OBJECTIVETo explore the quality of DNA with three methods of DNA extraction and the influence on RAPD-PCR.

METHODthe electrophoresis of total DNA, UV spectrophotometry and RAPD analysis were carried out on DNA extracted from three different methods.

RESULTThe DNA concentration and yields were different, which greatly influenced the result of RAPD-PCR.

CONCLUSIONThe higher quality DNA from Dendrobium can be obtained with the method of CTAB-free extraction medium before total DNA was isolated.

Cetrimonium Compounds ; DNA, Plant ; isolation & purification ; Dendrobium ; classification ; genetics ; Electrophoresis ; Plants, Medicinal ; classification ; genetics ; Quality Control ; Random Amplified Polymorphic DNA Technique ; Spectrophotometry, Ultraviolet

BACKGROUNDVitrification is a prospective technology in ovarian tissue cryopreservation, but it is still in an initial stage. This study was conducted to investigate a modified vitrification protocol for human ovarian tissue, which can be used as an alternative to preserve fertility for young women with cancer who have to undergo cytotoxic therapy and sterilization.

METHODSOvarian tissue samples were collected from 15 patients and randomly allocated to groups of fresh, vitrification, and conventional slow freezing. A modified carrierless vitrification method was applied. The proportion of morphologically intact follicles in fresh ovarian tissues was compared with that in warmed/thawed tissues. The initial growth of the follicles and the concentrations of estradiol and progesterone were detected to determine the viability and endocrine function of the cryopreserved tissues.

RESULTSThe proportion of morphologically intact primordial follicles in the fresh group (97.6%) was significantly higher than that in the other two groups (vitrification group 80.3% and slow-freezing group 72.6%, P < 0.001). In both the vitrification and slow-freezing groups, estradiol and progesterone were secreted continuously during 2-week culture in vitro, the proportion of primary follicles were both significantly increased compared to the fresh group. No statistically significant differences existed between the two groups after cryopreservation in the proportion of both primordial and primary follicles, and the concentrations of estradiol and progesterone (P > 0.05).

CONCLUSIONThe modified vitrification method for cryopreservation of human ovarian tissues is effective, simple, and inexpensive.

Adult ; Cryopreservation ; methods ; Estradiol ; biosynthesis ; Female ; Humans ; Ovary ; cytology ; metabolism ; Progesterone ; biosynthesis ; Tissue Culture Techniques

AIMTo determine the effects of azide methyl anthraquinone derivative (AMAD) on growth inhibition and inducing apoptosis of multidrug resistant (MDR) KBv200 cells and parental drug-sensitive KB cells.

METHODSCytotoxicity was determined by tetrazolium (MTF) assay. Reactive oxygen species (ROS) levels and mitochondrial membrane potential (deltapsi(m)) in cells were labeled with DCFH-DA and DiOC6 and tested by flow cytometry. Annexin V stain and DNA ladder were used to examine the apoptosis of KB and KBv200 cells induced by AMAD.

RESULTSAMAD was shown to inhibit the growth of KB and KBv200 cells significantly in a concentration-dependent manner, with mean IC50 of 0.36 and 0.45 micromol x L(-1), respectively. The generation of ROS increased obviously after the cells were treated with AMAD for 12 h, up to the peak in 24 h, meanwhile the levels of deltapsi(m) were time-dependently decreased. DNA fragmentation appeared on the agarose gel. Annexin V stain showed AMAD induced apoptosis of KB and KBv200 cells also in a concentration-dependent manner.

CONCLUSIONAMAD showed inhibitory effect on both MDR KBv200 cells and parental drug-sensitive KB cells. The mechanism of action was associated with the increase of the cellular ROS level and the decrease of the mitochondrial membrane potential induced by AMAD, which result in cell apoptosis.

Anthraquinones ; administration & dosage ; chemistry ; pharmacology ; Antineoplastic Agents ; administration & dosage ; chemistry ; pharmacology ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Humans ; KB Cells ; Mitochondria ; physiology ; Molecular Structure ; Mouth Floor ; Mouth Neoplasms ; pathology ; Reactive Oxygen Species ; metabolism ; Vincristine ; pharmacology

OBJECTIVETo observe the expressions of caspase-8 and caspase-9 mRNA, and explore the changes of apoptosis of bone marrow hematopoietic cells in patients with chronic mountain sickness (CMS).

METHODSOf 18 CMS patients and 16 controls were enrolled in this study. The apoptotic index (AI) of bone marrow mononuclear cells (BMMNC) was measured by TUNEL technique, the levels of caspase-8 and caspase-9 mRNA in BMMNC of CMS patients and controls were determined by RT-PCR. Results (1)The AI of BMMNC in patients with CMS (8.51 ± 3.35)% was lower than that in controls (16.00 ± 4.28)% (P < 0.01); (2) The values of caspase-8 and caspase-9 mRNA were (0.28 ± 0.07) and (0.23 ± 0.08) respectively, in CMS patients, which were significantly lower than those of (0.45 ± 0.09) and (0.41 ± 0.09) respectively, in the controls (both P < 0.01); (3) Hemoglobin (Hb) value was negatively correlated with levels of caspase-8 and caspase-9 mRNA (r values were -0.52 and -0.61 respectively, both P < 0.05) in CMS patients. There was a negative correlation between AI and Hb (r value was -0.89, P < 0.01) in CMS patients. However, the significant relationship was not found between AI and level of caspase-8 or caspase-9 mRNA (P > 0.05).

CONCLUSIONSThe results showed a decrease apoptosis of BMMNCs and reduced levels of caspase-8 and caspase-9 mRNA in CMS patients, the latter might be involved in the change of BMMNCs apoptosis.

Adult ; Altitude Sickness ; metabolism ; pathology ; Apoptosis ; Bone Marrow Cells ; metabolism ; Case-Control Studies ; Caspase 8 ; metabolism ; Caspase 9 ; metabolism ; Humans ; Male ; Middle Aged

【Objective】To form a new PTMAc- PEG- PTMAc triblock（PPP）copolymerhydrogel and to evaluate its sustained released performance and antiproliferative effects as an ocular drug carrier of Pirfenidone（PFD）【Methods】One type triblockcopolymers PPP hydrogel was chosen. The morphology of the material was evaluated by scanning electron microscopy（SEM）. The swelling properties of the PPP hydrogel was analyzed in PBS solution（37 ℃ ，pH 7.4）. The in vitro drug release of the pirfenidone loaded hydrogels were evaluated with non-dialysis method and the curves of drug release were drawed. We evaluated the adhesion，survival of human Tenon′s capsule fibroblasts（HTF）around hydrogels. The cell cytotoxicity of hydrogels and antiproliferative effects were evaluated through CCK-8 assay.【Results】The hydrogel has stable gelation conditions. The swelling rate decreased by increasing hydrogel concentration.The SEM images indicated the fibrous and porous structure. We also observed that the encapsulated pirfenidone were sustained released from hydrogels with an initial burst release at early stage（within 4 d）and then the release rate were declined for all hydrogels during the following 14 d. The PPP hydrogel can inhibit cell adhesion. The cell viability in hydrogels at four time point（24，48，72 and 96 h）were 85.7% ，93.0% ，82.0% ，81.6%. The inhibition rate of drug loaded hydrogel with two drug concentration（1 mg/mL or 2 mg/mL）are 25.8%，21.8%，55.4%，25.6%；44.6%，35.9%，55.5%，31.4%. While that of drug solution are 28.9% ，29.7% ，7.8% ，7.7%. The suppressive effects of the PFD loaded hydrogels on HTF proliferation followed a dose-dependent fashion and time-dependent fashion.【Conclusions】Such biocompatible copolymers hydrogel can be effectively used as an drug sustain released system. It can induce significant inhibition of HTF proliferation. With equal amount of drug，the inhibition effect of drug loaded gel was longer than that of drug solution.

OBJECTIVETo develop a real-time fluorescent PCR protocol suitable for the routine screening of AZFc/DAZ microdeletions on the Y chromosome in azoospermic and oligozoospermic male infertility patients.

METHODSA set of real-time fluorescent PCR was established. Eighty-seven azoospermic and ligozoospermic patients undergoing ICSI in the IVF center and 30 azoospermic men undergoing testicular biopsy in the clinic of urology surgery were screened for AZFc/DAZ microdeletions of Y chromosome.

RESULTSEleven cases (9.4%) of AZFc/DAZ microdeletions were found in 117 cases of azoospermic and oligozoospermic patients by screening of realtime fluorescent PCR. Four cases (6.6%) were found in 61 oligozoospermic patients, and 7 cases (12.5%) were found in 56 azoospermic patients.

CONCLUSIONThe real-time fluorescent PCR protocol presented in this study is an easy and reliable method for detection of AZFc/DAZ microdeletions on the Y chromosome, which yields identical results to those of the multiplex PCR.

Chromosome Deletion ; Chromosomes, Human, Y ; Deleted in Azoospermia 1 Protein ; Fluorescence ; Humans ; Infertility, Male ; genetics ; Male ; Polymerase Chain Reaction ; methods ; RNA-Binding Proteins ; genetics

OBJECTIVETo evaluate the efficacy and clinical significance of 64-multislice spiral computed tomography angiography(MSCTA) with image fusion for the anatomy of perigastric arteries.

METHODSA total of 53 patients underwent abdominal 64-MSCTA, among whom 26 patients with gastric cancer underwent gastrectomy. Using volume rendering techniques, computed tomography angiography(CTA) of perigastric arteries and the stomach were reconstructed respectively, and then the images were fused together. The branching pattern of the celiac trunk and the origins and courses along the stomach of the 10 perigastric arteries were assessed. The accuracy, sensitivity, and specificity of 64-MSCTA were determined based on intraoperative findings.

RESULTSCTA clearly showed the celiac trunk. The most common branching pattern of the celiac trunk was Michels type I( in 46 patients(86.8%). The anatomy of perigastric arteries and stomach could be clearly demonstrated from any angle according to image fusion. The left gastric artery and the right gastroepiploic artery were shown in 100%, the left gastroepiploic artery 94.3%(50/53), the right gastric artery 83.0%(44/53), short gastric artery 58.5%(31/53), posterior gastric artery 49.1%(26/53), the replaced left hepatic artery 15.1%(8/53). The accessory left hepatic artery, accessory left gastric artery and replaced right hepatic artery were all identified in 7.5%(4/53) patients. The accuracy of preoperative CTA in term of correctly identifying perigastric arteries ranged from 84.6% to 100%, the sensitivity 82.6% to 100%, and the specificity was 100% for all the perigastric arteries.

CONCLUSIONS64-MSCTA can clearly reveal individual perigastric arteries. The anatomy of the stomach and perigastric arteries can be shown in vivo by fused image, and can provide guidance for gastrectomy.

Adult ; Aged ; Angiography ; methods ; Arteries ; Female ; Humans ; Image Processing, Computer-Assisted ; Male ; Middle Aged ; Preoperative Care ; Sensitivity and Specificity ; Stomach ; blood supply ; Stomach Neoplasms ; diagnostic imaging ; surgery ; Tomography, Spiral Computed ; Young Adult

OBJECTIVETo develop a multiplex PCR protocol, which could be suitable for routine screening of microdeletions on the Y chromosome in azoospermic and oligozoospermic male infertility patients.

METHODSFive multiplex sets were established. Eighty-seven azoospermic and oligozoospermic patients undergoing intracytoplasmic sperm injection (ICSI) in the in vitro fertilization (IVF) center and 30 azoospermic men undergoing testicular biopsy in the clinic of Urology Surgery were screened for microdeletions of Y chromosome.

RESULTSA total of 19 (16.2%) cases of microdeletions were found in 117 azoospermic and oligozoospermic patients by screening of Y chromosome microdeletions. Of these, 11 cases (18.0%) were found in 61 oligozoospermic patients, and 8 cases (14.3%) were found in 56 azoospermic patients.

CONCLUSIONThe multiplex PCR protocol presented in this study is an easy-to-do and reliable method for detecting microdeletions on the Y chromosome. Routine screening of microdeletions on the Y chromosome for azoospermic and oligozoospermic patients is essential.

Chromosome Deletion ; Chromosomes, Human, Y ; genetics ; Female ; Genetic Testing ; methods ; Humans ; Infertility, Male ; diagnosis ; genetics ; Male ; Polymerase Chain Reaction

OBJECTIVETo investigate the therapeutic efficacy and its influencing factors of ultrasound-guided percutaneous radiofrequency ablation (PRFA) in the treatment of liver carcinoma.

METHODSWith a temperature-controlled multi-electrode needle, ultrasound-guided PRFA was employed to treat forty-seven patients with 67 tumor nodules, with a diameter of 2.6 +/- 1.1 cm (1.0 - 5.5 cm).

RESULTSA complete ablation (CA) rate of 80.6% was achieved in the present series, with a CA rate of 91.7% in the tumors < or = 3 cm in diameter, 75.0% in tumors from 3.1 to 4.0 cm, and 14.3% in tumors > 4 cm. The CA rate was significantly greater in tumors with a temperature rising up to 70 degrees C within the initial 2 minutes at ablation as compared with that longer than 2 minutes (P < 0.05). A markedly higher CA rate was obtained in tumors with an ablation-maintaining temperature of over 80 degrees C than that between 70 degrees C and 80 degrees C (P < 0.01). All patients were followed up with a mean time of 11.3 months. The local recurrence rate was 9.3% (5/54), and 1-year survival rate was 82.1%. Eighteen patients (38.3%) had a distant recurrence.

CONCLUSIONSThe tumor size, temperature-rising time and ablation-maintaining temperature represented the important factors affecting the therapeutic efficacy of PRFA.

Adult ; Aged ; Carcinoma, Hepatocellular ; diagnostic imaging ; surgery ; Catheter Ablation ; methods ; Female ; Humans ; Liver Neoplasms ; diagnostic imaging ; surgery ; Male ; Middle Aged ; Temperature ; Ultrasonography, Interventional

OBJECTIVETo investigate the relationship between cell apoptosis and the quality of early mouse embryos, understand the significance of apoptosis-regulatory genes in early embryonic development, and explore a new approach to improving the embryo quality.

METHODSThe levels of cell apoptosis and proliferation in early mouse embryos in different developmental status (morphologically normal embryos, arrested embryos and fragmented embryos) were analyzed with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), caspase in situ fluorescence and Bcl-2 immunofluorescence, and immunofluorescent detection of proliferating cell nuclear antigen (PCNA).

RESULTSThe cells in arrested embryos and embryonic fragments showed positive results in TUNEL assay with enhanced caspase activity and lowered expressions of Bcl-2 and PCNA.

CONCLUSIONCell apoptosis in early mouse embryos may be closely related to embryonic arrest and fragmentation.

Animals ; Apoptosis ; Caspases ; metabolism ; Embryo, Mammalian ; cytology ; Female ; Mice ; Mice, Inbred Strains ; Pregnancy ; Proliferating Cell Nuclear Antigen ; metabolism ; Proto-Oncogene Proteins ; metabolism ; Proto-Oncogene Proteins c-bcl-2