To establish induction and liquid culture system for hairy roots of Danshen (Salvia miltiorrhiza), Agrobacterium rhizogenes A4, LBA9402, 15834 as test bacterium were used to infect aseptic leaves of Danshen. The hairy roots were induced and positive transgenic hairy roots were selected with PCR using rolB and rolC as the target gene. Then hairy roots of S. miltiorrhiza were harvested and salvianolic acids were extracted with 70% methanol containing 1% formic acid. The content of salvianolic acid B (SalB) and rosmarinic acid (RA) were determined by HPLC. According to the above research results, the Danshen hairy roots induced by A. rhizogenes LBA9402 were inoculated into the following group of culture media: MSOH, MS, B5, and 6,7-V liquid media. Then the same methods of extraction and determination for the content of Danshen hairy roots were adopted. Last, the hairy roots of S. miltiorrhiza induced by A. rhizogenes LBA9402 were inoculated into the MSOH liquid media with different pH values. The content of salvianolic acid were extracted with 70% methanol containing 1% formic acid and determined by HPLC. As a result, three kinds of A. rhizogenes A4, LBA9402, 15834 could induce hairy roots and Ri plasmids were integrated into the genome of S. miltiorrhiza by PCR. Danshen hairy roots induced by A. rhizogenes LBA9402 and A4 produced much more salvianolic acid, which were (3.27 ± 0.37)% [including (1.04 ±0.36)% of RA and (2.22 ± 0.29)% of SalB] and (3.17 ± 0.20)% [including (0.92 ± 0.31)% of RA and (2.25 ± 0.26)% of SalB], respectively. Hairy roots induced by A. rhizogenes LBA9402 when they were cultured in MSOH liquid media produced much more salvianolic acid, which was (4.56 ± 0.36)%, including (1.12 ± 0.26)% of RA and (3.44 ± 0.23)% of SalB. Hairy roots induced by A. rhizogenes LBA9402 produced the most salvianolic acid when they were cultured in MSOH liquid media with the pH value 4.81, which was 4.85%, including 1.16% of RA and 3.69% of SalB. So Danshen hairy roots induced by A. rhizogenes LBA9402 and A4 produced much more salvianolic acid when they were cultured in MSOH liquid media with the pH value 4.81. The research had established the foundation on genetic engineering to improve the quality of S. miltiorrhiza.
Agrobacterium ; physiology ; Benzofurans ; analysis ; metabolism ; Cell Culture Techniques ; instrumentation ; methods ; Cinnamates ; analysis ; metabolism ; Culture Media ; chemistry ; metabolism ; Depsides ; analysis ; metabolism ; Drugs, Chinese Herbal ; analysis ; metabolism ; Plant Roots ; chemistry ; growth & development ; metabolism ; microbiology ; Salvia miltiorrhiza ; chemistry ; growth & development ; metabolism ; microbiology

Objective To investigate the expression and role of costimulatory molecule 4-1BB on T cells of patients with rheumatoid arthritis(RA).Metheds The expression of 4-1BB on T lymphocytes from 30 RA patients and 20 healthy controls were detected by flow cytometry.Results The expression of 4-1BB on CD4~+T and CD8~+T lymphocytes from RA patients was significantly higher than that of normal control(18.56?4.08,10.33?2.13 vs 1.24?0.12,0.87?0.09,P＜0.01).There was more expression of 4-1BB on CD4~+T and CD8~+ T lymphocytes stimulated by anti-CD3 antibody from RA patients(33?4 vs 21?8,P＜0.01).In addition,the ra- tio of CD4~+T/CD8+T in RA patients was higher than that of normal controls and was positively correlated with 4-1BB~+CD4~+T cell.The expression of 4-1BB on CD4~+T in RA patients was positively correlated with the level of ESR and IgA(r=0.476,P＜0.05;r=0.659,P＜0.05).Conclusion The costimulatory molecule 4-1BB is ab- normally expressed in T lymphocytes from patients with rheumatoid arthritis.The abnormal expression of 4-1BB on T lymphocytes may play an important role in the development of RA.The expression of 4-1BB on CD4~+T cell may take part in the inflammation of RA.

AIM:To investigate the effect of fluvastatin on the expression of serum and glucocorticoid inducible kinase 1 ( SGK1) and connective tissue growth factor ( CTGF) induced by aldosterone ( Ald) in rat mesangial cells (GMCs). METHODS:GMCs were divided into (1) control group; (2) aldosterone group with different concentrations and times; (3) Ald (10 -7 mol/L) + spironolactone (10 -9 mol/L) group; (4) Ald (10 -7 mol/L) + LY294002 (20 ?mol/L) group; (5) Ald (10-7mol/L) +SB203580 (20 mmol/L) group; (6) the group of Ald (10-7mol/L) + fluvas-tatin at different concentrations (10-7,10-6,10-5 mol/L); (7) Ald (10 -7mol/L) + fluvastatin (10 -5mol/L) + mevalonate (10 -4 mol/L) group; (8) Ald (10 -7 mol/L) + fluvastatin (10 -5 mol/L) + FPP (farnesyl pyrophosphate,10-4 mol/L) group; (9) Ald (10 -7mol/L) + fluvastatin (10 -5 mol/L) + GGPP (geranylgerany pyrophosphate,10 -4 mol/L) group. The protein levels of SGK1 and CTGF were determined by Western blotting. The levels of fibronection (FN),monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule-1 (ICAM-1) in the supernatants were determined by enzymelinked immunosorbant assay (ELISA). RESULTS:Aldosterone stimulated the protein expression of SGK1 and CTGF in cultured mesangial cells in a dose-dependent manner (P

Background: Acute mastitis is an acute infectious disease of breast. Antibiotic treatment is often unable to obtain a good effect, and we should actively look for a safe and effective non-drug therapy. Objective: To validate the clinical efficacy of kneading and dispersing manipulation in treatment of early-stage acute mastitis. Design, setting, participants and interventions: According to the multicenter randomized controlled trial design, 198 cases of acute mastitis from Yueyang Hospital of Integrated Traditional Chinese Medicine, Longhua Hospital, and Shanghai Yangpu Maternity and Child Health Hospital were randomly divided into treatment group and control group. There were 99 cases in each group. Patients in the treatment group were only treated with manipulation, and cefradine was orally administered to patients in the control group. Main outcome measures: The local breast lump size, clinical symptoms and the adverse reactions in the two groups were observed before and after the treatment. Results: The total response rates in the treatment and control group were 95.92% (94/98) and 80% (76/95) respectively. There was a significant difference in the total response rate between the two groups (P<0.05). There were significant differences in the score of breast lump size, and the score of signs and symptoms between the two groups (P<0.05). Conclusion: Kneading and dispersing manipulation has certain effects on early-stage acute mastitis, and the therapy is safe and repeatable.

italic>Salvia miltiorrhiza Bunge is a traditional Chinese medicinal herb widely used to treat cardiovascular and cerebrovascular diseases at clinic. Its main water-soluble components are rosmarinic acid (RA) and salvianolic acid B (SAB), which are produced by phenylpropanoid pathway. 4-Hydroxyphenylpyruvate reductase (HPPR) is a key enzyme in phenylpropanoid metabolism pathway. SmHPPR1 was cloned from S. miltiorrhiza and was constructed into plant expression vector pJR-SmHPPR1. On this basis, SmHPPR1 transgenic Arabidopsis plants were induced and the content of 4-hydroxyphenyllactic acid (pHPL) was determined. SmHPPR1-overexpressing (SmHPPR1-OE) hairy roots of S. miltiorrhiza were obtained and the concentration of active components and transcriptome analysis were performed. The results showed that the concentration of pHPL in SmHPPR1 transgenic Arabidopsis T1 was 0.594 mg·g^-1 dry weight. The concentration of RA, SAB and total salvianolic acid in SmHPPR1-OE-3 hairy roots were 1.09, 1.29, 1.15 times of that in control-3, respectively, and the content of Danshensu was 36.26% of that in control-3. Transcriptomic analysis revealed that overexpression of SmHPPR1 caused the upregulation of other phenylpropanoid pathway genes like SmTAT2. Protein-protein interaction indicated CYT (TR74706_c0_g1), NADP⁺ (TR26565_c0_g1) and NADP⁺ (TR68771_c0_g1) is the central node of the network and participated in metabolic process and cellular process. The tracking work in this study proved that SmHPPR1 could catalyze the reduction of 4-hydroxyphenylpyruvic acid to 4-hydroxyphenyllactic acid in SmHPPR1 transgenic Arabidopsis, and SmHPPR1-overexpressing in hairy roots of S. miltiorrhiza could increase the concentration of salvianolic acids through synergistically regulating other pathway genes.

OBJECTIVETo study the expression of lipoprotein related genes in subchondral bone of early experimental os-teoarthritis, which may play an important role in the pathogenesis of osteoarthritis.

METHODSAnimals are equally divided into two groups: experimental group and control group, both of which contain fifteen rats of similar weight. The right knee joints of experimental group underwent surgery,which involved in both medial collateral ligament(MCL) transaction and medial meniscectomy, while the control group was only carried out with a sham operation. Rats were killed at 1, 2 and 4 weeks postsurgery to obtain the right knee joints. Total RNA of the subchondral bone was extracted,and then hybridized to Agilent Whole Rat Genome Microarray. Differentially expressed genes analysis was used to study the chemokine signaling pathway.

RESULTSApoa5 expression was down-regulated at 1, 2 weeks post-surgery, Apoc2 expression was up-regulated at 1 week after surgery, Apol3 expression was up-regulated at 1 and down-regulated at 4 weeks post-surgery, Lrp1 expression was down-regulated at 1, 2 weeks after surgery. Lrp5 was down-regulated at 2 weeks after surgery. Gpihbp1, Lpl, Tfpi and Vldlr were up-regulated at 1 weeks after surgery. Lrpap1 and RGD1309808 were down-regulated at 4 weeks after surgery.

CONCLUSIONDysregulation of lipoprotein related genes plays an important role in pathogenesis of early experimental osteoarthritis.

Animals ; Disease Models, Animal ; Knee Joint ; metabolism ; Lipoproteins ; genetics ; Male ; Oligonucleotide Array Sequence Analysis ; Osteoarthritis ; genetics ; Rats ; Rats, Sprague-Dawley ; Transcriptome

Objective To summarize the SPECT/CT manifestation of spondylitis caused by Brucells infection and to evaluate the diagnostic value.Methods From June 2012 to October 2015,a total of 28 patients (14 males,14 females,average age 46.4 years) with Brucellosis spondylitis confirmed by laboratory test and pathology were included.The images of whole-body bone scan and SPECT/CT fusion imaging were retrospectively analyzed.According to the pathological and serologic test results,the diagnostic efficacy of imaging was calculated.x2 test was used.Results Most of the Brucellosis spondylitis happened in the lumbar(76.7％,43/56),and the most common locations were L3,L4,L5 (72.1％,31/43).Two or more involved consecutive vertebra were found in 71.4％ (20/28) of the patients.Moderate radioactive distribution was showed in 89.2％ (50/56) of lesions,high radioactive distribution was showed in 5.4％ (3/56) of lesions,and mild radioactive distribution was showed in the rest 3 lesions.Thirty-three lesions(58.9％,33/56) had diffuse increased radioactivity uptake in the affected vertebra,and 32.1％(18/56) showed diffuse increased radioactivity at the superior and inferior margin of the vertebra;only 8.9％ (5/56) of lesions were on one side of the vertebral bodies.The SPECT/CT results were as follows:(1) Bone destruction was showed in 80.4％ (45/56) of lesions,and the edge of the lesion was clear.(2) For 66.7％ (30/45) of lesions,bone hyperplasia was seen along with bone destruction and moderate radioactivity concentration on the edge of destruction area.(3) The damage of the intervertebral disc was mild,and the vertebral abscess was relatively rare (5.4％,3/56).The diagnostic accuracy of SPECT/CT was statistically higher than that of whole-body bone scan:67.8％(38/56) vs 96.2％(54/56);x2=13.1,P＜0.05.Conclusion SPECT/CT imaging has a higher diagnostic efficiency than whole-body bone scan in Brucellosis spondylitis.

ObjectiveTo explore the effect of blocking the JAK/STAT signal pathway on the activity of Caspase-3 in the synovial tissue of rheumatoid arthritis rats.MethodsFifty rat models of collageninduced arthritis,which had arthritis index more than 2 were divided into the model group,the low dosage of AG490 group,the medium dosage of AG490 group and high dosage of AG490 group.Inaddition,6 rats were treated as normal control group.Normal saline,AG490 1,5,10 mg·kg-1 ·d-1 were given by intraperitoneal injection.Then the volume claws and pathologic scores of the rat models were recorded and the activity of Caspase-3 in the synovial tissue were compared.The results were analyzed by one-way ANOVA and LSD-t or Tamhane's T2 test.ResultsThe arthritis of the CIA models progressed fast,the volume of the claws and the pathological score of them were significantly higher than those of the control group.At the same time,no Caspase-3 positive express could be detected in the control group,whilethe model group had slightly increased expression.After different dosages of AG490 were applied,the swollen of joints was significantly improved compared with the model group.The histopathological score of the medium AG490 dosage of group and high dosage group(2.7±0.8,1.8+0.9) were remarkably decreased than those of the model group(4.3+1.2),the differences were statistically significant (P＜0.01).In addition,the Caspase-3 expression in the low,medium and high AG490 dosage group ( 1.90±0.15,3.13±0.33,3.56+0.34) was significantly higher than that of the model group(1.48±0.18)(P＜0.05 or P＜0.01 ).ConclusionBlocking JAK/STAT signal pathway can increase the activity of Caspase-3,reduce the excessive proliferation of synovial tissue,and improve arthritis symptoms.

Immunohistochemical technique was an essential tool of conventional diagnosis,therefore,the application of immunohisto- chemistry in reformation of pathology laboratory teaching would boost pathological experimental teaching standards to a higher level.

Objective To study the effect of different concentration of 1,25-dihydroxyvitamin D3[1,25(OH)2D3] on cell proliferation,differentiation and the expression of vitamin D receptor (VDR) in mouse MC3T3E1 osteoblast.Methods Osteoblast were cultured in medium with different concentrations of 1,25(OH)2D3.Incubated for 48 h,cell proliferation of osteoblast were examined by MTT reduction assay (mono-nuclear cell direc cytotoxicity assay),the osteocalcin (OC) levels in cell medium were detected by ELISA,and the expression of VDR mRNA and protein were examined by using SYBR Green real-time PCR and Western blot,respectively.Results 1.After incubation with 1,25(OH)2D3 for 48 h,the number of MC3T3E1 osteoblast was significantly less than that in control group(P0.05).3.SYBR Green real-time PCR and Western blot results showed that the expression of VDR mRNA as well as VDR protein of osteoblast in 10-8,10-9 mol/L experimental groups were significantly higher than those in control group (Pa0.05).Conclusions Cell proliferation of mouse osteoblast can be inhibited,while the cell differentiation was promoted by 1,25(OH)2D3.1,25(OH)2D3 up-regulated the expression of VDR in mouse osteoblast,which suggested that the VDR signal pathway may play some role in proliferation and differentiation of osteoblast.