Endothelial progenitor cells (EPCs) can differentiate into mature endothelial cells and participate in postnatal vascular regeneration and impaired endothelium repair.Rearches in recent years on use of EPCs as seed cells in promoting angiogenesis,maintaining the integrity of endothelial function and constructing tissue engineered blood vessels are reviewed.

Objective To establish a mouse model stably replicating and expressing hepatitis B virus genotype C (HBV-C) prevailed in China. Methods The recombinant adeno-associated virus rAAV8-1.3HBV-C (adr serotype) was transduced into the human hepatocellular carcinoma Huh7 cells in vitro, and the expression of HBsAg and HBeAg in the cell culture supernatant was determined by ELISA. High expression recombinant virus rAAV8-1.3HBV-C was screened and injected via the tail vein into eight C57BL/6 mice (aged 6-8 weeks) as the experimental group; meanwhile the previously reported rAAV8-1.3HBV-D (ayw serotype) was injected into seven C57BL/6 mice as the control group. HBV DNA load, HBsAg and HBeAg levels in sera were assayed at weeks 2, 3, 5, 7 and 9 post viral injections. Mice were sacrificed 9 weeks post injection and Hematoxylin-eosin (HE) staining and immunohistochemistry were performed to evaluate the pathological changes, and HBsAg and HBcAg expressions in the liver tissue. Results The supernatant HBsAg and HBeAg were detectable in the HuH7 cells 72h after transduction in vitro. The fluorescence quantitative PCR results of the HBV DNA load in serum at week 2, 3, 5, 7, and 9 post viral injection suggested stable in vivo replication of HBV DNA in mice. The serum expression of HBeAg was stable while the serum expression of HBsAg fluctuated. No obvious inflammatory cell infiltration or abnormal structure of liver tissue was observed, while HBsAg and HBcAg expression in the liver tissue were detected for both groups. Conclusion By in vivo transduction with the recombinant virus rAAV8-1.3HBV-C, a mouse model that stably expressed and replicated HBV-C has been successfully established.

Objective: To investigate the effects of glycogen synthase kinase-3β (GSK3β)/eukaryotic extension factor kinase 2 (eEF2K) signaling pathway on the process of pulmonary fibrosis through in vivo experiments, and find new ideas for clinical treatment of pulmonary fibrosis. Methods: The pulmonary fibrosis model of C57BL/6 male mice was induced by bleomycin with intratracheal injection at the dose of 2 mg/kg. After 14 days of modeling, animals were divided into model group, negative inhibition group and inhibition group (n=5 for each group), and control group was not processed. The inhibition group was treated with TDZD-8 (4 mg/kg) after modeling, the negative inhibition group was given DMSO solution after modeling, and the samples were collected after 28 days. Hematoxylin-eosin staining method was used to detect lung fibrosis in mice and scored according to Ashcroft scale. Expression levels of GSK3β, p-GSK3β, eEF2K, p-eEF2K (Ser70, Ser392, Ser470), precursor protein of matrix metalloproteinase-2 (pro-MMP-2), matrix metalloproteinase-2 (MMP-2), collagen I (Col I), collagen Ⅲ (Col Ⅲ) and α-smooth muscle actin (α-SMA) were detected by Western blot. Results: Compared with control group, the fibrosis score was up-regulated, the expression levels of GSK3β, p-GSK3β, p-eEF2K (Ser70, Ser392, Ser470), pro-MMP-2, MMP-2, Col I, Col Ⅲ and α-SMA were increased, while that of eEF2K was decreased in model group (P＜0.05). Compared with model group, the fibrosis score, expression levels of GSK3β, p-GSK3β, p-eEF2K (Ser70, Ser392, Ser470), pro-MMP-2, MMP-2, Col I, Col Ⅲ and α-SMA were decreased, but the expression level of eEF2K was increased in inhibition group (P＜0.05). Conclusion: GSK3β can activate eEF2K by phosphorylation at the sites of Ser70, Ser392 and Ser470, increase the contents of fibrosis indicators, promote the formation of pulmonary fibrosis, and aggravate lung tissue lesions.
Animals ; Collagen ; Collagen Type I ; Elongation Factor 2 Kinase/metabolism* ; Eukaryota/metabolism* ; Fibrosis ; Glycogen Synthase Kinase 3 beta ; Male ; Matrix Metalloproteinase 2/metabolism* ; Mice ; Mice, Inbred C57BL ; Pulmonary Fibrosis/chemically induced* ; Signal Transduction

OBJECTIVETo investigate anticancer effects of 5-fluorouracil (5-FU) combined with CL, extract of Rosa roxburghii Tratt on human endometrial adenocarcinoma cell line (JEC).

METHODSJEC cells cultured in vitro in the logarithmic growth phase were seeded in the culture plate and divided into the control group (RPMI 1640), the positive group (10(-4) mol/L 5-FU), the CL groups (at the dose of 0.01, 0.1, 1, 10, and 100 microg/mL), and the CL (0.01, 0.1, 1, 10, and 100 microg/mL) combined with 5-FU groups. Effects of 5-FU combined with CL on JEC cell growth were drawn and measured by MTT and growth curves. Effects of CL combined with 5-FU on the JEC cell differentiation was analyzed by detecting the reduction capability of nitrobenzene thiocyanate (NBT) and lactate dehydrogenase (LDH) contents in the cultured medium. Effects of CL combined with 5-FU on the JEC cell apoptosis and cell proliferation cycle were detected by acridine orange (AO)/ethidium bromide (EB) fluorescent staining and flow cytometry (FCM).

RESULTSThe proliferation inhibitory effect of CL combined with 5-FU on JEC cells was enhanced when compared with that of CL or 5-FU alone (P<0.05). The percentages of NBT positive JEC cells and apoptotic JEC cells increased in the 5-FU combined with CL groups when compared with 5-FU group or the CL group alone (P<0.05). The LDH concentration of the JEC cell culture supernate decreased in 5-FU combined with CL groups (P<0.05). Furthermore, the percentage of G0-G1 phase JEC cells treated by 5-FU combined with CL was higher than that of 5-FU or CL alone (P<0.05).

CONCLUSIONCL could enhance anticancer effects of 5-FU. Its mechanisms might be correlated with reinforcing the cytotoxicity of 5-FU, inducing cell differentiation and apoptosis, and inhibiting cell proliferation and division.

Adenocarcinoma ; pathology ; Cell Differentiation ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Endometrial Neoplasms ; pathology ; Female ; Fluorouracil ; pharmacology ; Herb-Drug Interactions ; Humans ; Plant Extracts ; pharmacology ; Rosa ; chemistry

OBJECTIVETo observe the therapeutic effect of parthenolide (PTL) on rabbit knee arthritis (KOA) and its effects on serum expression of interleukin-1beta (IL-1beta) and contents of tumor necrosis factor-alpha (TNF-alpha).

METHODSEight rabbits were randomly selected from 40 healthy pure-bred New Zealand rabbits as the normal control group. The KOA model was established in the rest 32 rabbits by plaster cast fixation of the right hind limb extension position. After modeling they were randomly divided into 4 groups, i.e., the model control group, the high dose PTL group, the middle dose PTL group, and the low dose PTL group, 8 in each group. Serum contents of IL-1beta and TNF-alpha were detected using enzyme-linked immunosorbent assay.

RESULTSCompared with the model group, IL-1beta and TNF-alpha concentration decreased in the 3 PTL groups (P < 0.01). The decrement was positively correlated with PTL concentrations (IL-1beta: r = 0.55, P < 0.01; TNF-alpha: r = 0.56, P < 0.01). The inhibition reached the peak when the PTL concentration arrived at 20 micromol/L.

CONCLUSIONSPTL could down-regulate the blood IL-1beta and TNF-alpha concentrations of KOA rabbits. Besides, the decrement was positively correlated with the PTL concentration.

Animals ; Female ; Interleukin-1beta ; blood ; Male ; Osteoarthritis, Knee ; blood ; drug therapy ; Phytotherapy ; Rabbits ; Sesquiterpenes ; pharmacology ; therapeutic use ; Tumor Necrosis Factor-alpha ; blood

This study bioinformatically analyzed the non-VP1 capsid proteins (VP2-VP4) of Coxasckievirus A6 (CVA6), with an attempt to predict their basic physicochemical properties, structural/functional features and linear B cell eiptopes. The online tools SubLoc, TargetP and the others from ExPASy Bioinformatics Resource Portal, and SWISS-MODEL (an online protein structure modeling server), were utilized to analyze the amino acid (AA) sequences of VP2-VP4 proteins of CVA6. Our results showed that the VP proteins of CVA6 were all of hydrophilic nature, contained phosphorylation and glycosylation sites and harbored no signal peptide sequences and acetylation sites. Except VP3, the other proteins did not have transmembrane helix structure and nuclear localization signal sequences. Random coils were the major conformation of the secondary structure of the capsid proteins. Analysis of the linear B cell epitopes by employing Bepipred showed that the average antigenic indices (AI) of individual VP proteins were all greater than 0 and the average AI of VP4 was substantially higher than that of VP2 and VP3. The VP proteins all contained a number of potential B cell epitopes and some eiptopes were located at the internal side of the viral capsid or were buried. We successfully predicted the fundamental physicochemical properties, structural/functional features and the linear B cell eiptopes and found that different VP proteins share some common features and each has its unique attributes. These findings will help us understand the pathogenicity of CVA6 and develop related vaccines and immunodiagnostic reagents.
Amino Acid Sequence ; Capsid Proteins ; genetics ; immunology ; Computational Biology ; Enterovirus ; genetics ; pathogenicity ; Epitopes, B-Lymphocyte ; genetics ; immunology ; Humans

A high performance liquid chromatography (HPLC) method utilizing correction factors was established for the quantitative detection of related substances in flumazenil. Separation was achieved using an Agilent Pursuit XRs C18 column (250 mm × 4.6 mm, 5 μm) with an isocratic elution of dilute phosphoric acid, methanol, and tetrahydrofuran as the mobile phases. Correction factors calculated from a standard curve method were applied to determine the impurity content. The quantification of impurities in flumazenil was conducted using both external standard and correction factor methods, followed by validation and comparison of the two. For the identification of degradation products, a forced degradation approach was employed to prepare a flumazenil degradation solution, and the resulting impurities were confirmed by LC-MS analysis. The separation of flumazenil and its impurities was found to be efficient. The limits of quantification for impurities A, B, D, and E were established at 0.169 9, 0.314 7, 0.143 9, and 0.270 8 ng, respectively, with the limits of detection at 0.055 8, 0.096 9, 0.048 8, and 0.089 0 ng. These impurities demonstrated a strong linear relationship across the concentration ranges of 0.034 9-7.847 0, 0.038 7-8.710 7, 0.034 6-7.794 1, and 0.032 4-7.292 8 µg·mL^-1, respectively (n = 7). The method achieved average recoveries between 98.25% and 99.42%, with an RSD of less than 2.0% (n = 9), indicating high accuracy. The external standard and correction factor methods were used to determine the related substances in flumazenil, and the results of the two methods were consistent. The established HPLC method is characterized by its high accuracy, sensitivity, and repeatability, and is suitable for determining related substances in flumazenil.

ObjectiveTo investigate the role of protein kinase B （Akt） overexpression in the inhibition of human bladder cancer 5637 cell proliferation by erianin and related mechanisms. MethodThe 5637 cells stably over-expressing Akt were induced using the lentivirus vector. The 5637 cells infected with the empty vector were classified into blank group. Then the Akt group， empty vector combined with erianin （62.5 μg·L^-1） group， and Akt combined with erianin （62.5 μg·L^-1） group were set up. The cell viability was detected by cell counting kit-8 （CCK-8） assay， and the clone formation of 5637 cells in each group was determined in the clone formation experiment. The cell cycle distribution was detected by flow cytometry. Western blot was used to assay the protein expression levels of phosphorylated （p）-Akt， Akt， p21. The glycolysis of 5637 cells was determined in glucose uptake and lactate secretion assays. ResultCompared with the blank group， erianin inhibited the proliferation of bladder cancer 5637 cells （P<0.05）. Overexpression of Akt partially reversed the inhibitory effect of erianin on the proliferation of bladder cancer 5637 cells （P<0.05）. Clone formation assay showed that erianin inhibited the clone formation of bladder cancer 5637 cells （P<0.05）， which was partially reversed by the overexpressed Akt （P<0.05）. As revealed by comparison with the blank group， erianin arrested the bladder cancer 5637 cells in G₁ phase （P<0.05）， which was also reversed by the overexpressed Akt （P<0.05）. Western bolt showed that erianin promoted the expression of p21 but suppressed the expression of p-Akt and Akt （P<0.05）. By contrast， the overexpression of Akt down-regulated the elevated p21 protein expression induced by erianin （P<0.05）. Compared with the blank group， erianin inhibited the glucose uptake and lactate secretion of bladder cancer 5637 cells （P<0.05）. Overexpression of Akt weakened the inhibitory effect of erianin against the glycolysis of 5637 cells （P<0.05）. ConclusionErianin is able to inhibit the proliferation of bladder cancer 5637 cells， promote the expression of p21， and inhibit the expression of p-Akt. Overexpressed Akt reduces the inhibitory effect of erianin on the proliferation of bladder cancer 5637 cells， suggesting that Akt plays an important role in the inhibition of 5637 cell proliferation by erianin， which has provided a new target for the application of erianin in the treatment of bladder cancer.

Objective To express and purify recombinant and biologically active Clostridium difficile toxin B (rTcdB). Methods The genes of TcdB were amplified by polymerase chain reaction (PCR) using chromosomal DNA from a toxigenic strain, and cloned into a shuttle vector pHis1522.The sequences of TcdB genes in the vector were verified by DNA sequencing. The construction was transformed into Bacillus megaterium protoplasts and the protein expression was driven by a xylose promoter. The purified protein was tested for biological activity. Results rTcdB was successfully purified from bacterial crude extracts. Approximately 5-10 mg of highly purified recombinant toxin was obtained from one liter of bacterial culture. The expressed rTcdB had molecular mass similar to the native toxin, and its biological activity was proved to be similar to its native counterpart after an extensive examination. Conclusion rTcdB with biological activities is successfully expressed in Bacillus megaterium.

Objective To investigate difference of NKG2D receptor expression level on the surface of natural killer (NK) cells in the patients with hepatitis B and its clinical significance.Methods This was a four-arm study with different types of subjects,including patients with chronic hepatitis B (CHB,n =22),HBV carriers (HBVC,n=10),patients with acute hepatitis B (AHB,n=18) and healthy donors (HD,n=18).NKG2D protein and mRNA levels on the surface NK cells in the peripheral blood were examined by reverse transcription-polymerase chain reaction assay.The relationship between NKG 2D expression and serum hepatitis B virus (HBV) DNA level was analyzed.The data were compared by analysis of variance and linear regression.Results NKG2D mRNA expression levels in groups of HBVC, HD, AHB and CHB were 0.96±0.17, 1.03±0.12,1.53±0.30 and 1.51 ± 0.35,respectively; the differences among groups were statistically significant (q=7.586,7.485,7.920 and 7.880,respectively; all P＜0.01).NKG2D protein expression levels in groups of AHB,HD,CHB and HBVC were 0.87±0.14,0.89±0.17,0.67±0.09 and 0.59±0.13,respectively; the differences among groups were statistically significant (q=6.92,7.67,7.53and 8.16,respectively; all P＜0.01).The NKG2D mRNA expression levels on NK cells were negatively correlated with serum HBV DNA viral loads in patients with CHB,AHB or HBVC (r=-0.75,-0.66 and-0.69,respectively; all P＜0.01).The NKG2D protein levels on NK cells from patients with AHB and CHB were negatively correlated with serum HBV DNA levels (r=-0.47 and -0.45,respectively; both P＜0.05).Conclusion NKG2D mediated NK cytotoxicity may play a role in viral clearance in hepatitis B.