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

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

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.

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 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

Objective To investigate the safety and effectiveness of posterior approach laminectomy combined with localized resection of ossified posterior longitudinal ligament and dekyphosis for multilevel ossification of the posterior longitudinal ligament (OPLL) in the thoracic spine.Methods Thirty-one cases of thoracic multilevel OPLL was treated with this new technique between August of 2012 and August of 2016.Twenty-nine among the 31 cases were successfully followed up more than two years.Among these 29 cases,9 were male and the other 20 were female,with an average age of 48.5±7.1 years.The average segment number of OPLLwas 6.5±2.2 (range,3-11).The average segment number of laminectomy was 7.9±2.5 (range,4-13).There were 26 cases combined with ossification of the ligamentum flavum (OLF).Posterior approach laminectomy combined with localized resection of OPLL and dekyphosis for multilevel OPLL in the thoracic spine was applied to all cases.Firstly,en-bloc laminectomy was performed to all the segments of OPLL.Then the nearest segment of ossification to the kyphotic apex and the most stenotie level was selected and limitedly resected.Finally,wedge-shaped osteotomy was conducted to decrease the kyphosis.The outcomes including recovery rate of myelopathy and the radiological changes were recorded during the post-operative follow-up.Single group pre and post analysis was conducted by using paired t-test.Results Twenty-seven cases underwent one-level circumferential decompression,and the other two case underwent two-level localized resection of the ossified posterior longitudinal ligament.The average operation time was 245.2±75.1 min (range,131-423 min).The average blood loss was 1 307.9±1 457.7 ml(range,300-6 000 ml).The average follow-up time was 40.2± 14.9 months (range,25-69 months).The kyphotic angle of the stenotic segments decreased 11.4°±3.5° averagely after the surgery,from pre-operative 28.7°±9.6° to post-operative 17.3°±8.6°.The decreased kyphotic angle was 7.4°±3.1 ° at the final follow-up with an average kyphotic angel of 22.3°± 10.3°.The average length of the resected ossified posterior longitudinal ligament was 11.3±3.9 mm,and the average shortening length of the spinal column was 5.0±3.0 mm (range,0.4-13.8 mm).The pre-operative Japanese Orthopedic Association (JOA) score was 4.3±2.2 averagely (range,1-9),and the final JOA score increased to 9.3±2.3 (rang,3-11).The average recover rate was 85.7％ (range,-100％ to 100％),and the rate of excellent or good was 89.7％.Among the 29 cases,6 cases occurred post-operative transient deterioration and regained a satisfactory recovery eventually;one case occurred post-operative paraplegia and never recovered;19 cases occurred post-operative cerebrospinal fluid leakage and healed under conservative treatment.Conclusion For the thoracic multilevel OPLL,one-stage posterior approach laminectomy combined with localized resection of the ossified posterior longitudinal ligament and dekyphosis can significantly improve the outcomes of the myelopathy with low rate of post-operative paraplegia.Therefore,this new surgery technique is a safe and effective treatment for multilevel OPLL in thoracic spine.

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.

Objective To investigate serum levels of soluble matrix lysin 2 (sST2) in patients with different stages of heart failure and its relationship with prognosis. Methods Data of 300 patients with heart failure of stages A, B, C and D were included in this study. Thirty-three cases of healthy elderly population for physical examination were used as control group. The general information, echocardiography and related biochemical tests containing sST2 and NT-proBNP were collected in the two groups. The survival periods of patients were evaluated according to the Seattle heart failure mode (SHFM). Patients were followed up for 1 year to record the occurrence of adverse events. Results The sST2 level was higher in heart failure group than that of control group. The sST2 level began to increase in stage B, and which increased with the development of cardiac function staging. The sST2 levels were significantly higher in stages B, C and D than those of stage A, and which were significantly higher in stage D than those of stages B and C (P<0.05). There were significantly higher incidence rates of adverse events, left ventricular end diastolic diameter (LVEDD) and left ventricular mass index (LVMI) in the patients with high sST2 level than those of patients with lower sST2 level (P<0.05). Values of sST2, NT-proBNP, LVEDD and LVMI were significantly higher, and values of LVEF and SHFM life expectancy were significantly lower, in patients with adverse events than those of patients without adverse events (P<0.05). There was a negative correlation between sST2 and LVEF, and positive correlation between sST2 with NT-proBNP, LVEDD and LVMI (P<0.05). The size under ROC curve, which was used to predict the cardiovascular endpoint events judged by sST2 was 0.665 (95％CI:0.574-0.757, P<0.01), and the one by NT-proBNP was 0.790 (95％ CI: 0.731-0.848, P<0.01). The best cut-off value of predicting the clinical adverse events was 139.27μg/L by sST2 and 855.35μg/L by NT-proBNP. Conclusion The serum level of sST2 is early indicator of heart failure, which not only reflects the severity of ventricular remodeling but also is one of indicators to estimate the prognosis of heart failure in one year.