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

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 immunomodulating activity of Lonicera Japonica flavone by investigating immune enzymatic activity of serum and antoxidized activity of lymphoid organs in mice.

METHODSFifty KM mice were randomly divided into control group, model group, low dose group, middle dose group and high dose group(n = 10), respectively. And low dose group, middle dose group and high dose group were given Lonicera Japonica flavone with 100 mg/kg, 200 mg/kg and 400 mg/kg every day, respectively, while control group and model group were administered with NS. After continuously giving drug 7 weeks, other groups were injected with Dexamethasome (Dex: 25 mg /kg) for 3 days by subcutaneous injection, but the control group were treated with NS. And after giving Lonicera Japonica flavone 1 week simultaneously, organ indexes , the activity of acid phosphatase (ACP), alkaline phosphatase (AKP) and lysozyme (LSZ) in serum , and the content of monoamine oxidase (MAO), total antioxidant capacity (T-AOC), total superoxide dismutase (SOD) and malondialdehyde (MDA) in lymphoid organs in mice were tested, respectively.

RESULTSLonicera Japonica flavone could significantly improve the organ indexes, and significantly improve the activity of ACP, AKP and LSZ in serum, and significantly improve the contents of T-AOC and SOD, but reduce that of MAO and MDA in lymphoid organs in immunosuppressed mice.

CONCLUSIONIonicera Japonica flavone can significantly improve the activity of immune enzyme in serum and the antioxidized activity of lymphoid organs in mice. It suggests that Ionicera Japonica flavone has a good immunomodulatory effects.

Acid Phosphatase ; blood ; Alkaline Phosphatase ; blood ; Animals ; Antioxidants ; metabolism ; Flavones ; pharmacology ; Immunomodulation ; Lonicera ; chemistry ; Malondialdehyde ; metabolism ; Mice ; Monoamine Oxidase ; metabolism ; Muramidase ; blood ; Superoxide Dismutase ; metabolism

Objective To compare the effectiveness and safety of different intracranial stents assisted coil embolization of intracranial aneurysms and to discuss the selection of different stent assisted embolization of intracranial aneurysm.Methods From 2007 April to 2012 April,118 cases (a total of 128 wide-neck aneurysms) with intracranial aneurysms were analyzed retrospectively.This included the use of 70 neuroform,38 Enterprise,and 20 Solitaire AB stents forthe treatment of intracranial aneurysms.The successful use,aneurysm occlusion at the immediate post-operation,and early period of peri-operative complications were recorded from those clinical data in order to assess the effectiveness and safety of the different intracranial stents,which assisted coil embolization of intracranial aneurysms.Rank sum test and x2 test were used for statistics.Results Three aneurysms assisted with Neuroform stent were planted unsuccessfully,and the Enterprise and Solitaire stents were placed successfully.The embolism results of three stents after immediate postoperative angiography aneurysm: Neuroform stent occlusion rate was 40.0％ (28/ 70),the tumor residual rate was 38.6 ％ (27/70),and the partial embolization rate was 21.4 ％ (15/70) ; The Enterprise stent occlusion rate was 42.1％ (16/38),the tumor residual rate was 36.8 ％ (14/38),and the partial embolization rate was 21.1％ (8/38).The Solitaire AB stent occlusion rate was 40.0 ％ (8/20),the tumor residual rate was 35.0 ％ (7/20),and the partial embolization rate was 25.0 ％ (5/20).There were not significant differences in aneurismal occlusion (H =0.12,P ＞ 0.05).Early peri-operative complications results were: Neuroform stent occurred in 7,Enterprise frame in 4,and Solitaire AB stent occurred in 2.There were no significant differences in the incidence of complications in the early period after coiling (x2 =0,P ＞ 0.05).Conclusions Three kinds of intracranial stents assisted embolization of intracranial aneurysms are applied safely and effectively.The stent may be chosen according to morphology of parent artery and stent biological character.

Objective To assess the application of three-dimensional digital subtraction angiography (3D-DSA) in evaluation of ruptured intracranial aneurysm after clipping and to discuss the different variable use of vol-ume rendering(VR), gradient rendering (GR) and maximum intensity projection (MIP). Methods From January 2011 to December 2012 , 88 patients with 92 ruptured intracranial aneurysms were treated with clipping using titani-um clips in our hospital and followed up by both 2D-DSA and 3D-DSA. Residual aneurysms , Clips place, clips and parent arteries and stenosis of parent arteries were evaluated by volume rendering (VR), gradient rendering (GR) and maximum intensity projection (MIP). Results Among 92 clipped aneurysms, 23 residual aneurysms were found by 3D-DSA. Residual aneurysms were recorded according to the Sindou grade: 15 of gradeⅠ, 3 of gradeⅡ, 4 of grade Ⅲand 1 of grade Ⅳ. Three patients of grade Ⅲand 1 of grade Ⅳwith residual aneurysms were retreated by clipping or coiling, and 1 patient of grade Ⅲ was dead with rupture of residual aneurysm. The clips and number of clips were clearly visualized , and relationship between the clips and the aneurysms was well demonstrated by VR, GR and MIP images. VR, GR images showed the remnants clearly. Three-dimensional digital subtraction angiography did not showed accurate details of the stenosis of parent arties which required an analysis of 2D-DSA. Conclusion Three-dimensional digital subtraction angiography can be used for definite evaluation of resid-ual aneurysms after clipping, especially by VR, GR images. It is helpful to manage the residual ruptured aneurysms.

Objective To elucidate intracellular transcription factor activation of C_7～T_5 dorsal root ganglia in rats recurrently infected with respiratory syncytial virus (RSV). Methods Eighty 1～2 weeks old Sprague-Dawley rats were randomly divided into 2 groups: a control group and a RSV-infection group. The rats in the RSV-infection group were infected with 5 ×10~5 U/mL RSV once a week and the rats in the control group were treated with culture medium without RSV. Airway response was measured after 8 weeks. Lung tissue was submitted for HE staining and in situ hybridization. The C_7～T_5 dorsal root ganglia were obtained for the preliminary screening of the intracellular transcription factors by TranSignal~(TM) protein/DNA combo array. Nuclear protein of C_7～T_5 dorsal root ganglia were extracted and submitted to Western blot. Results Airway response in the RSV-infection group was higher than that in the control group (P＜0.05). HE staining showed inflammatory cell infiltration, and in situ hybridization demonstrated positive RSV RNA in the RSV-infection rat lung which was not present in the control group, thus validating the efficacy of our model. TranSignalTM protein/DNA combo array screening showed that 55 transcription factors increased by at least 2 folds in the C_7～T_5 DRG cells of the RSV-infection group. The transcription factors Smad and interferon regulatory factor (1 or 2) were the 2 most upregulated transcription factors identified by combo array screening (59 and 43 fold increase compared with the control, respectively). Western blot confirmed Smad(1/2/3) and IRF-1 upregulate while IRF-2 remained unchanged. Conclusion Respiratory syncytial virus infection results in airway hyperresponsiveness and transcription factor activation in C7～T5 spinal adorsal root ganglia in rats, which may contribute to airway nerve network dysfunction and airway hyperresponsiveness.

BACKGROUND: Cellulose is natural material, which has good chemical stability and biocompatibility. The modified material from cellulose has new properties besides its inherent properties.OBJECTIVE: Based on cytocompatibility evaluation tests, this study is designed to evaluate the cytocompatibiUty of materials synthesized from cellulose to make sure its reliability.DESIGN: Contrast study.SETTING: Department of High Polymer, Material Science and Engineering College, Beijing University of Technology.MATERIALS: This study was performed in the Material Science and Engineering College, Beijing University of Technology between March 2003 and June 2004. The material from cellulose prepared by Beijing University of Technology was white, granules, no taste, and no volatilization (density: 1.2 g/cm3, pH: 6-7). L-929 vigorous cells were provided by Chinese Military Academy of Medical Science. Ten male New Zealand rabbits were provided by Animal Center of Chinese Military Academy of Medical Science.METHODS: Based on GB/T16175-1996 standard, cytotoxicity of the materials from cellulose on L-929 cells was evaluated by growth suppression technique (MTr chromatometry). In addition, based on GB-T14233.2-1993 standard, hemolysis in vitro was evaluated through measuring erythrocyte lysis and ferrohemoglobin freeing degree with indirect contact method.MAIN OUTCOME MEASURES: Cytotoxicity test and hemolysis test.RESULTS: Cytotoxicity test showed that the materials from cellulose did not have obvious toxicity on L-929 cells, and the cytotoxicity graduation of the materials from cellulose was 1 or 0. Hemolysis test suggested that the materials from cellulose did not have obvious hemolysis reaction, and the hemolysis rate in vitro was 1.5 %, which was less than the national standard (5%).CONCLUSION: The materials from cellulose do not have obvious cytotoxicity but have great cytocompatibility.

Objective To investigate the influential factors on the effectiveness of the first 131I ablation therapy on thyroid remnant and of 131I treatment on metastatic lesions in differentiated thyroid cancer (DTC) patients. Methods Retrospectively,46 DTC cases (divided into complete-ablation group and incomplete-ablation group) of first 131I ablation were enrolled,and 40 DTC cases (divided into remission group and in-remission group) of consecutive 131I treatments on metastatic lesions were enrolled. Influential factors were analyzed (t-test,t'-test,x2-test,Fisher exact test) and logistic regression analysis was performed. Results For the first 131I ablation effectiveness,surgical method,remnant thyroid weight,thyroid stimulating hormone (TSH) level,interval between surgery and 131I ablation therapy,metastatic status were selected as influential factors (x2 = 5. 804,t' = - 5. 258,t' = 7. 376,x2 = 8. 867,x2 = 8. 615,all P ＜0. 05). After logistic regression analysis,formula was obtained as y = 3. 766 - 0. 947x1 ( remnant thyroid weight) -3. 149 x2 (lymph node metastasis) -3. 373 x3 (distant metastasis). For metastatic treatment effectiveness,remission rate of papillary DTC was higher than that of follicular DTC,remission rate of patients with lymph node metastasis was higher than that of distant metastasis,remission rate of total thyroidectomy was higher than that of other types of thyroidectomy ( Fisher exact test,x2 = 7. 278,P ＜ 0. 05 ). In remission group,serum TSH level was much higher and thyroglobulin (Tg) level was much lower before the first ablation therapy (t =4. 489,t' = -4.906,all P ＜0.01 ). After logistic regression analysis,formula was obtained as y = - 0. 363 + 0. 065 x4 ( TSH level) - 0. 250 x5 ( Tg level). Conclusions Influential factors of success rate of the first 131I ablation therapy included surgical method,remnant thyroid weight,TSH level,interval between surgery and 131I ablation therapy and metastatic status,while determinant factors were thyroid remnant weight,lymph node metastatic status and distant metastatic status. The influential factors of success rate of 131I treatment on metastatic lesions included pathological type,surgical method,metastatic status,TSH level and Tg level,while determinant factors were TSH level and Tg level before the first 131I ablation therapy.

Objective To provide information for higher nursing education by studying whether there are differences in learning achievements between the nursing students from arts and science student sources.Methods The complete curriculum results of the nursing students from arts and science student sources during 4-year studying in school were statistically analyzed.Results There are some differences of learning achievements in foundation curriculum performance between the nursing students from arts and science student sources,but there is no significant difference of learning achievements in specialized curriculum performance between them.Conclusions To make some appropriate teaching adjustments for those curriculums with achievement differences between the nursing students from arts and science student sources can eliminate the differences,and bring the students to achieve the same goal of teaching.It is feasible to enroll the arts and science students to pursue nursing subject meantime.

Objective To investigate the indication and result of parental artery occlusion, embolization with coils, stents in treatment of the traumatic carotid artery pseudoaneurysm. Methods There were six patients with traumatic carotid artery pseudoaneurysm including three patients of cavernous pseudoaneurysm combined with carotid-cavernous fistula (CCF), two with simple traumatic carotid artery pseudoaneurysm and one with traumatic carotid artery pseudoaneurysm that was found after CCF embolization with detachable balloon. The treatment included balloon occlusion for CCF and traumatic carotid pseudoaneurysm in one patient, coil embolization in three and intenal carotid artery balloon occlusion in two. The Clinical manifestations, imaging data, choice of treatment, clinical efficacy, follow-up data and literatures were analyzed to discuss the indications for three treatments. Results There was no cerebral ischemia or surgically-related complication. No epistaxis occurred. The eyeball protrusion restoration was found in three patients and intracranial bruit vanishing in three. Vision was improved one patient after half a year follow-up. The pupils shrank in three patients during follow - up. Digital subtraction angiography (DSA) showed no recurrence of pseudoaneurysm. Conclusions Endovascular treatment is the preferred choice of treatment for traumatic carotid artery pseudoaneurysm. The occlusion or parent artery balloon occlusion can be used when the pseudoaneurysm is combined with CCF. Coil embolization can be used for shoes with narrow neck, stent technology combined with coil embolization for those with wide neck and the covered stent for the rock section of the internal carotid artery.