The effect of gonadotropin-inhibitory hormone on steroidogenesis and spermatogenesis by acting through the hypothalamic-pituitary-testis axis in mice.

Gonadotropin inhibitory hormone (GnIH) is essential for regulating the reproduction of mammals and inhibiting testicular activities in mice. This study aimed to explore the mechanism of GnIH on spermatogenesis and steroidogenesis by acting through the hypothalamus-pituitary-testis axis of mice. Mice were subcutaneously injected with different doses of GnIH (1 µg/150 µL, 3 µg/150 µL, 6 µg/150 µL, 150 µL saline, twice daily) for 11 days. Subsequently, luteinizing hormone (LH), testosterone (T), and inhibin B (INH B) levels of peripheral blood were determined, and the expression of GnRH synthesis-related genes (GnRH-1, Kiss-1, NPY) and gonadotropin synthesis-related genes (FSH ß, LH ß, GnRH receptor) in the hypothalamus and pituitary gland were respectively detected. Additionally, the expression of steroidogenesis-related genes/proteins (P450scc, StAR and 3ß-HSD) and spermatogenesis-related proteins/genes including LH receptor (LHR), androgen receptor (AR), heat shock factor-2 (HSF-2) and INH B were analyzed using western blot and q-PCR. Results showed that GnIH treatment significantly reduced the concentration of LH in the peripheral blood. Further analysis revealed that GnIH treatment markedly reduced the expression of GnRHImRNA and Kiss-1 mRNA in the hypothalamus, and mRNA levels of FSH ß, LH ß, and GnRHR genes in the pituitary. We also observed that GnIH treatment significantly decreased T levels and expression of the P450scc, StAR, and 3ß-HSD proteins in the testis. Furthermore, GnIH treatment down-regulated LHR, AR proteins, and HSF-2 gene in the testis. Importantly, the INH B concentration of and INH ßb mRNA levels significantly declined following GnIH treatment. Additionally, GnIH treatment may induce germ cell apoptosis in the testis of mice. In conclusion, GnIH may suppress spermatogenesis and steroidogenesis by acting through the hypothalamus-pituitary-testis axis in mice.

Exosomes: The role in mammalian reproductive regulation and pregnancy-related diseases.

Exosomes are a kind of extracellular vesicles that are produced and secreted by different mammalian cells. They serve as cargo proteins and can transfer different kinds of biomolecules, including proteins, lipids, and nucleic acids, which consequently act on target cells to exert different biological effects. Recent years have witnessed a significant increase in the number of studies on exosomes due to the potential effects of exosomes in the diagnosis and treatment of cancers, neurodegenerative diseases, and immune disorders. Previous studies have demonstrated that exosomal contents, especially miRNAs, are implicated in numerous physiological processes such as reproduction, and are crucial regulators of mammalian reproduction and pregnancy-related diseases. Here, we describe the origin, composition, and intercellular communication of exosomes, and discuss their functions in follicular development, early embryonic development, embryonic implantation, male reproduction and development of pregnancy-related diseases in humans and animals. We believe this study will provide a foundation for revealing the mechanism of exosomes in regulating mammalian reproduction, and providing new approaches and ideas for the diagnosis and treatment of pregnancy-related diseases.

Integrative Analysis of miRNA-mRNA in Ovarian Granulosa Cells Treated with Kisspeptin in Tan Sheep.

Kisspeptin is a peptide hormone encoded by the kiss-1 gene that regulates animal reproduction. Our studies revealed that kisspeptin can regulate steroid hormone production and promote cell proliferation in ovarian granulosa cells of Tan sheep, but the mechanism has not yet been fully understood. We speculated that kisspeptin might promote steroid hormone production and cell proliferation by mediating the expression of specific miRNA and mRNA in granulosa cells. Accordingly, after granulosa cells were treated with kisspeptin, the RNA of cells was extracted to construct a cDNA library, and miRNA-mRNA sequencing was performed. Results showed that 1303 expressed genes and 605 expressed miRNAs were identified. Furthermore, eight differentially expressed miRNAs were found, and their target genes were significantly enriched in progesterone synthesis/metabolism, hormone biosynthesis, ovulation cycle, and steroid metabolism regulation. Meanwhile, mRNA was significantly enriched in steroid biosynthesis, IL-17 signaling pathway, and GnRH signaling pathway. Integrative analysis of miRNA-mRNA revealed that the significantly different oar-let-7b targets eight genes, of which EGR1 (early growth response-1) might play a significant role in regulating the function of granulosa cells, and miR-10a regulates lipid metabolism and steroid hormone synthesis by targeting HNRNPD. Additionally, PPI analysis revealed genes that are not miRNA targets but crucial to other biological processes in granulosa cells, implying that kisspeptin may also indirectly regulate granulosa cell function by these pathways. The findings of this work may help understand the molecular mechanism of kisspeptin regulating steroid hormone secretion, cell proliferation, and other physiological functions in ovarian granulosa cells of Tan sheep.

Long­chain non­coding RNA GAS5 promotes cell autophagy by modulating the miR­181c­5p/ATG5 and miR­1192/ATG12 axes.

The main aim of the present study was to explore the role of long­chain non­coding RNA (lncRNA) growth arrest­specific transcript 5 (GAS5) in macrophage autophagy. Firstly, the expression of lncRNA GAS5 during cell starvation or following treatment with 3­methyladenine was determined using reverse transcription­quantitative PCR (RT­qPCR). Additionally, fluorescent in situ hybridization (FISH) assay was utilized to determine the localization of the expression of lncRNA GAS5 in RAW264.7 cells. In vitro cell models were established through the transfection of LV5­lncRNA GAS5 (LV5­GAS5) or LV3­shRNA­lnc GAS5 (sh­GAS5), in order to overexpress or knockdown lncRNA GAS5 expression in RAW264.7 cells. The potential target microRNAs (miRNAs/miRs) of lncRNA GAS5 were analyzed using bioinformatics. The formation of autophagic bodies was detected with the use of laser confocal and transmission electron microscopy. Dual­luciferase reporter assay was performed to determine the target specificities of miR­181c­5p or miR­1192 to lncRNA GAS5 and autophagy­related gene (ATG) or ATG12. The mRNA levels of miR181c­5p, miR­1192, as well as ATG5 and ATG12 were detected using RT­qPCR. The protein levels of microtubule­associated proteins 1A/1B light chain 3B (LC3), p62, ATG5 and ATG12 were measured using western blot analysis. It was revealed that lncRNA GAS5 expression in RAW264.7 macrophages increased significantly during starvation­induced autophagy, and that lncRNA GAS5 overexpression was able to markedly promote the formation of autophagic bodies. Bioinformatics analysis demonstrated that miR­181c­5p and miR­1192 were potential targets of lncRNA GAS5, which was further confirmed by RT­qPCR, western blot analysis and the dual­luciferase reporter assay. Finally, it was confirmed that lncRNA GAS5 promoted autophagy by sponging miR­181c­5p and miR­1192, and upregulating the expression levels of the key autophagic regulators, ATG5 and ATG12. On the whole, the present study demonstrates that total, lncRNA GAS5 promotes macrophage autophagy by targeting the miR­181c­5p/ATG5 and miR­1192/ATG12 axes.

Nanobody-armed T cells endow CAR-T cells with cytotoxicity against lymphoma cells.

BACKGROUND: Taking advantage of nanobodies (Nbs) in immunotherapy, we investigated the cytotoxicity of Nb-based chimeric antigen receptor T cells (Nb CAR-T) against lymphoma cells. METHODS: CD19 Nb CAR-T, CD20 Nb CAR-T, and Bispecific Nb CAR-T cells were generated by panning anti-human CD19- and CD20-specific nanobody sequences from a natural Nb-expressing phage display library, integrating Nb genes with a lentiviral cassette that included other CAR elements, and finally transducing T cells that were expanded under an optimization system with the above generated CAR lentivirus. Prepared Nb CAR-T cells were cocultured with tumour cell lines or primary tumour cells for 24 h or 5 days to evaluate their biological function. RESULTS: The nanobodies that we selected from the natural Nb-expressing phage display library had a high affinity and specificity for CD19 and CD20. CD19 Nb CAR-T, CD20 Nb CAR-T and Bispecific Nb CAR-T cells were successfully constructed, and these Nb CAR-T cells could strongly recognize Burkitt lymphoma cell lines (Raji and Daudi), thereby leading to activation, enhanced proliferation, and specific killing of target cells. Furthermore, similar results were obtained when using patient samples as target cells, with a cytotoxicity of approximately 60%. CONCLUSIONS: Nanobody-based CAR-T cells can kill both tumour cell lines and patient-derived tumour cells in vitro, and Nb-based CAR-T cells may be a promising therapeutic strategy in future immunotherapy.

[Screening, expression and characterization of anti-human CD20 nanobodies].

Objective To screen the sequence of nanobodies against human CD20, and obtain anti-CD20-human IgG Fc nanobodies with high affinity and specificity. Methods Based on the naive phage display library, 4 rounds of liquid affinity screening were performed using biotinylated CD20 antigen as the target, and positive clones were identified by ELISA. Prokaryotic expression vector CD20-IgG Fc/pCZN1 was constructed and transformed into E.coli Arctic Express, and the expression of the recombinant protein was induced by IPTG at low temperature and purified by Ni column. The purified product was identified by ELISA and Western blot analysis. Results The specific CD20 nanobody showed good repeatability and hydrophilicity. The purity of anti-CD20-human IgG Fc nanobodies was higher than 85%. ELISA indicated that anti-CD20-human IgG Fc nanobodies had high affinity with CD20 antigen, and Western blot analysis demonstrated they could specifically recognize CD20 antigen. Conclusion The sequence of anti-CD20 nanobody was successfully obtained using the naive phage nanobody library. The purified anti-CD20-human IgG Fc nanobody has high affinity and specificity.

Bacillus pumilus improved drought tolerance in Glycyrrhiza uralensis G5 seedlings through enhancing primary and secondary metabolisms.

It has been reported that drought stress adversely affects the growth and yield of Glycyrrhiza uralensis, Chinese liquorice, in agricultural production. Bacillus pumilus, an important plant growth-promoting bacterium, play a significant role in improving plant tolerance to abiotic stress. However, the role of Bacillus pumilus G5 in resisting drought stress is largely unknown. In the present study, we found that drought stress significantly inhibited the growth and reduced the biomass of G. uralensis seedlings by restraining C- and N-metabolism, while this could be effectively reversed by B. pumilus G5 inoculation. Specifically, B. pumilus G5 significantly increased the content of primary metabolites such as soluble sugar, soluble protein, and free amino acids by regulating the C and N metabolic processes in G. uralensis seedlings. Moreover, B. pumilus G5 increased the content of glycyrrhizic acid, one of the important secondary metabolites, likely mediated through the increased content of primary metabolites and by recovering the expression of three key enzymes, HMGR, SQS, and ß-AS, in the biosynthesis of glycyrrhizic acid. Interestingly, the regulating effect of B. pumilus G5 inoculation on promoting the accumulation of glycyrrhizic acid and increasing the expression of synthesis-related genes is spatially selective. In summary, our findings suggest that B. pumilus G5 could alleviate adverse effects induced by drought stress on the growth of G. uralensis seedlings by regulating C- and N-metabolisms that further triggered the accumulation of secondary metabolites, and this finally improved the drought tolerance of cultivated G. uralensis seedlings.

[Construction of camelidae natural nanobody phage dispaly library and screening, production and identification of anti-CD19 nanobody].

Objective To construct and verify camelidae natural nanobody phage display library for selection of nanobodies against various antigens, and to obtain and identify the nanobody targeting CD19. Methods The total RNA of spleen of Bactrian camel was reverse transcribed and the variable region gene fragment of its heavy chain was obtained by nested PCR. It was constructed into the pCANTAB5e phagemid vector and electrotransformed into TG1 E. coli to develop the natural nanobody phage display library. After rescued by the KM13 helper phage, its capacity and diversity were analyzed and identified. Nanobody against CD19 was screened using biotinylated antigen combined with streptavidin magnetic beads, followed by ELISA, sequencing, exogenous expression and verification. Results The constructed natural phage nanobody display library had great diversity, and its fragment insertion rate was about 100%. The amino acid homology of 20 randomly selected clones was 65.85%, and the titer of the display library rescued by the helper phage was 9.0×1013 CFU/mL. After panning with CD19 as the antigen, the positive clones were sequenced and analyzed, and finally anti-CD19 nanobody sequences were obtained. The exogenously expressed anti-CD19 nanobody based on the sequences was verified having the ability to bind to CD19. Conclusion A camelidae natural nanobody phage display library with high titer and great diversity has been successfully constructed. Three anti-CD19 nanobody sequences have been obtained by panning with CD19. In addition, this study provides technical support for researching and developing diagnostic kits and antibody drugs targeting CD19, and it is a novel direction to improve CAR-T cells targeting CD19.

[Optimization of CAR-T cell culture system and lentivirus transduction conditions].

Objective To optimize the culture system of chimeric antigen receptor T (CAR-T) cells in vitro and lentivirus infection conditions. Methods Peripheral blood mononuclear cells (PBMCs) of healthy people and umbilical cord blood mononuclear cells (UCBMCs) of healthy pregnant women were isolated and purified by CD3 magnetic beads, and then they were cultured in different cell culture systems. There were eight cell culture systems containg different combinations of the following components: recombinant human interleukin 2 (rhIL-2), rhIL-12, rhIL-18, rhIL-7, rhIL-21, TWS119. Cell proliferation was detected by counting the cells at 0, 3, 5, 7, 10, 18 days after the cells were seeded into cell plates. Flow cytometry was used to detect the expression of programmed death 1 (PD-1), and ELISA was used to detect the expression of interferon-γ (IFN-γ). Cell culturing plates were coated with serial concentrations of recombinant human fibronectin fragment (RetroNectinr) (0, 20, 50 µg/mL), and antibodies against human CD3/CD28 (250, 500, 1 000 ng/mL). Then T cells cultured in the above plates were infected with negative control lentivirus at different multiplicity of infection (MOI=3, 5); 72 hours later, expression of green fluorescent protein (GFP) was observed under a fluorescence microscope to preliminarily determine virus infection efficiency. Flow cytometry was used to detect CD3/GFP positive rate to obtain lentivirus infection conditions. CD19 CAR lentivirus was packaged. Real-time quantitative PCR and Western blotting were performed to detect whether the CD19 CAR vector was successfully constructed. Finally, T cells were cultured in 1 µg/mL anti-human CD3/CD28 and 20 µg/mL RetroNectinr-coated culture plates, and rhIL-2, rhIL-12, rhIL-18 were added in the culture medium, then the cells were infected with CD19 CAR lentivirus at the optimized virus infection conditions. Results The cell culture system with the best proliferation ability was rhIL-2 combining with rhIL-18; the cell culture system with the strongest release of IFN-γ was rhIL-2 and rhIL-12 combined with rhIL-18. When the dose of antibodies against CD3/CD28 was 1 µg/mL, RetroNectinr was 20 µg/mL, and MOI was 3, the virus infection efficiency was optimal. The positive rate of CAR-T cells was 34% under the optimal condition. Conclusion The study achieved the optimal cell culture system of CD19 CAR-T cells in vitro and the conditions of lentivirus infection on primary T cells.

Therapeutic Protection Against H. pylori Infection in Mongolian Gerbils by Oral Immunization With a Tetravalent Epitope-Based Vaccine With Polysaccharide Adjuvant.

Urease is an effective target for design of a therapeutic epitope vaccine against Helicobacter pylori (H. pylori). In our previous studies, an epitope vaccine CTB-UE containing Th and B epitopes from H. pylori urease was constructed, and the CTB-UE vaccine could provide therapeutic effect on H. pylori infection in mice. However, a multivalent vaccine, combining different antigens participating in different aspects of H. pylori colonization and pathogenesis, may be more effective as a therapeutic vaccine than a univalent vaccine targetting urease. Therefore, a multivalent epitope vaccine FVpE, containing Th1-type immune adjuvant NAP, three selected functional fragments from CagA and VacA, and an urease multi-epitope peptide (UE) from CTB-UE, was constructed in this study and expected to obtain better sterilizing immunity than the univalent epitope vaccine CTB-UE. The therapeutic effect of multivalent epitope vaccine FVpE with polysaccharide adjuvant (PA) was evaluated in H. pylori-infected Mongolian gerbil model. The results showed that both FvpE and CTB-UE vaccine could induce similar levels of specific antibodies against H. pylori urease, and had similar inhibition effect on H. pylori urease activity. However, only FVpE could induce high levels of specific antibodies to CagA, VacA, and NAP. In addition, oral therapeutic immunization with FVpE plus PA significantly reduced the number of H. pylori colonies in the stomach of Mongolian gerbils compared with oral immunization with CTB-UE plus PA, or FVpE only, and the FVpE vaccine with PA even exhibited sterilizing immunity. The protection of FVpE was related to the mixed CD4+ T cell responses and epitope-specific antibodies against various H. pylori antigens. These results indicate that a multivalent epitope vaccine targetting various H. pylori antigens could be a promising candidate against H. pylori infection.

[Anti-breast cancer mechanism of flavonoids].

Flavonoids are a kind of ubiquitous natural products in plants and essential active ingredients of many medicinal plants. They have the characteristics of broad biological activity, high efficiency and low toxicity, with good prevention and cure effects on various types of tumors. Breast cancer is the commonest cancer disease and the main cause of cancer death in women worldwide. In China, the morbidity and mortality are still on the rise. Flavonoids can inhibit the occurrence and development of breast cancer in various aspects. The main mechanisms include the inhibition of aerobic glycolysis, the promotion of apoptosis, the retardation of cell cycle, the suppression of invasion and migration, the induction of DNA damage, and the inhibition of aromatase and microtubule production. Due to its chemical structure similar to estrogen, flavonoids have unique advantages in the prevention and treatment of breast cancer. In this paper, studies on anti-breast cancer of flavonoids in recent years were reviewed, which could provide reference for further studies.

Hepatoprotective Effects of Kaempferol-3-O-α-l-Arabinopyranosyl-7-O-α-l-Rhamnopyranoside on d-Galactosamine and Lipopolysaccharide Caused Hepatic Failure in Mice.

Fulminant hepatic failure (FHF), associated with high mortality, is characterized by extensive death of hepatocytes and hepatic dysfunction. There is no effective treatment for FHF. Several studies have indicated that flavonoids can protect the liver from different factor-induced injury. Previously, we found that the extracts of Elaeagnus mollis leaves had favorable protective effects on acute liver injury. However, the role and mechanisms behind that was elusive. This study examined the hepatoprotective mechanisms of kaempferol-3-O-α-l-arabinopyranosyl-7-O-α-l-rhamnopyra-noside (KAR), a major flavonol glycoside of E. mollis, against d-galactosamine (GalN) and lipopolysaccharide (LPS)-induced hepatic failure. KAR reduces the mouse mortality, protects the normal liver structure, inhibits the serum aspartate aminotransferase (AST) and alamine aminotransferase (ALT) activity and decreases the production of malondialdehyde (MDA) and reactive oxygen species (ROS) and inflammatory cytokines, TNF-α, IL-6, and IL-1ß. Furthermore, KAR inhibits the apoptosis of hepatocytes and reduces the expression of TLR4 and NF-κB signaling pathway-related proteins induced by GalN/LPS treatment. These findings suggest that the anti-oxidative, anti-inflammatory, and anti-apoptotic effects of KAR on GalN/LPS-induced acute liver injury were performed through down-regulating the activity of the TLR4 and NF-κB signaling pathways.

miR-1247-5p functions as a tumor suppressor in human hepatocellular carcinoma by targeting Wnt3.

Increasing evidence suggests that aberrant expression of certain microRNAs (miRNAs) may participate in the genesis and progression of tumors. Several studies have indicated that miR-1247-5p plays different roles in various types of cancer cells. The effects of miR-1247-5p on human hepatocellular carcinoma (HCC) cells are elusive. In the present study, we investigated the effects of miR-1247-5p on the progression of HCC. The transcript of miR-1247-5p was markedly downregulated in clinical samples of patients with HCC and HCC cell lines, and ectopic overexpression of miR­1247-5p markedly inhibited the proliferation and invasion of HepG2 cells, induced cell apoptosis in vitro, and suppressed the growth of transplanted tumors in vivo. Wnt3 was found to be a potential target of miR-1247-5p and overexpression of miR-1247-5p was able to significantly downregulate the expression of Wnt3 by directly targeting the 3'UTR of this gene, which was verified by luciferase reporter assay and western blotting. Furthermore, we found that the miR-1247-5p gene was hypermethylated in HepG2 cells, and the transcript of miR-1247-5p was increased significantly after treatment with the demethylation drug 5-azacytidine. These findings demonstrated that miR-1247-5p functions as a tumor suppressor in human HCC by targeting Wnt3 and that the expression of miR-1247-5p can be regulated by DNA methylation, which indicates that miR-1247-5p has the potential to be a therapeutic target as well as a diagnostic marker of HCC.

MicroRNA-144-3p inhibits autophagy activation and enhances Bacillus Calmette-Guérin infection by targeting ATG4a in RAW264.7 macrophage cells.

MicroRNAs (miRNAs) are small noncoding nucleotides that play major roles in the response of host immune cells. Autophagy plays a key role in activating the antimicrobial host defense against Mycobacterium tuberculosis (M. tuberculosis). Whether miRNAs specifically influence the activation of macrophage autophagy during M. tuberculosis infection is largely unknown. In the present study, we demonstrate that Mycobacterium bovis Bacillus Calmette-Guérin (BCG) infection of macrophages leads to increased expression of miR-144-3p, which targets autophagy-related gene 4a (ATG4a), to inhibit autophagy activation and antimicrobial responses to BCG. Overexpression of miR-144-3p significantly decreased both mRNA and protein levels of ATG4a, inhibited the formation of autophagosomes in RAW264.7 cells and increased intracellular survival of BCG. However, transfection with miR-144-3p inhibitor led to an increase in ATG4a levels, accelerated the autophagic response in macrophages, and decreased BCG survival in macrophages. The experimental results of this study reveal a novel role of miR-144-3p in inhibiting autophagy activation by targeting ATG4a and enhancing BCG infection, and provide potential targets for developing improved treatment.

Percutaneous transhepatic embolization of gastroesophageal varices combined with partial splenic embolization for the treatment of variceal bleeding and hypersplenism.

This study aims to evaluate the therapeutic results of percutaneous transhepatic embolization of gastroesophageal varices combined with partial splenic embolization in patients with liver cirrhosis, and to explore the role of this minimally invasive treatment as an alternative to surgery. 25 patients with liver cirrhosis were received percutaneous transhepatic embolization of gastroesophageal varices combined with partial splenic embolization. Another 25 patients with liver cirrhosis underwent Hassab's operation. They were followed up, and received endoscopy, B ultrasound, liver function and hematologic examination at 24 months after the therapy. In minimal invasive group, before treatment and after 24 month following up after treatment, improved varices, improved portal hypertension and improved hypersplenism were showed comparing with the surgery group, and that they were measured by endoscopic visualization, ultrasound and blood counts. the white blood cell and platelet count were 2.33±0.65 (10(9)/L) and 3.63±1.05 (10(10)/L), 7.98±3.0 (10(9)/L) and 16.3±9.10 (10(10)/L) (P<0.05); the diameter of the portal vein were 1.47±0.25 cm, 1.31±0.23 cm (P<0.05). Esophageal varices passed from grade III to lower grade II in 11 patients, and from grade II to lower grade I in 6 patients at 24 month following up. In surgical group, the white blood cell and platelet count were 2.2±0.60 (10(9)/L), 4.1±1.25 (10(10)/L) before treatment; 9.3±2.56 (10(9)/L), 32.1±12.47 (10(10)/L) after the treatment at 24 month following up (P<0.05). The diameter of the portal vein were 1.43±0.22 cm before the treatment and 1.28±0.18 cm after the treatment (P<0.05). Esophageal varices passed from grade III to lower grade II in 13 patients, and from grade II to lower grade I in 7 patients. The combination of PGEV and PSE can be considered as an option for the treatment of variceal bleeding with hypersplenism.

Immunological features and efficacy of a multi-epitope vaccine CTB-UE against H. pylori in BALB/c mice model.

Epitope vaccine is a promising option for prophylactic and therapeutic vaccination against Helicobacter pylori infection. Urease is an essential virulence factor and colonization factor for H. pylori. In this study, we constructed a multi-epitope vaccine named CTB-UE with mucosal adjuvant cholera toxin B subunit (CTB) and tandem copies of Th and B cell epitopes from H. pylori urease A and B subunits. The immunogenicity, specificity, ability to induce neutralizing antibodies against H. pylori urease, and prophylactic and therapeutic efficacy of the CTB-UE vaccine were evaluated in BALB/c mice model after purification. The experimental results indicated that CTB-UE could induce comparatively high levels of specific antibodies against native H. pylori urease, UreA, UreB, or the selected B cell epitopes UreA183₋203 and UreB327₋334 involved with the active site of urease and showed an effectively inhibitory effect on the enzymatic activity of urease. Besides, oral prophylactic or therapeutic immunization with CTB-UE significantly decreased H. pylori colonization compared with oral immunization with rUreB or PBS, and the protection was correlated with antigen-specific CD4⁺ T cells and IgG, IgA, and mucosal sIgA antibody responses. This CTB-UE vaccine may be a promising vaccine candidate for the control of H. pylori infection.

GSK3 inhibitor-BIO regulates proliferation of immortalized pancreatic mesenchymal stem cells (iPMSCs).

BACKGROUND: The small molecule 6-bromoindirubin-30-oxime (BIO), a glycogen synthase kinase 3 (GSK3) inhibitor, is a pharmacological agent known to maintain self-renewal in human and mouse embryonic stem cells (ESCs). However, the precise role of GSK3 in immortalized pancreatic mesenchymal stem cells (iPMSCs) growth and survival is not completely understood at present. RESULTS: To determine whether this molecule is involved in controlling the proliferation of iPMSCs, we examined the effect of BIO on iPMSCs. We found that the inactivation of GSK3 by BIO can robustly stimulate iPMSCs proliferation and mass formation as shown by QRT-PCR, western blotting, 5-Bromo-2-deoxyuridine (BrdU) immunostaining assay and tunel assay. However, we did not find the related roles of BIO on ß cell differentiation by immunostaining, QRT-PCR assay, glucose-stimulated insulin release and C-peptide content analysis. CONCLUSIONS: These results suggest that BIO plays a key role in the regulation of cell mass proliferation and maintenance of the undifferentiated state of iPMSCs.

[Construction and identification of recombinant retroviral vector of human ngn3 gene and its packaging cell line].

In order to construct the recombinant retrovirus vector of human ngn3 gene and its packaging cell line, we successfully amplified the open reading frame (ORF) of ngn3 gene from human fetal pancreatic tissue by RT-PCR. The PCR products of human ngn3 gene was subcloned into pMD18-T vectors and sequenced. Results showed that its sequence was fully consistent with the ngn3 gene published in GenBank(GenBank Accession No. BC126468). The correct fragment was digested by EcoR I and Hpa I from recombinant pMD18-T vector and inserted into the same restriction enzyme sites of retroviral vector pMSCV-neo. We got recombinant retrovirus vector pMSCV-ngn3, which was identified by double restriction enzyme digestion and then transfected into PT67 cells by lipofectamine 2000. We established the PT67-ngn3 packaging cell line by G418 selection, which was detected by RT-PCR and immunohistochemistry staining. The detection results showed that the Ngn3 expressed at the mRNA and protein level in the packaging cell line. RT-PCR detection and electronic microscope analysis showed that the recombinant retroviral vector pMSCV-ngn3 was packaged into infectious virus particles and released into the supernatant of the cells. These results demonstrated that a PT67-ngn3 packaging cell line was successfully established, and this could facilitate the study of differentiation of the human fetal pancreatic progenitor cells into insulin-producing cells by using the ngn3 gene.

Effect of 5-azacytidine induction duration on differentiation of human first-trimester fetal mesenchymal stem cells towards cardiomyocyte-like cells.

The aim of this study is to investigate effects of 5-azacytidine (5-aza) induction duration on differentiation of bone marrow mesenchymal stem cells (MSCs) from human first-trimester abortus (hfMSCs) towards cardiomyocyte-like cells. hfMSCs were stimulated with 10 micromol/l 5-aza for 24 h (group A), 48 h (group B) and 21 days (group C), respectively. During the induction, 30-40% of the cells gradually enlarged, elongated, connected with adjoining cells and formed myotube-like structures, branches and string-bead-like nuclei. Some of the cells congregated into cell clusters or strips. After the induction, numerous myofilaments in the cytoplasm and conjunction of intercalated disc-like structure between adjoining cells were observed. The induced cells expressed messenger ribonucleic acids (mRNAs) and proteins of myocardium-specific alpha-actin, sarcomeric beta-myocin heavy chain and troponin-T. The positive cell percentages for the three antigens in group C were each significantly higher than those antigens in group A and B (P<0.01) and the cell population doubling time (PDT) of group C was longer than those of group A and B (P<0.01). These indicate that 21-d induction with 10 micromol/l 5-aza slows down proliferation speed of hfMSCs but increases differentiation rate of hfMSCs into cardiomyocyte-like cells if compared with 24-48 h induction.