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.

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.

Objective To analyze and summarize the cause of false positive results of the cervical liquid-based preparation screening,to improve the accuracy of cervical cytology diagnosis.Methods 20 353 cases were col-lected.The cytological diagnosis was statistically analyzed.Test positive results contrast analysis of the histologic diag-nosis was conducted.Cytological diagnosis of positive and histology diagnosis of non -neoplastic to review the original cytology.Results The incidence of 637 cases of cytology screening for positive.Among the 388 cases with histologic control,228 cases of histological diagnosis of abnormal change.Include:low -grade squamous intraepithelial lesion (LSIL),high -grade squamous intraepithelial lesion (HSIL),cervical squamous cell carcinoma (SCC),cervical ade-nocarcinoma(ACC),endometrioid carcinoma,malignamt melanoma.The other 160 cases did not check out the abnor-mal lesions.Conclusion Incidence of false positive results in 160 cases,accounting for 41.2%.It almost focused on atypical squamous cells of undetermined significance (ASC -US)and LSIL for a variety of reasons.Standardized work process should be taken to strengthen the training of the doctors,summarize continuously improve,as far as possi-ble to avoid false positive diagnosis.

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.

BACKGROUND:Currently, antisense oligonucleotides (AS-ODN) have a good prospect in gene therapy, but AS-ODN with smal molecular weight cannot easily enter into the cels, which is susceptible to nuclease degradation. Therefore, there is stil a lack of fundamental understanding about how to improve their transfection efficiency, and target-based transferring. OBJECTIVE:To investigate whether a weak cationic and phosphorylcholine-containing diblock copolymer (MPC30-DEA70) can act as a carrier system to deliver a chemicaly synthesized transforming growth factor-β1 (TGF-β1) AS-ODN into myocardial cels. METHODS: MPC30-DEA70 was compounded with TGF-β1 AS-ODN at various N/P ratios and the MPC30-DEA70/TGF-β1 AS-ODN complexes were characterized by DNA electrophoresis. MTT assay was used to observe the biocompatibility. Confocal laser scanning microscope was used to observe the distribution and location of MPC30- DEA70/TGF-β1 AS-ODN in cells. Flow cytometry was used to detect the transfection efficiency and fluorescence intensity of MPC30-DEA70/TGF-β1 AS-ODN in cells. Western blot and RT-PCR methods were employed to measure the expression of TGF-β1 in cells. RESULTS AND CONCLUSION: Cell growth inhibition showed that the MPC30-DEA70 had low cytotoxicity to myocardial cells within the effective transfection dosage range (< 20 mg/L). Data from the flow cytometry test indicated a clear trend of increasing transfection efficiency with the increasing of N/P ratios. At high N/P ratios, the expression levels of TGF-β1 mRNA and protein in myocardial cells were significantly lower. This study shows that MPC30-DEA70 can work as an effective transgenic vector in myocardial cells. TGF-β1 AS-ODN can silence the expression of TGF-β1 gene efficiently and specially, and may antagonize TGF-β1-mediated biological function.

To enable students to better grasp the basic skills of pathogenic biology and im-munology experimental teaching , and make full use of the characteristics of experimental teaching to train students' scientific quality and innovative consciousness , the reform of pathogenic biology and immunology experiment teaching was explored. Microbiology, Parasitology and Immunology experiment were integrated into an experimental course , and corresponding laboratory was set up to take an in-dependent experimental teaching. Through renewing experiment teaching idea, some measures were taken such as modularization and personalization of the teaching content, the establishment of a com-plete management system , writing a new experimental course to match the experiment , improving teaching methods and developing students' innovative experiments to improve their enthusiasm and in-terest for experimental class learning, thus enhancing their innovation ability.

OBJECTIVETo study the clinicopathologic features, immunophenotype and gene rearrangement of primary cutaneous diffuse large B-cell lymphoma, leg type (PCLBCL).

METHODSSeven cases of PCLBCL were enrolled into the study. Clinicopathologic analysis, immunohistochemical staining and gene rearrangement for IgH and Igκ were undertaken in the study.

RESULTSAll 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.

CONCLUSIONSPCLBCL 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.

Aged ; Aged, 80 and over ; Animals ; Antigens, CD ; analysis ; Culicidae ; Gene Rearrangement ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin kappa-Chains ; genetics ; Immunophenotyping ; Insect Bites and Stings ; complications ; Leg ; Leg Injuries ; complications ; Lymphoma, Large B-Cell, Diffuse ; genetics ; metabolism ; pathology ; Male ; Middle Aged ; Prognosis ; Proto-Oncogene Proteins c-bcl-6 ; metabolism ; Skin Neoplasms ; genetics ; pathology

To investigate the chemical constituents of ethyl acetate from Cirsium setosum, fifteen flavonoids were obtained by column chromatography on silica gel, MCI, Sephadex LH-20, and preparative HPLC. Their structures were identified as 4',5,6-trihydroxy-7-methoxyflavone(1), 4',5-dihydroxy-7,8-dimethoxyflavone(2), sorbifolin-6-O-β-glucopyranoside(3), kaempferol-7-O-α-L-rhamnoside(4), kaempferol(5), quercetin-3-O-β-D-glucosyl-7-O-α-L-rhamnoside(6), myricetin(7), myricetin-3-O-β-D-glucoside(8), 5,7- dihydroxy -3',4'- dimethoxyflavone(9), 3',4',5- trihydroxy-3,7-dimethoxyflavone(10), 3',3,4',5-tetrahydroxy-7-methoxyflavone(11), 3'-hydroxy-4',5,7-trimethoxyflavone(12), 7-hydroxy-3',4',5-trimethoxyflavone(13), 4',5-dihydroxy-2',3',7,8-tetramethoxylflavone(14), and 5-hydroxy-2',3',7,8-tetramethoxylflavone(15) by spectroscopic data analysis. All compounds were isolated from this plant for the first time.Compounds(1-15) were evaluated for their hypoglycemic activities by PTP1B enzyme model. Among them, compounds 2, 12, and 14 showed significant PTP1B inhibitory activities with IC₅₀ values of 2.54, 1.85, 2.11 μmol•L⁻¹, respectively.