Objective To establish a system for rapidly detecting single nucleotide polymorphisms (SNPs) in mitochondrial DNA (mtDNA) using hybridization probes and melting temperature (Tm) curve analysis. This technique is suitable for population-based studies on the interaction between genetic factors and environmental exposures and the risk of Parkinson's disease (PD). Methods mtDNA was extracted from the blood. Rapid polymerase chain reaction (PCR) and melting curve analyses were performed with primers and fluorochrome-labeled probes on a LightCycler. Genotyping of 10 SNPs was based on the analysis of allele-specific Tm of detection probes. The results of melting curve analyses were verified by sequencing all 150 PCR products. Results Real-time monitoring showed optimal PCR amplification of each mtDNA fragment. The changes at nucleotide positions 1719, 4580, 7028, 8251, 9055, 10398, 12308, 13368, 13708, 16391 from wild-type to mutant genotype resulted in 6.51, 8.29, 3.26, 7.82, 4.79, 2.84, 2.73, 9.04, 8.53, 9.52℃ decline in Tm of the detection probes respectively. Genotyping of all detected genes was verified by 100% correspondence with the results of sequencing. Conclusion A rapid and reliable detection system for identifying mitochondrial polymorphisms and haplotypes has ben developed based on hybridization probe technology. This method may be suitable for mitochondrial genotyping of samples from large-scale epidemiology studies and may prove useful for exploring the molecular etiopathogenesis of PD, identifying markers of genetic susceptibility, and protecting susceptible individuals from PD.

Objective To explore the effect of BPDE on the expression of N-Ras in the human bronchial epithelial cell line. Methods The levels of mRNA and protein expression in BPDE transformed 16HBE cells(16HBE-T) and untransformed control 16HBE cells(16HBE-N) were examined by using RT-PCR and Western blot. Locations and expression levels of protein in both kinds of cells were analyzed by immunocytochemical method. Results Compared with 16HBE-N, the levels of mRNA and protein of N-Ras significantly increased to 3.616 and 1.600 times in 16HBE-T. Immunocytochemical method showed N-Ras protein in 16HBE-T and 16HBE-N expressed in the cytomembrane and cytoplasm, but the expression level of protein in 16HBE-T was significantly higher than that in 16HBE-N. Conclusion The up-regulated expression of N-Ras oncogene may play an important role in the malignant transformation of 16HBE induced by BPDE

Objective　To findout a good method for malignant transformation of human bronchial epithelial cells 16HBE cell induced by anti-benzo (a) pyrene-trans-7,8-dihydrodiol-9,10-epoxide (anti-BPDE) using the objective and simple index:colony forming frequency of cells in semisolid agar culture medium,and to establish a best model of malignant transformation of 16HBE cells.Methods　The tests on viable rate and colony formation of 16HBE cells were carried out to determine the exposure doses of anti-BPDE.The l6HBE cells were treated once or several times by anti BPDE at different doses and the transformed foci were observed and assessed at the different stages during the period of the whole experiment.The malignant features of transformed 16HBE cells with a good dose-response relationship were identified by the method of semisolid agar culture.The anti-BPDE induced colony forming frequencies of 16HBE cells in semisolid agar medium in each dose group were statistically compared.Results　The best method for malignant transformation of 16HBE cells induced by anti-BPDE was that the 16HBE cells were intermittently treated several times with anti-BPDE at doses of 0.1,0.5,1.0 and 2.0 μmol/L respectively and were inoculated for 15 generations,the related colony forming frequencies were 2.0‰,5.5‰,7.0‰ and 10.5‰respectively with a significant dose-response relation ship and a good linear correlation (r=0.9741,P＜0.05).Conclusion　The research infered that the malignant transformation of human bronchial epithelial cell-16HBE cells could be induced successfully by anti-BPDE,a suitable model could be established by anti-BPDE also.

OBJECTIVE To explore the dyna mic change of lncRNA expression during lung carcino-genesis induced by urethane.METHODS A total of 40 BALB/c mice received weekly ip injection of urethane 1 g·kg -1 for four continuous weeks,mice were euthanized at 12th week or 24th week after the first urethane treat ment,respectively.The RNA of lung tissues were isolated and used for microarray analysis.Based on the results of the microarray we selected lncRNA-AK017233 for additional qPCR analysis in individual mouse.RESULTS The incidence of lung cancer were 85% and 100% at 12th week and 24th week after the first ad ministration of urethane,respectively.The multiplicity and dia meter of lung tu mors in 24 weeks treated group were statistically significant fro m those in 12 weeks treated group (P<0.01 ),and pathological analysis showed that tu mors were classifiable as moderately differ-entiated adenocarcino ma.Total of 26 Down-regulated lncRNAs in which lncRNA-AK017233 stand for the most down-regulated lncRNA were identified by microcarray analysis.qPCR detected that the lncRNA-AK017233 was significantly altered by 0.33 ti mes in lung tu mors of urethane treated group at 12th week, co mpared to parallel lung tissues in urethane treated group at 12th week.AK017233 expression of ure-thane treated group was significantly reduced by 0.22 ti mes at 24th week,co mpared to parallel lung tis-sues in urethane treated group at 24th week.CONCLUSION LncRNA-AK017233 was consistently down-regulated during urethane induced lung carcinogenesis.

Environmental chemicals exposure is closely related to occurrence and development of diseases. The abnormal expression of non-coding RNAs is a key factor in biological effects of environmental chemicals. Non-coding RNAs play an important role in the normal function of cells and the occurrence and development of diseases. In addition, non-coding RNAs, as a biomarker, could be widely used in the diagnosis, prevention and treatment of diseases. This paper outlines the relationship between environmental chemical exposure and non-coding RNAs and the function and mechanism of non-coding RNAs. It also presents existing issues and future research directions.

OBJECTIVETo clone differentially expressed cDNA sequences involved in malignant transformation induced by benzo(a)pyrene metabolite dihydroxyepoxy benzo pyrene (BPDE).

METHODThe malignant transformation of human bronchial epithelial cell line 16HBE induced by BPDE in vitro was used as a model for comparing gene expression between the transformed cells and controls. cDNA representational difference analysis (cDNA-RDA) was performed to isolate differentially expressed cDNA fragment in transformed cells. The cDNA fragments were ligated to pGEM-T vector and transformed into JM109 bacteria. The plasmid DNA were sequenced and compared with data in GenBank by BLASTN.

RESULTSFive cDNA sequences were found to be novel ones and were registered in dbest database, which assigned accession numbers in GenBank are BG354691, BG354692, BG354693, BG354694 and BG354695, respectively. Eight of the remaining cDNA sequences showed sequence homology to those previously reported such as ribosomal protein S23, MLN137, ACTN4, transforming growth factor and G protein gene.

CONCLUSIONSThese 13 genes may be involved in BPDE-induced malignant transformation, but their biological characteristics and functions are left to further studies.

Benzopyrenes ; metabolism ; pharmacology ; Carcinogens ; pharmacology ; Cell Transformation, Neoplastic ; chemically induced ; genetics ; Cells, Cultured ; Cloning, Molecular ; DNA, Complementary ; analysis ; drug effects ; DNA, Neoplasm ; analysis ; Gene Expression ; drug effects ; Humans

In order to meet the needs of social development for public health talents and optimize the curriculum system and teaching content of preventive medicine, the course of "Health Emergency" should be set up. It integrates the related content in many courses, and there are 20 hours for theory and 18 hours for experiment or practice. The course takes the emergency response as the cutting point focuses on the projects with high demand of the industry improves the platform and expands the content of emergency skill training through the extracurricular activities and special lectures. Combining theory with practice, this paper explores teaching reform measures that are conducive to improving students' core competence in public health.

OBJECTIVE To explore the effects of irbesartan（Irb）combined with 5-fluorouracil（5-FU）on the proliferation and extracellular signal-regulated kinase （ERK）/peroxidase proliferator-activated receptor γ（PPARγ）signaling pathway of Lewis lung cancer cells. METHODS Lewis lung cancer cells from mice were divided into normal control （NC）group，Irb low-dose （LD）group（1×10－3 mmol/L），Irb high-dose （HD）group（1×10－1 mmol/L），5-FU group （10 μmol/L），Irb LD+ 5-FU group （Irb 1×10－3 mmol/L+5-FU 10 μmol/L）and Irb HD+ 5-FU group （Irb 1×10－1 mmol/L+5-FU 10 μmol/L）. MTT method was used to measure the activity of cell proliferation in each group. Plate colony formation experiment was used to determine the number of cell colonies formed in each group ；Western blot method was used to detect the expression levels of proliferating cell nuclear antigen （PCNA），p53，ERK1/2，p-ERK1/2 and PPAR γ protein in each group. RESULTS Compared with the NC group ，the cell proliferation activity ，the number of colonies formed and the protein levels of PCNA ，p-ERK1/2，and PPARγ were significantly reduced in the other five groups ，and the protein level of p 53 was significantly increased （P＜0.05）；the protein expression of ERK1/2 had no significant difference （P＞0.05）. The changes of above indexes in Irb LD+ 5-FU group and Irb HD+ 5-FU group were more significant than Irb LD group ，Irb HD group and 5-FU group （P＜0.05）. CONCLUSIONS Irb combined with 5-FU can inhibit the proliferation of Lewis lung cancer cell ，and the effect is better than that of the two alone. The mechanism may be related to the inhibition of ERK/PPARγ signal pathway.

AIM: To investigate the mechanism of elemene synergistic bortezomib against multiple myeloma based on ROS-NF-κB-p38MAPK signaling pathway. METHODS: CCK-8 assay was used to detect cell activity. Nude mice were randomly divided into control group, bortezomib (BTZ) group, elemene (ELE) group and combination group. Each group was treated with BTZ, ELE and BTZ combined with ELE, respectively. Tunel staining was performed to observe the apoptosis of tumor tissues. The expressions of Caspase-3, Bcl-2, NF-κB and p38 MAPK were detected by Western Blot. Cell cycle, apoptosis and reactive oxygen species (ROS) expression were detected by flow cytometry using human myeloma U266 cells. RESULTS: When 4.0 μmol/L ELE combined with 50 nmol/L BTZ treated U266, the cell activity was significantly reduced compared with that of NC, BTZ and ELE groups (P< 0.05). The tumor volume of nude mice in BTZ group, ELE group and combined group was significantly reduced compared with the control group (P <0.05), and the combined group was the smallest. Tunel staining results showed that the apoptosis level in the control group was lower than that in the BTZ group, ELE group and the combined group (P<0.05), and the combined group had the lowest apoptosis level. Compared with the control group, the expressions of Caspase-3 and p38 MAPK in BTZ group, ELE group and combination group were significantly increased, while the expression of Bcl-2 was significantly decreased. The apoptosis level and expression of ROS in BTZ group, ELE group and the combined group was significantly increased compared with the control group (P<0.05). CONCLUSION: ELE can enhance the role of BTZ in promoting apoptosis of myeloma cells, which may be achieved by regulating ROS/NF-κB/p38 MAPK signaling pathway to enhance the level of apoptosis of tumor cells to achieve anti-tumor effect.

Intervertebral disc degeneration (IDD) is one of major causes for intervertebral disc degenerative diseases, and patients with IDD usually suffer from serious low back pain. The current treatments for patients with IDD only relieve the clinical symptom rather than restore biological balance of IDD, leading to inadequate and unsatisfactory results. MicroRNAs (miRNAs) are endogenous, non-coding, single-stranded RNA molecules, which regulate the gene expression at the post-transcription levels. Research evidences support the involvement of miRNAs in many biological processes, such as lipid metabolism, apoptosis, differentiation and organ development. Accumulating evidences indicate that the expressions of miRNAs change significantly in degenerative tissues. In addition, dysregulated miRNAs contribute to multiple pathological process of IDD, including proliferation and apoptosis of nucleus pulposus and extracellular matrix components, inflammatory response and cartilage endplates degeneration. In this review article, we summarize the expression profiles and roles of miRNAs in IDD, which may provide a novel strategy of biological therapy for the disease.
Apoptosis ; Extracellular Matrix ; pathology ; Gene Expression ; Gene Expression Profiling ; Humans ; Intervertebral Disc Degeneration ; genetics ; pathology ; MicroRNAs ; genetics