This study was conducted to investigate the paclitaxel loaded by hydrazone bonds in poly(ethylene glycol)-poly(caprolactone) micelles (mPEG-PCL-PTX) on proliferation and apoptosis of human lung cancer A549 cells and its possible mechanisms of anti-tumor activity. The cell proliferation was measured with MTT assay. Flow cytometry were used to analyze the cell cycle. The cell apoptosis was analyzed using Hoechst/P staining. The expression levels of apoptotic genes expression in the mitochondrial apoptosis pathway were detected by RT-PCR and Western blotting, respectively. The mPEG-PCL-PTX could inhibit the proliferation of A549 cells and promote the apoptosis. The Bax, caspase-3 protein expression were increased while Bcl-2 protein expression was decreased in A549 cells. Results showed that the polymer containing hydrazone bond is non-toxic in vitro, the mPEG-PCL-PTX micelles can inhibit the proliferation and induce the apoptosis of A549 cells. Key words: paclitaxel; micelle; A549 cell; proliferation; cell cycle; apoptosis
Apoptosis ; Caspase 3 ; metabolism ; Cell Line, Tumor ; drug effects ; Cell Proliferation ; Humans ; Lung Neoplasms ; metabolism ; pathology ; Micelles ; Paclitaxel ; pharmacology ; Polyesters ; Polyethylene Glycols ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; bcl-2-Associated X Protein ; metabolism

OBJECTIVE: To investigate the bacteriostatic effect of platelet-rich plasma (PRP) and its derivative platelet gel (PG) supernatant on Escherichia coli in vitro and its relationship with platelet factor 4 (PF4). METHODS: Apheresis platelets donated by healthy volunteers were obtained from the Blood station of Lu an Blood Center as the source of PRP. The counts of platelet, white blood cell and red blood cell in PRP and its derivative PG supernatant were detected by automatic hematology analyzer. Bacterial growth of PRP and PG supernatants co-cultured with bacteria for different time was observed by plate coating culture method, and the contents of PF4 in PRP and PG supernatants were detected by ELISA. RESULTS: Apheresis platelets were collected from 28 healthy volunteers with a median age of 33 (21-56) years old. PRP can inhibit the growth of escherichia coli, but there were individual differences in antibacterial effect within 24 hours. PRP of 13 healthy volunteers had strong antibacterial effect at 24 hours, 7 cases had weak antibacterial effect at 24 hours, and 8 cases had no antibacterial effect at 24 hours. PG supernatant showed no significant individual difference, and all of them had bacteriostatic effect within 12 hours, but no bacteriostatic effect after 12 hours. There was no statistical difference in the bacteriostatic effect of PRP at 24 hours between healthy volunteers aged ≤30 years and >30 years (P>0.05), and there was no statistical difference between the white blood cell count ≤0.1×10⁹/L and (0.1-1) ×10⁹/L groups (P>0.05). There was significant difference in the bacteriostatic effect of PRP between the two groups with platelet content ≤1 000×10⁹/L and >1 000×10⁹/L (P<0.05). The platelet count in PRP was higher than that in PG supernatant ［(911.57±160.52) ×10⁹/L vs 0］. The PF4 level in PRP was higher than that in PG supernatant (23623.34±9822.14 vs 6664.74±4065.83, P<0.05). CONCLUSION Both PRP and PG supernatant have antibacterial effects in Escherichia coli. The bacteriostatic effect of PRP was better than that of PG supernatant, and the platelet and PF4 contents in PRP were higher than those in PG supernatant, suggesting that the platelet and PF4 levels play an important role in bacteriostasis.
Adult ; Humans ; Middle Aged ; Escherichia coli ; Platelet Factor 4 ; Platelet-Rich Plasma

Human Zona Pellucida(ZP), which is a complex matrix surrounding oocytes,is comprised of three immunologically distinct glycoproteins(hZP1, hZP2 and hZP3). Because hZP3 possesses the sperm receptor activity and the acrosome-inducing activity, it has long been used as a candidate antigen to develop an immunocontraceptive vaccine. However, a large amount of native hZP3 protein is unavailable. It is an effective way to express hZP3 protein directly in vitro. Nevertheless, it had been reported that the rhZP3 protein produced in Pichia pastoris was not secreted but accumulated in the cells and could only be purified after being solubilized by strong denaturants. More unfortunately, after purification the final product required 6mol/L urea to maintain solubility. An improved project was advanced with the aim to express secreted and soluble rhZP3 protein in yeast. In this study, the fragment of hZP3 cDNA coding for aa 23 - 408, which the N-terminal leader was removed and most of the C-terminal transmembrane-like domain was reserved, was amplified by two PCR primers including EcoR I and Not I sites respectively and a His6 codon cassette was added to 5'-terminal. The hZP3 insert was incorporated into expression vector pPIC9K. The resulting recombinant yeast expression vector was designated pPIC9K-rhZP3. Linearized pPIC9K-rhZP3 was transformed into Pichia pastoris. After G418 selection, the recombinant Pichia pastoris strains were identified by PCR and the rhZP3 was expressed following the manufacturer' s protocol. Following induction with methanol, the rhZP3 protein was secreted and dissolved into the culture supernatant. SDS-PAGE and Western blot analyses showed that the apparent molecular weight of the expressed rhPZ3 proteins in yeast was smaller and a little size heterogeneity than native ones; after purified with Ni-chelating affinity chromatography, the final product's apparent molecular weight was about 32 - 34KD and their yield more than 20mg/L. We supposed that the C-terminal transmembrane-like domain be useful for secretion of rhZP3 into the culture supernatant and the expressed rhZP3 protein be incompletely digested by proteinases of Pichia into shorter fragments which all were glycosylated inhomogeneously. Fortunately, the fragments of rhZP3 protein can be recognized in Western blot by the polyclonal antibodies to porcine ZP3 which has showed a cross-reactivity with human ZP in vitro. It will be expected that the rhZP3 protein expressed in Pichia pastoris not only has immunogencity, say, it can rise antibodies in vivo to prevent spermatozoa-ovum binding, but also does not contain ovarian factors that might be the cause of undesired side effects, e.g. ovaritis and can be used as a safe immunogen in human antifertility vaccine research.
Blotting, Western ; Chromatography, Affinity ; Egg Proteins ; genetics ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Genetic Vectors ; genetics ; Humans ; Membrane Glycoproteins ; genetics ; metabolism ; Pichia ; genetics ; metabolism ; Polymerase Chain Reaction ; Receptors, Cell Surface ; genetics ; metabolism ; Recombinant Proteins ; genetics ; isolation & purification ; metabolism ; Zona Pellucida Glycoproteins

To obtain the recombinant pZP3alpha protein for the study of the contraceptive vaccines, the DNA sequence (446-1423) encoding purified pZP3alpha was inserted into a vector--pPICZalphaA. The recombinant plasmid pPICZalphaA-pZP3alpha was linearized and then transformed into Pichia pastoris GS115 by electroporation. Engineering strains were attained by screening with zeocin and induced to produce rpZP3alpha in high-density fermentation. Then rpZP3alpha was purified by Cu2+ metal affinity column chromatography from the separated and concentrated fermentative supernatants. The purified rpZP3alpha was identified by SDS-PAGE and Western blot, and the quantity, purity and rate of recovery of the rpZP3alpha were analyzed by Quantity One software. One male rabbit was immunized with the Cu-NTA-purified rpZP3alpha. The antibody responses against rpZP3alpha and porcine ZP were detected by ELISA and the indirect immunofluorescence. Engineering strains expressing rpZP3alpha in secretion were constructed. A 46kD component named rpZP3alpha which can react with anti-pZP3 antibody was purified from fermentative supernatants of engineering strains and the average yield of purified rpZP3alpha obtained from fermentative supernatants was 8mg/L. The purity and the rate of recovery were up to 92% and 63% respectively. The anti-rpZP3alpha antiserum was prepared by immunization of a male rabbit with purified rpZP3alpha. This anti-rpZP3alpha antiserum could react with rpZP3alpha and purified pZP3 in ELISA and bind to porcine zona pellucida which produced bright green fluorescence in the indirect immunofluorescence. The rpZP3alpha (46kD) protein could be successfully expressed in the Pichia pastoris expression system. And this protein retained the immunogenic activity of natural pZP3.
Animals ; Egg Proteins ; genetics ; metabolism ; Electroporation ; Fermentation ; Immunization ; Male ; Membrane Glycoproteins ; genetics ; metabolism ; Pichia ; genetics ; metabolism ; Rabbits ; Receptors, Cell Surface ; genetics ; metabolism ; Recombinant Proteins ; genetics ; immunology ; secretion ; Swine ; Zona Pellucida Glycoproteins

OBJECTIVETo find a convenient and exact method for evaluating acrosome reaction in human spermatozoa.

METHODSThe semen of the normal male was mixed and then divided into 6 groups. Coomassie brilliant blue (CBB) staining, chlortetracycline (CTC) fluorescence staining and acid phosphatase (ACP) detection were used for morphological observation and data analysis of the acrosome status of the human sperm treated with or without progesterone.

RESULTSThere were obvious morphological differences between the acrosome-reaction and acrosome-intact spermatozoa in CBB staining and CTC fluorescence staining, and significant differences were observed between the experimental and control spermatozoa by the three methods (P < 0.05).

CONCLUSIONAll the three methods can be used to assess acrosome reaction in human spermatozoa, but Coomassie brilliant blue (CBB) staining is much more convenient and stable.

Acid Phosphatase ; Acrosome Reaction ; drug effects ; Cells, Cultured ; Chlortetracycline ; Humans ; Male ; Progesterone ; pharmacology ; Rosaniline Dyes ; Spermatozoa ; cytology ; Staining and Labeling ; methods

Objective To study the epidemiological characteristics of chronic pain following breast cancer surgery and analyze the related factors.Methods Nine hundred and twelve patients following breast cancer surgery were enrolled in the Department of Breast Surgery,Affiliated Hospital,Academy of Military Medical Sciences.The Douleur Neuropathique-4 (DN-4) questionnaire was used to identify neuropathic pain,and the related factors were statistically analyzed.Results The follow-up was completed in 821 patients,including 263 (32％) patients with chronic pain.DN-4 score of 47 (17.9％)patients was over 4 points.There was significant difference between the painful group and the non-painful group in methods of surgery and axillary lymph node dissection (P ＜ 0.05).Conclusion Findings suggest that chronic pain after breast cancer surgery is a significant problem clinically.Proper surgery and psychological buildup are of clinical value in preventing post-surgery chronic pain.

OBJECTIVETo analyze the effect of globular adiponectin on angiogenesis of ovarian microvascular endothelial cells (OMECs).

METHODSMouse OMECs were isolated and purified by density gradient centrifugation with Percoll and identified by immunofluorescence analysis of follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR), and endothelial cell marker von Willebrand factor (vWF). The capillary-like tube formation of OMECs was determined by vascular endothelial growth factor A (VEGFA) treatment in Matrigel matrix. OMECs treated with recombinant globular adiponectin protein were examined for cell proliferation with MTS assay and cell migration with scratch wound healing assay, and capillary-like tube formation was tested in Matrigel matrix. Western blotting was performed to detect the effect of globular adiponectin on AMPK phosphorylation.

RESULTSThe signals of LHR and vWF, but not that of FSHR, were detected in the isolated cells. VEGFA treatment of the cells induced capillary-like tube formation, indicating their properties of ovarian-specific endothelial cells. Treatment with 1 and 3 µg/mL of recombinant globular adiponectin significantly increased the number of OMECs by (158.72∓14.50) % and (186.50∓4.20)% (P<0.01) and resulted in scratch wound closure rates of (49.43∓3.43)% (P<0.05) and (69.67∓1.2) % (P<0.01) respectively. The cells treated with 3 µg/mL globular adiponectin formed a capillary-tube length 6.63∓0.66 folds greater than that formed by the control cells (P<0.01). Treatment of the cells with 3 µg/mL globular adiponectin for 15 and 30 min resulted in pAMPK/AMPK ratios of 0.86∓0.08 and 0.66∓0.13, respectively significantly higher than that in the control cells (0.13∓0.12, P<0.01). Compound C obviously suppressed the tube formation and AMPK phosphorylation induced by globular adiponectin.

CONCLUSIONGlobular adiponectin promotes angiogenesis of OMECs through activation of the AMPK signal pathway.

OBJECTIVETo explore the effect of nitric oxide (NO) on human sperm capacitation and acrosome reaction (AR).

METHODSDifferent concentrations of sodium nitroprusside (SNP) were added to the sperm suspension from 48 healthy fertile men, and the suspension was incubated in 1 x Earle at 37 degrees C for 1 hour. Progesterone was used to induce AR for 15, 30, 45 and 60 min, and then acid phosphatase (ACP) activity in the suspension before and after capacitation and at different time of AR was measured by p-nitrophenyl sodium phosphate assay. In the meantime, sperm motile parameters were assayed by CASA to observe sperm capacitation and AR.

RESULTSACP activity and sperm motile parameters increased in the 50 approximately 100 nmol/L NO concentration group, showed no significant variation in the 150 approximately 200 nmol/L group, and decreased in the 250 approximately 300 nmol/L group.

CONCLUSIONNO can facilitate sperm capacitation, AR and sperm motile parameters in low concentration and suppress them in high concentration. ACP activity assay of sperm is an objective and reliable method to evaluate sperm capacitation and AR in whole sperm population.

Acid Phosphatase ; metabolism ; Acrosome Reaction ; drug effects ; physiology ; Adult ; Dose-Response Relationship, Drug ; Humans ; Male ; Nitric Oxide ; physiology ; Nitric Oxide Donors ; pharmacology ; Nitroprusside ; pharmacology ; Sperm Capacitation ; drug effects ; physiology ; Sperm Motility ; drug effects ; physiology ; Spermatozoa ; enzymology

OBJECTIVETo investigate the effect of mesenchymal stem cell (MSC) transplantation in repairing ovarian injury in mice sensitized with porcine ovarian proteins.

METHODSWild-type female mice with ICR background (6-8 weeks old) were divided randomly into groups A, B and C (n=12). In groups B and C, the mice were treated with the total protein extract from porcine ovary to induce immunological injury of the ovary, while those in group A received no treatment. MSCs-derived from GFP transgenic mice were transplanted into the mice of group C, and equal volume of PBS was injected intraperitoneally in mice of the other two groups. PCR was used to detect GFP gene in the genomic DNA of the ovaries to assess MSCs homing in the ovary, and the reparative effect of MSCs on ovarian injury was evaluated using HE staining and TUNEL analysis.

RESULTSAfter transplantation, the MSCs could reach the injured ovaries to promote the repair of the ovarian injury, resulting also in reduced apoptosis of the granulosa cells (GCs) in the injured ovaries.

CONCLUSIONMSCs transplantation can promote the recovery of the immunological injury of the ovary in mice, the mechanism of which may involve reduced apoptosis of the GCs.

Animals ; Apoptosis ; Bone Marrow Cells ; Female ; Granulosa Cells ; cytology ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; Mice ; Mice, Inbred ICR ; Ovarian Diseases ; pathology ; surgery ; Ovary ; cytology ; pathology

OBJECTIVETo understand the epidemic status of Rickettsia in Xinyang areas of Henan province.

METHODSSamples including liver, spleen, kidney from mouse and chigger mites from Xinyang areas and serum samples were detected by nested-polymerase chain reaction (PCR) and indirect immunofluorescence assay (IFA).

RESULTSIn 62 viscus samples from mice organs, the positive rates were 16.13%, 8.06% and 6.45% for Orientia tsutsugamushi, R. typhii and Spotted fever group rickettsiae respectively. In blood clots samples from mice, the positive rates were 8.06%, 6.45% and 1.61 % for O. tsutsugamushi, R. typhii and Spotted fever group rickettsiae respectively. Three out of 26 mouse serum samples were positive for the predicted fluorexcent intensity O. tsutsugamushi.

CONCLUSIONUsing nested-PCR and IFA methods, O. tsutsugamushi, R. typhii and Spotted fever group rickettsiae were detected in the captured mice living in Xinyang areas of Henan province. Results showed that there were intensive natural reserviors of Rickettsia in Henan province, suggesting that the risk of outbreak of Rickettsia in these areas was high.

Animals ; China ; Fluorescent Antibody Technique, Indirect ; Humans ; Kidney ; microbiology ; Liver ; microbiology ; Mice ; Orientia tsutsugamushi ; classification ; genetics ; isolation & purification ; pathogenicity ; Phylogeny ; Polymerase Chain Reaction ; Rickettsia ; classification ; genetics ; isolation & purification ; pathogenicity ; Scrub Typhus ; epidemiology ; microbiology ; Spleen ; microbiology