GLuc (Gaussia luciferase) is a secreted luciferase with high sensitivity. In this study, we primarily compared expression character of PTTR with that of PCMV, relied on easy secretion, high sensitivity and simple and fast detection of GLuc. We firstly constructed two plasmids pAAV2-neo-TTR-GLuc and pAAV2-neo-CMV-GLuc. Then, 4 cell lines were transfected with the two plasmids in aid of Lipofectamine 2000, including Huh7 and HepG2, which are derived from liver cells, as well as HEK293 and HeLaS3 cells, which are non-liver cell lines. We monitored the expression of GLuc in the supernatant of these cell cultures at different time points post-transfection. Furthermore, we injected the two plasmids with different doses into BALB/c mice by the means of hydrodynamic delivery and monitored the GLuc expression in vivo with 2.5 microl tail tip blood since 2 h post-injection. The cell assay results suggested that the expression of GLuc driven by CMV promoter was significantly higher than that of GLuc driven by TTR promoter. And, the luciferase activity of GLuc driven by CMV promoter was 50-300 times higher than that of GLuc driven by TTR promoter in HEK293 and HeLaS3 cell lines, but less than 10 times higher than that of GLuc driven by TTR promoter in the HepG2 and Huh7 cell lines, indicating the relative liver-specificity of TTR promoter. In the animal assay, the higher luciferase activity was determined in CMV promoter group than in TTR promoter group at different doses of the two plasmids. But the expression patterns for the two promoters differed obviously. The expression of GLuc driven by CMV promoter reached the maximum 10 hours post-injection and declined rapidly; while the expression of GLuc driven by TTR promoter reached the maximum 48 hours after delivery, and declined very slowly. These results implied that PTTR could keep expression of driven gene in a long time although its expression intensity is lower than PCMV's. Thus, it is more suitable for maintaining longer expression of target genes in liver.
Animals ; Cell Line ; Cytomegalovirus ; genetics ; metabolism ; Gene Expression ; Gene Transfer Techniques ; Genes, Reporter ; Humans ; Luciferases ; genetics ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Prealbumin ; genetics ; metabolism ; Promoter Regions, Genetic

OBJECTIVETo investigate the association of Coxsackie B virus (CBV) with habitual abortion.

METHODSCBV IgM antibody, viral RNA and virions were detected in 86 women with habitual abortion and 40 with induced abortion by means of enzyme-linked immunosorbent assay (ELISA), RT-PCR and virus isolation, respectively.

RESULTSThe positivity rate of CBV IgM were 87.2% and 35% in the two groups, respectively, and the detection rate of the viral RNA was 53.5% and 17.5% in blood lymphocytes, and 59.3% and 17.5% in the placentas. The virions were found in the placentas in 41.9% and 6.9% of the women, respectively. The positivity rates of CBV IgM, viral DNA and virions showed significant difference between the two groups (P<0. 01).

CONCLUSIONCBV might be one of the causes responsible for habitual abortion.

Abortion, Habitual ; blood ; etiology ; Adult ; Antibodies, Viral ; blood ; Coxsackievirus Infections ; complications ; virology ; Enterovirus B, Human ; immunology ; isolation & purification ; Female ; Humans ; Immunoglobulin M ; blood ; Lymphocytes ; ultrastructure ; virology ; Microscopy, Electron ; Pregnancy ; Pregnancy Complications, Infectious ; blood ; virology ; RNA, Viral ; blood

Objective To study the cytotoxicity of multiple gliomaassociated antigens sensitized dentritic cell activated cytotoxic T lymphocytes (GDC-CTL) on the human glioma cell line U87 in vitro and the anti-tumor effect of GDC-CTL on the BALB/c nude mouse model of malignant glioma in vivo.Methods Multiple glioma-associated antigens sensitized dentritic cell (GDC) and GDC-CTL were prepared and then analyzed with the phenotypes by flow cytometry.Cytotoxicity of GDC-CTL on U87 cells was determined by CCK8 assay and the level of interferon-γ (IFN-γ) secreted from GDC-CTL co-culturing with U87 cells for 48 h was detected by ELISA at different effect/target ratios (5∶ 1,10∶1,20∶1).The T lymphocytes without activation with GDC were evaluated as the control group.The BALB/c Nude mice tumor model established by the subcutaneous injection of U87 cells was adopted to assess the anti-tumor effect.The mice were randomly divided into four groups:the control group receiving subcutaneous injection with 0.9％ NaCl 0.2 mL,the model,intravenous treatment and local treatment groups receiving subcutaneous injection with 1 × 107 U87 cells in 0.2 mL Dulbecco's Modified Eagle Medium (DMEM).When the diameter of tumor tissue reached 3 mm,the model group was subcutaneously injected with 0.9％ NaC1 0.2 mL surrounding the tumor,while the intravenous treatment group and local treatment group were injected with 0.2 × 107 GDC-CTL in 0.2 mL phosphate buffer saline (PBS) through the tail vein and subcutaneous injection into the surrounding area of the tumor respectively,3 times a week for 2 weeks.The tumor volume was calculated and the pathological changes in the tumor tissues were observed for comparison.Results Matured GDC expressing the high levels of CD83,CD1a and HLA-DR successfully activated GDC-CTL in which 93.00％ of CD3 + T lymphocytes and 69.00％ of CD3 + CD8 + T lymphocytes were detected.In vitro experiments proved that the killing rates of GDC-CTL and T lymphocytes on U87 cells were (24.35 ±1.12)％ vs (15.21 ±0.91)％,(38.57±2.10)％ vs (23.35 ±1.30)％,(59.44±3.79)％ vs (35.23 ± 2.33) ％,and the IFN-γlevels secreted from GDC-CTL and T lymphocytes co-culturing with U87 cells were (405.36±27.65) vs (371.11 ±23.23) pg · mL-1,(1509.22 ±97.16) vs (913.54 ±48.35) pg · mL-1,(2429.57 ±183.18) vs (1814.97 ± 123.24) pg · mL-1,at the different effect/target ratios of 5∶1,10∶1 and 20∶1 respectively.There were significant differences between this two groups at the effect/target ratios of 10∶1 and 20∶1 (P ＜0.05).The results obtained from the in vivo experiments showed that the tumor volumes in the intravenous treatment group and local treatment group shrank 34.83％ and 45.37％ respectively,when comparing with the model group (100.00％,P ＜ 0.05).The pathological changes of tumor tissues showed that the tumor cells in the local treatment group and intravenous treatment group were significandy decreased.Conclusion The experimental results that GDC-CTL can significantly inhibit the growth of ghoma provide more evidences to further study the effective targeting therapy on glioma.

OBJECTIVEMedically unexplained dyspnea is common in adult and accounts for 14% patients complaining of dyspnea. Its occurrence in children is seldom recognized. In the present paper, 34 children with medically unexplained dyspnea (age 10 to 18 years) seen in Peking Union Medical College Hospital from 1996 to 2002 are reported.

METHODSThe diagnosis of medically unexplained dyspnea was clinical: it was based on the presence of dyspnea and other complaints which cannot be explained by an organic disease. The patients answered Nijmegen questionnaire and state and trait anxiety (STAI), and performed hyperventilation provocation test. Twenty sessions of breathing therapy were applied and 13 out of 34 children were followed up after the therapy.

RESULTSAmong the children, 75% started to have symptoms at the age of 13 to 16 years, though the age of first episode could be as early as 8 years. In most of the cases, the course was chronic clinically. In addition to marked dyspnea, their clinical profile included symptoms of hyperventilation i.e. blurred vision, dizziness, tingling, stiff fingers or arm. The symptoms of anxiety were less frequent in children and accordingly the level of anxiety evaluated by means of STAI was lower in children compared to adult patients. The precipitating psychological factors appeared to be related to middle school competition. Pressure from exams, reprimand from stern and unsympathetic teachers coupled with high parental expectation could be emotionally damaging to psychologically susceptible children. Thirteen patients were followed up after 2-3 months of breathing therapy with emphasis on abdominal breathing and slowing down of expiration. After therapy, the sum score of the Nijmegen Questionnaire was markedly decreased. Dyspnea and symptoms of hyperventilation were improved. The level of anxiety was minimally modified.

CONCLUSIONThe cases illustrated the need for careful diagnostic evaluation and treatment because of the high rate of chronicity of the disorder.

Adolescent ; Child ; Dyspnea ; etiology ; pathology ; therapy ; Female ; Humans ; Hyperventilation ; Male ; Prognosis ; Treatment Outcome

OBJECTIVETo evaluate the efficacy and safety of selective serotonin re-uptake inhibitors (SSRIs) in the treatment of premature ejaculation (PE).

METHODSFrom MEDLINE (Jan, 1950-Mar, 2008), EMBASE (Jan, 1980-Mar, 2008), The Cochrane Library (Issue 1, 2008) and CNKI (Jan, 1979-Mar, 2008), we retrieved and screened the randomized controlled trials (RCT) and randomized crossover trials (RT) as well as various related data, published and unpublished, on the treatment of PE with SSRIs. The methodological quality of the included trials was evaluated by 2 reviewers. Meta-analyses were conducted with RevMan 5.0 on the homogeneous studies.

RESULTSTotally 22 studies on 4 291 patients were included. Meta-analyses showed that after treated with sertraline, fluoxetine, paroxetine, citalopram, dapoxetine and fluvoxamine, the WMD (95% CI) values of the changes in intravaginal ejaculatory latency time (IELT) were 2.63 (1.80, 3.46), 2.21 (1.50, 2.92), 4.31 (2.71, 5.91), 3.82 (3.39, 4.25), 1.57 (1.31, 1.84) and 0.01 (0.71, 0.73) respectively; the RR (95% CI) values of the sexual satisfaction rate of the patients were 1.65 (1.12, 2.43), 2.93 (0.50, 17.31), 3.08 (2.27, 4.17), 2.48 (1.99, 3.09) and 2.93 (2.36, 3.65), and those of their partners were 1.47 (0.98, 2.21), 2.88 (0.38, 21.77), 4.81 (3.15, 7.36), 5.38 (3.75, 7.72) and 2.91 (1.09, 7.78) respectively for sertraline, fluoxetine, paroxetine, citalopram and dapoxetine.

CONCLUSIONAll the known SSRIs but fluvoxamine could prolong IELT, and some could improve the sexual satisfaction of both the patients and their partners, but their adverse effects should be noted. The moderate possibility of selection bias and publication bias in the included studies might have a negative impact on the evidence intensity of our results. We expect more reliable evidence from more randomized controlled trials.

Humans ; MEDLINE ; Male ; Randomized Controlled Trials as Topic ; Serotonin Uptake Inhibitors ; adverse effects ; therapeutic use ; Sexual Dysfunction, Physiological ; drug therapy ; Treatment Outcome

OBJECTIVETo investigate the clinical manifestation of patients with renal injury induced by chronic mercury intoxication and the application of the diagnostic criteria of occupational mercury poisoning.

METHODSThe clinical data of 8 patients with chronic occupational mercury intoxication were analysed and evaluated.

RESULTSAll the observed clinical signs of chronic mercury intoxication correspond with the items of the diagnostic criteria of occupational mercury poisoning. The increasing beta2-MG was one of the clinical manifestations of renal injury induced by chronical mercury intoxication. The renal injury obviously was dose-dependent and reversible.

CONCLUSIONSThe national diagnostic criteria of occupational mercury poisoning is practically valuable. The renal injury induced by chronic mercury intoxication should not be neglected.

Adult ; Female ; Humans ; Male ; Mercury Poisoning ; diagnosis ; Middle Aged ; Occupational Diseases ; diagnosis ; Reference Standards

Gastrodia elata Bl. is a famous and costful traditional Chinese medicine. Their genomic DNA fingerprints were investigated using a modified Randomly Amplified Polymorphic DNA method. DNA fragments common to all or to fine populations were identified and recovered. Five DNA fragments were proven not to be reported through DNA cloning, PCR identifying, nucleotide sequencing and bioinformatics analyses and were received in and recorded by NCBI GenBank. Gastrodine contents of the Gastrodia tuber samples were determined using high performance liquid chromatography technique. The distribution of the five DNA fragments in 9 Gastrodia elata Blue populations and the correlation with gastromedicine content were studied. The results show the distribution of these DNA sequences varied greatly among the populations whereby DNA Sequence 1 was the common and distinguishing molecular marker for all the populations studied and DNA Sequence 2 may relate to higher gastrodine content. In conclusion, these DNA marker sequences can be employed to identify genuine gastrodia tubers, better varieties and optimize their selection and cultivating.
Base Sequence ; Benzyl Alcohols ; analysis ; Cloning, Molecular ; Computational Biology ; DNA, Plant ; chemistry ; Gastrodia ; genetics ; Glucosides ; analysis ; Plant Tubers ; genetics

Dental Pulp Cavity ; anatomy & histology ; Humans ; Male ; Maxilla ; Middle Aged ; Molar ; anatomy & histology ; Odontometry ; Tooth Root ; anatomy & histology

OBJECTIVETo find sensitive and specific laboratory examination items for early diagnosing and monitoring liver transplantation reject reaction.

METHODSRandomly investigate 41 liver transplantation patients, among them there were 16 patients with reject reaction (including 12 with acute rejection, 4 with chronic rejection). Plasma soluble thrombomodulin (STM) and von Willebrand factor (vWF) levels were measured before operation and every other day after operation.

RESULTSPlasma STM level increased significantly after operation, two days before rejection and after acute rejection (5.58 ng/ml +/- 0.42 ng/ml, 5.93 ng/ml +/- 0.45 ng/ml, and 7.88 ng/ml +/- 0.29 ng/ml, respectively), so did vWF level (101.2% +/- 4.68%, 104.3% +/- 5.78%, and 127.7% +/- 5.74%, respectively). STM level was much higher in acute rejection than that in chronic rejection (7.88 ng/ml +/- 0.29 ng/ml vs. 6.35 ng/ml +/- 0.54 ng/ml, t = 2.46, P < 0.05), in no reaction group after impacting therapy than in effective group (8.30 ng/ml +/- 0.19 ng/ml vs. 3.82 ng/ml +/- 0.22 ng/ml, t = 12.98, P < 0.01), and in dead group after treatment than in living group (7.98 ng/ml +/- 0.18 ng/ml vs. 6.51 ng/ml +/- 0.41 ng/ml, t = 3.39, P < 0.01).

CONCLUSIONSPlasma STM and vWF can be taken as laboratory items for monitoring liver transplantation rejection. Plasma STM can act as not only an early prognosticating marker, but also suitable to distinguish acute from chronic reject reaction, and as a marker for monitoring impacting therapy effect and judging prognosis.

Adolescent ; Adult ; Biomarkers ; blood ; Female ; Graft Rejection ; Humans ; Liver Transplantation ; adverse effects ; Male ; Middle Aged ; Thrombomodulin ; blood ; von Willebrand Factor ; analysis

Objective To evaluate the efficacy and safety of oxiracetam in the treatment of neurological deficits resulting from brain injury through the comparison of oxiracetam for injection and piracetam for injection in clinical trials. Methods A multiple-center, randomized, double-blind,parallel study was performed on 239 patients; these patients were divided into experimental group (oxiracetam for injection, n=120) and control group (piracetam, n=119). National institutes of health stroke scale (NIHSS), Glasgow coma scale (GCS), myodynamia grading, mini-metal state examination (MMSE) were employed to evaluate the therapeutic effects; electrocardiogram and laboratory examination were performed, and the side effects were also observed. Results The scores of NIHSS,GCS and myodynamia grading after treatment in the 2 groups were all significantly higher than those before treatment (P＜0.05); however, no significant differences on these scores were noted between the experimental group and control group (P＞0.05). No serious adverse events were noted in both groups.Conclusion Oxiracetam, the same as piracetam, is safe and effective in the treatment of neurological deficits secondary to brain injury.