Objective:To construct the yeast expressive vector of rmFⅦ, in which mFⅦ was mutated to inhibit coagulation without affecting the affinity for TF, and express it in Pichia pastoris. Methods:The full length cDNA encoding mFⅦ was amplified from a mouse liver by RT-PCR method, site-direct mutated and restriction enzyme digested as design. Cloning into pPIC9K, electroporation of Gs115, in vivo screen of multiple inserts by G418 resistance, BMGY/BMMY are used for induction and expression of rmFⅦ in pichia pastoris. These proteins were also screened for functional activity. Results: Three different rmFⅦ-pPIC9K yeast expression vectors and it's aim protein were obtained,two kinds of proteins were found to be functional active as design. Conclusion:rmFⅦ protein can be expressed in pichia pastoris and it might facilitate the development of tumor-target molecule, and novel anti-agiogenesis drug study.

AIM: To study the quality control of Fuyankang Capsule(Radix et Rhizoma Salviae Miltiorrhizae,Radix Paeoniae Rubra,Radix Scutellariae,etc.) METHODS: The presence of Radix Paeoniae Rubra,Herba Taraxaci,Radix Scutellariae,Rhizoma Polygoni Cuspidati were identified by TLC.The content of hydroxybenzyl laxticacid,dansensu in the capsule was assayed by HPLC. RESULTS: Linearity of dansensu was found in the range from 0.04 ?g to 0.20 ?g.The average recovery was 98.8% and RSD was 1.5%. CONCLUSION: The method is highly sensitive,simple,precise and reproducible and can be used for the quality control of the capsule.

Aging can cause degenerative changes in the function of multiple tissues and organs in the body. Gastrointestinal diseases and intestinal dysfunction are very common in the elderly people. The purpose of this study is to explore the effect of the total extract of Astragalus membranaceus (Fisch.) Bge. on intestinal function and gut microbiota homeostasis in natural aging mice, which will provide clues for further mechanism study. The natural aging mice model is established and animal experiments follow the regulations of the Animal Ethics Committee of Nanjing University of Traditional Chinese Medicine. The overall health of the mice was evaluated by the "frailty index" scoring method. The intestinal absorption and transport function were measured by detecting intestinal glucose absorption capacity, transport time, lipase and amylase activities of aging mice. Intestinal inflammation was assessed by detecting inflammatory cytokines by enzyme-linked immunosorbent assay (ELISA). The pathological changes in the intestines of aging mice were tested by hematoxylin-eosin (H&E) staining and alizarin blue (AB) staining. The qRT-PCR method was used to explore the gene transcription level related with the proliferation and differentiation of intestinal stem cells. Microbiota analysis based on 16S rDNA were used to evaluate the composition of gut microbiota. The results showed that Astragalus had a tendency to reduce the "frailty index" of aging mice, but did not show a significant difference. In some indicators of aging phenotype, Astragalus has the most significant effect on hair loss and physical fitness. In terms of intestinal function, Astragalus could increase intestinal glucose absorption capacity, shorten intestinal transportation time and promote lipase secretion in aging mice. The levels of inflammatory cytokines such as tumor necrosis factor-α (TNF-‍α) in the aging intestinal tissue were reduced after Astragalus administration. Astragalus also ameliorated the pathological degeneration of the intestinal tissue of aging mice by increasing the length of small intestinal villi, the thickness of colonic mucosa and goblet cell number. In addition, Astragalus elevated the expression of genes associated with the proliferation and differentiation in jejunum and modulated gut microbiota, especially restoring the abundance of Lachnospiraceae. Taken together, the above research results demonstrate the total extract of Astragalus as a key factor improving the intestinal function and gut microbiota homeostasis of aging mice.

In this study, the ameliorative effects of Flos Abelmoschus manihot on mice with chronic inflammatory bowel disease (IBD) were investigated and its effects on the structure of the intestinal flora as well as the lipid profile in feces of IBD mice were analyzed. All animal welfare and experimental procedures followed the regulations of the Animal Ethics Committee of Nanjing University of Chinese medicine. A mouse model with chronic IBD induced by dextran sulfate sodium (DSS) was used to evaluate changes in body weight, disease activity index (DAI), colonic histopathological damage as well as gene expression levels of inflammatory factors in the colon. Fecal samples from mice in each group were collected and subjected to Illumina high-throughput sequencing to detect the abundance of intestinal flora; samples were analyzed by UHPLC-Q-Exactive^® HF Quadrupole-Orbitrap^® of untargeted lipidomics, which detects lipid content in feces. Administration of Flos Abelmoschus manihot could significantly restore the body weight and ameliorate colonic histopathological damage in IBD mice. Sequencing of the gut microbiota revealed that the species diversity and richness of the gut microbiota in IBD mice were decreased, with a significant increase in the abundance of Verrucomicrobia and a significant decrease in the abundance of Bacteroidetes; Flos Abelmoschus manihot significantly increased the richness and diversity of intestinal microbiota in IBD mice, increased the number of taxa species at each level, and restored the abundance of bacteria in the phylum Bacteroidetes. Analysis of fecal lipid profiles identified the most significant changes in sphingolipid and glycerophospholipid metabolic pathways in IBD mice, with Flos Abelmoschus manihot inhibiting ceramide and sphingomyelin synthesis in sphingolipid metabolism. In summary, Flos Abelmoschus manihot can effectively improve the disease condition of mice with chronic IBD, and it has the effect of regulating intestinal flora homeostasis and lipid metabolism, but the related mechanism between the two still needs to be deeply explored.

OBJECTIVETo study the effects of sodium tanshinone II A sulfonate (STS) on proliferation of fibroblasts (Fbs) in human hypertrophic scar (HS), the mRNA and protein expressions of transforming growth factor beta 1 (TGF-β1) and alpha smooth muscle actin (α-SMA), and to investigate the scar inhibition mechanism of STS.

METHODSFbs were isolated from HS tissues that were removed from eight patients after burn injury, and they were cultured in vitro. Cells from the 3rd to the 6th passages were used in the experiment. Fbs were divided into control group and experimental group according to the random number table, and cells in the experimental group was divided into 0.050, 0.075, 0.100, 0.125, 0.150, 0.200 mg/mL STS subgroups. Cells in each subgroup were cultured with the corresponding concentration of STS, and cells in control group were cultured in equal volume of serum-free medium. After being cultured for 24 and 48 h, cell morphology was observed with inverted phase contrast microscope; cell proliferation was determined with MTT method and the proliferation inhibition rate (IR) was calculated. After being cultured for 48 h, the protein levels of TGF-β1 and α-SMA were determined with Western blotting; the mRNA expressions of TGF-β1 and α-SMA were determined with RT-PCR (no 0.200 mg/mL STS subgroup was set for these two indicators). Data were processed with factorial analysis of variance; differences between groups were processed with LSD test or Games-Howell test for unequal variances.

RESULTS(1) Fbs grew well in control group, but reduction in adherence and disorderly arranged Fbs were observed in experimental group. The cells in experimental group became smaller and round, with increasing intracellular particles and necrosis. A large amount of necrotic debris of cells was observed in 0.200 mg/mL STS subgroup. (2) The absorbance value of Fbs in each experimental subgroup was significantly lower than that in control group (with P values all below 0.01). Along with the increase in the concentration of STS and extension of culture time, the IR value increased, showing a certain degree of time-concentration dependence. After being cultured with STS for 24 and 48 h, IR values of cells in the experimental subgroups were respectively 23.58%, 32.11%, 37.56%, 57.98%, 79.53%, 96.69% and 34.72%, 38.48%, 47.62%, 64.40%, 89.70%, 98.01%. (3) Except for the 0.050 mg/mL STS subgroup, the protein levels of TGF-β1 and α-SMA in the other subgroups were significantly lower than those in control group (with F values respectively 57.674, 47.795, P values all below 0.001). The protein levels of TGF-β1 and α-SMA reached the nadir in 0.150 mg/mL STS subgroup, respectively 0.34 ± 0.06, 0.33 ± 0.07. The relative expression amounts of TGF-β1 and α-SMA mRNA in the experimental subgroups were obviously decreased compared with those in control group (with F values respectively 68.548, 47.522, P values all below 0.001), which was most significant in 0.150 mg/mL STS subgroup, with TGF-β1 mRNA and α-SMA mRNA respectively 0.39 ± 0.07 and 0.42 ± 0.08.

CONCLUSIONSSTS can inhibit the proliferation of Fbs, reduce the protein and mRNA expressions of TGF-β1 and α-SMA, which may be beneficial to ameliorate the formation and contracture of HS, and it is assumed as a potential drug for treating scars.

Actins ; genetics ; metabolism ; Adult ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cicatrix ; metabolism ; pathology ; Female ; Fibroblasts ; cytology ; drug effects ; metabolism ; Humans ; Male ; Middle Aged ; Phenanthrenes ; pharmacology ; RNA, Messenger ; genetics ; Transforming Growth Factor beta1 ; genetics ; metabolism ; Young Adult

Objective To understand the relationship of tendency on drug abuse and related influencing factors for middle school students.Methods A total of 603 students wcre assessed with self-made copying style scale,consciousness to resistance of smoking,drinking and resistance ability through a questionnaire.A relationship model was set up by structure equation modeling.Results Copying style and resistance ability had significantly direct influence on tendency of drug abuse(with standardized coefficients were 0.202 and 0.092 respectively).Consciousness to resistance of smoking and drinking had significantly indirect influence on tendency of drug abuse.The goodneSS of fit for the structural equation model was satisfactory,with GFI,AGFI,RMSEA as 0.981,0.972 and 0.014 respectively.Conclusion It Was meaningful to grasp some copying skills and resistance ability to lower the tendency of drug abuse in middle school students.

Electrocoagulation ; methods ; Female ; Follow-Up Studies ; Humans ; Male ; Middle Aged ; Treatment Outcome ; Trigeminal Neuralgia ; surgery

OBJECTIVETo explore the types and clinical characteristics of traumatic subdural hydroma (TSH).

METHODOne hundred and ninety-two cases of TSH were classified into four types: The types of resolution, steadiness, development and evolution on the basis of their clinical characteristics and dynamic observation of CT scanning.

RESULTSThe patients in the resolution type often occurred in the prime of life. They had normal intracranial pressure and good prognoses using the conservative therapy. The majority of the elderly patients was in the steadiness type. Their main clinical manifestations included headache, dizziness, nausea, vomit, abnormal mentality and so on. Generally, there was no positive nervous systemic sign related to TSH. The prognoses of patients with the steadiness type treated by conservative therapy were satisfactory. The development type was common in the babies and children. This was mainly manifested as progressive increased intracranial pressure, mild hemiplegia, aphasia and abnormal mentality. The patients with development type often needed surgical treatment and might die once in a while due to accompanying cerebral parenchymal damage or postoperative complications. The evolution type was characterized by the polarized age, chronic increased intracranial pressure, often happening between 22 and 100 days after TSH and in the cases of small hydromas treated conservatively and mild accompanying cerebral damage, which always have a good prognosis by the treatment of surgery.

CONCLUSIONSThe mechanism, clinical characteristics, treatment methods and prognoses varied with different types of TSH.

Adolescent ; Adult ; Aged ; Child ; Child, Preschool ; Craniocerebral Trauma ; complications ; Female ; Humans ; Infant ; Male ; Middle Aged ; Prognosis ; Subdural Effusion ; classification ; diagnostic imaging ; therapy ; Tomography, X-Ray Computed

OBJECTIVETo explore the features of the clinical manifestations, imaging, pathology and microsurgery on the patients with gliomas of limbic and paralimbic system.

METHODSThe clinical data of 28 patients with gliomas of limbic and paralimbic system were analyzed respectively.

RESULTSSeizure was the most common symptom, presented in 24 cases. CT scans showed hypodensity in 19 cases and isodensity in 9 cases. MRI scans were achieved in 23 cases, showing hypointense signal on T(1)WI and hyperintense signal on T(2)WI. Total resection was achieved in 19 cases, subtotal in 6 cases and partial in 3 cases. All patients had excellent postoperative recovery, except 5 patients who developed temporary frontal aphasia and 6 patients who developed contralateral hemiplegia. The patients had a postoperative follow-up ranging from 6 months to 5 years, and 23 cases still survived.

CONCLUSIONHigh-resolution CT and MRI may reveal the size, location, confines and sharp demarcation of the tumors. Gliomas can be microsurgically removed with considerable results.

Adolescent ; Adult ; Brain Neoplasms ; diagnostic imaging ; pathology ; surgery ; Child ; Female ; Glioma ; diagnostic imaging ; pathology ; surgery ; Humans ; Limbic System ; Magnetic Resonance Imaging ; Male ; Microsurgery ; Middle Aged ; Tomography, X-Ray Computed

The purpose of this research is to study the effect of small molecule compound piceatannol (PIC) on host inflammation in adenine induced chronic kidney disease (CKD) mice, and then to explore its mechanism based on the regulation of gut microbiota. All procedures were approved by the Institutional Animal Care and Use Committee of the Nanjing University of Chinese Medicine. The level of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) was detected by enzyme linked immunosorbent assay (ELISA); UPLC-TQ/MS technology was used to monitor the level of proinflammatory uremic toxin indoxyl sulfate (IS) and p-cresol sulfate (PCS); the expression of occludin was tested by Western blot; in vitro anaerobic culture of gut bacteria was used to produce indole; the abundance of gut microbiota was evaluated by 16S rDNA sequencing. The results showed that PIC had no effect on inflammatory infiltration in kidney tissue of CKD mice, but could decrease IL-6 level in blood and IL-6/TNF-α level in colon tissue. PIC did not improve intestinal occludin protein expression in CKD mice; while it could significantly reduce the levels of IS and PCS in blood and liver of CKD mice. Further mechanism studies showed that PIC could inhibit the synthesis of IS precursor indole in gut bacteria. Moreover, PIC could decrease the abundance of gut bacteria which producing uremic toxin, such as reducing the abundance of indole and p-cresol producing gut bacteria. In conclusion, PIC could regulate gut microbiota and inhibit the synthesis of uremic toxin precursor, thereafter reducing the accumulation of IS and PCS in vivo, ultimately relieving the inflammation of CKD mice.