AIM: To investigate the use of therapy ultrasound to enhance nonviral gene delivery. METHODS: Endothelial cells (EC) and vascular smooth muscle cells (VSMC) were cultured in 6-well plates. Plasmid (pcDNA3.1/His/LacZ) with or without microbubbles at the different concentrations was transfected into the cells with the use of ultrasound for 1 min at 2 MHz, 1.8 mechanical index (MI). Additional controls included ultrasound alone, microbubble alone and microbubble plus plasmid. The rate of blue cells and the activities of ?-Gal were measured. In addition, cell viability was detected with different time from 1 to 30 min of ultrasound irradiation and the different concentrations of microbubbles. RESULTS: In the group of ultrasound with microbubble, the rate of blue cells and activity of ?-Gal markedly increased by 60% and 9-fold, respectively. Microbubbles at concentration of 10% led to the highest transfection effect. Ultrasoud exposure at 1 to 30 minute had no cell toxic effects, while microbubbles at the concentration of 50% had significant effect on cell survival. CONCLUSIONS: Albumin-coated microbubbles markedly enhance gene delivery by therapeutic ultrasound-mediated microbubble destruction, which can be used as a safe and practicality vectors in gene therapy.

A bacterium strain BFHM2002 is isolated from Lake Donghu, Wuhan. BFHM2002 has advantages that it can produce melanin with a high rate and high yield in the absence of tyrosine. Induced by tyrosine, melanin yield can be dramatically increased. BFHM2002 may be identified as a new strain in Bacillus firmus, for melanin-production.

OBJECTIVETo study the effect of ursolic acid (UA), apentacyclic triterpene acid, on MCF-7 cell apoptosis, and probable mechanism involved by detecting the expressions of caspase-3 and poly ADP-ribose polymerase(PARP) at protein level.

METHODMCF-7 cells were cultured with different concentrations of UA. Growth inhibition of UA on MCF-7 cells was evaluated by MTT assay. Cell cycle and sub-G1 peak were performed by FCM. Morphologic changes of UA-treated cells were observed by light microscope. Apoptotic cells with condensed or fragmented nuclei were visualized by Ho 33258 staining by a fluorescence microscope (EX: U. V.). The protein expression of caspase-3 and PARP was analyzed by immunofluorescence cell staining (SABC-Cy3).

RESULT24 hours after UA treatment, inhibition of MCF-7 cell growth was concentration-dependent. The IC50 value for UA was (22.6 +/- 3.0) micromo x L(-1). Cell cycle anaysis by FCM showed that 50 micromol x L(-1) of UA arrested MCF-7 cell cycle at G0 - G1 phase. Morphological changes of MCF-7 Cells exhibited many of the hallmark features of apoptosis, including chromatin clumps and aggregation and DNA fragmentation. UA increased caspase-3 protein expression.

CONCLUSIONThe results suggest that UA evokes MCF-7 cell apoptosis is correlation with the up-regulation of caspase-3. Our study indicated that UA might be a potential Chinese medical component for breast neoplasm.

Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Breast Neoplasms ; enzymology ; pathology ; Caspase 3 ; Caspases ; metabolism ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Female ; Humans ; Poly(ADP-ribose) Polymerases ; metabolism ; Triterpenes ; pharmacology

Objective To investigate the clinical efficacy of finger reconstruction in child with second toe transplantation,and evaluate the postoperative appearance and function regarding the reconstructed donor feet.Methods From June 2002 to May 2011,sixteen cases were reconstructed in sub-emergency with second toe transplantation.Two thumbs,eight index fingers,and 6 middle fingers were reconstructed.All patients were followed-up from 12 to 24 months.The functions of reconstructed fingers were analysed.Results All the reconstructed fingers survived.Vascular crisis occurred in 1 patient,and survived after re-anastomosis.Necrosis of skin grafts at the domon site with exposed tedons was seen in 1 ease,and healed after changing dressings.All the reconstructed fingers showed good in growth and development,and performed good functions as grabbing,grasping and nipping.Two-point discrimination was between 6 mm and 10 mm.The donor site of the foot had normal gait,without obvious influence on walking.Also,no pain was complained.Conclusion The method of transplanting the second toe can reconstruct the appearance and function of the finger defects in child,and has little effect on the appearance and motion of feet.It is an effective treatment method.

OBJECTIVE: To establish a 15-plex rapid STR multiplex amplification system. METHODS: Fourteen auto-chromosome loci and one sex-chromosome were selected to compare the situations of allelic losses and nonspecific amplication under different conditions. FastStart Taq DNA polymerase and DNA standard sample 9947A were used during amplification and optimization process.15-plex rapid STR amplification system was achieved by performing various experiments including selection of amplification conditions and the volume of DNA polymerase, adjustment of inter-locus balance, optimization of rapid amplification, screening of reaction buffers, selection of reaction volume, and a variety of additives. RESULTS: Using 10 μL rapid PCR system, including 1 ng DNA templates, 0.4 μL polymerase and 10xFastStart high fidelity reaction buffer, a complete and well-balance DNA profile of 15 STR loci for standard genomic DNA was obtained in 32 minutes, without the allele drop-out and non-specific amplicons. Meanwhile, 5% glycerinum, 0.01% gelatin, 0.05% gelatin and 5 mmol/L ammonium sulfate could be used as the reactive additive during the amplification procedure. CONCLUSION The 15-plex rapid STR multiplex amplification system can be used to decrease reaction time and enhance sample throughput.
Alleles ; Chromosome Mapping ; DNA/genetics* ; DNA Fingerprinting/methods* ; Forensic Genetics/methods* ; Humans ; Microsatellite Repeats/genetics* ; Polymerase Chain Reaction/methods* ; Racial Groups/genetics* ; Sensitivity and Specificity ; Tandem Repeat Sequences

OBJECTIVETo establish a primary culture method of rat testis Leydig cell.

METHODSThe primary rat Leydig cells were treated with or without 4 U/ml human chorionic gonadotropin (hCG), and testosterone in culture medium was detected by radioimmunoassay. The morphology and biological characteristics of Leydig cell were observed.

RESULTSThe culture cells were highly homogeneous, proliferative and had a high differentiation rate. The high purified Leydig cells were verified by their dynamic morphological changes and 3beta-hydroxysteroid dehydrogenase delta4-delta5 isomerase (3beta-HSD) histochemical staining. The testosterone secretion induced by hCG significantly increased (P < 0.05) 24 hours after inoculation than that induced without hCG in the control.

CONCLUSIONIt suggests that the Leydig cell cultured in vitro may secrete high concentration of testosterone, and this study laid the basis of androgen replacement therapy for partial androgen deficiency in aging male.

3-Hydroxysteroid Dehydrogenases ; Animals ; Cell Culture Techniques ; Cells, Cultured ; Chorionic Gonadotropin ; pharmacology ; Humans ; Leydig Cells ; cytology ; drug effects ; secretion ; Male ; Rats ; Rats, Sprague-Dawley ; Testis ; cytology ; drug effects ; Testosterone ; metabolism

OBJECTIVETo compare the effects of Bushen Jiedu Recipe (BJR) and Jianpi Jiedu Recipe (JJR) containing plasma on dendritic cells (DCs) of chronic hepatitis B virus (HBV) infection patients under different immune states.

METHODSRecruited were 36 chronic HBV infection outpatients from First Affiliated Hospital of Hunan University of Traditional Chinese Medicine from April 2010 to January 2011. They were assigned to the immune tolerance group (18 cases) and the immune clearance group (18 cases).Another 10 healthy subjects were recruited as the healthy control group. Their anticoagulated peripheral venous blood was respectively collected. The peripheral blood mononuclear cells (PBMCs) were isolated and further extracted for incubating DCs. The DCs were intervened by BJR and JJR containing plasma. The morphology of DCs was identified. The expressions of CD1alpha, CD80, CD86, and HLA-DR were detected. The level of interferon-alpha (IFN-alpha) in the supernatant was observed by ELISA.

RESULTSThe CD80 expression level was lower in the immune clear group than in the healthy control group before intervention (P < 0.05). The expression levels of CD80, CD86, and HLA-DR were lower in the immune tolerance group than in the healthy control group before intervention (P < 0.05).The IFN-alpha expression level was lower in the immune tolerance group and the immune clearance group than in the healthy control group before intervention (P < 0.05). The expression levels of CD80, HLA-DR, and IFN-alpha were lower in the immune tolerance group than in the immune clearance group before intervention (P < 0.05). Compared with the same group before intervention, the CD80 expression significantly increased in each treatment group (P < 0.05). After intervention the expression levels of CD80 and HLA-DR were higher in the immune tolerance group than in the immune clearance group in the same time phase, and the CD86 expression level was higher in the BJR group than in the immune clearance group in the same time phase, showing statistical difference (P < 0.05).

CONCLUSIONSThe middle dose BJR and the small dose JJR both could promote the recovery of DCs in chronic HBV infection patients. Besides, BJR showed more prominent effects on the function of DCs in chronic HBV infection patients in the immune tolerance stage.

Adult ; B7-1 Antigen ; metabolism ; B7-2 Antigen ; metabolism ; Case-Control Studies ; Dendritic Cells ; drug effects ; immunology ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Female ; HLA-DR Antigens ; metabolism ; Hepatitis B, Chronic ; blood ; drug therapy ; immunology ; Humans ; Immune Tolerance ; drug effects ; Interferon-alpha ; metabolism ; Male ; Phytotherapy ; Plasma ; Young Adult

OBJECTIVETo observe the functional changes of dendritic cells (DCs) after infection by recombinant retrovirus carrying human telomerase reverse transcriptase (hTERT) gene fragment.

METHODSInterleukin-12 (IL-12) levels in DC culture supernatant was determined by enzyme-linked immunosorbent assay (ELISA). The abilities of DCs infected with recombinant retrovirus carrying hTERT gene (hTERT-DCs) and non-infected DCs (N-DCs) to stimulate allogeneic lymphocyte proliferation were evaluated with mixed leukocytes reaction (MLR), and the surface markers of DCs including CD80, CD83, CD86 and HLA-DR were detected by flow cytometry. Cytotoxic T lymphocyte (CTL) assay was performed with CytoTox 96 non-radioactive cytoxicity assay.

RESULTSCompared with N-DCs, hTERT-DCs showed no significant changes in IL-12 secretion and capacity to stimulate allogeneic lymphocytes reaction, but had significantly lower CD83 expression. Specific CTLs induced by hTERT-DCs resulted in higher cytotoxicity against telomerase-positive target cells than that against the negative target cells.

CONCLUSIONInfection with the recombinant retrovirus carrying hTERT fragment may jeopardize the maturation of DCs, which, however, still retain their capacity to activate and stimulate lymphocyte proliferation and to prime autologous T lymphocytes to generate specific CTL against hTERT.

Cells, Cultured ; Dendritic Cells ; cytology ; immunology ; virology ; Genetic Vectors ; Humans ; Interleukin-12 ; biosynthesis ; Recombination, Genetic ; Retroviridae ; genetics ; metabolism ; T-Lymphocytes, Cytotoxic ; immunology ; Telomerase ; biosynthesis ; genetics

The formation of antisperm antibodies (AsAb) results from the disruption of the blood-testis barrier by a variety of mechanisms, which leads to exposure of immunogenic sperm antigens to the immune system and initiates an immune response. AsAb can impair the fusion of sperm and egg and even the embryo development, resulting in infertility. The etiology of AsAb, effect of AsAb on assisted reproduction and treatment of AsAb in the literature are reviewed in this article.
Antibodies ; immunology ; Antibody Formation ; Humans ; Infertility, Male ; etiology ; Male ; Reproductive Techniques ; Spermatozoa ; immunology

OBJECTIVETo investigate the role of recombinant human growth hormone (rhGH) in the growth of Bel-7402 human hepatic carcinoma cell line (Bel-7402 line) in vitro and its effects on GHR expression.

METHODSTumor cell count, MT assay and colony forming test were performed to determine the responses of Bel-7402 to different concentrations of rhGH (0, 1, 10, 100, 1000, 10 000 ng/ml). Metabolism of DNA in tumor cells was analyzed with the method of mixture of 3H-TdR. Radioreceptor assay was used to detect the GHR expression of the hepatic carcinoma cell lines and its relation to different rhGH concentrations.

RESULTSrhGH accelerated the proliferation of the Bel-7402 line when the concentration of rhGH was over 100 ng/ml (P < 0.05). Other rhGH concentrations had also positive effects, but with reduced effect as compared with that of 100 ng/ml. After 24 h of rhGH addition of concentration of 10 ng/ml and 100 ng/ml, GHR site number was significantly higher than that in control group, while the 10,000 ng/ml group showed a significantly lower GHR site number.

CONCLUSIONSDifferent concentrations of rhGH might result in variable effects on the growth of Bel-7402 hepatic carcinoma cell line. Certain concentrations of rhGH might stimulate the growth of the cell line. rhGH can regulate the expression of GHR in the cell line.

Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Human Growth Hormone ; pharmacology ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Receptors, Somatotropin ; genetics ; metabolism