AIMTo construct the RNAi eukaryotic vector of inhibitory member of the prohibitin (PHB-1) gene and observe the interfering effect in HEK293 cell line after the vector transfection.

METHODSThe specific Mi RNA sequence was designed according to the PHB-1 sequence in GenBank, complementary single-strand DNA oligonucleotides were designed and synthesized, and annealed the single-stranded oligonucleotides to generate a double strands oligonucleotides , cloned the oligonucleotides into pcDNATM6.2-GW/EmGFP-MiR-PHB to obtain an entry clone and then sequence analysis was performed. The recombinant plasmid was transfected into HEK293 cell by liposome. PHB-1 expression was detected by Western blotting.

RESULTSThe DNA sequence of interest clone to the vector was constructed to generate an entry clone and an expression clone successfully, which were proved by sequence determination. Western blotting analysis demonstrated that PHB-1 MiR RNA expression construction could suppress the expression of PHB-1.

CONCLUSIONA RNAi eukaryotic vector containing prohibitin gene was successfully constructed.

Genetic Vectors ; genetics ; HEK293 Cells ; Humans ; MicroRNAs ; biosynthesis ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Repressor Proteins ; biosynthesis ; genetics ; Transfection

OBJECTIVETo compare the efficacy difference in the treatment of insomnia between scraping technique of stuck needle and conventional acupuncture at Anmian (Extra).

METHODSOne hundred and thirty one cases were randomized into an Anmian group (68 cases) and a conventional acupuncture group (63 cases). In the Anmian group, Anmian (Extra) was selected. After arrival of qi, the stuck needling was adopted by rotating the needle gently in single direction, 2-3 rounds till the needle body was stuck tightly. Afterwards, the needle tail was touched gently with the index finger to fix the needle body and the needle handle was scraped gently with the thumbnail from bottom to up. The needle was retained for 30 min. In the conventional acupuncture group, Sanyinjiao (SP 6), Shenmen (HT 7) and Baihui (GV 20) were selected and stimulated with reducing technique by rotating the needles. The needles were retained for 30 min. The treatment was given once every day, continuously for 2 weeks in both groups. The score of each factor and the total score in Pittsburgh sleep quality index (PSQI) were assessed before and after treatment in the two groups. Additionally, the efficacies of two groups were evaluated.

RESULTSFor the patients in the conventional acupuncture group, the sleep quality and time of falling into spleen after treatment were improved as compared with those before treatment (all P < 0.01). The improvement of sleep time, sleep efficiency, sleep disturbance, hypnotic drug and daytime dysfunction, and PSQI total score did not present statistically significant difference as compared with those before treatment (all P > 0.05). After treatment, for the patients in the Anmian group, the factor score and total score in PSQI were apparently improved as compared with those before treatment (P < 0.01, P < 0.05). And the improvements of the above items were superior to the conventional acupuncture group (P < 0.01, P < 0.05). The total effective rate was 91.2% (62/68) in the Anmian group and was 74.6% (47/63) in the conventional acupuncture group. The clinical efficacy in the Anmian group was apparently superior to the conventional acupuncture group.

CONCLUSIONThe scraping technique of stuck needle at Anmian (Extra) achieves the superior effect on insomnia as compared with the conventional acupuncture.

Acupuncture Therapy ; instrumentation ; methods ; Adult ; Aged ; Female ; Humans ; Male ; Middle Aged ; Needles ; Sleep ; Sleep Initiation and Maintenance Disorders ; physiopathology ; therapy ; Treatment Outcome ; Young Adult

OBJECTIVETo explore whether the nerve growth factor has protective effects on PC12 cells from injury induced by 2, 5-hexanedione.

METHODSWith PC12 cells as the model of neurons, different concentrations of NGF were added into the culture of PC12 cells. Then cell viability was tested with MTT. The DNA fragment was observed with agarose gel electrophoresis. The apoptosis ratio was tested with flow cytometry (FACS). The p53 protein was detected with western blot. The differences among the groups were compared.

RESULTSCell viabilities were increased with the increase of the concentrations of NGF (P < 0.05). The DNA fragment, the apoptosis ratio and the expression of p53 were all decreased with the increase of the concentrations of NGF (P < 0.05).

CONCLUSIONThe NGF might have direct nutritional effects on PC12 cells, and protect them from injury induced by 2, 5 HD. Moreover, it might also have anti-apoptosis effect to some extent.

Animals ; Apoptosis ; drug effects ; DNA Fragmentation ; drug effects ; Dose-Response Relationship, Drug ; Electrophoresis, Agar Gel ; Flow Cytometry ; Hexanones ; toxicity ; Mice ; Nerve Growth Factors ; pharmacology ; PC12 Cells ; Rats ; Tumor Suppressor Protein p53 ; biosynthesis

OBJECTIVETo investigate abnormal liver function associated with polymorphism of GSTT1, GSTM1 and CYP2E1 in workers exposed to N, N-dimethylformamide.

METHODSSixty-nine workers with abnormal liver function in a synthetic leather factory were recruited as case. One hundred and twenty five control subjects with similar work tasks were selected from the same factory. Genotypes for GSTT1 and GSTM1 were determined by multiplex PCR, and for CYP2E1 PstI by PCR-RFLP assay.

RESULTSThe frequency of positive GSTM1 was 59.42% in cases and 38.40% in control, with an odds ratio (OR) of 2.34,95% CI: 1.29-4.29 (P=0.005). For GSTT1 and CYP2E1 PstI, the frequencies of genotypes showed no significant difference between case and control.

CONCLUSIONGSTM1 positive genotype may be genetic risk factors for development of abnormal liver function in workers exposed to N, N-dimethylformamide.

Adult ; Chemical and Drug Induced Liver Injury ; etiology ; genetics ; Cytochrome P-450 CYP2E1 ; genetics ; Dimethylformamide ; adverse effects ; Female ; Genotype ; Glutathione Transferase ; genetics ; Humans ; Male ; Occupational Exposure ; adverse effects ; Polymorphism, Genetic

OBJECTIVETo investigate the hepatotoxic effects of N, N-dimethylformamide (DMF) in the workers of a synthetic leathers factory, and the effects on liver function of covariates such as alcohol consumption and other factors.

METHODSThe workers were classified into three groups (low, high and the control) by the concentration of DMF in workplace which was determined in the past two years. A questionnaire was drawn up for relevant demographic characteristics and other factors influencing liver function. The bloods were collected for laboratory test which included parameters especially relevant to the liver (ALT AST and gamma GT).

RESULTSLow and high-exposure groups were significantly associated with elevated ALT and gamma GT, and high-exposure group was significantly associated with elevated Liver index. Modeling by stepwise regression analysis demonstrated that high concentration of DMF and BMI were associated with and elevated ALT, gamma GT and Liver index, besides DMF and BMI, the elevation of ALT was also associated with high TRIG. AST was only associated with alcohol consumption. The AST/ALT ration < 1 was present in 86.7% of the exposure workers of liver function abnormal.

CONCLUSIONDMF can cause liver function alternations even if air concentration of DMF was below PC-TWA. Besides the levels of DMF exposure, obesity (BMI) and alcohol consumption are covariates alternating liver function. Liver index can be a parameter for assessment liver function, and the AST/ALT ration < 1 may serve as markers of risk in health screening programs.

Adult ; Alanine Transaminase ; blood ; Dimethylformamide ; toxicity ; Female ; Humans ; Liver ; metabolism ; physiopathology ; Liver Function Tests ; Male ; Occupational Exposure

The aim of this study was to investigate the expression of transferrin receptor 2 (TfR2) mRNA in bone marrow mononuclear cells (BMMNC) of children with hyperplastic anemia (HA), to analyze the correlation of TfR2 mRNA expression level with Hb level, bone marrow erythroid hyperplasia, iron status in body and underlying diseases, and to evaluate the role of TfR2 in erythroid hemopoiesis and the useful value in diagnosis of HA. The experiment was divided into 2 groups: test group, in which 40 patients with HA were enrolled, and control group in which 10 patients without erythroid disorders and hematological malignancies confirmed by bone marrow examination were enrolled. The bone marrow samples of patients in mentioned above 2 groups were collected, the TfR2 mRNA expression in BMMNC of patients with HA was detected by fluorescence-quantitative PCR, the correlation of HA with bone marrow erythroid hyperplasia, iron status of body and underlying diseases was analyzed. The results showed that the relative level of TfR2 mRNA expression in HA patients was significantly higher than that in control patients. The TfR2 mRNA expression level negatively correlated with Hb level in peripheral blood (r = -0.715), while it positively correlated with ratio of bone marrow erythroblasts (r = 0.533). It is concluded that TfR2 mRNA expression in HA patients increases and closely correlates with hyperplasia status of bone marrow and anemia level in peripheral blood.
Adolescent ; Anemia ; metabolism ; pathology ; Bone Marrow Cells ; metabolism ; Case-Control Studies ; Child ; Child, Preschool ; Erythroid Precursor Cells ; metabolism ; Female ; Humans ; Infant ; Male ; RNA, Messenger ; metabolism ; Receptors, Transferrin ; metabolism

OBJECTIVETo evaluate cord blood stem cell transplantation (CBT) in the treatment of X-linked agammaglobulinemia, and observe the courses of the hematopoietic and immune reconstitution.

METHODSA 14-year-old male patient with agammaglobulinemia received CBT from a 1/6 HLA-mismatched unrelated cord blood. The conditioning regimen was Bu/Cy/anti-CD3 antibody. CsA was given together with MMF and MTX for prophylaxis of GVHD. The patient received 0.42 x 10(8) nucleated cells/kg, containing 0.35 x 10(6) CD34(+) cells/kg.

RESULTSThe recipient showed hematopoietic reconstitution on day 30 post-transplantation when ANC was 0.5 x 10(9)/L and BPC 20 x 10(9)/L. Sex chromosome analysis showed engraftment (donor 46, XX/recipient 46, XY = 4:1) on day 45. The recipient's blood group changed from AB to O, IgG from 1.1 g/L to 3.5 g/L, sex chromosome from 46, XY to full 46, XX, and mature B cells in peripheral blood from 0 to 5% on day 100, indicating immune reconstitution. At the last follow-up of 360 days, the patient without acute or chronic GVHD showed normal hemogram and myelogram, IgG 13.5 g/L and 10% mature B cells in peripheral blood, indicating the hematopoiesis and immune persistent reconstitution. No acute or chronic GVHD was developed.

CONCLUSIONThis is the first case report of successful treatment of X-linked agammaglobulinemia by HLA-mismatched unrelated CBT.

Adolescent ; Agammaglobulinemia ; surgery ; Cord Blood Stem Cell Transplantation ; methods ; Graft vs Host Disease ; prevention & control ; HLA Antigens ; immunology ; Humans ; Male ; Transplantation Conditioning ; Treatment Outcome

OBJECTIVETo evaluate the effect of a 10-day course of triptolide (TP) on rat cytochrome (CY) P3A4 activity, and on the pharmacokinetics of cyclophosphamide (CPA).

METHODSIn the pharmacokinetics experiment, rats were orally given 0.9% NaCl solution (n=5) and TP [1.2 (mg/kg·d)] for 10 days and a single dose of CPA was administered intravenously (100 mg/kg) to rats on day 11. Blood samples were collected up to 4 h at predetermined time intervals, the plasma concentration of CPA was determined by high performance liquid chromatography (HPLC) and pharmacokinetic parameters were determined. In the in vitro CYP3A4 activity inhibition research, rat blank liver microsomes were divided into 3 groups: a control group, a TS (5 μL, 200 μmol/L) with TP (5 μL, 12.5 μmol/L) group, a TS with ketoconazole (5 μL, 1 μmol/L) group. Concentration of 6β-hydroxylated testosterone (6β-OHT) in liver microsomes was measured by HPLC and the activity of CYP 3A4 was calculated through the following formula: Einhibitor/Econtrol × 100%=Cinhibitor/Ccontrol × 100%.

RESULTSCompared with the control group, the area under the plasma concentration-time curve (AUC0-∞) of CPA was significantly increased by 229.05% pretreated with TP (P<0.01). Peak plasma concentrations (Cmax) of CPA was significantly increased and plasma half-life was correspondingly extended. The CYP3A4 activity was significantly inhibited by ketoconazole 93.5%±0.2% and TP 84.6%±0.3% compared with the control group (P<0.01 and P<0.05, respectively).

CONCLUSIONOur results strongly suggested that long-term oral intake of TP can distinctly inhibit the CYP3A4 activity and this inhibition evidently decrease the formation of toxic metabolites of CPA.

Animals ; Cyclophosphamide ; pharmacokinetics ; Cytochrome P-450 CYP3A ; metabolism ; Cytochrome P-450 CYP3A Inhibitors ; pharmacology ; Diterpenes ; pharmacology ; Epoxy Compounds ; pharmacology ; Herb-Drug Interactions ; Hydroxytestosterones ; metabolism ; Immunosuppressive Agents ; pharmacokinetics ; Injections, Intravenous ; Ketoconazole ; pharmacology ; Male ; Microsomes, Liver ; drug effects ; metabolism ; Phenanthrenes ; pharmacology ; Rats, Sprague-Dawley

Mitochondrial ferritin (MtF), a new player in iron metabolism, first identified in 2001, is highly homologous to ferritin both structurally and functionally. Preliminary studies have suggested that MtF might play very important roles in the regulation of mitochondrial iron homeostasis. Leukemic cells, just like other malignant cells, demand more iron for their greater proliferation potential. However, little is known about what roles MtF might play in leukemic cell iron metabolism and cell proliferation. The aim of this study was to investigate the expression of MtF, transferrin receptor 1 (TfR1) and ferritin (Fn) mRNAs in K562 leukemic cells during TPA-induced cell differentiation and to explore the interrelationship between the expression levels of these iron metabolism-related molecules. K562 cells cultured with or without TPA (16 nmol/L) were collected at 24, 72 and 120 hours respectively. Cell differentiation toward monocyte lineage was confirmed by microscopic study (Wright's staining) and flow cytometry. Semiquantitative RT-PCR was performed to determine mRNA expression, with house-keeping gene beta-actin as control reference. This study revealed that over 95% of K562 cells showed morphological features of monocyte/macrophage, and the expression of CD64 on cell surface increased significantly at day 5 with TPA treatment. K562 cells could express a certain level of MtF before TPA-induced differentiation. With increase of TPA-induced cell differentiation, MtF mRNA expressions were downregulated progressively. After 5 days of induced cell differentiation, expression levels of MtF and TfR1 mRNA were just 50.3% and 68.2% of that before TPA treatment. While Fn mRNA expression increased to 1.97 folds of that before TPA treatment. It is concluded that MtF expression is downregulated during TPA-induced K562 cell differentiation, with concomitant downregulation of TfR1 and upregulation of Fn. The coordinated expression regulation of these key iron metabolism-related molecules during cell differentiation may in turn inhibit TfR1-mediated iron uptake via endocytosis and thus adversely affect cell proliferation potential.
Antigens, CD ; metabolism ; Cell Proliferation ; Cell Transformation, Neoplastic ; drug effects ; Ferritins ; biosynthesis ; genetics ; Humans ; Iron-Regulatory Proteins ; metabolism ; K562 Cells ; Mitochondria ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, Transferrin ; metabolism ; Tetradecanoylphorbol Acetate ; pharmacology

BACKGROUNDPain has a substantial impact on patients' activities and overall quality of life, but current conventional drugs have debilitating side effects, including gastrointestinal disorders. Thus there is a pressing need for new therapies with fewer side effects to alleviate cancer pain. We recently developed a topical herbal formula Xiaotan Tongluo analgesic gel (XTTL gel) based on the principles of traditional Chinese herbalism, and we have received positive feedback from bone cancer pain patients. The aim of this study was to determine the analgesic effects and explore the mechanisms of XTTL gel in a rat model of bone cancer pain.

METHODSThe rat model of bone cancer pain was established by inoculating Walker-256 rat carcinoma cells directly into the right tibial medullary cavity of Wistar rats. The rats were randomly assigned to three groups (n = 10 per group): (1) sham bone cancer control (sham group): vehicle (PBS) inoculation without carcinoma cells plus topical administration of blank gel; (2) Sham treatment control (vehicle group): Walker-256 cell inoculation plus topical administration of blank gel; (3) XTTL gel treatment (treatment group): Walker-256 cell inoculation plus topical administration of XTTL gel. XTTL gel treatments were applied daily for 7 days starting on day 14 following inoculation. Outcomes were assessed 21 days after inoculation by mechanical allodynia, histological staining, and by measuring concentrations of type I collagen carboxy-terminal telopeptide (ICTP) and bone-specific alkaline phosphatase (BAP) in serum.

RESULTSFourteen days after cancer cell incubation, significant mechanical allodynia in the ipsilateral hind paw and tumor growth in proximal end of the tibia were observed in the vehicle and treatment groups but not in the sham group. At day 21, mechanical withdrawal thresholds in treatment group rats were significantly higher ((4.8557 +/- 0.8336) g) compared with those of the vehicle group ((1.8630 +/- 1.4369) g, P < 0.05). ICTP and BAP levels increased significantly in vehicle group rats ((101.5176+/- 11.0694) U/L and (370.7838 +/- 12.8273) U/L, respectively) compared with those of the sham group ((11.7553 +/- 1.1885) U/L and (185.7338 +/- 3.6761) U/L, respectively; P < 0.05). XTTL gel decreased the level of blood serum ICTP ((41.8998 +/- 6.4970) U/L, P < 0.05) but had little effect on blood serum BAP ((365.5338 +/- 18.5361) U/L, P > 0.05).

CONCLUSIONTopical use of XTTL gel may have an analgesic effect on bone cancer pain, an effect mediated by lowering of ICTP levels and inhibiting bone resorption.

Alkaline Phosphatase ; metabolism ; Animals ; Body Weight ; Bone Neoplasms ; complications ; Cell Line, Tumor ; Collagen Type I ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Pain ; drug therapy ; etiology ; Peptide Fragments ; blood ; Peptides ; Procollagen ; blood ; Random Allocation ; Rats ; Rats, Wistar