Objective To examine expression of PTEN gene in ovarian cancer cisplatin-sensitive cell line OV2008 cells and cisplatin-resistant cell line C13K cells,and evaluate the effect of wild-type PTEN gene on reversing cisplatin-resistance of C13K cells and underlying mechanisms.Methods The expression of PTEN mRNA and protein in OV2008 and C13K cells were detected by semi-quantitative RT-PCR and western blot.Recombinant eukaryotic expression plasmid containing human wild-type PTEN gene was transfected into C13K cells by lipofectamine 2000.The expression of PTEN mRNA was monitored by RT- PCR and the expression of PTEN,protein kinase B(AKT),phospho-AKT(p-AKT)protein were analyzed by western blot in PTEN transfected and untransfected C13K cells.Proliferation and chemosensitivity of cells to cisplatin were measured by methyl thiazolyl tetrazolium(MTT),and cell apoptosis was detected by flow cytometry after treatment with cisplatin.Results(1)The expression of PTEN mRNA and protein(1.02 ?0.05,1.02?0.07)in OV2008 cells were significantly higher than those in C13K cells,which were 0.45 ?0.03 and 0.55?0.03 respectively(P

Background and Objective: Mutations in DNA repair system are related to carcinogenesis.This study was to evaluate the correlations of polymorphisms and haplotypes of XPD gene with individual susceptibility to gastric cancer. Methods: Genomic DNA were extracted from peripheral blood leukocytes of 207 gastric cancer patients and 212 healthy controls. Genotypes at codon 312 and codon 751 polymorphic sites were identified by amplification refractory mutation system-polymerase chain reaction(ARMS-PCR) or polymerase chain reaction-restriction fragment length polymorphism (PCRPFLP), respectively. Results: At codon 312, the frequency of GA or AA genotype was higher in the gastric cancer patients than in the healthy controls (P＜0.01,OR=3.41, 95% CI: 2.06-4.79; P＜0.01, OR=3.47,95% CI:1.39-8.68). No significant difference was found in the distribution of the polymorphism at codon 751 between the two groups(P＞0.05). By the haplotype AA (codon 312A-codon 751A) analysis, the frequency of heterozygote(-/AA) or homozygote (AA/AA) was higher in the patients than in the controls (P＜0.01 ,OR=2.81, 95% CI:1.82-4.34;P=0.02,OR=3.92, 95%CI:1.31-11.70,respectively). Whereas there were no significant differences of the other three haplotypes between the patients and the controls (P＞0.05).Conclusions: The polymorphism of XPD at codon 312 might contribute to the etiology of gastric cancer.The haplotype AA (codon 312A-codon 751A) would be a critical risk factor of the susceptibility to gastric cancer.

AlM:To discuss the protective effect ofα-mangostin on retinal light damage in mice.METHODS:Totally 30 Balb/c mice, aged 6~8wk, were randomly divided into the control group, light-exposure group and α-mangostin group. Every group contained 10 mice. Mice of α-mangostin group were treated with alpha-mangostin at the dose of 30mg/( kg · d ) body weight by intragastric administration daily for 7d, and then exposed to white light at the 5th d. The light-exposure group and α-mangostin group were exposed to 5 000 ± 200lx white light-emmiting diodes (LEDs) for continuously 1h to establish the mice model of retinal light damage. Flash -electroretinograme was recorded 72h after light exposure. The changes in retinal morphology of mice were observed by light microscopy. Retinas were extracted to detect the malondialdhyde ( MDA ) content change of the retinal homogenate.RESULTS: Flash-electroretinogram ( F-ERG ) showed that retinal dysfunction was less severe in α-mangostin group than in light-exposure group ( P<0. 05 ). Light microscopy test showed that retina structural damage was less severe in α-mangostin group than in light-exposure group (P<0. 05). The level of MDA in retinal tissue of α-mangostin group was significantly lower when compared with light-exposure group (P<0. 05).CONCLUSlON: α-mangostin inhibits lipid peroxidation induced by light damage and protect retina against light damage.

Objective: The current study was designed to distinguish Phase G0/G1， S， G2/M cell groups in the whole cell cycle by DNA flow cytometry. Method: The authors developed a multiparameter flow cytometry of cyclin E＋ A/DNA for MOLT-4. Result: The data showed that the method could distinguish Phase G0, early G1, late G1, S, G2,and M cells. The method could be used in molecular cell biology, especially in cell kinetic study.

OBJECTIVE: To develop a multiplex allele-specific PCR (AS-PCR) assay with three-color fluorescence labeling for mitochondrial DNA (mtDNA) SNP typing. METHODS: Based on the principle of AS-PCR, the primer sets were designed for 20 SNP located on the coding region of mtDNA and divided into 2 groups labeled with FAM and HEX fluorescence, respectively. A primer set included two forward (reverse) allelic specific primers with different sizes and a generic reverse (forward) primer. Blood samples from 200 unrelated individuals were analyzed by AS-PCR and capillary electrophoresis. Three random samples at least for each SNP site were examined and verified by direct sequencing. The haplotype frequency was investigated. RESULTS: Distinct electropherograms of 200 blood samples were obtained successfully. The typing results of direct sequencing were identical to those obtained from AS-PCR. The minimum detectable DNA concentration was 0.2 pg under the system of 10 microL. The sensitivity of the DNA concentrations ranged from 0.5 to 5 pg. The 200 individuals were assigned into 15 haplotype, and the haplotype diversity was 0.906 0. CONCLUSION AS-PCR is a simple, rapid and efficient method for mtDNA SNP typing, and can be applied to forensic practice.
Alleles ; DNA ; DNA Primers ; DNA, Mitochondrial/analysis* ; Electrophoresis, Capillary ; Haplotypes ; Humans ; Mitochondria ; Polymerase Chain Reaction/methods* ; Polymorphism, Single Nucleotide ; Sequence Analysis, DNA

We investigated brucellosis incidence trends in Kashgar,Xinjiang in 2005-2016 for further prevention and control,and analyzed the brucellosis epidemiological characteristics there,by using the descriptive epidemiology method.A total of 767 cases were reported,with an average annual incidence of 1.68/100 000,and incidence of annual report showed a relatively stable trend (Z:29.49,P＜0.001).The maximum number of reported cases was mainly concentrated from May to July.Incidence ratio of the male and female was 1.81:1.Cases were identified in each age group,the minimum age was five months,and the maximum age was 85 years old,with the median of 39.The highest proportion of reported cases was peasant.The top five average annual incidence counties (cities) were the Markit County,Yopurga County,Tashikuergan Tajik Autonomous County,Bachu County and Kashgar City.The brucellosis incidence increased year by year,especially during 2012-2016.We need further analysis for the data from increasing brucellosis outbreak and further strengthen the prevention and control of that in Kashgar area.

Objective:To investigate the influence of metastasis suppressor gene KAI1 on the proliferation,invasion and metastasis of endometrial carcinoma cell line AN3CA and HEC-1-B.Methods:The KAI1 cDNA was transfected into human endometrial carcinoma cells AN3CA and HEC-1-B via Lipofectamine 2000.The expression of KAI1 protein was ex- amined by Western blotting and flow cytometry before and after transfection.The proliferation ability of AN3CA and HEC- 1-B cells was observed by MTT assay and anchorage-independent growth assay.The changes of cell invasive ability were studied by transwell assays.Results:Stable expression of KAI1 protein was observed in AN3CA and HEC-1-B cells and on their surface after transfection with pcDNA3-KAI1 plasmid.Cells transfected with blank plasmid formed more colonies and had a larger size,with the colony forming rates being(54.2?3.1)% for AN3CA cells and(52.7?4.3)% for HEC- 1- B cells;the doubling time of AN3CA and HEC-1-B cells were 21.3 h and 20.1 h,respectively.Cells transfected with pcDNA3-KAI1 formed less colonies and had a smaller size,with the colony forming rates being(37.4?5.1)% for AN3CA cells and(32.1?3.7)% for HEC-1-B cells;the doubling time of AN3CA and HEC-1-B cells were 43.7h and 45.2 h,respectively.The cell proliferation abilities and colony-forming ability were significantly different between the two groups(P

Objective To investigate the permeability of brain microvascular endothelial cells under the condition of high glucose exposure. Methods The bEnd.3 cell line was chosen to detect the value of trans- endothelial electrical resistance (TEER), the activity of alkaline phosphatase (ALP) and γ-glutamyl transferase (γ-GT).Hence, the characteristics of blood-brain barrier in cell model were identified.The permeability of brain microvascular endothelial cells on high glucose exposure was evaluated by cell morphology, cell viability, intracellular lactate dehydrogenase activity and relative expression of ZO-1 and Occludin genes. Results The value of TEER, the activity of ALP and γ-GT increased gradually with increasing incubation time.The observation of cell morphology showed that the number of cells decreased significantly under high glucose exposure, and the adherence was unstable.Cell viability decreased with higher concentration of glucose or longer exposure time under high glucose exposure.The activity of lactate dehydrogenase was also decreased, and there were significant differences among the dose groups (P < 0.05).In addition, the expression levels of tight junction protein ZO-1 and Occludin were further detected.It was found that high glucose exposure inhibited the expression of ZO-1 and Occludin genes in a dose-dependent manner. Conclusion The bEnd.3 cell line has the characteristics of blood-brain barrier.High glucose exposure inhibited the expression of tight junction protein ZO-1 and Occludin. The results might be related to the change of the permeability in brain microvascular endothelial cells

OBJECTIVE: To express and purify the extra cellular full-length human apolipoprotein M(ApoM). METHODS: The ApoM gene fragment was amplified from the human liver cDNA library by PCR. The resulting product was cloned into pGEXT vector and sequenced. Then the confirmed canstatin cDNA was cloned into plasmid E.coli JM109 and then transformed into E.coli DL21(DE3) where it was induced to express protein by IPTG. RESULTS: The ApoM gene was cloned by PCR and a 560 bp DNA fragment was shown on the agarose electrophoresis. The cloned gene was sequenced and demonstrated to have the same sequence as that of human ApoM gene in GenBank. Then ApoM cDNA gene fragment was induced by IPTG, and a 24 kD recombinant ApoM protein was tested on SDS-PAGE. CONCLUSION Human ApoM gene is successfully cloned and its recombinant proteins are expressed.
Apolipoproteins ; biosynthesis ; genetics ; isolation & purification ; Apolipoproteins M ; Base Sequence ; Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; genetics ; metabolism ; Humans ; Lipocalins ; Molecular Sequence Data ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification

OBJECTIVETo investigate effect of AP-1 and Ets binding site adjacent to matrix metalloproteinase-9 (MMP-9) promoter on activation of MMP-9 transcription of nasopharyngeal carcinoma cells transfected with EBV-encoded latent membrane protein 1 (LMP1), and to ascertain if cross-talk between c-Jun and Ets1 is involved in LMP1-regulating expression of MMP-9.

METHODSSite-directed mutagenesis technique was used to establish a series of mutants, including MMP-9-CAT-Ets(-540)mt, MMP-9-CAT-AP-1(-533)mt and MMP-9-CAT-AP-1(-533)/Ets(-540)mt. After the mutants were transfected into LMP1-expressing NPC HNE2 cells regulated by Tet-on system (pTet-on-LMP1 HNE2), CAT activity of these mutants were assayed with induction of LMP1. With blockade of c-Jun or Ets1 antisense oligonucleotides, the activity of MMP-9 induced by LMP1 was assayed with gelatin zymography.

RESULTSThe CAT activity of MMP-9-Ets(-540)mt-CAT, MMP-9-AP-1(-533)mt-CAT, MMP-9-AP-1(-533)/Ets(-540) mt-CAT decreased significantly compared to MMP-9-CAT wt. After blockade with c-Jun or Ets1 antisense oligonucleotides, activity of MMP-9 induced by LMP1 decreased significantly, especially with combined blockade of c-Jun and Ets1.

CONCLUSIONThe results suggest that transcription factor AP-1 and Ets play an crucial role in activation of MMP-9 transcription induced by LMP1, and cross-talk between c-Jun/Ets1 is involved in expression of MMP-9 mediated by LMP1.

Herpesvirus 4, Human ; genetics ; Humans ; Matrix Metalloproteinase 9 ; biosynthesis ; genetics ; Nasopharyngeal Neoplasms ; metabolism ; virology ; Proto-Oncogene Protein c-ets-1 ; genetics ; Proto-Oncogene Proteins c-jun ; genetics ; Transfection ; Tumor Cells, Cultured ; Viral Matrix Proteins ; genetics