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Objective:To develop an efficient and rapid method for the isolation and cultivation of human scalp dermal papilla cells from small specimens.Methods:Hair-bearing skin specimens measuring 0.5 cm × 0.5 cm -0.5 cm × 1 cm in size were obtained from the scalp of 3 patients with pigmented nevus and 6 with sebaceous nevus during surgery in Department of Dermatology, the First Hospital Affiliated to Army Medical University from September 2018 to January 2019. The subcutaneous fat layer containing hair follicles was cut out of the specimens, and hair follicles were sorted with ophthalmic forceps, which were subsequently digested with 0.6% dispase Ⅱ for 30 minutes, then with 0.2% collagenase Ⅳ at 37 ℃ for 30 - 60 minutes, and were centrifuged to obtain hair papillae. Morphological observation was performed on the isolated hair papillae, and dermal papilla cells were cultured, passaged and identified.Results:Under the microscope, the hair papillae isolated by two-step enzyme digestion of small scalp specimens were intact, and showed an inverted pear-like shape, and residual dermal sheaths could be observed around some hair papillae. However, no hair papilla was isolated by one-step enzyme digestion. With the two-step enzyme digestion method, the hair papilla separation rate was 60.8% ± 2.1%, the adherence rate of the dermal papilla cells at 72 hours was 86.6% ± 3.9%, the time for cells to emigrate out of hair papillae was 0.5 - 3.0 days, the total operation duration was 2.0 - 3.0 hours, and the actual operation duration after subtraction of digestion duration was 1.0 - 1.5 hours. The dermal papilla cells isolated by the two-step enzyme digestion method could grow in an aggregative pattern in early stage, but grew in a non-aggregative pattern after 8 passages.Conclusion:The two-step enzyme digestion of small specimens is a simple and efficient method for isolating human scalp dermal papilla cells.
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Objective To investigate an efficient rapid method for the isolation and cultivation of human axillary dermal papilla cells.Methods Skin specimens with hair follicles were obtained from the axillary area of patients who received bromhidrosis surgery in the Department of Dermatology of the First Affiliated Hospital to Army Medical University from October 2015 to May 2016.The axillary dermal papilla cells were isolated by two-step enzyme digestion method,one-step digestion method and micro-dissection method separately.Then,axillary dermal papilla cells were cultured and identified.Differences in the operative procedure,separation efficiency and adhesion efficiency of dermal papilla cells,cell emigration duration,total operation duration and actual operation duration were compared among the above 3 methods.Results Compared with the one-step digestion method and micro-dissection method,the two-step enzyme digestion method showed simpler operative procedure,more than 30% separation rate and 96% adhesion rate of dermal papilla cells after 1 week.Moreover,the cell emigration duration was shortened by 3-4 days by the two-step enzyme digestion method.The two-step enzyme digestion method also showed longer total operation duration,but shorter actual operation duration compared with the one-step digestion method and micro-dissection method,as well as lower contamination rate compared with the micro-dissection method.Cultured axillary dermal papilla cells grew in an aggregative pattern in the early stage,but grew in a nonaggregative pattern after 6 passages.Immunofluorescence assay showed positive staining for laminin and collagen Ⅳ in axillary dermal papilla cells.Conclusion The modified two-step enzyme digestion method is a kind of simple,efficient and rapid method for the isolation of human axillary dermal papilla cells,and axillary dermal papilla cells can be harvested through this method by using a few specimens.
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Objective To evaluate the efficacy of combined therapies of ablative fractional Er ∶YAG laser (2,940 nm) and ALA-PDT on refractory verruca plana.Methods 120 cases of refractory verruca plana patients were randomly divided into two groups:60 cases in the control group and 60 cases in the experimental group.The control group used 10% ALA-PDT with LED irradiation of a power density 70 of mW/cm2 at a distance of 20 cm,which lasted for 20 min each time.The experimental group was treated with ablative fractional Er ∶ YAG laser (2,940 nm) first with the fluence of 500P/cm2 and short pulse duration,and then treated 10% ALA-PDT as mentioned before.Every patient was treated twice at two-week intervals.Three independent investigators evaluated subject outcomes at 3 months post-treatment including efficacy and side-effects.Results The effective rate of the experimental group was 86.44 % at 3 months post-treatment after one to two times.Meanwhile,the control group was 59.65 %.The clinical outcome of experimental group was better than the control group.There was significant difference between the two groups (P<0.05).The recurrent rate of experimental group was 3.39% and 12.30% in control group.There was significant difference between the two groups (P<0.05).There were no obvious side-effects in both groups.Conclusions Ablative fractional laser with low fluence promotes the transdermal absorption of ALA and enhances the efficacy of PDT.
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<p><b>OBJECTIVE</b>To study the clinicopathologic features, immunophenotype and gene rearrangement of primary cutaneous diffuse large B-cell lymphoma, leg type (PCLBCL).</p><p><b>METHODS</b>Seven cases of PCLBCL were enrolled into the study. Clinicopathologic analysis, immunohistochemical staining and gene rearrangement for IgH and Igκ were undertaken in the study.</p><p><b>RESULTS</b>All the seven cases were male, and the median age was 72 years. Patients usually presented with multiple purple tumors, nodules, papules and infiltrative plaques. Two patients had a history of leg injury before onset, and one had mosquito bites. Histologically, the tumor involved the dermis and subcutis with dense and diffuse infiltrative pattern composing of centroblasts and/or immunoblasts. Immunohistochemical staining showed that seven cases (7/7) expressed CD20, six (6/6) expressed bcl-2, four (4/4) expressed MUM-1, four (4/5) expressed CD79a, four (4/5) expressed PAX-5 and four (4/6) expressed bcl-6, respectively. All cases did not express CD3ε, CD45RO, CD10 and CD30. IgH gene rearranged bands were detected in three (3/6) cases and Igκ was detected in one (1/5) case. Six of the seven cases died and the remaining patient, who was 44-year-old, was alive after 22 months of follow-up.</p><p><b>CONCLUSIONS</b>PCLBCL is rare, predominantly affects elderly male patients. PCLBCL has poor prognosis and high mortality, but younger patients seem to have better prognosis. Some cases had a history of trauma or mosquito bites. The relationship between the history and the onset of PCLBCL needs further evaluation.</p>
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Anciano , Anciano de 80 o más Años , Animales , Humanos , Masculino , Persona de Mediana Edad , Antígenos CD , Culicidae , Reordenamiento Génico , Cadenas Pesadas de Inmunoglobulina , Genética , Cadenas kappa de Inmunoglobulina , Genética , Inmunofenotipificación , Mordeduras y Picaduras de Insectos , Pierna , Traumatismos de la Pierna , Linfoma de Células B Grandes Difuso , Genética , Metabolismo , Patología , Pronóstico , Proteínas Proto-Oncogénicas c-bcl-6 , Metabolismo , Neoplasias Cutáneas , Genética , PatologíaRESUMEN
Objective To evaluate the efficacy and safety of 308-nm excimer light in the treatment of stable vitiligo. Methods Thirty patients with stable vitiligo were enrolled in this clinical trial. All the subjects received the treatment with 308-nm excimer light on a twice-weekly schedule for 3 months. Results The repigmentation rate was 95.0%, 75.0% and 66.7% for lesions in the face and neck, trunk and limbs, with the treatment sessions averaging 10.22 ± 1.60, 19.10 ± 2.38, 37.74 ± 3.06, respectively, and accumulative irradiation dose averaging 7.50 ± 3.45, 10.60 ± 1.01, 18.56 ± 3.05 J/cm2 respectively. Significant differences were observed in the repigmentation rate and treatment sessions between the lesions in the face and neck, trunk and limbs (all P < 0.05). No severe side effects were seen during the treatment. Conclusion 308-nm excimer light is effective and safe for the treatment of vitiligo.
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Objective To study the survival and melanogenic potential of human melanocytes reversibly immortalized via SV40T antigen gene and Cre/loxP system in Guinea pigs. Methods The supernatants of retrovirus vector Cre-ERT2 were used to infect melanocytes which had been successfully transfected by SV40TAg gene (MCT), then the expression of Cre recombinase was induced with tamoxifen in infected cells; subsequently, the surviving cells, which were named as MCTC, were subjected to expansion culture. Guinea pigs were utilized to establish animal models of vitiligo, then MCTC and primary melanocytes were transplanted respectively into the animal models. The repigmentation at the transplanted area was observed with naked eyes successively until 3 months after the transplantation when tissue samples were obtained from implanted area and nonimplanted area of guinea pigs and subjected to Masson-Fontana silver stain and Hematoxylin-eosin stain for the analysis of melanocyte distribution and melanin deposition in epidermis. Results Repigmentation started 4 weeks after the transplantation, and dark or brown patches, which ranged in size from 0.5 to 1 cm, were observed in the implanted area 3 months after the transplantation. The repigmentation rate was of no significant difference between pigs transplanted with MCTC and those with primary melanocytes (82.5% vs 76.7%, P > 0.05). Pathological examination revealed melanin deposition in the basal layer of epidermis and some hair follicles in transplanted area. Conclusions SV40T antigen gene combined with Cre/loxP site-specific recombinase system can induce the reversible immortalization of human melanocytes, and the immortalized melanocytes have a favorable profile of biological safety and similarity in survival rate and melanogenic potential to primary melanocytes.
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Objective To investigate the expression of T cell factor-4 (TCF4) gene in dermal papilla cells of hair follicles.Methods The expression of TCF4 gene was examined by in situ hybridization in scalp tissues of patients with alopecia areata and normal human controls,The protein and mRNA exprcssions of TCF4 were detected by immunochemistry and RT-PCR method,respectively,in aggregated and non-aggregated human dermal papilla cells.ResultsAs shown by in situ hybridization,TCF4 gene was expressed in the dermal papilla cells from healthy controls,but not in those from patients with alopecia areata.Both cell immunochemistry and RT-PCR showed that TCF4 gene expressed in aggregated dermal papilla cells,but not in non-aggregated dermal papilla cells.ConclusionsTCF4 gene is expressed in dermal papilla cells.The growth cycle Of follicles may be related to wnt signal.
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OBJECTIVE To design and construct eukaryocyte expression vector of SV40 virus large T antigen and induce its targeted expression in eukaryocyte.METHODS SV40 large T gene which excised intron was cloned by SOE(splicing by overlapping extension) and digested with restricted enzymes EcoR Ⅰ and BamH Ⅰ.By the same methods,we got the digested product of pEGFP-N1.After that,the two fragments were ligated to form SV40(TEGFP) by Ligation Kit,and sequenced by TaKaRa ABI Prism Terminator Cycle Sequence Kit.The reconstructed vector was transfected into primary cultured human fibroblast using a Lipofectin transfection method.At 48 h(after) transfection,the expression of SV40T was detected with PCR and RT-PCR using specific primer of T gene.(RESULTS) The restricted enzymes digested and sequencing results showed that SV40 large T gene had cloned into pEGFP-N1 vector successfully.The genome DNA and total RNA were isolated from the positive cells.With these samples,the specific 288 bp fragment was amplified using PCR and RT-PCR.CONCLUSIONS The recombinant plasmid SV40TEGFP will be a stable and valuable molecular tool for human eukaryocyte study.
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The dermal papilla plays pivotal roles in hair follicle cycle and dermal papilla cells (DPCs) with aggregative behavior have more obviously inductive ability. In the present study, the suppression subtractive hybridization method was employed to identify the differentially expressed genes in dermal papillae cells with aggregative behavior. Following mRNA isolation of DPC with and without aggregative behavior, cDNA of both populations were prepared and subtracted by suppression PCR. Sequencing of enriched cDNAs identified five genes differentially expressed including capping protein, paladin, and vascular endothelial growth factor. Interestingly, HSPC016, first cloned from CD34+ hematopoietic stem/progenitor cells (HSPC), was identified by SSH, cDNA dot blot and Northern blot, which showed that this gene was differentially expressed in DPC with aggregative behavior. The full-length cDNA of HSPC016 was shown to be 366 bp, and the possible function of HSPC016 related to transcriptional regulation.
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Perfilación de la Expresión Génica , Regulación de la Expresión Génica/genética , Folículo Piloso/citología , Folículo Piloso/metabolismo , Proteínas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Agregación Celular , Clonación Molecular , Humanos , Datos de Secuencia Molecular , Proteínas/química , Homología de Secuencia de AminoácidoRESUMEN
BACKGROUND: Earthworms are one of the most important constituents in the ecosystem and become an environmental information carrier between terrestrial organisms and soil ecosystem. They were known as important non-target terrestrial soil organisms for assessing the general impact of pollution on the soil community. Protein content and growth rate in response to organic and heavy metal contaminants in soil are often used to assess the soil ecotoxicity.OBJECTIVES: To investigate the effects of 1, 8-dihydroxyanthraquinone,one representative of hydroxyanthraquinones, on the earthworm eisenia foetide under laboratory conditions of the growth rate and protein content.DESIGN: Experimental study based on the experimental animals.SETTING: Eco-toxicological laboratory and genetic laboratory in a university.MATERIALS: The experiment was conducted in the Eco-toxicological Laboratory and the Genetic Laboratory, Dalian University of Technology from February to July 2004. Eisenia foetide, a kind of international standard earthworm, were maintained in a soil mixture before experiments. Earthworms used in this study were healthy adults with 1 -2 month old and 200 - 300 mg mass and the number of the earthworms in each analytical experiment was 10.METHODS: The earthworms were removed from the soil 12 hours before use and stored in Petri dishes on damp filter paper to void gut contents.Then, these earthworms were exposed for various durations to soils contaminated. The earthworm weight and protein content were determined respectively at every 7 days interval.MAIN OUTCOME MEASURES: Effects of 1, 8-dihydroxyanthraquinone on growth rate and protein content of earthworms.RESULTS: No lethal effect of 1, 8-dihydroxyanthraquinone was observed even at the highest concentration(1.0 g/kg soil) of exposure. The sub-lethal effect, however, was evident at all the concentration scale. 1,8-dihydroxyanthraquinone caused a significant reduction in the growth rate (maximum -22. 5% ) at the dosage of 1.0 g/kg and 28 days contact time.Additionally a reduction in total soluble protein was observed in all treated worms(maximum- 39.6% ) at the dosage of 0. 8 g/kg and 7 days contact time.CONCLUSION: 1, 8-dihydroxyanthraquinone was potentially dangerous to the soil ecosystem and more ecological risk assessment of this chemical material should be thoroughly carried out.
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To train medical students' clinical thinking ability is an important aspect of clinical probation practice and also the common goal of dermartology probation training. This article analyzes the problems around the cultivation of clinical thinking ability during probation practice in our department,and proposes the corresponded measures to improve it.
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Objective To isolate melanocytes from epidermis and identify their biological characters. Methods Taking TPA/bFGF/IBMX as basic supplements in medium DMEM/F12(1∶1), the cultured cells from the resected foreskin were purified with trypsin digestion and G418 selection, and the cellular structure and function were observed by Dopa-staining, melanin content assay, tyrosinase activity assay and immunohistochemical staining. Results Dopa-staining showed that the cultured melanocytes had melanin synthesis, melanin content assay and tyrosinase activity assay showed the tyrosinase function of the cells was normal, immunohistochemical staining demonstrated the cell differentiation was normal. Conclusion Melanocytes could be cultured by TPA/bFGF/IBMX and would maintain normal structure and biological function under such conditions.
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Objectives To construct a cDNA subtractive library of dermal papilla cells (DPCs) in anagen with suppression subtractive hybridization (SSH) and clone differentially expressed genes related to DPCs in anagen. Methods Total RNA was isolated from DPC of anagen and telogen follicles. Then ds cDNAs were synthesized in turn using SMART cDNA synthesis technique. After cDNAs from anagen and telogen follicle DPCs were hybridized with each other twice and underwent two rounds of nested PCR, PCR products were ligated with arms of T/A plasmid vectors to set up the subtractive library. Selected clones were verified by reverse Nothern blot and DNA sequencing, and the acquired sequences were analyzed for homology based on Genbank nucleotide database. Results cDNA subtractive library of DPCs in anagen follicle was set up successfully with high subtractive efficiency. Thirty-five genes were identified with 22 known functional genes and 13 unknown functional genes. Conclusions These results demonstrate the effectiveness and sensitivity of SSH in detecting differentially expressed genes from a small amount of clinical samples. Information about such alterations in gene expression might be useful for elucidating the genetic events in hair follicle growth regulation.
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Objective To screen and analyze genes differentially expressed within dermal papillae cells(DPC)with aggregative behavior.Methods Total RNA was extracted from DPC with and without ag-gregative behavior,and double-stranded cDNA were synthesized by using SMART cDNA synthesis.The cD-NA fragments of differentially expressed genes in dermal papillae cells with aggregative behavior were isolat-ed by suppression subtractive hybridization,sequencing,and then subtracted library was set up.Positive clones were screened by PCR method and verified by cDNA dot blot and then analyzed through homologous retrieving.Results A subtractive cDNA library of DPC with aggregative behavior was successfully construct-ed.The results of screening and cloning of the library showed that DPC with aggregative behavior could ex-press genes related to homologous aggregation,regnlation of growth,differentiation and development,and sig-nal transduction proliferation and cycle control,which included known genes(capping protein,paladin,vas-cular endothelial growth factor),hematopoietic stem/progenitor cells(HSPC)related genes(HSPC011and HSPC016)and a new gene.Conclusions The construction of subtractive library of DPC lays solid founda-tion for screening and cloning new and specific genes related to aggregative behavior of DPC.Several genes may cooperatively involve in homologous aggregation,and regnlation of growth of DPC.Among these genes,capping protein and palladin may be closely related to aggregative behavior of DPC,and VEGF and HSPC re-lated clones may be responsible for the status of higher proliferation of DPC.
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Objective To identify the expression of gene HSPC011(HSPC,hematopoietic stem/pro-genitor cell)in the dermal papilla cells of hair follicle and clone its cDNA.Methods The expression of gene HSPC011was confirmed by intracellular mRNA hybridization in situ;the objective cDNA was amplified by RT-PCR(reverse transcription PCR).Results By in situ hybridization,it was shown that gene HSPC011expressed in the coagulated dermal papilla cells,but not in the non-coagulated dermal papilla cells and the dermal fibroblast;The objective cDNA with a fragment of430bp was amplified by RT-PCR,and a recombi-nant eukaryotic expressing plasmid pCI-neo/HSPC011was constructed.By enzyme cutting and sequencing analysis,the objective cDNA was completely identical with gene HSPC011.Conclusion Gene HSPC011was clearly shown to express in the dermal papilla cells,and the expression of HSPC011was possibly related with the differentiation and functional status of the dermal papilla cells.
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Objective To explore the inhibitory effects of He Ne laser repeated irradiation on the collagen synthesis of cultured scar fibroblasts. Method Cultured fibroblasts derived from hypertrophic scars(HS) were irradiated with He Ne laser for 30 minutes at various power densities(10,50,100 and 150 mW/cm2),once a day for 3 consecutive days.In 24 hours after repeated irradiation collagen production and type I procollagen mRNA level of fibroblasts were measured with the incorporation of 3H proline and blot hybridization techniques espectively.Results Collagen synthesis and type I procollagen mRNA level remained unchanged when the laser was irradiated at the power density of 10 mW/cm2 or 50 mW/cm2.Compared with control,collagen synthesis and type I procollagne mRNA level were significantly decreases at the power density of 100 mW/cm2 or 150mW/cm2(P< 0.05).Type I procollagen mRNA level at the power densityof 150 mW/cm2 was lower than that at the 100 mW/cm2 (P< 0.05).Conclusion Repeated He Ne laser irradiation at the power density of 100 mW/cm2 or 150 mW/cm2 can suppress collagen synthesis of cultured fibroblasts in HS.The cause of suppression may be associated with down regulation of type I procollagen mRNA expression.
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Objective To construct a cDNA substractive library of dermal papilla cell (DPC) in patchy and normal area of alopecia areata with technique called suppression substractive hybridization. Methods Total mRNA was isolated form DPC of patchy and normal area. Moreover, double strand cDNAs were synthesized in turn. cDNA from patch and normal area then were restricted and divided into two groups and ligated to the specific adaptor 1 and adaptor 2 respectively. After cDNAs from patchy and normal area hybridized with each other twice and underwent two times of nested PCR, then PCR products were ligated with arms of T/A plasmid vectors to set up the substractive library. Results cDNA substractive library of DPC in patchy and normal area of alopecia areata was set up successfully with highly subtractive efficiency. The amplified library contained 120 positive clones, in which 90 positive subclones contained 100 500bp inserts. Conclusion The cDNA substractive library of DPC in patchy and normal area of alopecia areata is a highly efficient one and lays a solid foundation for screening and cloning new and specific genes from patchy and normal DPC of anagen hair follicle in DPC of alopecia areata.
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Objective To investigate the immortalization of human melanocytes by transfection with SV40 T antigen ( SV40T ). Methods By using SofastTM,a gene transfection reagent, the reconstructed eukaryotic expression vector SV40T-pEGFP was stably transfected into cultured primary human melanocytes, then the positive cells were selected with G418. After the positive cells were expanded in culture, the expression of SV40T gene was detected by RT-PCR and PCR, and the protein expression of SV40T by Western blotting. Results The genome DNA and total RNA were isolated from the positive cell clones, and a 288 bp fragment, which was specific for the SV40T antigen gene, was amplified. The results of immunohis-tochemistry and Western blotting confirmed the expression of SV40T protein in transfected cells. Conclusion SV40T antigen gene can successfully induce the immortalization of human melanocytes.
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Objective To identify the relationship between expression of hematopoeitic stem/progenitor cell(HSPC)-related gene HSPC016 and aggregative behavior of the dermal papilla cells of hair follicles. Methods The aggregated human dermal papilla cells, non-aggregated human dermal papilla cells and the human dermal fibroblasts were used in this study. Expression of HSPC016 mRNA was investigated in the three cell groups by intracellular mRNA hybridization in situ and RT-PCR. Results By in situ hybridization, it was shown that gene HSPC016 specifically expressed in the aggregated dermal papilla cells, but not in the non-aggregated human dermal papilla cells and human dermal fibroblasts. A 200bp fragment of target cDNA was amplified from RNAs of the aggregated human dermal papilla cells by RT-PCR, but could not from RNAs of other two cell lines. Conclusions Gene HSPC016 was specifically expressed in the aggregated dermal papilla cells, and expression of HSPC016 might be related to the differentiation and functions of dermal papilla cells.
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Objective To introduce the amino acid substitution for HP V16E749-57(HLA-A2-restricted CTL epitope) and to identify the novel epitopes.Me thods Quantitative method was used to evaluate the affinity of the substituted peptides.To determine the peptide candidates to be synthesized and identified,the molecular models of the HLA-A2-peptide complex and CTL epitope candidates b ound to the HLA-A2 molecule were established by computer molecular modeling.Pep tides were synthesized and purified with standard Fmoc assay,lactate dehydrogen ase (LDH) release assay was used to determine their abilities of inducing the ge neration of specific CTLs.Results Modified peptides met the requirements of H LA-A2-restricted CTL epitopes.Peptide RLHYNIVTF had the abilitiy of inducing th e generation of specific CTLs.Conclusions Compared with HPV17E749-57 the mod ified peptide RLHYNIVTF has a higher antigenicity and affinity to HLA-A2.So,pe ptide RLHYNIVTF may be used as one of the HLA-A2-restricted candidate epitopes,instead of HPV17E749-57,for peptide vaccine in the treatment of HPV infection.