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.

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.

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.

ObjectiveTo clone TCF4 (T cell factor 4) gene and construct its eukaryotic expression vector. MethodsThe total RNA was extracted from the aggregated human dermal papilla cells. The full length cDNA encoding TCF4 was obtained by RT- PCR, digested by restriction enzyme, then inserted in the eukaryotic expression vector pcDNA3.0. The sequence and reading frame were confirmed by two restriction enzymes and sequencing. The recombinant vector TCF4/pcDNA3.0 was stably transfected into dermal papilla cells, and the expression changes of TCF4 gene were detected. ResultsTCF4 gene was cloned from dermal papilla cells and its eukaryotic expression vector was constructed. After the identification and sequencing, the reconstructed plasmid was confirmed containing the correct and full nucleotide sequence of TCF4 gene. After stable transfection, the mRNA and protein level of TCF4 gene were up-regulated in dermal papilla cells and the proliferation of dermal papilla cells was promoted. ConclusionThe expression vector TCF4/pcDNA3.0 was constructed successfully and could be expressed in the dermal papilla cells. TCF4 gene can promote the proliferation of the dermal papilla cells.

In this research a strain of isolated Pseudomonas alcaligenes which causes degradation of dexamethasone was acclimated further and its proteins of every position in the bacterium were separated by the osmotic shock method. The separated intracellular proteins which had the highest enzyme activity were extracted by the salting out with ammonium sulfate and were purified with the cation exchange chromatography and gel chromatography. The purified proteins which was active to cause degradation of dexamethasone had been detected were cut with enzyme and were analyzed with mass spectrometry. The results showed that the degradation rate to dexamethasone by acclimated Pseudomonas alcaligenes were increased from 23.63% to 52.84%. The degrading enzymes were located mainly in the intracellular of the bacteria and its molecular weight was about 41 kD. The specific activity of the purified degrading enzymes were achieved to 1.02 U x mg(-1). Its 5-peptide amino acid sequences were consistent with some sequences of the isovaleryl-CoA dehydrogenase. The protein enzyme may be a new kind degrading enzyme of steroidal compounds. Our experimental results provided new strategies for cleanup of dexamethasone in water environment with microbial bioremediation technique.
Amino Acid Sequence ; Bacterial Proteins ; chemistry ; Chromatography ; Dexamethasone ; chemistry ; Molecular Weight ; Pseudomonas alcaligenes ; enzymology

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.

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.

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.

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.

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.