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 correlation between serum HCY (Homocysteine) and carotid artery stenosis,plaque stability in patients with ischemic cerebrovascular disease.Methods 154 patients with ischemic cerebrovascular disease in Tangdu Hospital were enrolled in the study from June to December 2016.The serum levels of HCY were detected.CT angiography (CTA) was uesd for patients with neck vascular scanning.According to the difference of serum HCY level,patients were divided into 80 cases of high HCY group (observation group) and 74 cases of normal HCY group (control group).The degree of carotid artery stenosis,number and stability of plaque were compared between the two groups and the correlation between serum HCY level and degree of carotid artery stenosis and plaque stability were analyzed.Results The total stenosis rate in the observation group was significantly higher than that in the control group,the moderate stenosis rate and severe stenosis rate in the observation group were significantly higher than those in the control group,with the statistically significant differences (x2 =5.594～ 22.506,all P＜0.05).The levels of serum HCY in mild,moderate and severe stenosis group were 13.16 ± 6.73,15.19± 5.93 and 26.13 ±11.18 μmol/L respectively.The levels of H CY in moderate stenosis group and severe stenosis group were significantly higher than that in mild stenosis group,and the levels of HCY in severe stenosis group was significantly higher than that in moderate stenosis group,with the statistically significant differences (t=2.684～ 5.270,all P＜0.01).The rate of carotid plaque in the observation group was significantly higher than that in the control group,and the differences statistically significant (x2 =25.053,P＜0.01).The rate of unstable plaque and mixed plaque in the observation group was significantly higher than that in the control group,and the rate of stable plaque was significantly lower than that in the control group (x2 =4.067～ 14.95,all P＜0.05).The levels of serum HCY in stable plaque group,mixed plaque group and unstable plaque group were 16.14±5.49,21.91 ± 6.32 and 26.74 ± 10.59 μmol/L respectively.The levels of HCY in mixed plaque group and unstable plaque group were significantly higher than that in stable plaque group,and the differences were statistically significant (t=4.370,4.628,all P＜0.01).The level of HCY in unstable plaque group was significantly higher than that in mixed plaque group,and the difference was statistically significant (t =2.249,P＜ 0.05).Conclusion Serum HCY levels were closely related to carotid artery stenosis and plaque stability.Hyperhomocysteinemia can increase the incidence and degree of carotid artery stenosis as well as the number of carotid plaques and unstable plaques.

Objective To establish the determination method of total polyphenols in vitis amurensis seed extract.Methods Using gallic acid as reference substance,the content of total polyphenols in Vitis amurensis seed extract was determined by the optimum conditions of Folin-Ciocalteu chromatometry.Results There was a good linearity between gallic acid concentration and absorbance in the range 0.002-0.010 g?L-1(Y=86.243X+0.007,r=0.9996);the precision RSD was 0.14%;the stability RSD was 1.77%;the repeatability RSD was 1.44%;the average recovery rate was 102.00%(RSD=1.37%).Conclusion This method is simple,fast and has high sensitivity and good reproducibility.

Objective To investigate the role and possible mechanism of P120 catenin involving in the hemodynamic changes by inducing vascular endothelial cells injury through an in vitro experiment. Methods The hemodynamic environment under the different hemodynamic conditions at the vascular bifurcations was simulated through a T-shaped flow chamber system designed by ourselves. The human umbilical vein endothelial cells (HUVEC)cultured in vitro were stimulated and used the HUVEC cells of the small interfering RNA (SiRNA)after P120ctn gene fragments being knocked out. After loading flow rate of 250 and 500 ml/ min respectively and acting on for 12 h,the HUVEC morphology,growth pattern,and expression of P120ctn and other proteins were observed. Results (1)Normal HUVEC:500 ml/ min was loaded for 12 h,the cells were fused excessively at the impinging point,the cell gaps became narrowed,the cell density decreased and the morphology was elongated in the high wall shear stress (WSS)and wall
shear stress gradient (WSSG)regions. A part of cells migrated downstreamly,and their arrangement direction was consistent with the direction of impinging flow. Compared with the unloaded impinging flow field,after the 2 kinds of impinging flows being loaded for 12 h,the expression levels of P120ctn,vascular endothelial calcium (VEC),Kaiso,α-catenin,and other proteins were decreased. The expression level of matrix metalloproteinase 2 (MMP-2)was increased. There were significant differences (all P < 0. 05). (2)HUVEC after P120ctn being knocked out:Under the impact of the impinging flow,the cell growth time was reduced to 60 min. 250 ml/ min being loaded for 60 min,the impinging point and its surrounding cells still maintained the polygon,but some cells in the high WSS and high WSSG regions began to move downstreamly and aggregated,the cell arrangement mode partly arranged along with the direction of the flow;500 ml / min being loaded for 60 min,the cell density in the high WSS and high WSSG regions were decreased significantly and the morphology was elongated. A large number of cells migrated downstreamly and aggregated. The arrangement mode was parallel and consistent with the direction of the impinging flow. Compared with the unloaded impinging flow field,after the 2 kinds of velocities being loaded for 60 min,the expression levels of VEC,Kaiso,α-catenin proteins were decreased. The expression level of MMP-2 was increased,There were significant differences (all P < 0. 05) Conclusions The hemodynamic change may induce the changes in vascular endothelial cell morphology,growth pattern,and expression of P120ctn and other related proteins, leading to the decrease of vascular endothelial cell adhesion connection stability and the expression changes of related proinflammatory factors. The knockout of P120ctn may result in a further decrease of the vascular endothelial cell adhesion connection stability.

Objective To express Schistosoma japonicum Mago nashi(SjMago)gene,and prepare its specific polyclonal antibody.Methods SjMago gene was amplified by PCR from Schistosomulum cDNA library and subcloned into pET28a(+)vector,its recombinant proteins were expressed with IPTG.Rabbits were immunized with the polyacrylamide gel particles containing the recombinant proteins for polyclonal antibody preparation,the sera were detected for antibody specificity by Western blot and titer by ELISA assay.Results SjMago prokaryotic expression plasmid was successfully recombined and the target proteins was induced by IPTG in a molecular weight of 17 X 103，the high titer(1∶40 960)polyclonal antibody was isolated from the immunized rabbit，specific rotein band was detected by Western blot.Conclusion SjMago protein has been successfully expressed and its specific polyantibody is prepared,which lays the foundation for further study.

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.

The genetic diversity of three Tibetan herbs, i. e., Sang-Di, E-Dewa and Ye-Xingba (Tibetan names), was studied based on the field collection, specimen identification and DNA sequence analysis. Swertia hispidicalyx, Gentiana lhassica and Scrophularia dentata, as the original plants of the three Tibetan herbs, were collected and identified. The regions of ITS, matK, rbcL, rpoC1, trnL(UAA), psbA-trnH, atpB-rbcL, trnS (GCU)-trnG(UCC), rpl20-rps12, trnL(UAA)-trnF(GAA) and nadl 2nd intron were amplified and sequenced. The ITS regions of S. hispidicalyx and S. dentata were cloned and sequenced, and the sequences were classified into different genotypes. All the sequences were analyzed and compared with those of closely related species. Our studies may provide reference for the genetic diversity analysis and molecular identification of the three Tibetan herbs.
Genetic Variation ; Gentiana ; classification ; genetics ; Phylogeny ; Plant Proteins ; genetics ; Plants, Medicinal ; classification ; genetics ; Scrophularia ; classification ; genetics ; Swertia ; classification ; genetics ; Tibet

ObjectiveTo compare clinical and laboratory outcomes of in vitro maturation (IVM) with in vitro fertilization-embryo transfer (IVF-ET) in treatment of infertility associated with polycystic ovary syndrome (PCOS).MethodsFrom Jan.2007 to Dec.2010,infertile patients with PCOS underwent 701 cycles in First Affiliated Hospital of Wenzhou Medical College were studied retrospectively.Those were divided into 293 cycles of IVM group and 408 cycles of IVF/intra-cytoplasmic sperm injection ( ICSI ) group.The average transplantation rate,mean number of retrieval oocytes,maturation rate,fertilization rate,cleavage rate,high quality embryo rate,embryo implantation rate,pregnancy rate per transfer,pregnancy outcomes and incidence of ovarian hyperstimulation syndrome (OHSS) of the two methods of treatment were compared between two groups.ResultsThere were 275 cycles in IVM group and 342 cycles in IVF/ICSI group established embryo transfer.The transplantation rate was 93.9％(275/293)in IVM group and 83.8％ (342/408) in IVF/ICSI,which reached statistical difference (P ＜ 0.01 ).The maturation rate of 56.64％,cleavage rate of 88.08％,high quality embryo rate of 38.72％ and embryo implantation rate of 17.8％ in IVM group were significantly lower than 91.09％,94.91％,51.50％ and 25.4％ in IVF/ICSI group (all P ＜ 0.01 ).The clinical pregnancy rate per transfer were 37.8％ (104/275) in IVM group and 44.2％ (151/342) in IVF/ICSI group,which did not show statistical difference (P＞0.05).The mean number of oocytes ( 12.9 ±6.5 vs.12.9 ±7.9) and fertilization rate (76.52％ vs.70.75％ ) didn't show significant difference between IVM group and IVF/ICSI group ( P ＞ 0.05 ).The 21.3％ ( 87/408 ) cycles presented mild to moderate OHSS and 2.0％cycles(8/408)presented severe OHSS in IVF/ICSI group.While,no OHSS cycles were observed in IVM group.Conclusion IVM could get similar clinical pregnancy rates compared with IVF/ICSI in patient with PCOS,however,it can avoid occurrence of OHSS.

OBJECTIVETo study the correlation between arachidonic acid (AA) and acute red blood cells damage in rats, and to build a model with hidden blood loss in vivo, and to explore the pathological mechenism of hidden blood loss.

METHODSA total of 50 male adult Sprague-Dawley rats weighing (200 ± 20) g were randomly divided into five groups (n = 10): control group and four experimental groups. The rats in the experimental groups were given 0.5 ml different concentrations of AA dilu- ents, 5, 10, 20, 40 mmol/L respectively. The blood samples were collected from orbital venous at the beginning and 24, 48, 72 hours after administration. Then the changes of hemoglobin (Hb) ,red blood cell count (RBC), glutathione peroxidase (GSH- PX) activity, total superoxide dismutase (T-SOD) activity and hydrogen peroxide (H202) in the blood samples were tested.

RESULTSSignificant hidden blood loss occurred when the concentration was 10 mmol/L in the experimental group, with the RBC and Hb sharply reduced in blood samples. The Hb and RBC were reduced in all the experimental groups and control group at 24 hours after administration, while in the experimental groups they changed more obviously. The GSH-PX activity, T-SOD activity and H₂O₂were also significantly reduced in all groups, and the changes showed significant differences. The Hb and RBC were relatively stable in the control group and the experimental groups at 48 hours after administration; while GSH-PX activity, T-SOD activity and H₂O₂were all significantly decreased, and the changes in the experimental groups were more notable.

CONCLUSIONElevated levels of AA in the blood causes oxidative stress in the red blood cells, leading to the damage of red blood cells and hemoglobin, which is responsible for hidden blood loss.

Animals ; Arachidonic Acid ; toxicity ; Erythrocytes ; drug effects ; metabolism ; Glutathione Peroxidase ; blood ; Hemoglobins ; analysis ; Male ; Rats ; Rats, Sprague-Dawley ; Superoxide Dismutase ; blood

ObjectiveTo evaluate the clinical efficacy of cutaneous branches of reverse second and third dorsal metacarpal artery fasciocutaneous flaps for repair of distal- and middle-segment finger soft tissue defects. MethodsA total of 14 patients with distal- and middle-segment finger soft tissue defects complicated by exposure of the phalanx or tendon were repaired by using cutaneous branches of second and third dorsal metacarpal artery fasciocutaneous flaps ranging between 2.0 cm × 4.5 cm and 3.0 cm × 7.0 cm.ResuitsAll of the skin flaps survived after surgery.Follow-up data during a 6-40 month period showed that the flaps exhibited a satisfactory appearance.They were not fat or clumsy,with a 2-point discrimination of 59 mm,and there was good recovery of finger function.The donor site was able to be directly sutured without dermoplasty.Pigmented linear surgical streaks appeared in the donor site.Conclusion The cutaneous branches of Second and third dorsal metacarpal artery fasciocutaneous flaps provide a good approach for the repair of distal- and middle-segment finger soft tissue defects and functional reconstruction because of convenient dissection,little trauma,sufficient use of the dorsal metacarpal artery.