Fourteen compounds were isolated from 95% ethanol extract by silica gel, MCI, and ODS column chromatography. These compounds were respectively identified as quercetin (1), kaempferol (2), (+)-catechin (3), fraxin (4), protocatechuic acid (5), gallic acid (6), methyl gallate (7), ethyl gallate (8), apocynol A (9), baccatin (10), cerevisterol (11), ellagic acid (12), 3, 3',4'-tri-0-methylellagic acid(13) and N-benzoyl-L-phenylalaninyl-N-benzoyl-L-phenylalaninate(14) by analyzing their spectral data and comparing with the previously reported literatures. Except for gallic acid (6), all other compounds were isolated from this plant for the first time. Compounds 1, 2 and 6 showed moderate anti-proliferation activities on tumor cells.
Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Drugs, Chinese Herbal ; chemistry ; toxicity ; Euphorbiaceae ; chemistry ; Humans ; Plants, Medicinal ; chemistry ; Spectrometry, Mass, Electrospray Ionization

To knock out β-lactoglobulin (BLG) gene and insert human lactoferrin (hLF) coding sequence at BLG locus of goat, the transcription activator-like effector nucleases (TALEN) mediated recombination was used to edit the BLG gene of goat fetal fibroblast, then as donor cells for somatic cell nuclear transfer. We designed a pair of specific plasmid TALEN-3-L/R for goat BLG exon III recognition sites, and BLC14-TK vector containing a negative selection gene HSV-TK, was used for the knock in of hLF gene. TALENs plasmids were transfected into the goat fetal fibroblast cells, and the cells were screened three days by 2 μg/mL puromycin. DNA cleavage activities of cells were verified by PCR amplification and DNA production sequencing. Then, targeting vector BLC14-TK and plasmids TALEN-3-L/R were co-transfected into goat fetal fibroblasts, both 700 μg/mL G418 and 2 μg/mL GCV were simultaneously used to screen G418-resistant cells. Detections of integration and recombination were implemented to obtain cells with hLF gene site-specific integration. We chose targeting cells as donor cells for somatic cell nuclear transfer. The mutagenicity of TALEN-3-L/R was between 25% and 30%. A total of 335 reconstructed embryos with 6 BLG-/hLF+ targeting cell lines were transferred into 16 recipient goats. There were 9 pregnancies confirmed by ultrasound on day 30 to 35 (pregnancy rate of 39.1%), and one of 50-day-old fetus with BLG-/hLF+ was achieved. These results provide the basis for hLF gene knock-in at BLG locus of goat and cultivating transgenic goat of low allergens and rich hLF in the milk.
Animals ; Animals, Genetically Modified ; genetics ; Female ; Fibroblasts ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Goats ; genetics ; Humans ; Lactoferrin ; genetics ; Lactoglobulins ; genetics ; Milk ; chemistry ; Nuclear Transfer Techniques ; Plasmids ; Pregnancy ; Transfection

Human tissue kallikrein-binding protein (Kallistatin, KAL), a secretory protein that participates in the regulation of multiple signaling pathways by binding to the extracellular receptor, however, at present has not been reported about the intracellular activity, and whether it has the similar biological activity with extracellular activity. Here we constructed no signal peptide KAL (NSK) into the adeno-associated virus vector to explore the intracellular activity of KAL. Both the endothelial cell and lung cancer cells could express KAL, but not secreted after rAAV2-NSK transfection. The proliferation and migration of human umbilical vein endothelial cells (HUVECs) were inhibited, but the apoptosis rate was not affected. The proliferation rates, mobility and tubule formation of all the three tested lung cancer cells, such as NCI-H446, NCI-H460 and A549, were inhibited to different extents. This cellular study not only confirmed the intracellular activity, but also suggested it may serve as a kind of "balance factor" in multi-targeted controlling, which may provide a new train of thoughts to explain the regulatory contradiction in PI3K-Akt signaling pathways by KAL.
Apoptosis ; Cell Proliferation ; Dependovirus ; Genetic Vectors ; Human Umbilical Vein Endothelial Cells ; metabolism ; Humans ; Lung Neoplasms ; metabolism ; Serpins ; metabolism ; Signal Transduction ; Transfection

The purpose of this study was to fabricate decelluarized valve scaffold modified with polyethylene glycol nanoparticles loaded with transforming growth factor-β1 (TGF-β1), by which to improve the extracellular matrix microenvironment for heart valve tissue engineering in vitro. Polyethylene glycol nanoparticles were obtained by an emulsion-crosslinking method, and their morphology was observed under a scanning electron microscope. Decelluarized valve scaffolds, prepared by using trypsinase and TritonX-100, were modified with nanoparticles by carbodiimide, and then TGF-β1 was loaded into them by adsorption. The TGF-β1 delivery of the fabricated scaffold was measured by asing enzyme-linked immunosorbent assay. Whether unseeded or reseeded with myofibroblast from rats, the morphologic, biochemical and biomechanical characteristics of hybrid scaffolds were tested and compared with decelluarized scaffolds under the same conditions. The enzyme-linked immunosorbent assay revealed a typical delivery of nanoparticles. The morphologic observations and biological data analysis indicated that fabricated scaffolds possessed advantageous biocompatibility and biomechanical property beyond decelluarized scaffolds. Altogether this study proved that it was feasible to fabricate the hybrid scaffold and effective to improve extracellular matrix microenvironment, which is beneficial for an application in heart valve tissue engineering.

Objective To investigate the expression level of Gas6 in patients with Immune Thrombocytopenia (ITP) and its clinical significance.Methods The experimental group was peripheral blood samples collected from 35 cases diagnosed with ITP in hematology department of Changhai Hospital in Shanghai from October 2013 to December 2015.Control group was peripheral blood from 35 healthy examined individuals at the same time.After separating plasm from the two group samples,the protein level of Gas6,IFN-α,IL-4,IFN-γ and IL-17 were measured by Enzyme-linked immunosorbent assay (ELISA),comparison of expressional level of the two groups was measured by t test.Pearson correlation analysis was used to decide the relation between Gas6 and cytokines such as IFN-α.Results The expression level of Gas6 in experimental and control groups was 27.28±7.56 ng/ml vs 20.51±5.39 ng/ml (t=4.314,P＜0.000 1);IFN-γ was 221.67±57.64 pg/ml vs 45.32 ±16.79 pg/ml (t=17.38,P＜0.000 1);IL-4 was 113.86±26.48 pg/ml vs 49.87±14.98 pg/ml (t=12.44,P＜0.000 1);IL-17 was 168.96±47.88 pg/ml vs 109.56±28.97 pg/ml (t=6.28,P＜0.000 1);IFN-α was 34.83±8.12 pg/ml vs 29.89 ± 5.76 pg/ml (t=2.936,P=0.004 5),all with statistical differences (P＜0.05).Pearson analysis showed that Gas6 was positively related with IL-17,IL-4,IFN γ (r=0.564,0.486,0.449,P＜0.05) and there was difference statistically,but Gas6 was not correlated with IFN-α.Conclusion Gas6 may participate in the disease formation of ITP through affection on cytokines secreted by Th cell subsets,and it was the potential therapeutic and predicted target for this disease clinically.

Aged ; Biopsy, Needle ; methods ; Coal Mining ; Humans ; Lung ; pathology ; Lung Neoplasms ; complications ; diagnosis ; Middle Aged ; Pneumoconiosis ; complications

OBJECTIVETo study the possible association between C-reactive (CRP) and insulin resistance (IR) as well as the pathogenesis of type 2 diabetes macrovascular complication.

METHODSSerum CRP was measured by ultrasensitive immuoturbidimetric, which was determined on type 2 diabetes (T2DM) with or without macrovascular complication (70 and 60 cases respectively) and on normal controls (90 cases). IR was estimated by homeostasis model assessment (HOMA-IR).

RESULTSResults showed that the concentration of CRP was higher in T2DM with or without macrovascular complications than that in the healthy subjects (P < 0.01), while it was higher in diabetic patients with macrovascular complications than that in diabetic patients without macrovascular complications (P < 0.01). In diabetic patients with macrovascular complications, person correlation analysis indicated that there existed positive correlations between CRP and FINS, HOMA-IR, triglyceride (TG) while stepwise linear regression showed that usCRP and HOMA-IR, TG having linear correlation.

CONCLUSIONCRP seemed to play a role in the initiation and progression of atherosclerosis in type 2 diabetes, possibly was by the way of IR.

C-Reactive Protein ; metabolism ; Case-Control Studies ; Diabetes Complications ; blood ; metabolism ; pathology ; Diabetes Mellitus, Type 2 ; complications ; metabolism ; physiopathology ; Female ; Homeostasis ; Homocysteine ; blood ; Humans ; Inflammation Mediators ; metabolism ; Insulin Resistance ; Linear Models ; Male ; Middle Aged

OBJECTIVETo explore the biological mechanisms underlying Angelica sindsis polysaccharide (ASP) -induced aging of human-derived leukemia stem cells (LSCs) in vitro.

METHODAcute myelogenous leukemia stem cells were isolated by magnetic activated cell sorting (MACS). The ability of LSC proliferation treated by various concentration of ASP(20-80 mg · L(-1)) in vitro for 48 hours were tested using cell counting Kit-8 ( CCK8) , colony forming were evaluated by methylcellulose CFU assay. The ultra structure changes of AML CD34+ CD38- cells were analyzed by transmission electron microscopy. The aging cells were detected with senescence-β-galactosidase Kit staining. Expression of aging-related p53, p21, p16, Rb mRNA and P16, Rb, CDK4 and Cyclin E protein were detected by quantitative reverse transcription polymerase chain reaction( qRT-PCR) and Western blotting, respectively.

RESULTThe purity of the CD34 + CD38 - cells is (91.15 ± 2.41)% after sorted and showed good morphology. The proliferation of LSC was exhibited significantly concentration-dependent inhibited after exposure to various concentration of ASP. Treated by 40 mg · L(-1) ASP for 48 hours, the percentage of positive cells stained by SA-β-Gal was dramatically increased (P < 0.01) and the colony-formed ability has been weakened (P < 0.01). The observation of ultrastructure showed that cell heterochromatin condensation and fragmentation, mitochondrial swelling, lysosomes increased in number. Aging-related p53, p21, p16, Rb and P16, Rb were up-regulated, protein regulatory cell-cycle CDK4 and Cyclin E were down-regulated. ASP may induce the senescence of LSCs effectively in vitro, P16-Rb cell signaling pathway play a significant role in this process.

CONCLUSIONASP can induce human leukemia stem cell senescence in vitro, the mechanism involved may be related to ASP regulation P16-Rb signaling pathways.

Angelica sinensis ; chemistry ; Cell Cycle ; drug effects ; Cell Cycle Proteins ; genetics ; metabolism ; Cells, Cultured ; Cellular Senescence ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Gene Expression Regulation, Leukemic ; drug effects ; Humans ; Leukemia ; drug therapy ; genetics ; metabolism ; physiopathology ; Neoplastic Stem Cells ; cytology ; drug effects ; Polysaccharides ; pharmacology ; Signal Transduction ; drug effects

OBJECTIVE We have recently reported that cysteinyl leukotriene (CysLT) signaling plays an important role in microglial interleukin (IL)-1β secretion and subsequent neurotoxicity. The present study aimed to examine microglial morphological changes and the upstream molecular underlying IL-1βproduction in CysLT receptor agonist leukotriene D4 (LTD4)-treated BV2 microglia in vitro. METHODS Twenty-four hours after murine microglial BV2 cells were stimulated with LTD4 (1-100 nmol·L- 1), the cell proliferation and morphology were observed. The expression level of cysteinyl aspartate-specific protease 1 (CASP1) protein was measured by Western blotin BV2 cells. In addition, BV2 cells were pretreated with or without CysLT1 receptor antagonist montelukast for 1 h and the effects of monte-lukaston LTD4-stimulated microglial activation and CASP1 expression were evaluated. RESULTS The number of BV2 cells had an increasing tendency after 24 h treatment with LTD4, but no significant differences were observed between the control and LTD4-treated cells (P>0.05). Under basal and resting conditions, BV2 microglial cells displayed a ramified morphology. However, LTD4 at 100 nmool · L- 1 drove microglial morphological changes from a ramified towards an amoeboid shape. The expression of CASP1 protein was significantly upregulated in 100 nmool·L-1 LTD4-treated BV2 microglia (P<0.01). Furthermore, pretreatment with CysLT1 receptor antagonist montelukast prevented cell morphological changes and suppressed the increased CASP1 expression in LTD4-treated BV2 cells (P<0.05). CONCLUSION CysLT receptor agonist LTD4 induces morphological changes and CASP1 expressionin BV2 microglia, which can be inhibited by CysLT1 antagonist. These results suggest the involvement of CysLT signaling in microglial morphological changes and CASP1 expression.

[Objective]To investigate the influence of healthy walking intervention on risk factors of noncommunicable chronic disease in occupational population, and to explore the suitable mode of exercise intervention for occupational population in Shanghai. [Methods]Before and after healthy walking intervention were compared the changes of body weight, BMI, waist circumference, body fat rate, visceral fat index, over-weight and obesity rate, central obesity rate, blood-pressure controlling rate. [Results]Weight, BMI, waist circumference, body fat percentage, viscera index, SBP and DBP all reduced after100 days of healthy walking, and the results were (1.52 ± 2.75) kg (Z =-21.99, P < 0.01) , (0.55 ±1.03) kg/m2 (Z =-21.64, P<0.01) , (2.10±5.27) cm (Z =-17.62, P<0.01) , (0.31±4.59) ％ (Z=-3.48, P < 0.01) , (0.12 ± 1.99) (Z =-2.70, P < 0.01) , (2.51 ± 10.87) mm Hg (Z =-9.35, P <0.01) and (1.67±8.26) mm Hg (Z =-9.06, P < 0.01). The rate of over-weight and obesity decreased7.86％, the rate of central obesity decreased 6.92％, and the rate of blood-pressure controlling increased2.72％. There were significant difference between the three indicators before and after healthy walking (χ2= 916.48, P< 0.01; χ2= 585.90, P < 0.01; χ2= 366.37, P < 0.01). [Conclusion] Healthy walking could reduce occupational population' s over-weight and obesity rate, central obesity rate, and increase blood-pressure controlling rate. The risk factors of un-communicable chronic disease have improved significantly. Healthy walking plays a positive role in the prevention and control of chronic diseases.