Cell Differentiation ; drug effects ; Cells, Cultured ; Fetal Blood ; cytology ; Fibroblast Growth Factor 4 ; pharmacology ; Hepatocyte Growth Factor ; pharmacology ; Hepatocytes ; cytology ; Humans

To understand the structure of the soluble region of Enterovirus 68 3A protein, we construct a prokaryotic expression vector expressing the soluble region of EV-D68 3A protein, and identify the forms of expression product after purification. The EV-D68 3A(1-61) gene was amplified by PCR and then cloned into the expression vector pET-28a-His-SUMO. The recombinant plasmid was transformed into Escherichia coli BL21 induced by IPTG to express the fusion protein His-SUMO-3A(1-61). The recombinant protein was purified by Ni-NTA Agarose and cleaved by ULP Protease to remove His-SUMO tag. After that, the target protein 3A(1-61) was purified by a series of purification methods such as Ni-NTA, anion exchange chromatography and gel filtration chromato- graphy. Chemical cross-linking reaction assay was taken to determine the multiple polymerization state of the 3A soluble region. A prokaryotic expression vector pET28a-His-SUMO-3A(1-61) expressing the solution region of EV-D68 3A was successfully constructed and plenty of highly pure target proteins were obtained by multiple purification steps . The total protein amount was about 5 mg obtained from 1L Escherichia coli BL21 with purity > 95%. At the same time, those results determined the homomultimer form of soluble 3A construct. These data demonstrated that the expression and purification system of the soluble region of 3A were successfully set up and provide some basic konwledge for the research about 3A crystal structure and the development of antiviral drugs targeted at 3A to block viral replication.
Amino Acid Sequence ; Enterovirus D, Human ; chemistry ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Models, Molecular ; Molecular Sequence Data ; Protein Structure, Tertiary ; Sequence Alignment ; Viral Proteins ; chemistry ; genetics ; metabolism

ObjectiveTo detect specific antigens of neural cells in amniotic epithelial cells(AECs) in rats. MethodsAECs were dissociated and purified from the amnion of pregnancy 12—14 d rats. The expression of specific markers of neural stem cells (Nestin, Musashi) and differentiated cells (MAP-2,NSE,GFAP) and ChAT, NT-3 in the AECs were detected by immunocytochemistry. ResultsThe cultured AECs displayed positive immunoreactivity to MAP-2, NSE, GFAP, Nestin and Musashi. In addition, the cells also demonstrated immunoreactivity to ChAT and NT-3. ConclusionAECs are similar with neural cells and it may be useful as a sustained source to improve outcome of neural stem cells transplantation.

ObjectiveTo investigate an effective method to isolate neural stem cells(NSCs).MethodsNSCs were dissociated by digestion with trypsin, EDTA and different doses of Dispase,and serum-free culture techniques and immunohistochemistry techniques were used to verifying the dissociated.ResultsA lot of single neural stem cells were obtained by using Dispase to digest neurosphere, and the cells could keep its structure and morphology.ConclusionIt is an ideal method by using Dispase to digest neurosphere for isolating NSCs.

The HPLC fingerprint determination method of Jinzhen oral solution was established to provide a new method for quality control of Jinzhen oral solution. RP-HPLC was used for phenomenex Luna C18 (4.6 mm x 250 mm, 5 μm) chromatographic column, with 0.1% H3 PO4 water solution and acetonitrile as the mobile phase for gradient elution. The detection wavelength was 280 nm. HPLC fingerprint of Jinzhen oral solution was established to identify 17 common peaks in Jinzhen oral solution. The similarity of fingerprints of 10 batches of finished products was more than 0. 90. The established HPLC fingerprint has a better precision, reproducibility and stability, and can be applied in quality control of Jinzhen oral solution.
Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; chemistry ; Quality Control

The aim of this study was to investigate and evaluate the antitumor effects of a novel poly(ADP-ribose) polymerase (PARP) 1/2 inhibitor, YHP-836, in combination with temozolomide (TMZ) for the treatment of glioblastoma (GBM). The cytotoxicity of YHP-836 was tested alone or in combination with TMZ using MTT assay. Immunoblotting and flow cytometry were also employed to assess the combination activity of YHP-836 and TMZ in multiply GBM cell lines. Further, the antitumor activity of YHP-836 and TMZ was evaluated using subcutaneous and orthotopic mice xenograft tumor models. All procedures were approved by the Ethics Committee for Animal Experiments of the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College and conducted under the Guidelines for Animal Experiments of Peking Union Medical College. The approval number is 00009138. It was demonstrated that the combination of YHP-836 and TMZ increased the cytotoxicity against GBM cells and upregulated histone H2AX phosphorylation (γH2AX) expression levels compared to TMZ treatment alone. The combination also led to the S-phase cell cycle arrest. Moreover, YHP-836 significantly enhanced TMZ antitumor effects without significantly increasing chemotherapy drug toxicity in vivo, whereas YHP-836 alone showed limited therapeutic efficacy against GBM. In conclusion, the novel PARP1/2 inhibitor, YHP-836, sensitizes TMZ and provides a basis for further investigation into its mechanism of action. These findings suggest that YHP-836 may be a potential candidate for combination therapy with TMZ in patients with TMZ resistance.

Objective To observe the effect of Ti-6AL-4V particles on morphological and func- tional changes of osteoclast in vitro.Methods Mature osteoclasts separated from New Zealand Rabbits were cultured on glass slices and cortical bone slices.The experimental group was stimulated by Ti-6AL- 4V particles at concentration of 0.1 mg/ml.The cells were stained with TRAP at different culture time to observe the morphological variety.The bone resorption pits on bone slices were stained by toluidine blue and the resorption areas analyzed by computer image analysis software.Results Osteoclasts phagocy- tosed the particles,with irregular shapes,deeper TRAP stain and earlier apoptosis.Stimulation by Ti- 6AL-4V particles brought about larger area of bone absorption lacuna.Conclusion Osteoclasts have the ability to phagocytose Ti-6AL-4V particles,which leads to morphological and functional changes and enhances bone resorption.

Prostate cancer (PCa) is one of the most common malignancies in the urinary system of males. A growing number of studies have shown that microRNAs, as small ribonucleic acid molecules and a class of non-coding small RNAs, are closely related with PCa and a variety of microRNAs are abnormally expressed in it. This article focuses on the roles of microRNAs in the occurrence and progression of PCa, with a description of differentially expressed microRNAs in PCa and an analysis of their association with its prognosis as well as their correlation with chemotherapy, androgen receptors, and metastasis of PCa.
Disease Progression ; Humans ; Male ; MicroRNAs ; metabolism ; Prognosis ; Prostatic Neoplasms ; chemistry ; genetics ; metabolism ; Receptors, Androgen ; metabolism

To investigate the pharmacokinetic characteristics and absolute bioavailability of ginkgolide A (GA), ginkgolide B (GB) and bilobalide (BB) in rats. In this experiment, a high-performance liquid chromatography-tandem mass spectrometry (LC-MS/ MS) method was established to determine the plasma concentrations of GA, GB and BB in rats after rats were administrated with the three drugs through ig and iv respectively. The main pharmacokinetic parameters and absolute bioavailability of three ginkgolide compounds were obtained by using pharmacokinetic software DAS 2. 0. After the inject of GA, GB and BB, the results showed Cmax at (513.9 ± 116.9), (701.3 ± 76.0), (5,255.6 ± 476.8) µg · L(-1) and AUC0.24h of (960.9 ± 268.5), (779.5 ± 140.6), (7,409.3 ± 1,181.1) µg · h · L(-1), respectively; after the oral administration, the results showed Cmax at (522.9 ± 39.9), (146.8 ± 31.6), (2,711.9 ± 588.9) µg · L(-1) and AUC0-24 h of (1,760.4 ± 300.7), (636.6 ± 180.3), (16,651.4 ± 1,306.5) µg · h · L(-1), respectively. The absolute bioavailability of GA, GB and BB in rats was (61.1 ± 10.4)%, (27.2 ± 7.7)%, (56.2 ± 4.4)%, respectively. The method established in this experiment has a good specificity and sensitivity and so can be used to study the pharmacokinetics and absolute bioavailability of GA, GB and BB in rats.
Animals ; Biological Availability ; Chromatography, High Pressure Liquid ; Cyclopentanes ; pharmacokinetics ; Furans ; pharmacokinetics ; Ginkgolides ; pharmacokinetics ; Lactones ; pharmacokinetics ; Male ; Rats ; Rats, Sprague-Dawley ; Tandem Mass Spectrometry

Objective To investigate the expression of survivin and bcl-2 in human squamous cell carcinoma (SCC) lesions and cell line SCL-1. Methods Tissue samples from 60 patients with SCC and 10 normal human controls were immunohistochemically stained to detect the expressions of survivin and bcl-2.Western blot was used to measure the expressions of bcl-2 and survivin proteins in HaCaT human keratinocytes and SCL-1 human squamous cell carcinoma cells. Results In normal control tissues, there was no expressions of survivin or bcl-2, while in SCC, the expression rates of bcl-2 and survivin were 70% and 60%, respectively,and there was no statistical correlation between the expressions of bcl-2 and survivin (P >0.05). Neither the expression of survivin nor that of bcl-2 was correlated to patients' age, gender or lesional site (all P >0.05). A statistical correlation was observed between the pathological stage in patients and expression of bcl-2 as well as between lymph node metastasis and expression of survivin (both P < 0.05). Western blot analysis revealed a significant increase in the expression of survivin and bcl-2 in SCL-1 cells compared with HaCaT cells. Con-clusion In SCC, survivin and bcl-2 seem to play their roles via different anti-apoptotic pathways.