Objective To study the expression of ABC gene family of MCF 7/ADM cells as compared to parental cell line MCF 7/S, and its reversion by realgar of the expressed genes. Methods Expression of ABC family genes was examined by RT PCR, and cell growth by MTT test. Results The expression of ABC gene family was positive in MCF 7/ADM cells compared to MCF 7/S cells; Realgar can down regulate the expression ABC gene family in MCF 7/ADM cells; the IC 50 of ADM to MCF 7/ADM cells was decreased.Conclusion ABC gene family was responsible for the induced resistance of MCF 7/ADM cells to ADM; Realgar is able to reverse partly the multidrug resistance of the human breast cancer cell line MCF 7/ADM. [

Objective To establish a novel multiplex amplification system which comprises 24 Y-STR loci.Methods Total 24 Y-STR gene loci, concluding DYS531,DYS630,DYS622,DYS552,DYS510, DYS449, DYS459a/b, DYS446, DYS443, DYS635, DYS587, DYS527a/b, DYS460, Y-GATA-A10, DYS520, DYS557,DYS522,DYS481,DYS570,DYS385a/b,DYS444, were chosen for establishing the fluorescence multiplex amplification system. The specificity, identity, sensitivity, balance of the amplification, anti-in-terference and accuracy of the system were detected and the gene diversity was investigated in the popu-lation of Guangdong.Results No band was found in nonhuman and female samples that were tested by the established multiplex amplification system. The same genotyping results were obtained from different tissues of the same person. Complete profiles could be obtained from more than 0.1 ng of the standard sample 9948. The loss of alleles was found when the common inhibitors such as hemoglobin and calci-um ion were added 120-200μmol/L and 1.5-2.0 mmol/L respectively to the system which with a strong anti-interference to the indigo, humic acid and EDTA. The typing of 24 Y-STR system could give the reliable results when 146 unrelated male individuals were detected and compared with the Yfiler system parallelly. The haplotype diversity(HD)of the population in Guangdong reached 0.99972 that was better than the result retained from Yfiler system, which the HD was 0.99858.Conclusion The fluorescence amplification system with 24 Y-STR loci established in present study has a wildly application prospect and can be used for cases inspection, paternity tests and Y-STR database construction.

OBJECTIVETo construct a RNA interference lentiviral vector for human glutathione peroxidase 2 (GPX2) gene and observe the effect of GPX2 knockdown on cell apoptosis.

METHODSThe sequence of the small interfering RNA (siRNA) for GPX2 interference was inserted into the pSicoR vector. HepG2 cells were infected by the packaged si-GPX2 lentivirus and the expression of GPX2 in the infected cells was detected by both RT-PCR and Western blotting. Changes of cell apoptosis following the infection were analyzed by flow cytometry.

RESULTSThe lentiviral particles pSicoR-GPX2 were successfully packaged. The expression of GPX2 in the infected cells was obviously down-regulated at both RNA and protein levels. GPX2 knockdown caused increased apoptosis rate, increased Bax expression and lowered Bcl-2 expression in HepG2 cells.

CONCLUSIONWe have successfully constructed the lentiviral vector for RNA interference of human GPX2 gene.

Apoptosis ; Down-Regulation ; Genetic Vectors ; Glutathione Peroxidase ; genetics ; Hep G2 Cells ; Humans ; Lentivirus ; RNA Interference ; RNA, Small Interfering

OBJECTIVETo construct a lentiviral vector for RNA interference (RNAi) of human glycerol kinase (GK) gene to stably down-regulate GK expression in human hepatocytes.

METHODSThe sequence of siRNA for GK interference were cloned into the pSicoR vector. Following packaging in 293T cells, the lentivirus was titrated using fluorescence activated cell sorting. Human hepatocyte L02 cells was infected with the lentivirus and the expression of GK was analyzed using Western blotting.

RESULTSThe lentiviral particle pSicoR-GK was successfully packaged with a virus titer reaching 3×10(7) pfu/ml. The expression level of GK protein was down-regulated to 20% of the control level in L02 cells infected with the lentivirus.

CONCLUSIONThe lentiviral vector for RNAi of human GK gene has been successfully constructed, which can significantly down-regulate GK expression in human hepatocytes.

Cell Line ; Gene Expression Regulation ; Genetic Vectors ; Glycerol Kinase ; genetics ; Hepatocytes ; enzymology ; Humans ; Lentivirus ; genetics ; Plasmids ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection

Objective To construct a RNA interference lentiviral vector for human glutathione peroxidase 2 (GPX2) gene and observe the effect of GPX2 knockdown on cell apoptosis. Methods The sequence of the small interfering RNA (siRNA) for GPX2 interference was inserted into the pSicoR vector. HepG2 cells were infected by the packaged si-GPX2 lentivirus and the expression of GPX2 in the infected cells was detected by both RT-PCR and Western blotting. Changes of cell apoptosis following the infection were analyzed by flow cytometry. Results The lentiviral particles pSicoR-GPX2 were successfully packaged. The expression of GPX2 in the infected cells was obviously down-regulated at both RNA and protein levels. GPX2 knockdown caused increased apoptosis rate, increased Bax expression and lowered Bcl-2 expression in HepG2 cells. Conclusion We have successfully constructed the lentiviral vector for RNA interference of human GPX2 gene.

Objective To construct a RNA interference lentiviral vector for human glutathione peroxidase 2 (GPX2) gene and observe the effect of GPX2 knockdown on cell apoptosis. Methods The sequence of the small interfering RNA (siRNA) for GPX2 interference was inserted into the pSicoR vector. HepG2 cells were infected by the packaged si-GPX2 lentivirus and the expression of GPX2 in the infected cells was detected by both RT-PCR and Western blotting. Changes of cell apoptosis following the infection were analyzed by flow cytometry. Results The lentiviral particles pSicoR-GPX2 were successfully packaged. The expression of GPX2 in the infected cells was obviously down-regulated at both RNA and protein levels. GPX2 knockdown caused increased apoptosis rate, increased Bax expression and lowered Bcl-2 expression in HepG2 cells. Conclusion We have successfully constructed the lentiviral vector for RNA interference of human GPX2 gene.

Objective To investigate the mechanism underlying gasdermin E(GSDME)-mediated pyroptosis in radiation-induced intestinal injury and to find out whether gasdermin(GSDM)family members regulate pyroptosis through similar signaling pathways.Methods Human normal colon epithelial cells(NCM460)and human colon cancer cells(HT-29)were exposed to radiation of different doses and durations before pyroptosis indicators were evaluated by observing pyroptotic bubbles,cell survival,and the cleavage of pyroptosis execution proteins.HT-29 cells overexpressing GSDME were subjected to radiation,followed by enrichment analysis of pyroptosis-related differentially expressed genes using RNA-seq.Results Radiation induced substantial pyroptosis in NCM460 cells.Overexpression of GSDME in HT-29 cells resulted in substantial radiation-induced pyroptosis.The pyroptosis state of human intestinal cells was simulated in the HT-29 model cell line.Overexpressions of GSDME-N and GSDMD-N resulted in the expression of more than 50％ of the differentially expressed genes in the pyroptosis state.Sequencing analysis showed that the genes in the pyroptosis state were mainly overrepresented in immune response,inflammatory response,and Rapl signaling pathway.Conclusion GSDME activation can mediate radiation-induced pyroptosis by producing GSDME-N fragments.GSDM family members participate in pyroptosis in a similar mode of regulation.Furthermore,radiation-induced activation of GSDME/D may regulate pyroptosis through immune response,inflammatory response,and Rap1 signaling pathway.

Objective To investigate the protective effect and mechanism of glutathione peroxidase 3-(GPx3)modified mesenchymal stem cells(MSC)against radiation-induced lung injury(RILI).Methods GPx3-modified MSCs were injected into the tail vein of mice whose lungs were irradiated with 20 Gy.Lung tissues were collected and sections were stained to observe pathological changes.The expression levels of inflammation-related factors were detected by real time quantitative PCR(qPCR),while the levels of malondialdehyde(MDA),H2O2,and 8-hydroxyguanine(8-OHG)were detected via biochemical experiments.Additionally,RNA damage was assessed by reverse transcription blocking combining with double primer PCR.Results GPx3-modified MSCs significantly improved the pathological damage in post-radiation lung tissues and inhibited the fibrosis process and inflammatory response.GPx3-modified MSCs were able to scavenge reactive oxygen species(ROS)more effectively,resulting in a reduction of lipid peroxidation products such as MDA and oxidative damage to RNA formation of 8-OHG.Conclusion By decreasing ROS accumulation,GPx3-modified MSCs can potentially reduce oxidative damage and attenuate RILI.GPx3-modified MSCs can improve the therapeutic efficacy against RILI.

Objective : The CRISPR / dCas9-SAM system was used to explore genes related to the proliferation of gastric cancer cells AGS，and their role in the occurrence and development of gastric cancer was analyzed． Methods : sgRNA was designed for genes with differential expression between gastric cancer and normal gastric tissue， and a lentiviral library was obtained after packaging was constructed．The AGS cells at different time points after the library was infected with AGS cells were used as the screening pressure，and the AGS cells at three time points on days 0，7 and 14 were collected．High-throughput sequencing analyzed sgRNA enrichment in AGS cells at dif- ferent time points after infection to obtain differential genes related to AGS cell proliferation. Results : Bioinformat- ics showed that compared with the 0 d group，42 and 45 negative screening differential genes and 59 and 40 posi- tive screening differential genes were obtained in the 7 d group and 14 d group，respectively．Among them，the 7 d group and the 14 d group had 11 genes in the negative screening and the positive screening． Conclusion In this study，11 genes inhibiting the proliferation of AGS cells were screened，of which 5 were protein-coding genes and 6 were long non-coding RNA ( lncRNA ) genes． 11 candidate genes that promoted AGS cell proliferation were screened，of which 3 were protein-coding genes and 8 were lncRNA genes．It laid a foundation for further function- al verification and comprehensive analysis of the occurrence and development process of gastric cancer.