The electrospray ionization-ion mobility spectrometric (ESI-IMS) technique has the potential as an analytical separation tool in analyzing polypeptides and amino acids for fast screening unknown samples in anti-chemical and biological terror attacks. A method for detecting several polypeptides and amino acids was developed based on ESI-IMS using air as drift gas at room temperature. The ion mobility of four amino acids and two polypeptides dissolved in methanol was determined on the system at elution rate of 2 mL/ min. The spectra of these compounds had characteristics of finger-printing maps. The limit of detection of this instrument for Substance P could reach 855 ng / mL in 1 min. The results showed that a small, self-contained ESI-IMS instrument with reservoirs of air could be used to quickly detect and accurately identify polypeptides and amino acids.

Human nerve growth factor (NGF) is a nerve cell growth regulation factor, which can provide nutrition for the neurons and promote the neurites outgrowth. In order to produce large-scale recombinant human nerve growth factor (rh-beta-NGF), we constructed a plasmid vector, which can stably express the rh-beta-NGF in the HEK293 cell lines. First, the plasmid of pCMV-beta-NGF-IRES-dhfr was constructed and transformed into HEK293 cells. Then MTX pressurized filter and limiting dilution methods were used to obtain monoclonal HEK293 cell lines. After stepwise reducing serum in culture media, the cells eventually adapted to serum-free medium and secreted rh-beta-NGF. SDS-PAGE analysis revealed that the expression product owned a molecular weight of about 13 kDa and a purity of more than 50%. The peptide mapping sequencing analysis demonstrated the sequences of rh-beta-NGF matched with the theoretical ones. Later we purified this protein by ion exchange and molecular sieve chromatograph. Finally, our experimental results exhibited that the recombinant cell lines can stably express rh-beta-NGF with a high efficiency of more than 20 pg/cell x day. In addition, this protein could successfully induce differentiation of PC12 cells. In summary, our recombinant HEK293 cells can express bio-active rh-beta-NGF with great efficiency and stability, which supply a valid basis to large-scale production of rh-beta-NGF.
Cell Differentiation ; Genetic Vectors ; HEK293 Cells ; Humans ; Nerve Growth Factor ; biosynthesis ; Plasmids ; Recombinant Proteins ; biosynthesis

OBJECTIVETo observe the effect of gastric dynamics by transcutaneous electrical acupoint stimulation (TEAS) combined general anesthesia when controlled hypotension dropped to 60% of the mean arterial prenssure (MAP) baseline, and to provide experimental evidence for organ protection in clinical controlled hypotension.

METHODSEighteen male beagles were randomly divided into three groups, the general anesthesia group (blank), the general anesthesia induced controlled hypotension group (control), and the general anesthesia combined TEAS induced controlled hypotension group (experiment), 6 in each group. Controlled hypotension was performed in the latter two groups with isoflurane inhalation and intravenous injection of sodium nitroprusside (SNP). The mean arterial pressure (MAP) was lowered to 60% of the MAP baseline and kept for 60 min. Controlled hypotension was not performed in Beagles of the control group. For Beagles in the experiment group, TEAS [2/100 Hz, (4 ± 1) mA] was applied to bilateral Hegu (LI4), Quchi (LI11), Zusanli (ST36), and Sanyinjiao (SP6) from stable physiological conditions to the end of maintaining stages. Changes of EGG frequencies and EGG amplitudes were monitored. Serum levels of gastrin (GAS) and motilin (MTL) were also detected at corresponding time points during and after experiment.

RESULTSAs for the pressure control effect of TEAS combined general anesthesia in the controlled hypotension, during the process of controlled hypotension (T1-T4), MAP levels of two controlled pressure groups remained relatively stable, and were kept at 60% of the MAP baseline. When the blood pressure dropped to the target low MAP and maintained at 60 min (T1-T4), EGG amplitudes of Beagles in all the three groups showed decreasing tendency. But it was more obviously lower than its basic level in the control group (P <0.05), while it was not obviously decreased in the experiment group (P < 0.05). EGG frequencies of Beagles in all the three groups showed no obvious change during this stage. By the end of the MAP rising stage (T8), the EGG amplitude of the experimental group was significantly higher than that of the control group and the blank group (P < 0.05), while it didn' t show any obvious increase in the control group. During this period, EGG frequencies of the two controlled hypotension groups decreased more than those of the blank group. Two h after rising blood pressure (at T9), EGG amplitudes and frequencies in the two controlled hypotension groups basically restored to their respective baselines and levels of the blank group at T9. At 2 h (T9) after controlled hypotension, serum levels of GAS and MTL were lower than those of basic levels in the two controlled hypotension groups (P <0.05). However, serum levels of GAS and MTL had an increasing trend in the two controlled hypotension groups at 24-72 h (T10-T12). Besides, the increasing speed and amplitude was better in experiment group than in the control group at T10-T12. However, there was no statistical difference between the two groups (P > 0.05). At 72 h (T12) serum levels of GAS and MTL had basically restored to their basic levels in the two controlled hypotension groups and that of the blank control group.

CONCLUSIONEGG amplitudes could be effectively improved in TEAS combined general anesthesia for controlled hypotension at 60% of the MAP baseline, the recovery of the serum GAS level accelerated, gastric power improved and stomach protected.

Acupuncture Points ; Anesthesia ; methods ; Anesthesia, General ; Animals ; Arteries ; Dogs ; Gastrins ; Hypotension, Controlled ; Male ; Motilin ; Nitroprusside ; Transcutaneous Electric Nerve Stimulation

OBJECTIVETo evaluate the influence of big Y chromosome on the outcomes of in vitro fertilization and embryo transfer.

METHODSData of 127 cycles of IVF/ICSI-ET, performed in our Reproductive Medicine Center from March 2001 to June 2003 were analyzed. The patients were divided into two groups according to the length of chromosome: Group A, 56 cycles with big Y chromosome

RESULTSNo significant difference was observed in the quality of embryos and in the and Group B, 71 cycles with normal karyotype. rates of fertilization, cleavage, clinical pregnancy, implantation, miscarriage, ectopic pregnancy, dead infant delivery, malformation,

CONCLUSIONBig Y chromosome has no significant influence on the baby boy delivery and baby girl delivery between the two groups. development of embryos and the outcome of pregnancy.

Chromosomes, Human, Y ; Embryo Implantation ; Female ; Humans ; Infertility, Male ; genetics ; therapy ; Male ; Pregnancy ; Pregnancy Rate ; Prospective Studies ; Sperm Injections, Intracytoplasmic

OBJECTIVETo compare 3 common sperm counting chambers by the computer-assisted sperm analysis (CASA) system and evaluate their precision in analyzing sperm density and motility.

METHODSWe used latex bead solution at (20 +/- 5) x 10(6)/ml as analogue semen samples and analyzed the samples with Makler, Leja and Microcell counting chambers, 30 times with each chamber. And the average (x +/- s) and the coefficient of variation of sperm density were calculated by the CASA system. Meanwhile 54 semen samples collected from the outpatients analyzed with the 3 sperm counting chambers by the CASA system for the rates of forward movement and motility of the sperm.

RESULTSThe averages of sperm density obtained with Makler, Leja and Microcell chambers were (25.90 +/- 3.97) x 10(6)/ml, (18.74 +/- 1.62) x 10(6)/ml and (20.35 +/- 2.55) x 10(6)/ml, the coefficients of variation were 15.31%, 8.64% and 12.54%, the rates of sperm forward movement were (46.54 +/- 17.09)%, (30.65 +/- 14.88)% and (30.49 +/- 13.21)%, and the rates of sperm motility were (59.75 +/- 16.12)%, (46.76 +/- 14.11)%, (43.11 +/- 14.02)% respectively. There were significant differences in average sperm density among the 3 groups (P < 0.05). The rates of sperm forward movement and motility obtained with the Makler chamber were significantly higher than those achieved with the Leja chamber and Microcell chamber (P < 0.05), but there were no significant differences between the latter two (P > 0.05).

CONCLUSIONThe rates of sperm density obtained with the 3 sperm counting chambers differed significantly. In the analysis of sperm motility, a higher rate can be achieved with the coverslip-pressed chamber than the capillary-drawn chamber.

Evaluation Studies as Topic ; Humans ; Image Processing, Computer-Assisted ; instrumentation ; methods ; Male ; Sperm Count ; instrumentation ; methods ; Sperm Motility

Objective To observe the clinical effect of stemless hip replacement.Methods The stemless hip replacement was performed in 40 cases and the effect evaluated.Results Follow-up for average 24.6 months(6-48 months)showed satisfactory outcomes in all cases after the stemless hip re- placement.Based on harris scale(averaging 90.6),the excellent result was,seen in 28 case(70%),good in eight(20%)and common in four(10%),with total excellence rate of 90%.Conclusions Stemless hip replacement is characterized by little trauma,less blood loss,less complications and fitting for revision. The general effects of this technique are satisfactory.It is especially suitable for the old and weak cases or the young cases who need hip replacement.The long term outcomes need further evaluation.

Objective To discuss experiences in reconstruction of severe tibial shaft fractures by using different flaps and external fixations.Methods The study involved 190 patients with type GustiloⅢB Ⅲ C (160 patients) and GustiloⅢC (30 patients) tibial shaft fractures treated from 1990 to 2007.There were 169 males and 21 females,at average age of 42.5 years.The injury causes included traffic accidents in 132 patients,machine accidents in 32 and stone smashing in 26.The management procedure consisted of administration of antibiotics,serial debridement and different flap grafting (including free thoracoumbilical flaps in 20 patients,sural neurocutaneous vascular flaps in 108,saphenous neurocutane ous vascular flaps in 12,superficial peroneal neurocutaneous flap in two,fasciocutaneous flaps in 26 and gastrocnemius muscular flaps in 22) and different external fixators (half-ring fixators in 84 patients,unilateral axial dynamic fixators in 12,AO fixators in 10,Weifang fixators in 42 and hybrid fixators in 40).The average follow-up was 7.3 years.Results All flaps survived.Of all,186 patients obtained fracture healing,with mean fracture healing time varying in different patients treated with different external fixators:7.5 months for 84 patients treated with half-ring fixators,11.2 months for eight with unilateral axial dynamic fixators,8.5 months for 12 with AO fixators ,8.1 months for 42 with Weifang fixators and 7.8 months for 40 with assembly fixators.Except for half-ring fixation,the other fixators needed necessary bone graft.Four patients treated with unilateral axial dynamic fixators resulted in nonunion due to osteo myelitis.The latest follow up showed that the function of the ankle and knee was normal,with no pain.Conclusion Combination of half-ring external fixators with various flaps provides good method for treatment of Gustilo ⅢB and ⅢC tibial shaft fractures.

BACKGROUNDIt is essential to establish an animal model for the elucidation of the biological behaviors of stem cells in vivo. We constructed a chimeric animal model by in utero transplantation for investigation of stem cell transplantation.

METHODSThis chimerism was achieved by injecting the stem cells derived from the bone marrow of green fluorescence protein (GFP)-transgenic mice into fetal mice at 13.5 days of gestation. Several methods such as polymerase chain reaction (PCR), real-time PCR, fluorescence-assisted cell sorting (FACS) and fluorescence in situ hybridization (FISH) were used for the observation of donor cells.

RESULTSUnder a fluorescence microscope, we observed the GFP cells of donor-origin in a recipient. PCR, FACS analysis and FISH indicated chimerism at various intervals. Real-time PCR indicated that some donor cells existed in chimera for more than 6 months.

CONCLUSIONSAllogenic stem cells may exist in recipients for a long time and this allogenic animal model provides a useful tool for studying the behavior of hematopoietic stem cells and also offers an effective model system for the study of stem cells.

Animals ; Female ; Flow Cytometry ; Hematopoietic Stem Cell Transplantation ; In Situ Hybridization, Fluorescence ; Mice ; Models, Animal ; Polymerase Chain Reaction ; Transplantation Chimera ; Transplantation, Homologous

ObjectiveTo investigate the mechanism of action of Sini powder in the treatment of liver cancer based on network pharmacology and molecular docking. MethodsTraditional Chinese Medicine Systems Pharmacology Database and Analysis Platform was used to obtain the compound and target of Sini powder, and the corresponding gene Symbol was obtained through Uniprot. The disease genes of liver cancer were obtained from Human Genome Database, and the genes with intersection with the target genes of Sini powder were screened out. Cytoscape3.7.1 software was used to draw the map of “traditional Chinese medicine (TCM)-compound-target” network. STRING was used to construct a protein-protein interaction (PPI) network, R studio software was used to conduct gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses on therapeutic targets, and then the results were visualized. The active component with the highest number of targets was selected as the ligand, and the target with the highest degree in the PPI network was selected as the receptor, so as to predict the structure of receptor-ligand complex and the amino acid residues that bind to each other. ResultsIn this study, 91 core targets and 141 relevant active components of Sini powder were screened out, among which quercetin and kaempferol were the main active components in the treatment of liver cancer. TP53 and HSP90AA1 were the main therapeutic targets. The GO enrichment analysis obtained 1007 items which met the screening criteria, which were mainly involved in the biological processes of antioxidation reaction, activity regulation of protein serine and threonine kinase, and cellular stress response. The KEGG enrichment analysis obtained 102 pathways, which mainly regulated the hepatitis B pathway and the PI3K-Akt signaling pathway in the prevention and treatment of liver cancer. The results of molecular docking showed a synergistic antitumor effect between the crystal structure domains VAL147, CYS220, GLU221, and PRO222 of quercetin-TP53. ConclusionThis study reveals the mechanism of action of Sini powder in the treatment of liver cancer by acting on multiple targets and signaling pathways, which provides a theoretical basis for biological experiments.

Objective To observe the effect of Bushen Jianpi Prescription,a prescription with the actions of tonifying the kidney and strengthening the spleen,combined with short-term intensive insulin therapy on dedifferentiation of pancreatic beta-cells in patients with type 2 diabetes mellitus(T2DM)of spleen and kidney deficiency type.Methods Seventy patients with T2DM of spleen and kidney deficiency type were randomly divided into control group and observation group,with 35 cases in each group.The control group was treated with intensive insulin therapy,and the observation group was treated with Bushen Jianpi Prescription on the basis of treatment for the control group.The course of treatment lasted for 2 weeks.The changes of TCM syndrome score,and levels of blood glucose,serum high mobility group protein B1(HMGB1),interleukin 1β(IL-1β),tumor necrosis factor α(TNF-α)and the ratio of proinsulin to insulin(PI/I)in the two groups were observed before and after treatment.Results(1)During the treatment,one case in the control group and 2 cases in the observation group fell off.Eventually,34 cases in the control group and 33 cases in the observation group were included in the efficacy statistics.(2)After treatment,the scores of TCM syndromes in the two groups were significantly lower than those before treatment(P＜0.05),and the decrease in the observation group was significantly superior to that in the control group(P＜0.05).(3)After treatment,the levels of fasting plasma glucose(FPG)and 2-hour postprandial plasma glucose(2hPG)in the two groups were significantly lower than those before treatment(P＜0.05),and the decrease in the observation group was significantly superior to that in the control group(P＜0.05).(4)After treatment,the level of HMGB1 in the two groups was significantly lower than that before treatment(P＜0.05),and the decrease in the observation group was significantly superior to that in the control group(P＜0.05).(5)After treatment,the serum levels of inflammatory factors of IL-1β and TNF-α in the two groups were significantly lower than those before treatment(P＜0.05),and the decrease in the observation group was significantly superior to that in the control group(P＜0.05).(6)After treatment,the PI/I ratio of the two groups was significantly lower than that before treatment(P＜0.05),and the decrease of the observation group was significantly superior to that of the control group(P＜0.05).Conclusion Bushen Jianpi Prescription combined with short-term intensive insulin therapy can effectively improve the islet function of patients with T2DM of spleen and kidney deficiency type.The mechanism may be related to the relief of high-glucose toxicity and the decrease of the inflammatory factor levels,thus to inhibit the dedifferentiation of pancreatic beta-cells.