The objective of this study was to screen out the effective shRNA which can inhibit the gene expression of tumour necrosis factor-alpha (TNF-alpha), to construct the recombinant plasmid and to determine its sequence so as to provide the new approach for searching gene therapy of TNF-alpha related diseases. The primary macrophages were added into 15% DMEM, then cells were adjusted as 2 x 10(7) cells/L and were inoculated in 6-well plate with 3 ml/well, and were cultured at 37 degrees C in a fully humidified atmosphere with 5% CO(2). Cells were stimulated with lipopolysaccharide (LPS) and the concentration of TNF-alpha in the supernatant at different time points was determined by enzyme-linked immunosorbent assay (ELISA). The 5 synthesized DNA sequences which can be transcripted into shRNA were transfected into cells with lipofectamine 2000, then the cells were stimulated with LPS for 24 hours. The concentration of TNF-alpha in the supernatant and the expression of TNF-alpha mRNA were determined by ELISA and reverse transcription polymerase chain reaction (RT-PCR) respectively. The most effective shRNA was inserted into plasmid, and the recombinant plasmid was identified by sequence analysis. The results showed that the concentration of TNF-alpha in the supernatant reached peak after the stimulation with LPS for 24 hours. In the RNA interference group, the shRNA 1 was the most effective one, which could inhibit the expression of TNF-alpha by 59.46% and the expression of TNF-alpha mRNA by 61.2%. The recombinant plasmid was cloned and the sequence of interest was obtained. In conclusion, the most effective shRNA targeting TNF-alpha was successfully screened out and the recombinant plasmid was constructed. The recombinant plasmid may be helpful to search new gene therapy for TNF-alpha related disease.
Animals ; Cells, Cultured ; Gene Expression ; Genetic Therapy ; Genetic Vectors ; Macrophages ; cytology ; Male ; Mice ; Mice, Inbred C57BL ; Plasmids ; RNA Interference ; RNA, Messenger ; genetics ; RNA, Small Interfering ; genetics ; Recombination, Genetic ; Transfection ; Tumor Necrosis Factor-alpha ; genetics

Objective To evaluate the clinical efficacy and safety of enalapril or irbesartan combined with hydrochlorothiazide in the treatment of medium or severe hypertension.Methods Sixty -nine patients with moderate to severe essential hypertension were randomly divided into control group ( n=34) with enalapril 10 mg +hydrochlorothiazide 12.5 mg orally bid and treatment group ( n =35 ) with irbesartan 50 mg +hydrochlorothiazide 12.5 mg orally bid with 2 months.After treatment, the clinical efficacy, serum level of creatinine, acidum uricum and potassium and the ratio of adverse event were compared between the two groups. Results After 2 months treatment, the blood pressure was significant decreased in control and treatment groups. The clinical efficacy were 88.24% and 88.57% with not statistical difference ( P >0.05 ) .The serum level of creatinine, acidum uricum and potassium had no obvious difference before and after the treatment in the two groups (P>0.05).Four cases of cough were observed in the control group and the treatment group, respectively, without statistical difference (P>0.05).Conclusion There was no clinical efficacy difference for enalapril or irbesartan combined with hydrochlorothiazide in the treatment of medium or severe hypertension.But irbesartan combined with hydrochlorothiazide had less cough adverse event.

Objective To investigate the feasibility of using low Kv,low iodine contrast Agent concentration (dual low)CT scan techniques in Coronary CTA (CCTA).Methods Seventy-six patients undergoing CCTA were divided into Group A and Group B , randomly.Group A (38 patients)was the dual-low group,which was scanned with tube voltage of 100 kVp,and injected with iso-osmolarity contrast agent visipaque 270 (270 mg I/mL),with iterative reconstruction technique (ASIR 40%).Group B (38 pa-tients)was scanned with 120 kVp,and low osmolarity contrast agent omnipaque 350 (350 mg I/mL)and FBP reconstruction,The images are assessed double-blindly by two experienced radiologists.Five ROIs were placed onto the ascending root of aorta (AO), left main artery(LM),left anterior descending (LAD),left circumflex artery(LCX),right coronary artery (RCA),and the image qualities are evaluated objectively using CT values,noise,signal noise ratio (SNR),contrast noise ratio (CNR),and compared sta-tistically using Paired t-test.The radiation dosages,such as CTDIvol,DLP and ED were also recorded and compared with Paired t-test.Results CTDIvol,DLP and ED of Group A (dual low)decreased 35.7%,38.6% and 38.6% respectively compared with Group B,the iodine intake decreased 22.9%.While the image qualities of the two groups were not significantly different,all images are good enough for diagnosis,with Group A slightly better than Group B in radiologists’scores.Conclusion Voltage 100 kVp, combined with low contrast agent concentration of 270 mg I/mL can fully satisfy the diagnostics need in CCTA,and significantly lower both the radiation dosage and iodine intake.

The objective of this study was to investigate the function and mechanism of hyperbaric oxygen (HBO) in antagonizing acute graft-versus-host disease (aGVHD) and improving the rate of survival. The lethally irradiated C57BL/6 recipients were injected with bone marrow and lymphocyte of spleen from BALB/c donors and were treated with HBO, cyclosporine A (CsA) and methotrexate (MTX). T lymphocytes and subsets, adhesion molecules and cytokines were detected by flow cytometry, ELISA and RT-PCR respectively. The results showed that the survival rate in HBO group was much higher than that in allogenetic bone marrow transplantation (allo-BMT) group and CsA + MTX group; the numbers of CD3(+), CD4(+), CD8(+), CD4(+)CD11a(+), CD4(+)CD18(+), CD8(+)CD11a(+), CD8(+)CD18(+) lymphocytes in spleen were decreased markedly by HBO and CsA + MTX (p < 0.05); the levels of IL-2 and TNFalpha mRNA and their serum concentrations in HBO group were much lower than those in allo-BMT group but were higher than those in CsA + MTX group; the levels of IL-4 and IL-10 mRNA in HBO group were much higher than those in allo-BMT group and CsA + MTX group. It is concluded that HBO has more remarkable advantage in improving the rate of survival than CsA + MTX, its mechanism of anti-aGVHD is tightly correlated with the transform of T cell and its subsets and the expression of adhesion molecules and cytokines.
Acute Disease ; Animals ; Bone Marrow Transplantation ; adverse effects ; Cytokines ; biosynthesis ; Female ; Graft vs Host Disease ; etiology ; therapy ; Hyperbaric Oxygenation ; Lymphocyte Transfusion ; adverse effects ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; T-Lymphocytes ; immunology ; Whole-Body Irradiation

The aim of this study was to explore the effect of hyperbaric oxygen (HBO) preconditioning on the rejection of skin allograft in mice and its molecular mechanism. BALB/c donor mice and C57BL/6 recipients received hyperbaric oxygen preconditioning once a day for 7 days. After skin transplantation, the recipients were treated with cyclosporine A (CsA) intraperitoneally. Immunofluorescent staining technique and flow cytometry were used to observe the influence HBO on percentage of spleen lymphocytes CD3+, CD4+, CD8+ and cell adhesion molecule LFA-1 (CD11a/CD18). The results showed that as compared with control, the numbers of CD3+, CD4+, CD8+, CD4+CD11a+, CD4+ CD18+, CD8+CD11a+, CD8+CD18+ lymphocytes of spleen decreased in HBO preconditioning groups and CsA group, and decreased markedly in HBO preconditioning combined with CsA group (p<0.05); the general state of recipient mice in HBO preconditioning combined with CsA group was better than that of recipient mice received HBO preconditioning or CsA only. It is concluded that the method of HBO preconditioning combined with traditional immunosuppressive agent CsA has remarkable advantage in inhibiting the rejection of skin graft. Its molecular protective mechanism is correlated with the expression of adhesive molecules on T cell subsets.
Animals ; Cell Adhesion ; Cell Adhesion Molecules ; pharmacology ; Cyclosporine ; pharmacology ; Female ; Graft Rejection ; prevention & control ; Graft Survival ; Hyperbaric Oxygenation ; Lymphocyte Count ; Lymphocytes ; cytology ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Skin Transplantation ; immunology ; Spleen ; cytology ; Transplantation Conditioning ; methods

OBJECTIVETo attempt to visualize the endolymph in patients with Meniere's disease by applying non-invasive intratympanic gadolinium through eustachian tube and three-dimensional fluid-attenuated inversion recovery magnetic resonance imaging (3D-FLAIR MRI).

METHODSWith a 3 Tesla magnetic resonance imaging (MRI) unit, 3D-FLAIR imaging was performed 24 hours after intratympanic gadolinium through eustachian tube in two patients with medically active and intractable Meniere's disease. Pure tone test and tympanometry were performed 24 hours before and after the administration of gadolinium.

RESULTSThe gadolinium appeared in almost all parts of the perilymph inside the inner ear; moreover, the border between the perilymph and the endolymph was visible so endolymphatic space was clearly shown on 3D-FLAIR. No change in pure tone test and tympanometry was noted.

CONCLUSIONS3D-FLAIR MRI with intratympanic gadolinium through eustachian tube can clearly reveal the visualization of endolymph in patients with Meniere's disease. Intratympanic gadolinium therapy through eustachian tube is a safe and effective.

Adult ; Endolymph ; diagnostic imaging ; Female ; Humans ; Imaging, Three-Dimensional ; Magnetic Resonance Imaging ; Male ; Meniere Disease ; diagnostic imaging ; Middle Aged ; Radiography

OBJECTIVETo analyse the video-oculographic findings of positional tests and evaluate the efficacy of canalith repositioning procedure (CRP) in patients with paroxysmal positional vertigo ( BPPV) of the anterior semicircular canal (ASC).

METHODSA retrospective study of 31 patients with ASC BPPV. Then the CRP was performed.

RESULTSTwenty-two individuals (70.97%) presented a unilateral positional nystagmus during the Dix-Hallpike test, in 17 individuals had torsional nystagmus component, 5 individuals only had pure positional down beat nystagmus. Nine patients presented bilateral positional nystagmus, 7 individuals had torsional component positional nystagmus, in 2 patients the direction of the torsional component were the same during right and left Dix-Hallpike test, in 4 patients the torsional component were concurrent with positional down beat nystagmus but the direction could not be ascertained clinically, in 2 patients had pure positional down beat nystagmus. Nineteen patients (61.29%) had unilateral lesion, 11 patients had the left ASC BPPV, 8 patients had right ASC BPPV. Eleven patients had with both ASC and PSC BPPV in the ipsilateral. Twenty-one patients (67.74%) were cured, 29 patients (93.55%) were improved, 2 (6.45%) patients were inefficacy. CRP effectively resolved the nystagmus and vertigo in 14 patients (45.16%) when applied only once, The average number of CRP was 1.7 times, there were 5 patients recurrence during the follow-up.

CONCLUSIONSASC BPPV was not a common condition. The torsional nystagmus component of ASC BPPV might be weak during the Dix-Hallpike test. The positional nystagmus of ASC BPPV was triggered bilaterally. Based on these findings, CRP could be one of the most effective treatment methods for ASC BPPV.

Adult ; Aged ; Benign Paroxysmal Positional Vertigo ; Female ; Humans ; Male ; Middle Aged ; Retrospective Studies ; Semicircular Canals ; Vertigo ; diagnosis ; therapy

OBJECTIVETo establish a new method using AOTF-Near infrared spectroscopy (NIRS) for quick determination of tanshinone II(A) and salvianolic acid B in Fufang Danshen tablets.

METHODHPLC was used as a reference method to determine the contents of tanshinone II(A) and salvianolic acid B in Fu Fang Dan Shen tablets. Multivariate calibration models based on PLS1 algorithm were developed to correlate the spectra and the corresponding values determined by the reference methods.

RESULTRMSECV (root-mean-square error of cross-validation) of the models for tanshinone II(A) and salvianolic acid B were 0.0103 and 0.1868 respectively. The correlation coefficients of the calibration models were 0.9873 and 0.9832 respectively. External validation with external validation samples proved that the relative coefficient of the predicted value and the truth value were R2 = 0.9743 and R2 = 0.9886 respectively, with measured recycle rates of 103.0% and 99.0%.

CONCLUSIONNIRS can be used in the determination of tanshinone II(A) and salvianolic acid B, which sets up the foundation of product-line control of Fuang Danshen tablets.

Benzofurans ; analysis ; Diterpenes, Abietane ; Drugs, Chinese Herbal ; chemistry ; Phenanthrenes ; analysis ; Reproducibility of Results ; Spectroscopy, Near-Infrared ; methods ; Tablets ; chemistry

Objective To evaluate the pharmacodynamics of saikosapo-nin d ( SSd) in the treatment of hepatoma model rat.Methods Sixty SD rats with succeed modeling were randomly divided into model group ( n=20 ) , experimental group ( n=20 ) and control group ( n=20 ) , 20 SD rats without modeling were conducted to placebo group.The experi-mental group was treated with SSd 10 mg? kg -1 once a day, the control group was treated with thalidomide 200 mg? kg -1 once a day, the place-bo group and model group were treated with 0.9% NaCI 2 mL once a day.Liver function index were compared by Biochemical analysis me-thod, proliferating cell nuclear antigen ( PCNA) and MKI-67 gene en-codes nuclear protein(Ki-67) were compared by immunohistochemistry after 6 weeks.Results Compared with the model group, liver function index as well as the expression of PCNA and Ki-67 of the control group and the experiment group were lower (P<0.05).Compared with the control group, liver function index with expression of PCNA and Ki-67 of the experiment group were lower ( P<0.05).Pathological display:the pathological sections of the liver in the placebo group showed that the structure of the normal liver was complete, the liver cells were neat, the liver cells were arranged regularly, and the nu-cleus was clear.The model group showed that the tumor foci were in the shape of the necrosis, and the structure of the normal liver was destroyed, and the white nodules were the liver cancer cells.A small amount of low malignant degree, focal necrosis of cancer cells and fewer mitoses could be observed in control group, a small amount of blood and rare mitotic figures could be seen in the experiment group.Conclusion SSd can significantly improve liver function, repair the damage of liver cells, and reduce protein expression of PCNA and Ki-67 in model rats.

OBJECTIVETo study the cyclodextrin inclusion complexes of Dragon's blood for improving the drug solubility and the preparation.

METHODThe inclusion complexes were prepared with beta-cyclodextrin, HP-beta-cyclodextrin, SBE-beta-cyclodextrin and confirmed by DTA. The activity of the inclusion complex was tested by animal experiments. Inclusion complexes tablets were prepared and the dissolution test was performed.

RESULTThe solubility of inclusion complexes was increased to 13. 75-168. 39 times. The activity of the inclusion complex was markedly improved, and dissolution rate was 78.69%.

CONCLUSIONThe cyclodextrin inclusion complexes of Dragon's blood have a good solubility, dissolution rate and pharmacological activity.

2-Hydroxypropyl-beta-cyclodextrin ; Cyclodextrins ; chemistry ; Drug Stability ; Drugs, Chinese Herbal ; chemistry ; Plant Extracts ; chemistry ; Solubility ; Tablets ; chemistry ; beta-Cyclodextrins ; chemistry