Objective To clone ORF of DcR3 gene and insert it into eukaryotic expression vector and express it in COS-7 cells. Methods Encoding sequence of human DcR3 gene was cloned by PCR and sequenced. The sequenced ORF was cloned into eukaryotic expression vector pAAV-IRES-hrGFP to construct recombinant plasmid. COS-7 cells were transfected with recombinant plasmid by lipofectamine2000. Expression of recombinant DcR3 gene was verified by Western blotting and confocal microscopy. Results A 1 000-bp gene segment was obtained by PCR and inserted into pAAV-IRES-hrGFP to construct recombinant plasmid. The gene segment was proved to be encoding sequence of human DcR3 gene by sequencing. DcR3 expression in COS-7 cells was verified by Western blotting and confocal microscopy. Conclusion DcR3 gene was successfully cloned and expressed in COS-7 cells.

Objective To investigate the expression and clinical significance of MUC1,MUC2,MUC4 and MUC5AC in pancreatic ductal adenocarcinoma(PDA).Methods To analyze the expression profiles of MUC1,MUC2,MUC4 and MUC5AC in PDA(n=26),chronic pancreatitis(CP,n=4),normal pancreas(n=16)and intraductal papillary-mucinous neoplasm(IPMN)(n=2),solid-pseudo-papillary tumor of pancreas(SPT)(n=4),serous cystic neoplasm(SCN)(n=1)using immunohistochemistry.Results Positive staining with MUC1 Was exclusively found in normal pancreas and CP tissues(100%);the expression of MUC1,MUC4 and MUCSAC in PDA was 100%,88.5%(23/26)and 76.9%(20/26)in PDA tissue;MUC2 and MUC5AC were expressed in 2 samples of IPMN;none of the four mucins were expressed in Sfrr and SCN.There was no association between the expression of MUC4,MUC5AC and the clinicopathologic parameters in PDA(P＞0.05).Conclusions Multiple mucins were expressed in PDA.Measurement of the mucin profile including all 4 mueins(MUC1,MUC2,MUC4,and MUC5AC)may be helpful in the diagnosis and differential diagnosis of PDA.

The purpose of this study is to investigate the intracellular transporters effect and the cytotoxicity of carboxyl nanodiamond (CND) - podophyllotoxin (PPT). Nanodiamond (ND) was treated with mixed carboxylic acid and finally got 64 nm CND by centrifugation, and then it was reacted with PPT to form CND-PPT. UV spectrophotometry was used to calculate the content of PPT in CND-PPT, the particle size distribution and zeta potential were measured by Dynamic laser scattering instrument. CND, PPT, CND-PPT and CND + PPT (physical mixture of CND and PPT) were characterized by Fourier transform infrared spectroscopy, at the same time, thermal analysis and element analysis were used to estimate the content of the PPT in CND-PPT. The affect of CND, PPT, CND-PPT on HeLa cell was measured with MTT assay. The results showed that content of PPT combined with CND accounted for about 10%. MTT assay showed that CND has low cytotoxicity and CND-PPT can increase the water soluble of PPT. As a conclusion, CND as a hydrophilic pharmaceutical carrier combined with PPT is able to increase the water solubility of PPT, at low concentration, CND-PPT can enhance the antitumor activity in comparison with PPT, so CND can be used as a potential anticancer drug carrier.
Antineoplastic Agents, Phytogenic ; administration & dosage ; chemistry ; pharmacology ; Carboxylic Acids ; chemistry ; Drug Carriers ; HeLa Cells ; drug effects ; Humans ; Nanodiamonds ; chemistry ; Particle Size ; Podophyllotoxin ; administration & dosage ; chemistry ; pharmacology ; Solubility ; Spectrophotometry, Ultraviolet ; Spectroscopy, Fourier Transform Infrared

PURPOSETo investigate the clinical significance and management of ASCUS/LSIL.

METHODS254 patients who were examined with cervical cytology in the Cancer Institute and Hospital Chinese Academy of Medical Sciences were ASCUS/LSIL, of whom 136 cases underwent colposcopy, Data were analyzed retrospectively according to the golden criterion of pathology outcome.

RESULTS140 cases were ASCUS, and 114 cases were LSIL. Cervical intra-epithelial neoplasia (CIN) were diagnosed in 51.5% of patients with ASCUS, compared with 59.6% of patients with LSIL (P>0.05). High-grade cervical intraepithelial neoplasia were diagnosed in 22.9% of patients with ASCUS, compared with 30.7% of patients with LSIL (P >0.05). In the 136 patients examined with colposcopy, inflammation was found in 47 cases, low-grade intraepithelial lesion in 53 cases, High-grade intraepithelial lesion in 36 cases. The pathological results show inflammation in 55 cases, low-grade intraepithelial lesion in 41 cases, High-grade intraepithelial lesion in 40 cases (Kappa=0.314, U=0.064, P less than 0.05). CIN were diagnosed in 79% (67/84) of HPV-positive patients identified by pathology, compared with 43.5% (74/170) of HPV-negative patients (chi2=29.88 P less than 0.05). 83.5% of 254 patients were between 35 to 55 years old, and that was consistent with HPV-positive women age peak.

CONCLUSIONPatients with ASCUS should be paid the same attention with LSIL patients and colposcopy examination should be done immediately to avoid missed diagnosis and missed follow-up examination, especially for HPV positive patients between 35 to 55 years old.

Adult ; Cervical Intraepithelial Neoplasia ; diagnosis ; therapy ; virology ; Colposcopy ; Cytodiagnosis ; instrumentation ; methods ; Female ; Humans ; Middle Aged ; Neoplasms, Squamous Cell ; diagnosis ; therapy ; virology ; Papillomaviridae ; isolation & purification ; Papillomavirus Infections ; diagnosis ; therapy ; virology ; Retrospective Studies ; Uterine Cervical Neoplasms ; diagnosis ; therapy ; virology ; Young Adult

OBJECTIVEPrader-Willi syndrome (PWS) is an example of a human genetic disorder that involves imprinting genes on the proximal long arm of chromosome 15 and SNRPN gene as a candidate gene for this syndrome. The purpose of this study was to show the molecular genetic defects and genomic imprinting basis in Chinese PWS patients and to evaluate the clinical applications of a differential diagnostic test for PWS.

METHODSFluorescence in situ hybridization (FISH) and methylation-specific PCR (MSPCR) techniques were applied for 4 clinically suspected PWS patients. Using three probes, including SNRPN probe for identification of the critical locus in PWS region, D15Z1 and PML control probes for identification of the 15p arm and 15q arm, the authors detected the deletions 15q in PWS. MSPCR was based on sodium bisulfite treatment of DNA and PCR primers specific for the maternal and paternal allele.

RESULTSWhen hybridized with mixed probes, it was found in 2 patients that the central specific signal was absent, but both the flanking control signals were retained, indicating SNRPN gene deletion of chromosome 15q11-13. Bisulfite-modified DNA from all PWS children amplified with methylated allele-specific primer pair showed only maternal 131bp PCR product, indicating the maternal uniparental disomy (UPD15).

CONCLUSIONGenomic imprinting plays an important role in the molecular pathogenesis of PWS that caused by paternal microdeletions of 15q11-q13 or maternal UPD of chromosome 15. The basic defect seemed to be an absence of function of PWS genes that are normally expressed only from the paternal chromosome 15. MSPCR is a rapid and simple PCR-based assay compared with other cyto-molecular tests and its results were consistent with the clinical diagnosis of PWS, so it seems to be a reliable diagnostic method for PWS patients who show abnormal methylation at SNRPN. The genetic differential tests for PWS are important in determining familial recurrence risk.

Adolescent ; Autoantigens ; Chromosome Deletion ; Chromosomes, Human, Pair 15 ; genetics ; Gene Deletion ; Genomic Imprinting ; genetics ; Humans ; In Situ Hybridization, Fluorescence ; Male ; Polymerase Chain Reaction ; methods ; Prader-Willi Syndrome ; genetics ; Ribonucleoproteins, Small Nuclear ; genetics ; snRNP Core Proteins

OBJECTIVETo study the segregation of two novel RHD alleles in Chinese pedigrees.

METHODSThe Rh antigens of the samples were identified by using monoclonal antibodies. The 10 exons of the RHD gene for the 2 probands and their family members were amplified separately and sequenced. The parents of proband 2 were analyzed by sequence specific primer-polymerase chain reaction (SSP-PCR).

RESULTSThe two probands were RhD negative and the RHD was D/d type. After alignment with the nucleotide sequence in GenBank, a deletion of nucleotide C at position 78 in exon 1 of proband 1 was detected, and her sister also had the deletion, which was confirmed by sequencing. The sequencing results of proband 2 showed a 10 nucleotide deletion in exon 8 as well as a RHD 520 G to A mutation in exon 4. The results of SSP-PCR and sequencing showed that the proband's mother also carried RHD 520 G to A and RHD 1080 del 10 mutation, which was transmitted to proband 2. The sequences of the novel alleles have been submitted to GenBank (accession No. GQ477180 and GU362076).

CONCLUSIONThe two novel RHD alleles, RHD 78delC and RHD 520 G to A+1080 del 10, were both pseudo genes and stably transmitted.

Adult ; Alleles ; Base Sequence ; Exons ; Female ; Genotype ; Humans ; Male ; Pedigree ; Rh-Hr Blood-Group System ; genetics ; Young Adult

OBJECTIVETo establish molecular typing of Listeria monocytogenes isolates by pulsed-field gel electrophoresis (PFGE) for studying the epidemiologic characteristics of Listeria monocytogenes isolated from foodstuff in Guangdong province and to build up PFGE typing database of Listeria monocytogenes isolates for identifying the infectious resource of the outbreaks and other epidemiologic investigation.

METHODS"Standardized Protocol for Molecular Subtyping of Listeria monocytogenes by PFGE" was followed. BioNumerics software was applied on image analysis, database establishment, comparative and corresponding analysis.

RESULTS107 Listeria monocytogenes isolates were typed by PFGE, 41 PFGE types were observed among the isolates. The PFGE types were dispersive among these isolates. Listeria monocytogenes isolates were most frequently isolated in raw chicken while the most PFGE types were found in this type of food. The positive rate was relatively high in cold and iced foods. Only 1-2 DNA fragment difference occurred in 26 Listeria monocytogenes isolates by PFGE, so high degree of relatedness remained among these isolates. There were unique PFGE patterns in the regions of Shaoguan and Huizhou. From time to time, a number of isolates remained close relationship.

CONCLUSIONPFGE typing of the 107 Guangdong Listeria monocytogenes isolates demonstrated relative genetic diversity but a number of the isolates showed close relatedness.

Electrophoresis, Gel, Pulsed-Field ; methods ; Food Microbiology ; Genetic Variation ; genetics ; Genotype ; Listeria monocytogenes ; classification ; genetics ; isolation & purification ; Phylogeny

OBJECTIVESTo investigate the prevalence of oncogenic type of human papillomavirus (HPV) infection and identify the high risk population for conducting immuno/chemoprevention of cervical cancer.

METHODSAll married women aged 30 to 50 with no history of hysterectomy, pelvic radiation and non-pregnant from certain villages of Xiangyuan and Yangcheng County were invited. This study was conducted through two phases. In phase one, subjects sampled the vaginal secretions using the collectors after signing the informed consent. And physicians sampled exfoliated cells from cervix in the phase two. All the specimens were tested with the Hybrid Capture 2 test. The data was managed and analyzed by VFP and SPSS software.

RESULTSThere were 9,683 women participated in this study. Local women welcomed this study and population compliance rate was 75.4%. In tested population, we found 2,666 subjects of HPV DNA positive and HPV prevalence was 27.5%. The rates of different age group were 24.5% (30-34 yrs), 27.4% (35-39 yrs), 28.2% (40-44 yrs), 27.4% (45-50 yrs) respectively and had no significant differences (P = 0.604). The rates were slightly increased with the higher education level and had no differences (P = 0.106). The rate in mountain areas was higher than that in half-mountain areas (P = 0.001).

CONCLUSIONSThe prevalence of HPV infection is indeed high in this region. Local women and health professionals welcome the activities of cervical cancer screening and prevention. It is an emergent task to improve their sanitary condition and prevent them from cervical cancer in these women. A women health cohort is established successfully among high HPV exposed women in rural China. The extensive biologic specimen repository has been successfully established to simultaneously study the etiology, early detection, and immuno/chemoprevention of cervical cancer.

Adult ; China ; epidemiology ; DNA, Viral ; analysis ; Female ; Humans ; Mass Screening ; Middle Aged ; Papillomaviridae ; isolation & purification ; Papillomavirus Infections ; epidemiology ; prevention & control ; Prevalence ; Tumor Virus Infections ; epidemiology ; prevention & control ; Uterine Cervical Neoplasms ; epidemiology ; prevention & control ; virology ; Vaginal Smears

To investigate the effect of the total flavonoids in Scutellaria barbata(TF-SB) against autophagy in tumor cells in vivo, and further determine whether the mechanism is correlated with the PI3K/AKT/mTOR pathway, which lead to autophagy-induced tumor cell death. Melanoma-bearing mice were prepared and divided into control group, rapamycin group (Rap 1.5 mg•kg⁻¹), and high, middle and low-dose TF-SB (200, 100, 50 mg•kg⁻¹) groups. The groups were given drugs once a day for successively 11 days. The inhibitory effect of TF-SB on the growth of melanoma was determined by measuring tumor volume and tumor inhibition rate. TUNEL method was used to detect the apoptosis of tumor cells to further verify the antitumor activity of TF-SB. The protein expressions of LC3-Ⅰ and LC3-Ⅱ were detected by Western blot, and the relative expression of LC3-Ⅱ was calculated based on LC3-Ⅱ/LC3-Ⅰ. In addition, the effect of TF-SB on autophagy of tumor cells, the underlying molecular mechanism of TF-SB in inducing autophagy and PI3K/AKT/mTOR pathway marker protein phosphorylation were also studied. According to the results, TF-SB effectively inhibited melanoma growth in mice, reduced tumor volume, increased the tumor inhibition rate, and significantly increased tumor cell apoptosis index and the ratio of LC3-Ⅱ/LC3-I (P<0.05, P<0.01 or P<0.001). The protein expressions of p-PI3K, p-AKT and p-mTOR were also suppressed dramatically compared with those in control group (P<0.05, P<0.01 or P<0.001). In conclusion, the total flavonoids in S. barbata could inhibit the growth of melanoma in vivo by inducing autophagy and apoptosis of tumor cells, which may be correlated with suppression of PI3K/AKT/mTOR pathway.

Objective:To explore the protective effect and the mechanism of Danggui Shaoyaosan（DSS） on angiotensin Ⅱ （AngⅡ）/transient receptor potential cation channel 6 （TRPC6） pathway in nephrotic syndrome （NS） rats. Method:In animal experiments， doxorubicin （4 mg·kg^-1 for the 1^st week and 2 mg·kg^-1 for the 2^nd week） was injected twice to the tail vein of rats to induce NS model in 160 rats， which were then randomly divided into model group （normal saline）， losartan group （30 mg·kg^-1·d^-1）， and low-（4.3 g·kg^-1·d^-1）， medium-（8.6 g·kg^-1·d^-1）， and high-dose （17.2 g·kg^-1·d^-1） DSS groups. Besides， a normal group was also set. After intervention for four weeks， ultrastructure changes of the kidney were identified by transmission electron microscopy （TEM）. The 24-hour urine protein was detected by kits. Radioimmunoassay was used to detect the content of AngⅡ and Calcineurin （CaN） in plasma. Western blot was used to detect the protein expression of TRPC6， angiotensin Ⅱ type 1 receptor （AT1R）， podocyte slit diaphragm-specific protein （Nephrin）， and cysteine-aspartic acid protease-3 （Caspase-3） in the renal cortex. Immunohistochemistry was used to detect the expression of TRPC6 and AT1R in the slit diaphragm. In cell experiments， AngⅡ stimulated MPC5 podocytes. The cells were randomly divided into a normal group， an AngⅡ group， an AngⅡ+SAR7334 （TRPC6-specific inhibitor） group， an AngⅡ+5%DSS group， an AngⅡ+10%DSS group， and an AngⅡ+15%DSS group. Western blot was used to detect the protein expression of TRPC6， AT1R， Nephrin， and Caspase-3 in podocytes. Result:Compared with the normal group， the model group showed increased 24-hour urine protein content （P<0.01） and AngⅡ and CaN in plasma （P<0.01）， incomplete glomerular structure， the extensive fusion of podocyte process with elevated fusion rate （P<0.01）， increased expression distribution of AT1R and TRPC6 in the renal cortex， and up-regulated protein expression of AT1R， TRPC6， and Caspase-3 in renal tissues （P<0.01）， and reduced Nephrin protein expression （P<0.01）. Compared with model group， the losartan group and the high-dose DSS group exhibited decreased 24-hour urine protein content （P<0.01） and the content of AngⅡ and CaN in plasma （P<0.01）， improved glomerular structure， reduced fusion rate of podocyte process （P<0.01）， diminished expression distribution of TRPC6 and AT1R in the renal cortex， declining protein expression of AT1R， TRPC6 and Caspase-3 in renal tissues （P<0.01）， and elevated Nephrin protein expression （P<0.01）. Additionally， compared with the normal podocytes， AngⅡ-stimulated podocytes showed increased protein expression of AT1R， TRPC6 and Caspase-3 （P<0.01）， and decreased expression of Nephrin （P<0.01）. Compared with the AngⅡ group， the AngⅡ+SAR7334 group displayed reduced protein expression of AT1R， TRPC6， and Caspase-3 （P<0.01） and increased protein expression of Nephrin （P<0.01）. Conclusion:DSS can improve the pathological characteristics of NS presumedly by inhibiting the interaction between AngⅡ and TRPC6 in podocytes and improving the structural integrity of podocytes to repair the damage of glomerular molecular barrier and slow down the progression of NS-induced proteinuria.