OBJECTIVETo investigate molding technology of total alkaloids from Sophora alopecuroides freeze-dried powders and observe its inhibition effects on liver transplantation tumor in mice.

METHODWith color, clarity, water-soluble and formability as indexes, single factor tests were adopted to screen type and amount of filler, the concentration of total alkaloids in drug liquid, pH in order to determine optimum prescription of total alkaloids from S. alopecuroides freeze-dried powders, the lowest melting point was determined and freeze drying curve was drafted. Mice hepatoma H22 ascites tumor strain was sterile inoculated in right axillary subcutaneous of mice, and antitumor effect of total alkaloids from S. alopecuroides freeze-dried powders on liver transplantation tumor H22 in mice.

RESULTWhen selected 80 g x L(-1) as mannitol as filler, the concentration of total alkaloids in drug liquid was 25 g x L(-1) and pH 6.5-7.5, freeze-dried effect was optimum with fast reconstitute speed. Average inhibition rate of the big (120 mg x kg(-1)) and medium (60 mg x kg(-1)) dose group of total alkaloids from S. alopecuroides freeze-dried powders on liver transplantation tumor H22 in mice were 56.08% and 35.49%, respectively.

CONCLUSIONPreparation technology was reasonable, reproducible and stable, total alkaloids from S. alopecuroides freeze-dried powders had significant antitumor effect and showed a dose-effect relationship.

Alkaloids ; chemistry ; pharmacology ; Animals ; Antineoplastic Agents, Phytogenic ; chemistry ; pharmacology ; Cell Line, Tumor ; Color ; Freeze Drying ; Humans ; Liver Neoplasms ; drug therapy ; Mice ; Powders ; chemistry ; Sophora ; chemistry ; Tumor Burden ; drug effects

Field avian infectious bronchitis virus (IBV) designated as JS/9 5/03, which was isolated from Jiangsu province of china, was cultivated in chicken emb ryo. It's single strain RNA was extracted from purified virus and worked as temp late of reverse transcription polymerase chain reaction (RT-PCR), a pair of pri mer designed according to megalign results of published IBV sequences in Genbank was used to amplify the neucleocapsid gene, the RT-PCR product was sequenced d irectly. Sequence analysis revealed that the sequence of JS/95/03 is most homolo gized with that of M41 strain.

Altitude ; Animals ; CA1 Region, Hippocampal ; metabolism ; physiology ; Male ; Neural Cell Adhesion Molecules ; metabolism ; Rats ; Rats, Sprague-Dawley ; Time Factors

Objective To establish a method of gold nanoprobe-based solution hybridization (GNBSH) to detect nucleic acid sequence-based amplification (NASBA) products for the rapid diagnosis of invasive aspergillosis (IA).Methods The Aspergillus specific 18S rRNA was amplified by NASBA and then the amplified products were hybridized with the gold nanoprobes which were modified with thiol compounds at the 5'end.Serum samples from 106 patients,including 14 with a definite IA,32 with suspected IA and 60 without IA,were detected by the established method,and the obtained results were compared with that of galactomannan (GM) test to evaluate its accuracy.Results The gold nanoprobes only hybridized with Aspergillus NASBA products but not other non-Aspergillus strains.The sensitivity,specificity and the area under the ROC curve (AUCROC) of the established GNBSH method for detecting 106 clinical samples were 82.61％ (38/46),81.67％ (49/60) and 0.890,respectively.The sensitivity,specificity and AUCROC of GM test were 56.52％ (26/46),83.33％ (50/60) and 0.723,respectively.Conclusion The established GNBSH method to detect Aspergillus NASBA products has high sensitivity and specificity and simple operation,which may be used to detect the infection of Aspergillus by clinical laboratories.

Objective To investigate the effect of combined use of insulin and acarbose on glucose excursion in type 1 diabetic patients.Methods 120 cases were randomly divided into control group and observation group.The control group received preprandial ultra-short effect insulin and long-acting insulin before bedtime while the observation group received acarbose 50 mg added to the medicine taken by the control group.Continuous Glucose Monitoring System (CGMS) was used to watch the blood glucose fluctuations.Data related to blood glucose level,glucose excursions after meals and hypoglycemia at night were compared between patients in the two groups.Results The average blood glucose (9.37 ± 1.70) mmol/L,the largest amplitude of glycemic excursions (LAGE) ( 11.42 ± 2.73 ) mmol/L,hyperglycemia-area under curve 0.89 ± 0.54,mean amplitude of glycemic excursions (MAGE) (5.13 ± 2.23) mmol/L,M-value (18.93 ± 11.43) mmol/L and insulin dosage (42.11 ± 14.42)U/day of observation group were significantly lower than in the control group (P＜0.05 ).Glucose excursions after meals and the times( 0.33 ± 0.50 )/day,the maintenance time (43.75 ± 43.50)/min and low glycemic index ( LBGI ) (0.005 ± 0.002 ) mmol/L of hypoglycemia at night were also significantly lower than in the control group,with statistically significant (P＜0.05) differences.Conclusion The blood glucose fluctuation was significantly improved,with the decrease of insulin dosage while both glucose excursions and hypoglycemia at night reduced in patients with typel diabetes mellitus after the acarbose treatment.We suggested that this program deserve further observation.

AIM: To analyze the effect of pigment epithelium derived factor (PEDF) on nitrogen monoxide (NO) and expression of cysteine-containing, aspartate-specific proteases-3 (caspase-3) in retinal tissues of model rats with optic nerve crush injury.METHODS: A total of 60 SD rats were randomly divided into the blank control group, model group and PEDF group, with 20 rats in each group.Except the blank control group, the optic nerve crush injury rat models were established in the other groups, and left eyeballs were taken as samples.After successfully modeling, the model group were treated with intravitreal injection of 5μL of balanced salt solution while PEDF group were treated with intravitreal injection of 5μL of PEDF (0.2μg/μL).Two weeks later, the retinal tissues were collected, and changes of shape were observed under microscope after HE staining.The changes of NO level were measured by colorimetry assay, the expression of caspase-3 mRNA and caspase-3 protein was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western-blot.RESULTS: HE staining showed that retinal tissues of the blank control group arranged neatly and clearly.Retinal ganglion cells (RGCs) arranged in a monolayer, and cells were oval, uniform in size and distribution, the cell nuclei were clear, closely arranged, with clear boundaries.The retinal tissues of the model group were sparse in shape, RGCs showed vacuolar changes, the overall number of cells was reduced, and cell nuclei of residual RGCs showed pyknosis and uneven staining.RGCs in PEDF group were with slightly edema and arranged closely, and the degree of injury was significantly milder than that in the model group.Levels of Caspase-3 mRNA and protein and NO levels in the three groups showed the model group > PEDF group > blank control group (all P < 0.05).CONCLUSION: The application of PEDF can down regulate the expression of Caspase-3 and NO in rates with optic nerve injury and reduce RGCs injury.

OBJECTIVETo assess the influence of polymorphisms of methylenetetrahydrofolate reductase (MTHFR) gene on response to antidepressant treatment.

METHODSTwo hundred and eight one Chinese Han patients have received single antidepressant drugs for at least 6 weeks, among whom 275 were followed up for 8 weeks. Hamilton depression scale 17 (HAMD-17) was used to evaluate the severity of depressive symptoms and therapeutic effects. Single nucleotide polymorphisms (SNPs) of the MTHFR gene were determined using gene chips. Associations of single loci and haplotypes with response to treatment were analyzed using an Unphased 3.0.13 software.

RESULTSNo significant differences in gender, age, year of education, family history, episode times, and antidepressant agents were found between responders and non-responders (all P U+003E 0.05), while the baseline scores of HAMD-17 was significantly different(t=2.891, P=0.004). There was also no significant difference between age, years of education, family history, baseline scores of HAMD-17 and antidepressant agents between remitters and non-remitters (both P U+003E 0.05), while proportion of male patients was significantly higher in non-remission group than remission group (t=2.381, P=0.018), and episode times in non-remission group was significantly higher (t=-1.983, P=0.049). Single locus association analysis has found no significant association between SNPs rs1801131 and rs1801133 in the MTHFR gene with antidepressant response (P U+003E 0.05). On the other hand, haplotype A-C of MTHFR gene (rs1801131 and rs1801133) was significantly associated with antidepressant response in total group (U+03C7 2=11.39, P=0.0007), male subgroup (U+03C7 2=8.767, P=0.003) and serotonin noradrenaline reuptake inhibitors (SNRIs) subgroup (U+03C7 2=10.51, P=0.001).

CONCLUSIONParticular haplotype of MTHFR gene may be related with antidepressant effect, in which the haplotype (rs1801131, rs1801133) A-C type may be associated with better antidepressant efficacy, particularly in males and patients receiving SNRIs drugs.

Adult ; Alleles ; Antidepressive Agents ; therapeutic use ; Depressive Disorder, Major ; drug therapy ; genetics ; Female ; Haplotypes ; Humans ; Male ; Methylenetetrahydrofolate Reductase (NADPH2) ; genetics ; Middle Aged ; Polymorphism, Single Nucleotide ; Serotonin Uptake Inhibitors ; therapeutic use ; Sex Factors ; Treatment Outcome ; Young Adult

OBJECTIVETo construct wild-type and mutant pEGFP SPAST vectors and to explore the molecular mechanism of hereditary spastic paraplegia.

METHODSMutant SPAST vector was constructed using overlap PCR method following construction of wild-type SPAST vector. Wild-type and mutant constructs were transfected to COS7 cells and subcellular localization of spastin was observed. Co-localizations of spastin and microtubule, spastin and mitochondria were viewed by immunofluorescence staining.

RESULTSWild-type spastin is localized in plasma, and mutant spastin did not change its cellular localization. Wild-type and mutant spastins did not co-localize with microtubules and mitochondria by immunofluorescence analysis.

CONCLUSIONWild-type and mutant SPAST constructs were successfully generated. Mutant spastin did not change its localization in cells. Spastin does not co-localize with microtubules and mitochondria. This study may facilitate further studies on molecular mechanism of hereditary spastic paraplegia.

Adenosine Triphosphatases ; genetics ; metabolism ; Animals ; Base Sequence ; Cell Line ; Genetic Vectors ; genetics ; Humans ; Mitochondria ; genetics ; metabolism ; Mutation ; Spastic Paraplegia, Hereditary ; genetics ; metabolism ; Spastin

The effects of over-expression of testis-specific expressed gene 1 (TSEG-1) on the viability and apoptosis of cultured spermatogonial GC-1spg cells were investigated, and the immortal spermatogonial cell line GC-1spg (CRL-2053™) was obtained as the cell model in order to explore the function of TSEG-1. We transfected the eukaryotic vector of TSEG-1, named as pEGFP-TSEG-1 into cultured spermatogonial GC-1spg cells. Over-expression of TSEG-1 inhibited the proliferation of GC-1spg cells, and arrested cell cycle slightly at G0/G1 phase. Transfection of TSEG-1 attenuated the transcript levels of Ki-67, PCNA and cyclin D1. In addition, over-expression of TSEG-1 induced early and late apoptosis, and reduced the mitochondrial membrane potential of GC-1spg cells. Moreover, transfection of TSEG-1 significantly enhanced the ratio of Bax/Bcl-2 and transcript levels of caspase 9, and decreased the expression of Fas and caspase 8 in GC-1spg cells. These results indicated over-expression of TSEG-1 suppresses the proliferation and induces the apoptosis of GC-1spg cells, which establishes a basis for further study on the function of TSEG-1.

Objective: To observe the changes of PD-1 expression, mRNA level and cytotoxic activity of CD19 CAR-T cells during the culture process of CAR-T cells. Methods: The peripheral blood T cells of 6 lymphoma patients with high expression of PD-1 and 6 healthy volunteers were the source of CAR-T cells. The expression of PD-1 was analyzed by flow cytometry. The mRNA level of PD-1 was analyzed by PCR. The cell proliferation was analyzed by CCK-8 assay. The cytotoxicity was analyzed by LDH assay. Results: ①The transfection efficiency of high PD-1 expression T cells and healthy volunteer T cells were as the same (P>0.05) . ②The cell proliferation capacity of CD19 CAR-T cells from high PD-1 expression T cells or healthy volunteer T cells, with or without PD-1 inhibitor were as the same (P>0.05) . ③The cytotoxicity to lymphoma cells of high PD-1 expression T cells and CAR-T cells were lower than that of these two T cells combined with PD-1 inhibitor and the CAR-T cells from healthy volunteer T cells (P<0.001) . There was no difference of the cytotoxicity between the CAR-T cells from high PD-1 expression T cells combined with PD-1 inhibitor and the CAR-T cells from healthy volunteer (P>0.05) . ④There was no difference of the expression of PD-1 in all CAR-T cell groups during the culture process (P>0.05) . There was no difference of mRNA level of PD-1 in all groups during the culture process (P>0.05) . ⑤The PD-1 expression of CAR-T cells increased by the time of culture after contacting with lymphoma cells (P<0.001) . The PD-1 inhibitors could antagonize this effect. There was no difference of mRNA level of PD-1 in all groups after contacting with lymphoma cells (P>0.05) . Conclusion: The PD-1 expression of CAR-T cells from high PD-1 expression T cells increased by the time of culture after contacting with lymphoma cells. However, the mRNA level of PD-1 of all groups did not change, even if PD-1 inhibitor was applied.
Antigens, CD19 ; Humans ; Programmed Cell Death 1 Receptor/genetics* ; RNA, Messenger ; Receptors, Antigen, T-Cell ; T-Lymphocytes