨＰＶ的特征性指标。     

口腔鳞状细胞癌高危型人乳头状瘤病毒感染的检测

目的 观察口腔鳞癌及口腔粘膜高危型人乳头状瘤病毒（HPV）感染情况,探讨HPV感染与临床及病理资料的关系。方法 用多聚酶链反应（PCR）检测73例口腔鳞癌及40例正常口腔粘膜石蜡包埋组织中HPV16、HPV18的DNA。统计分析其与临床及病理资料的关系。结果 口腔鳞癌HPV16／HPV18 DNA阳性率为74％（54／73）,正常口腔粘膜为55％（22／40）。口腔鳞癌与正常口腔粘膜HPV阳性率存在显著性差异（P＝0．040）。统计分析显示：HPV感染与患者的性别、年龄有关,与其它因素（肋瘤发生部位、嗜烟酒情况、肿瘤病理分级、临床分期）无关。结论 高危型HPV感染与口腔鳞癌的发生有关。口腔粘膜中HPV感染普遍存在,提示HPV在口腔肿瘤的发生中并非独立发挥作用。