The purpose of this study was to set up an approach for expansion of the peripheral blood gammadeltaT cells from normal subjects in order to explore the characteristics of gammadeltaT cells. Peripheral blood mononuclear cells (PBMNC) were separated from 5 - 10 ml peripheral blood and stimulated by the low molecular peptide derived from Mycobacterium tuberculosis (MTb-Ag), and expanded in rIL-2-containing medium. The relative amount of gammadeltaT cells were measured by anti TCR gammadelta-PE staining and flow cytometry. The Cytotoxicity were detected by gammadeltaT assay. The results showed that after stimulation and expansion for 10 days, gammadeltaT cells increased to 69.2% of the total PBMNC and demonstrated significant cytotoxicity against K562 cells. In conclusion, this is a simple, rapid and specific method for expansion of peripheral blood gammadeltaT cells in vitro.
Antigens, Bacterial ; immunology ; Cell Separation ; methods ; Flow Cytometry ; Humans ; Mycobacterium tuberculosis ; immunology ; Receptors, Antigen, T-Cell, gamma-delta ; analysis ; T-Lymphocyte Subsets ; cytology

Objective To evaluate the therapeutic effect of oral and external application of Chinese medicine combined with Ilizarov external fixator and the accordion technique for severe chronic osteomyelitis of the tibia.Methods A total of 78 patients with severe chronic osteomyelitis of the tibia were randomized into a routine treatment group and a combined treatment group, 39 in each group. All the patients in the two groups received external fixation by use of Ilizarov external fixator and the accordion technique. All the patients in the combined treatment group received oralGuyu decoction. The patients who had large wound received vacuum-sealing drainage in the routine treatment group, and vacuum-sealing drainage combined with external application ofShengji-Yuhong plaster in the combined treatment group. All the patients were treated for 8 weeks and followed up for 2 years. The time to wound healing and fracture healing, and the drainage time were compared between the two groups. Functional and radiologic findings were evaluated according to Paley's criteria. The functions of the knee joint and ankle joint were evaluated using the Hospital for Special Surgery (HSS) knee score and the scoring system of Baird and Jackson, respectively.Results The time to wound healing (17.33 ± 6.21 dvs. 22.27 ± 8.12 d;t=3.018,P=0.004) and the time to fracture healing (32.25 ± 6.02 weeks vs. 36.37 ± 7.75 weeks;t=2.623,P=0.011), and the drainage time (17.01 ± 4.66 dvs. 21.51 ± 5.23 d;t=4.012, P<0.001) in the combined treatment group were significantly shorter than those in the routine treatment group. According to Paley's criteria, the patients who achieved a score of excellent or good for fracture healing (84.6%vs. 53.8%;χ2=7.282,P=0.007) and function (92.3%vs. 66.7%;χ2=6.369,P=0.012) in the combined treatment group were significantly more than those in the routine treatment group. The scores of the HSS knee score (84.56 ± 7.42vs. 78.81 ± 5.33;t=3.391,P=0.002) and the scoring system of Baird and Jackson (85.01 ± 8.21vs. 79.21 ± 6.78;t=3.402,P=0.024) in the combined treatment group were significantly higher than those in the routine treatment group.Conclusion OralGuyu decoction and external application ofShengji-Yuhong plaster combined with Ilizarov external fixator and the accordion technique can promote recovery of the joint function, fracture healing and wound healing.

Objective To synthesize 99Tcm- (hydrazinonictinamide- [Lys3] -bombesin) (tricine)(trisodium triphenylphosphine-3,3',3"-trisulfonate) ((HYNIC-[Lys3]-BBS) (tricine) (TPPTS)) and evaluate its biodistribution and binding capability with tumor tissue in Balb/c nude mice bearing human pancreatic cancer xenografts. Methods HYNIC was conjugated to the [Lys3] -BBS at pH = 9.0 with SnCl2 as reducing agent and both tricine and TPPTS as coligands for 99Tcm-labeling. 99Tcm-HYNIC-[Lys3]-BBS)(tricine) (TPPTS) was purified by Sep-Pak C18 cartridge and was analysed by HPLC. The radiochemical purity and radiolabeling yield were measured. The stability of 99Tcm-(HYNIC-[Lys3]-BBS) (tricine)(TPPTS) in serum, biodistribution (% ID/g) in the normal mice and imaging of the Balb/c nude mice bearing human pancreatic cancer xenografts in vivo were studied. Results The radiolabeling yield was (90 ±2)% and the radiochemical purity was over 95%. The radiochemical purity after 4 h in serum was over 85%. The distribution in normal mice showed rapid clearance from blood (the uptake was (0.07 ±0.01) %ID/g at 2 h postinjection). 99Tcm-(HYNIC-[Lys3]-BBS) (tricine) (TPPTS) was excreted mainly via the kidney with little radioactivity accumulation in the liver and gastrointestinal tract (the uptake of liver, stomach, intestine was (0.27 ±0.03), (0.06 ±0.03), (0.04 ±0.00) %ID/g at 2 h postinjection). Marked uptake of radioactivity was found in tumor tissue of the Balb/c nude mice bearing human pancreatic cancer with maximum T/NT ratio of 3.71 ± 0.57 at 2 h postinjection. Conclusions 99Tcm-(HYNIC-[Lys3]-BBS)(tricine) (TPPTS) can be easily prepared with high radiolabeling yield and radiochemical purity. The stability in serum and good biodistribution charateristics make it useful for the diagnosis of human pancreatic cancer with over-expression of the gastric-releasing peptide(GRP) receptor.

Hyoscyamine 6 beta-hydroxylase (H6H) is the last rate-limiting enzyme directly catalyzing the formation of scopolamine in tropane alkaloids (TAs) biosynthesis pathway. It is the primary target gene in the genetic modification of TAs metabolic pathway. Full-length cDNA and gDNA sequences of a novel H6H gene were cloned from Datura arborea (DaH6H, GenBank accession numbers for cDNA and gDNA are KR006981 and KR006983, respectively). Nucleotide sequence analysis reveals an open reading frame of 1375 bp encoding 347 amino acids in the cDNA of DaH6H, while the gDNA of DaH6H contains four exons and three introns, with the highest similarity to the gDNA of H6H from D. stramonium. DaH6H also exhibited the most identity of 90.5% with DsH6H in amino acids and harbored conserved 2-oxoglutarate binding motif and two iron binding motifs. The expression level of DaH6H was highest in the mature leaf, followed by the secondary root, and with no expression in the primary root based on qPCR analysis. Its expression was inhibited by MeJA. DaH6H was expressed in E. coli and a 39 kD recombinant protein was detected in SDS-PAGE. Comparison of the contents of scopolamine and hyoscyamine in various TAs-producing plants revealed that D. arborea was one of the rare scopolamine predominant plants. Cloning of DaH6H gene will allow more research in the molecular regulatory mechanism of TAs biosynthesis in distinct plants and provide a new candidate gene for scopolamine metabolic engineering.
Cloning, Molecular ; DNA, Complementary ; Datura ; enzymology ; genetics ; Escherichia coli ; Hyoscyamine ; chemistry ; Mixed Function Oxygenases ; genetics ; Plant Leaves ; enzymology ; Plant Roots ; enzymology ; Recombinant Proteins ; genetics ; Scopolamine Hydrobromide ; chemistry

Traumatic optic nerve injury is common in patients with traumatic brain injury,mainly in young adults.The optic nerve has no regenerative function,leaving problems of visual field defect and loss of vision and causing high disability rate among patients with traumatic brain injury.The current clinical treatments of traumatic optic nerve injury are hormonotherapy and decompression of optic nerve canal,lacking definite clinical efficacy and treatment norm.In recent years,a series of brand new treatments,such as neuroprotective drugs,neurotrophic factor therapy,nerve transplantation,stem cell therapy and gene therapy,have offered new perspectives for solving the problem of high disability rate of traumatic optic nerve injury clinically.This article attempts to summarize the status quo and latest progress in clinical treatment of traumatic optic nerve injury based on relevant literature in recent years,which can serve as reference for clinicians in choosing the optimal therapeutic regimen.

Chemical and Drug Induced Liver Injury ; Hair Dyes ; toxicity ; Humans ; Liver ; drug effects ; pathology ; Liver Diseases ; diagnosis ; drug therapy ; etiology ; Male ; Young Adult

AIMTo explore the effects of different doses of tyrosine modulation on behavioral performances in open field test of psychological stress rats.

METHODSThe animal model of psychological stress was developed by restraint stress for 21 days. Wistar rats were randomly assigned to five groups (n = 10) as follows: control group (CT), stress control group (SCT), low, medium and high-doses of tyrosine modulation stress groups (SLT, SMT and SIT). The changes of behavioral performances were examined by open-field test. Serum levels of cortisol, norepinephrine and dopamine were also detected.

RESULTSThe levels of serum cortisol were all increased obviously in the four stress groups, and their bodyweight gainings were diminished. The behavioral performances of SCT rats in open-field test were changed significantly in contrast to that of CT rats. However, The behavioral performances of SMT and SHT rats were not different from that of CT rats. In addition, the serum levels of norepinephrine and dopamine were downregulated obviously in SCT and SLT groups, and no differences were observed in other groups.

CONCLUSIONPsychological stress can impair body behavioral performances, and moderate tyrosine modulation may improve these abnormal changes. The related mechanisms may be involved with the changes of norepinephrine and dopamine.

Animals ; Behavior, Animal ; drug effects ; Dopamine ; blood ; Male ; Norepinephrine ; blood ; Random Allocation ; Rats ; Rats, Wistar ; Restraint, Physical ; psychology ; Stress, Psychological ; drug therapy ; physiopathology ; Tyrosine ; therapeutic use

AIMTo evaluate the effects of different doses of zinc on the expression of metallothionein isoforms in stressed hippocampal neurons in vitro.

METHODSThe cell stress model was developed by corticosterone. The cultured hippocampal neurons were assigned to seven groups as follows: control group, zinc deficiency group, and their corresponding stressed groups, as well as three different levels of zinc complementarity groups.

RESULTSIn zinc deficiency group, the expressions of metallothionein and MT-1 mRNA, MT-3 mRNA were downregulated. On the other hand, inductions of metallothionein and it's mRNAs in stressed zinc complementarity group were increased. In addition, the levels of supernatant IL-6 and NO were increased clearly in zinc deficiency group and corticosterone stressed groups.

CONCLUSIONOur results suggest that zinc deficiency may decrease while zinc complementarity increase the expressions of metallothioneins and MT-1 mRNA, MT-3 mRNA in stressed hippocampal neurons in vitro.

Animals ; Animals, Newborn ; Hippocampus ; cytology ; metabolism ; In Vitro Techniques ; Metallothionein ; metabolism ; Neurons ; metabolism ; RNA, Messenger ; Rats ; Zinc ; pharmacology

AIMTo observe the impairment of homocysteine (Hcy) on neurons in vitro and the related mechanisms.

METHODSWe examined the consequences of treatment of cultured rat cortical and hippocampal neurons with Hcy and detected the neurons' apoptosis, calcium influx, DNA damage and oxidative injury.

RESULTSPrimary cortical and hippocampal neurons were treated with Hcy (250 micromol/L) for 4 h resulted in apoptosis time-dependently. S-adenosyl methionine (SAM) could significantly, but MK-801, an NMDA receptor inhibitor, couldn't repress the Hcy induced neuron apoptosis. Hcy could induce neuron calcium overload through activating the NMDA receptors. The DNA of neurons was damaged by Hcy because the methylation reactions were inhibited. Hcy treatment also induced MDA level significantly increased, but did not affect the neurons' T-AOC.

CONCLUSIONThese findings indicate that Hcy compromises neuronal homeostasis by multiple, divergent routes, including DNA damage, neuron exitotoxicity, and oxidative injury. Hcy mediated neuron apoptosis was mainly due to DNA damage.

Animals ; Apoptosis ; Calcium ; metabolism ; DNA Damage ; Hippocampus ; drug effects ; pathology ; Homocysteine ; metabolism ; pharmacology ; Neurons ; drug effects ; metabolism ; Oxidative Stress ; Rats ; Rats, Wistar

Objective To study the physical-chemical parameters of antiviral drug GZ 914 and provide data for the preparation design .Methods The appearance , crystal structure and solubility of GZ 914 were investigated .An HPLC method was established to determine the content of GZ 914 in vitro before oil/water partition coefficient and solubility in different pH experiments were calculated .Results GZ914 was a straw yellow powder with a crystalline structure , low water-solubility and good lipotropy .The HPLC method had a good linear relationship within the range of 12-60 μg/ml (r=0.9998).The oil/water partition coefficient of GZ914 was 1.9.Conclusion This analytical method is accurate and reliable.The oil/water partition coefficient indicates that the drug could be formulated as an oral solid preparation.