OBJECTIVE:To determine the concentration of dehydroisoandrosterone sulfate (DHEAS) in human plasma by LC-MS.METHODS:With estrogen sulfate (ES) served as an internal standard,the plasma samples were deproteinized with acetonitrile,extracted by solid phase,hydrolyzed and derivatized.Then the concentration of DHEAS was determined by HPLC-MSD on Agilent SB C18 with column temperature kept at 40℃.The mobile phase consisted of acetonitrile-water (in gradient elution).Atmosphere pressure chemical ion source in negative ion detection model was employed.The ions selected for SIM (selected ion monitoring) quantitative analysis included m/z 490.0 (DHEAS ) and m/z 472.1(ES)[M-H]-.RESULTS:The linear range of DHEAS was 250.0～320.0 ng?mL-1(r=0.999 4).The extraction recovery of the simulated human albumin samples ranged from 71.1%～78.9% and its relative recovery ranged from 98.3%～101.4%.Both the intra-day RSD and inter-day RSD were less than 10%.The mean concentration of DHEAS in 15 health aged male volunteers was (981.6?353.4) ng?mL-1.CONCLUSION:The method is simple,practical,accurate and sensitive,and it is applicable for the determination of plasma concentration of DHEAS.

Objective To observe the clinical effect of a silicone tractor in the treatment of nipple inversion. Methods 15 patients were treated with this new type of tractor. Of all the patients, 11 were with mild to moderate nipple inversion, and 4 were serious affected patients. In the seriously affected group, the tractor was used after operation. Patients in the mild to moderate nipple inversion group was treated with the silicone tractor only. Results The result in all the patients was satisfactory after follow-ing-up for 6 to 12 months. The appearance of the nipples was well. Conclusion Continuously pulling with silicone tractor is an ideal method in nipple inversion treatment. It is simple, safe and effective.

Objective To explore the relationship between brain-derived neurotrophic factor (BDNF)gene polymorphisms and the susceptibility to pediatric epilepsy.Methods BDNF polymorphisms in 128 patients with pediatric epilepsy and 132 healthy controls were analyzed with polymerase chain reaction restriction and fragment length polymorphism (PCR-RFLP).Results There were significant differences between pediatric epilepsy and controls on genotype frequency of BDNF-270C/T (X2 =7.08,P =0.03 ).The CC genotype was positively associated with pediatric epilepsy (OR =3.91,95％CI =1.26 ～ 12.14).No differences in genotype or allele frequencies of the other polymorphisms were found between patients and controls.The frequencies of haplotypes did not show significant differences between patients and controls.Conclusion These findings support the hypothesis that BDNF-270C/T polymorphism may contribute to the risk of developing pediatric epilepsy.

Some studies indicate that adipose derived stem cells (ADSCs) can differentiate into adipogenic, chondrogenic, myogenic, and osteogenic cells in vitro. However, whether ADSCs can be induced to differentiate into neural cells in vitro has not been clearly demonstrated. In this study, the ADSCs isolated from the murine adipose tissue were cultured and transfected with the EGFP gene, and then the cells were induced for neural differentiation. The morphology of those ADSCs began to change within two days which developed into characteristics of round cell bodies with several branching extensions, concomitantly expressing EGFP fluorescence. Approximately 60% of the total cell populations were bipolar or multipolar in shape. Some of them appeared to make contact with their neighboring cells. RT-PCR, Western blot and Immunocytochemistry revealed that the expression levels of the markers of neurons and oligodendrocytes such as MAP2, NF-70, Neu N and RIP upon neural induction were increased, but the expression of the special marker of astrocytes, GFAP, was undetectable until 96 h after induction when a small signal was observed. It was concluded that the ADSCs transfected with EGFP possessed the ability to undergo morphologic and phenotypic changes consistent with neural differentiation in vitro. It suggests that these cells might provide an ideal source for further stem cell research with possible therapeutic application for spinal cord injury.

Objective To investigate the effect of direct intercell contact on the bone mesenchymal stem cells(MSCs)differentiate into nucleus pulposus cells(NPs)when cocultured with NPs in constant magnetic field.Methods The primary NPs labeled by DAP1 were cocultured with the 3 rd generation of MSCs through direct and indirect intercell contact in the presence or absence of constant magnetic field(0.05,0.10,0.50 and 1.00 mT,respectively). Observation of morphological changes was performed every 24 hours.The method of MTT was employed to evaluate the level of proliferation.The gene expression of collagen Ⅱ,Sox-9 and Aggrecan was measured by using RT-PCR. Results MSCs cocultured with direct intercell contact with the NPs rounded up and presented a round ring-like structure appearance.The expression of marker genes including Collagen type Ⅱ,Aggrecan and Sox-9 were significantly increased when cells cocultured in constant magnetic field of 0.05 mT compared with those without constant magnetic field(P＜0.05).There were no significantly changes with regard to the expression of the above genes in 0.10 mT field(P＞0.05).The growth of NP-like cells was suppressed when the intensity of magnetic field was higher than 0.10 mT(P＜0.05).Conclusion It is suggested that 0.05 mT constant magnetic field and direct intercell contact facilitate differentiation of MSCs into NPs.

Objective To compare the effects of sodium hyaluronate (SH) and celecoxib (CO) administration on the treatment of knee osteoarthritis (OA) and to investigate their influences on levels of uPA and MMP-3 in synovial fluid. Methods One hundred and thirty-six knee osteoarthritis (OA) patients from January 2010 to October 2011 were randomly enrolled into two groups: the SH group and the CO group. In the SH group, patients were injected with 2 mL sodium hyaluronate intra articulation once a week for 5 weeks. In the CO group , patients were given oral administration of celecoxib daily at a dosage of 200 mg for 5 weeks. Before and at 1 ,6 months after treatment, Lequesne′s index and VAS-pain were detected to assess the clinical results of these two drugs. The levels of uPA and MMP-3 in synovial fluid were measured by using ELISA assay. Results All patients were followed up for 6 months to 12 months. The Lequesne′s index and VAS-pain score were lowered at 1 and 6 moths after treatment in both the SH group and the CO group(P<0.05). In the CO group, however, higher Lequesne′s index and VAS-pain score were obtained at 6 months compare to that obtained at 1 month after treatment (P<0.05). In contrast, no significant difference of Lequesne′s index and VAS-pain score were shown at 6 months and at 1 month after treatment in the SH group (P>0.05). Dramatic reduction of the levels of uPA and MMP-3 in synovial fluid were observed in the SH group after treatment(P<0.01), while marginal changes were found in the CO group(P>0.05). Conclusion The redueced levels of uPA and MMP-3 in synovial fluid after treatment of sodium hyaluronate may contribute to its longer-lasting effect than that of celecoxib. Therefore , the combination of sodium hyaluronate with celecoxib may lead to better therapeutic effect on OA patients.

Objective To investigate the characteristic sonographic and pathological features of breast ductal carcinoma in situ (DCIS) without microcalcifications on mammography (MG).Methods Forty cases of DCIS without microcalcifications on MG were retrospectively reviewed.The 40 lesions were classified into mass and non-mass groups according to their sonographic findings.The pathological subtypes and nuclear grades of these cases were also analyzed.Fisher exact test was used to compare the differences of the sonographic accuracy rate,sonographic microcalcification rate,pathological nuclear grade and subtype rate between mass and non-mass groups.Results No abnormal finding was found in sixteen cases (40.0％)on MG and only one case (2.5％) on ultrasonography (US),respectively.The most common sonographic feature of DCIS without microcalcifications on MG were masses (75.0％,30/40),and other sonographic findings were round/oval and irregular shape,microlobulated margin,heterogeneous hypoechogenicity and isoechogenicity,and posterior acoustic feature.Ductal dilatations and heterogeneous isoechogenicity were present in most non-mass lesions of DCIS without microcalcifications on MG (22.5％,9/40).The ultrasonographic microcalcifications were found in 5 cases of DCIS without microcalcifications on MG.The common pathological features of DCIS without microcalcifications on MG were medium-low nuclear grade (85.0％,34/40) and noncomedo (87.5％,35/40).The difference of US accuracy rate in mass and non-mass groups was statistically significant [73.3％ (22/30) vs 33.3％ (3/9),P=0.047].The differences of US microcalcification rate,pathological subtype and nuclear grade were not significant (P=1.000,0.070).Conclusions The mass appearance and medium-low nuclear grade were most common sonographic findings and pathological features of DCIS without microcalcifications on MG.Ultrasonography should be an helpful tool for improving the diagnostic sensitivity ofmammography in breast DCIS.

Sox9 gene was cloned from immortalized precartilaginous stem cells and its eukaryotic expression vector constructed in order to explore the possibility of bone marrow-derived stromal cells differentiation into precartilaginous stem cells induced by Sox9. A full-length fragment of Sox9 was obtained by RT-PCR, inserted into pGEM-T Easy clone vector, and ligated with pEGFP-IRES2 expression vector by double digestion after sequencing. The compound plasmid was transfected into born marrow-derived stromal cells by Lipofectamine 2000, and the transfection efficacy and the expression of Sox9 and FGFR-3 were observed. Flow cytometry was used to identify the cell phenotype, and MTT was employed to assay proliferative viability of cells. Sequencing, restrictive endonuclease identification and RT-PCR confirmed that the expansion of Sox9 and construction of Sox9 expression vector were successful. After transfection of the recombinant vector into bone marrow-derived stromal cells, the expression of Sox9 and FGFR-3 was detected, and proliferative viability was not different from that of precartilaginous stem cells. It was concluded that Sox9 gene eukaryotic expression vector was successfully constructed, and the transfected bone marrow-derived stromal cells differentiated into the precartilaginous stem cells.
Base Sequence ; Bone Marrow Cells/*cytology ; Cartilage/*cytology ; Cell Differentiation/genetics ; Cells, Cultured ; Cloning, Molecular ; Genetic Vectors/genetics ; Molecular Sequence Data ; Receptor, Fibroblast Growth Factor, Type 3/metabolism ; Recombinant Proteins/biosynthesis ; Recombinant Proteins/genetics ; SOX9 Transcription Factor/biosynthesis ; SOX9 Transcription Factor/*genetics ; Stem Cells/*cytology ; Stromal Cells/*cytology ; Transfection

Objective To study the roles of icariin and cyclic tensile strain (CTS) in promoting the osteogenic differentiation of adipose-derived stem cells (ASCs) and the molecular mechanisms involved.Methods ASCs were isolated from Sprague-Dawley rats and treated either with icariin (10-7 mol/L) or with 1000 μ,2000 μ or 3000 μ of CTS for 7 days,or with icariin plus CTS at 2000 μ for three days.Alkaline phosphatase (ALP) activity was detected after 3 and 7 days of intervention.Western blotting was performed to detect the expression of Runt-related transcriptional factor 2 (Runx2),Yes-associated protein (YAP) and connective tissue growth factor (CTGF) after the third day of the intervention.A reverse transcription polymerase chain reaction was performed at 7 days to detect the expression of osteopontin (OPN) and collagen la and after 3 days to detect the expression of the YAP target gene,CTGF and ankyrin repeating domain 1 (Ankrd1).Results Icariin and CTS at 1000 μ,2000 μ or 3000 μ could all significantly promote the expression of ALP protein.CTS at 2000 μ was the nost effective.The co-treatment with icariin and CTS significantly promoted ALP protein expression compared with icariin or CTS treatment alone.It also significantly promoted the expression of Runx2 and CTGF protein.Icariin or CTS (2000 μ) alone could not promote the expression of YAP protein,but icariin combined with CTS (2000 μ) promoted it significantly.Either icariin or CTS (2000 μ) could significantly promote ALP activity after 3 and 7 days,but icariin combined with CTS had the most obvious effect.Both icariin and CTS (2000 μ) could also significantly promote the expression of the osteogenesis-related genes OPN and collagen la,as well as the YAP targeted genes CTGF and Ankrdl.However,the combination of icariin and CTS had the greatest effect in promoting the expression of OPN mRNA,collagen la mRNA,CTGF mRNA and ankrdl mRNA.Conclusion Icariin and CTS co-treatment may promote osteogenic differentiation of ASCs via activating YAP expression.

Objective To establish a doxycycline-controlled immortalized pre-cartilaginons stem cells (IPCSCs) strains, clone parathroid hormone-related peptide[PTHrP(1-36)] gene and construct re- sponsive plasmid, pTRE-PTHrP (1-36). Methods Plasmid pTet-on was transfected into IPCSCs by using LipoinfectaminTM 2000 and then the stable clones were obtained by G418 screening. The doxycyc- line was added into the medium of monoclonal cells that were transiently transfected with plasmid pTRE- 2Hyg-Lue. The total RNA was extracted from PCSCs and the PTHrP(1-36) gene obtained by RT-PCR method. Then, the PTHrP (1-36) gene was subcloned to plasmids of Tet-responsive element with the se- lection marker of hygromycin pTRE-2Hyg to construct recombinant eukaryotic expression plasmid pTRE- PTHrP(1-36). After transferred into E. coli-DH5α, the clone was amplified, the recombinant plasm0ids were purified and identified by double-enzyme digestion. Results The doxycycline induced IPCSCs line was obtained, with 50 times higher than the non-induced cell line. Double enzyme digestion analysis and sequencing showed that the target gene was cloned into recombinant plasmid. Conclusions The induced IPCSCs line can be used to highly express alien genes. The responsive plasmid containing PTHrP (1-36) gene may be premising for rigorous control of PTHrp (1-36) gene expression.