Reversible transfection of human melanocytes mediated by Cre/loxP site-specific recombination system and SV40 large T antigen.

OBJECTIVE: To study the reversible transfection of human melanocytes mediated by simian virus 40 large T antigen (SV40LTAg) and Cre/loxP site-specific recombination system. METHODS: The reconstructed SV40LTAg-EGFP-neo-loxP vector was transfected into primary cultured human melanocytes with Sofast(TM) transfection reagent and the positive cells were selected using G418. After expanding culture of these positive cell clones, the expression of SV40LTAg was detected by polymerase chain reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescent method. After that, these positive cells were infected by virus supernatant of Cre-ER(T2) retrovirus vector and Cre recombinase was induced to act by tamoxifen. On the 6th and 10th day after Cre recombinase acting, the expression of SV40LTAg was detected using the same methods as above, and cell tumorigenicity was studied using soft agar assay, athymic mouse study and karyotype analysis. On 10th day after tamoxifen treatment, cell biological characters were identified with immunofluorescent staining and transmission electron microscopy. Then these cells were transplanted into vitiligo animal model to observe their melanogenesis ability in vivo. RESULTS: The genome DNA and total RNA were isolated from the positive cells transfected by SV40LTAg (designated as MCT) and specific 288 bp fragment was amplificated using PCR and RT-PCR methods. The results of immunofluorescence confirmed the expression of SV40LTAg in cell nucleus. On the 6th day after tamoxifen treatment in infected cells by Cre-ER(T2) retrovirus vector (designated as MCT-Cre), there could be detected SV40LTAg expression, but on 10th day, there could not be detected SV40LTAg expression in cells. These results showed that the excised efficiency of Cre recombinase increased along with time prolongation, and would obtain complete recombination efficiency. The identification of MCT-Cre cell biological characters showed that these cells had normal parent-cell-like cell phenotype and no tumorigenicity in vitro. The pigmentation started in 4 weeks and formed black macula in 3 months after grafting. The pathological results showed that there had been significant melanocytes and melanin accumulation in epidermis and some hair follicle in transplanted area, which confirmed that MCT-Cre had melanogenesis function in vivo. CONCLUSION: Human melanocytes could be mediated by reversible transfection by SV40LTAg and Cre/loxP site-specific recombination system, which had stable parent-cell-like phenotypic characters and no tumorigenicity in vitro; moreover, these cells still had melanogenesis function in vivo.

Identification of differentially expressed genes in anagen dermal papilla by suppression subtractive hybridization / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>We constructed a cDNA subtractive library of dermal papilla cells (DPCs) in anagen with suppression subtractive hybridization (SSH) technique and clone differentially expressed genes related to DPCs in anagen.</p><p><b>METHODS</b>Total mRNA was isolated from DPCs of anagen and telogen follicles. Moreover, single-strand (ss) and double-strand (ds) cDNAs were synthesized in turn using SMART PCR cDNA synthesis technology. ds cDNAs then were digested with Rsa I and divided into two groups, and ligated to the specific adaptor 1 and adaptor 2R, respectively. After cDNAs 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 demonstrated by reverse Northern blot and sequenced. The acquired sequence data were aligned against the Genbank nucleotide database.</p><p><b>RESULTS</b>cDNA subtractive library of DPCs in anagen follicles was set up successfully with high subtractive efficiency. Thirty-five genes were identified in this study with 22 known functional genes and 13 unknown functional genes.</p><p><b>CONCLUSIONS</b>All results confirm 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 could be useful for elucidating the genetic events in hair follicle growth regulation.</p>

Effect of anti-human VEGF monoclonal antibody on VEGF level in serum of nude mice transplanted buccal carcinoma: a quantitative study / 华西口腔医学杂志

<p><b>OBJECTIVE</b>Quantitative study of the effect of anti-human VEGF mAb E11 to VEGF level in serum of nude mice transplanted buccal carcinoma.</p><p><b>METHODS</b>E11 was administered into BALB/c nu/nu mice which were transplanted human buccal carcinoma. The saline was administrated as negative control. Mice were killed at 18 days. The VEGF level in serum of mice was determined by improved indirect ELISA.</p><p><b>RESULTS</b>Compared with the VEGF level in serum of mice in saline group, it was dramatically decreased in E11 group. The VEGF level in serum of mice treated E11 by subcutaneous was lowest and only reached (1.17 +/- 0.13) microg/L.</p><p><b>CONCLUSION</b>It demonstrated that the anti-human VEGF mAb could reduce the VEGF level in serum by binding VEGF, and block its biological activity. It indicates that VEGF in serum of malignant tumor patient is a new tumor marker.</p>

Study on serum-free culture of dermal papillae cells of human hair / 中华皮肤科杂志

Objective To explore the feasibility of culturing dermal papillae cells (DPC) of hu- man hair in a serum-flee medium,and to observe the growth characteristics of these cells.Methods Cell culture flasks (plates) were pretreated with fibronectin,and DPC (2nd passage) were incubated with Williams E serum-flee medium supplemented with insulin-transferrin-selenite (ITS).Cells were observed by an inverted phase-contrast microscope.Proliferation of DPC was evaluated with 3-(4,5-dimethylthia- zol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and by their growth curve.Results In a serum-free medium,2nd passage DPC adhered to the flask surface within two to four hours of incubation; two to three days later,confluence,of the cells was observed,without noticeable proliferation.Four days later,cell connection was interrupted,isolated cells or cell clusters were seen,and detachment of some cells from the flask surface was observed.One to two weeks later,most cells had died.After incubation with 4% bovine serum for ten hours,cell proliferation was observed surrounding the remaining viable cell colonies. DPC growth curve showed stagnant phase and slow growth phase;however,log growth phase was not ob- served.Conclusion DPC could be successfully cultured in serum-free medium.However,the culture con- dition needs to be further optimized.