Objective To investigate the role of mutated BRAFV599E gene in the growth of malignant melanoma cells. Methods In the previous study, plasmids containing small hairpin RNAs (shRNAs), braf1 and braf2 specific for mutated BRAFV599E gene, were designed and used to transfect A375 cells to inhibit the expression of BRAF gene in these cells. In this study, four kinds of A375 cells, including Abraf1 (transfect ed with braf1), Abraf2 (transfected with braf2), Aneg (transfected with negative plasmid) and A375 (untransfected) cells, were chosen and cultured in 96-well plate. MTT assay, plate clone forming assay, flow cytometry were applied to test the growth, clone formation, cell cycle and apoptosis of these cells respectively. Results Compared with A375 and Aneg cells, inhibited proliferation (F=25.48, P<0.001) and clone-forming rate (F=90.06, P<0.001) were observed in Abraf1 and Abraf2 cells; furthermore, flow cytometry showed a decrease in S-phase population(F=147.87, P<0.001) but an increase in G1-phase population (F=9.14, P<0.05)in Abraf1 and Abraf2 cells. However, neither Abrafl nor Abraf2 cells exhibited a significant increase in apoptosis ratio (F=2.27, P>0.05). Conclusions Mutated BRAFV599E gene could induce the switch from G1 phase to S phase in melanoma cells, subsequently accelerate the growth of melanoma cells, but it has no obvious influence on the apoptosis of these cells.

Objective Trying to predict the degree of GVHD after partly matched hematopoietic stem cell transplantation. Methods Analysis of the relationship between three-dimensional structure differences of donor-patient unmatched HLA and the GVHD levels after hematopoietic stem cell transplantation. Results GVHD levels were related to donor-patient unmatched HLA structure differences. The HLA structure differences forⅠ - Ⅱ degree GVHD were much smaller than that for Ⅲ - Ⅳ degree GVHD. Conclusion Prediction of GVHD by HLA structure differences is simple, rapid, specific and could help select proper conditioning regimens before transplantation and the proper immune suppressive agents after transplantation.

Objective To assess the environmental contact allergens in patients with allergic contact dermatitis (ACD).Methods Totally,167 patients with ACD were included in this study.All the patients underwent patch test.Results Of these patients,92 (55.1％) were diagnosed as facial ACD,and 148 showed positive patch test results (88.6％).The six most common allergens in a decreasing order were nickel sulfate,fragrance mix,paraphenylenediamine,thimerosal,octanoates and amerchol L 101.Conclusion Patch test may be an efficient way to confirm the cause of ACD.

Objective To construct the short hairpin RNA (shRNA)-expressing plasmid vectors specific for BRAF gene, and to test their effects in BRAF knockdown in human melanoma cell lines. Methods Two pairs of specific BRAF shRNA oligoes and a pair of randomly synthesized non-specific shRNA oligo were synthesized and inserted into plasmid pGenesil-1. Their fidelity was confirmed by double endonuclease digestion and sequencing. The constructed plasmids were transfected into human melanoma cell lines A375 and M14. The expression of BRAF mRNA and BRAF protein were detected by RT-PCR and Western blotting, respectively. Results The designed shRNA oligoes were precisely cloned into the plasmid pGenesil-1. The expression of BRAF mRNA and protein were down-regulated by specific plasmid braf 1 and braf 2, except to non-specific plasmid neg. The plasmid braf 1 was more effective, reducing BRAF gene expression by 90 per cent. Conclusions Plasmid mediated shRNA could successfully knockdown BRAF expression in human melanoma cells, and the suppression of the gene expression could maintain for 1 month at least.

Objective To observe the relationship between expression changes of γH2AX and the radiosensitivity of lung cancer cells in vitro. Methods The radiosensitivity of lung cancer cell lines A549 and SBC-3 was measured by clone forming assay. The DSBs damage of lung cancer cell lines A549 and SBC-3 was determined by Western blot assay. Re-sults The clone forming rates of lung cancer cell lines A549 and SBC-3 were gradually decreased with the increased radia-tion dose.γH2AX expression was related to the cell radiosensitivity 1 hour and 6 hours after radiated. Conclusion The phosphorylated histoneγH2AX is a powerful tool to monitor DNA DSBs and to predict the radiosensitivity in lung cancer ra-diotherapy.

Objective To investigate the effect of BRAFV600E mutation on the invasion capacity of a human melanoma cell line, A375. Methods Plasmids containing short hairpin RNAs (shRNA) specific for BRAF gene were prepared in previous study, and used to transfect A375 cells. Those cells transfected with negative plasmid and untransfected cells served as the controls. Transwell chambers were used to examine the invasion ability of melanoma cells in vitro. RT-PCR and Western blotting were performed to detect the mRNA and protein expressions of matrix metalloproteinase 2 (MMP-2) and vascular endothelial growth factor (VEGF), respectively, before and after the transfection. The activity of MMP-2 was also studied with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results Compared with the negative control, the specific shRNA decreased the mRNA and protein expressions of MMP-2 by 35% and 85%, respectively, and those of VEGF by 45% and 14%, respectively. Additionally, the number of cells invading through Matrigel chambers reduced by 69% in those cells transfected with the positive plasmid. Conclusions The mutant BRAFV600E has the potential to enhance the invasion capacity of melanoma cells, whereas specific shRNA could suppress the increase in metastasis capacity likely by inhibiting the production of VEGF and MMP.

Objective To explore the effect of Chaihu Shugan Powder (CSP) on the content of 5-hydroxytryptamine (5-HT) in the hippocampus and the expression of tryptophan hydroxylase 2(TPH2) in median raphe nuclei in depression rats.Methods SD rats were randomly divided into normal group(n=12),model group (n=12),fluoxetine group (n=12),low-dose and high-dose CSP group (n =12,respectively).Depression model was made by reserpine intraperitoneal injection.During the experiment,the weight and the open-field scores were calculated;the content of 5-HT was detected by ELISA.The expression of TPH2 in median raphe nuclei was detected by Western blot.Results Compared with the weight ((225.02±5.23) g),the open-field scores ((12.6± 5.1)score) and content of 5-HT ((1.09±0.27) ng/ml) in the model group,high-dose CSP showed significantly improve the depressive rats in weight,open field score and content of 5-HT ((238.78±5.16) g,(15.6±7.8) score and (1.80±0.58) ng/ml,respectively;P＜0.05 or P＜0.01).The expression of TPH2 (0.66±0.21) in median raphe nuclei in the high-dose CSP group was apparently increased compared with that in the model group(0.16±0.04) (P ＜0.01).Conclusion CSP have the effects of anti-depression,which could be related with the increase of the 5-HT content in the hippocampus and the expression of TPH2 in median raphe nuclei.

OBJECTIVETo explore the inhibitory effect of targeting miRNA on the expression of vascular endothelial growth factor (VEGF) and cell proliferation in malignant melanoma (MM) SKmel-28 cells.

METHODSRecombination miRNA plasmid vectors targeting VEGF gene were transfected into SKmel-28 cells via Lipofectamine 2000. The integrity of the inserted fragments was detected using colony PCR and sequence analysis. The expression of VEGF mRNA and protein in SKmel-28 cells was detected by RT-PCR and Western blotting, respectively. MTS assay was used to determine the inhibitory effect of a selected targeting miRNA on SKmel-28 cell proliferation, and the apoptosis of SKmel-28 cells was detected using flow cytometry.

RESULTSTransfection with the targeting miRNAs significantly down-regulated the expressions of VEGF mRNA and protein in SKmel-28 cells (P<0.01), and the miRNA construct X-26-2n-1 showed the highest inhibitory effect. The miRNA X-26-2n-1 significantly suppressed SKmel-28 cell proliferation in a time-dependent manner (P<0.01) and increased the early, late and overall apoptosis rates of the cells (P<0.01).

CONCLUSIONThe targeting miRNA we constructed can effectively suppress the cell proliferation and induce apoptosis of SKmel-28 cells by down-regulating the expressions of VEGF gene.

Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; Humans ; Melanoma ; genetics ; metabolism ; pathology ; MicroRNAs ; genetics ; Skin Neoplasms ; Transfection ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

BACKGROUND:Currently, neural stem celltransplantation can be performed through three main approaches:local lesions, blood circulation, and cerebrospinal fluid.
OBJECTIVE:To review the transplantation of neural stem cells or neural precursor cells via the cerebrospinal fluid in the treatment of central nervous system diseases.
METHODS:A computer-based search of PubMed and CHKD databases was performed to retrieve articles concerning transplantation of neural stem cells via the cerebrospinal fluid, and its application and therapeutic mechanism in the treatment of central nervous system diseases in both animal experiment and clinic study published from 2000 to 2009.
RESULTS AND CONCLUSION:It is suitable for neural stem cellsurvival, proliferation, and differentiation in the cerebrospinal fluid. Transplantation of neural stem cells via the cerebrospinal fluid is effective and feasible to treat central nervous system diseases. However, some problems have not been solved, such as the source of neural stem cells, the optimal time window and celldose, the safety and the long-term effect. Further studies are needed to pave the way for the intrathecal injection of neural stem cells in the treatment of central nervous system diseases.