Effects of hyaluronic acid on differentiation of human amniotic epithelial cells and cell-replacement therapy in type 1 diabetic mice.

Our hypothesis is that hyaluronic acid may regulate the differentiation of human amniotic epithelial cells (hAECs) into insulin-producing cells and help the treatment of type 1 diabetes. Herein, a protocol for the stepwise in vitro differentiation of hAECs into functional insulin-producing cells was developed by mimicking the process of pancreas development. Treatment of hAECs with hyaluronic acid enhanced their differentiation of definitive endoderm and pancreatic progenitors. Endodermal markers Sox17 and Foxa2 and pancreatic progenitor markers Pax6, Nkx6.1, and Ngn3 were upregulated an enhanced gene expression in hAECs, but hAECs did not express the ß cell-specific transcription factor Pdx1. Interestingly, hyaluronic acid promoted the expression of major pancreatic development-related genes and proteins after combining with commonly used inducers of stem cells differentiation into insulin-producing cells. This indicated the potent synergistic effects of the combination on hAECs differentiation in vitro. By establishing a multiple injection transplantation strategy via tail vein injections, hAECs transplantation significantly reduced hyperglycemia symptoms, increased the plasma insulin content, and partially repaired the islet structure in type 1 diabetic mice. In particular, the combination of hAECs with hyaluronic acid exhibited a remarkable therapeutic effect compared to both the insulin group and the hAECs alone group. The hAECs' paracrine action and hyaluronic acid co-regulated the local immune response, improved the inflammatory microenvironment in the damaged pancreas of type 1 diabetic mice, and promoted the trans-differentiation of pancreatic α cells into ß cells. These findings suggest that hyaluronic acid is an efficient co-inducer of the differentiation of hAECs into functional insulin-producing cells, and hAECs treatment with hyaluronic acid may be a promising cell-replacement therapeutic approach for the treatment of type 1 diabetes.

Hyaluronic acid promotes osteogenic differentiation of human amniotic mesenchymal stem cells via the TGF-ß/Smad signalling pathway.

AIMS: This study investigated the effects of hyaluronic acid (HA), a commonly used osteogenic medium referred to as DAG, and the combined administration of HA and DAG (CG) on the osteogenic differentiation of human amniotic mesenchymal stem cells (hAMSCs), and the underlying mechanism. MAIN METHODS: The phenotype of hAMSCs was detected by flow cytometry and immunocytochemical staining. Alkaline phosphatase (ALP) and calcium deposition assays were employed for evaluating the osteogenic differentiation of hAMSCs. The expression of osteogenesis-related genes and proteins was determined by quantitative reverse transcription PCR (qRT-PCR) and Western blotting, respectively. Meanwhile, the molecular mechanism of osteogenic differentiation of hAMSCs was detected by PCR array and qRT-PCR. KEY FINDINGS: The results showed that treatment with CG could significantly stimulate hAMSC ALP activity and calcium deposition compared to treatment with DAG, while HA had little effect. The expression of osteogenesis-related molecules and stemness-related molecules was up-regulated at the mRNA and protein levels in all three groups, and this up-regulation was most significant in the CG group. In addition, treatment with CG significantly increased the gene expressions involved in regulation of the TGF-ß/Smad signalling pathway compared to treatment with DAG. Furthermore, the pro-osteogenic differentiation effects as well as the up-regulated expression of genes observed in the CG treatment group were significantly inhibited when the cells were pre-treated with SB431542, an inhibitor of the TGF-ß/Smad pathway. SIGNIFICANCE: These results suggest that HA in combination with DAG could significantly enhance the osteogenic differentiation of hAMSCs, potentially via the TGF-ß/Smad signalling pathway.

Synergistic antitumor efficacy of antibacterial helvolic acid from Cordyceps taii and cyclophosphamide in a tumor mouse model.

The antibacterial agent helvolic acid, which was isolated from the active antitumor fraction of Cordyceps taii, showed potent cytotoxicity against different human cancer cells. In the present study, the in vivo antitumor effect of helvolic acid was investigated in murine sarcoma S180 tumor-bearing mice. Doses of 10 and 20 mg/kg/day helvolic acid did not exert significant antitumor activity. Interestingly, co-administration of 10 mg/kg/day helvolic acid and 20 mg/kg/day cyclophosphamide (CTX) - a well-known chemotherapy drug - showed promising antitumor activity with a growth inhibitory rate of 70.90%, which was much higher than that of CTX alone (19.5%). Furthermore, the combination markedly prolonged the survival of tumor-bearing mice. In addition, helvolic acid enhanced the immune organ index. The protein expression levels of ß-catenin, cyclin D1, and proliferating cell nuclear antigen were significantly suppressed in mice treated with 20 mg/kg/day helvolic acid and in those receiving combination therapy. Taken together, these results indicated that helvolic acid in combination with CTX showed potent in vivo synergistic antitumor efficacy, and its mechanism of action may involve the Wnt/ ß-catenin signaling pathway.

Hyaluronic acid enhances proliferation of human amniotic mesenchymal stem cells through activation of Wnt/ß-catenin signaling pathway.

This study investigated the pro-proliferative effect of hyaluronic acid (HA) on human amniotic mesenchymal stem cells (hAMSCs) and the underlying mechanisms. Treatment with HA increased cell population growth in a dose- and time-dependent manner. Analyses by flow cytometry and immunocytochemistry revealed that HA did not change the cytophenotypes of hAMSCs. Additionally, the osteogenic, chondrogenic, and adipogenic differentiation capabilities of these hAMSCs were retained after HA treatment. Moreover, HA increased the mRNA expressions of wnt1, wnt3a, wnt8a, cyclin D1, Ki-67, and ß-catenin as well as the protein level of ß-catenin and cyclin D1 in hAMSCs; and the nuclear localization of ß-catenin was also enhanced. Furthermore, the pro-proliferative effect of HA and up-regulated expression of Wnt/ß-catenin pathway-associated proteins - wnt3a, ß-catenin and cyclin D1 in hAMSCs were significantly inhibited upon pre-treatment with Wnt-C59, an inhibitor of the Wnt/ß-catenin pathway. These results suggest that HA may positively regulate hAMSCs proliferation through regulation of the Wnt/ß-catenin signaling pathway.

Supramolecular assembly-induced yellow emission of 9,10-distyrylanthracene bridged bis(pillar[5]arene)s.

9,10-Distyrylanthracene has been introduced to bridge two pillarenes to form a dimeric host, which can assemble into a linear supramolecular polymer upon cooperatively binding to a neutral guest linker, exhibiting yellow fluorescence emission in solution and solid states.

Stimuli-responsive metal-organic frameworks gated by pillar[5]arene supramolecular switches.

Spurred on by recent advances in materials chemistry and drug delivery, a new stimuli-responsive theranostic hybrid platform, based on mechanized monodisperse nano metal-organic frameworks (NMOFs) gated by carboxylatopillar[5]arene (CP5) switches with bio-friendly pH-triggered cargo release capabilities, has been constructed for the first time. This nanoscale smart cargo delivery system showed pH- and/or competitive binding agent-triggered controlled cargo release with negligible premature release, large pore sizes for drug encapsulation, low cytotoxicity, good biodegradability and biocompatibility, and potential application in cell imaging, which offers a new tool in targeted drug delivery and the controlled release of therapeutic agents.

Stimuli-responsive blue fluorescent supramolecular polymers based on a pillar[5]arene tetramer.

A tetraphenylethene-bridged pillarene tetramer with aggregation-induced emission properties forms an A4/B2-type supramolecular polymer and a gel with a symmetric neutral guest linker, showing a remarkable fluorescence emission enhancement in solution and the solid state and a good responsiveness to temperature and solvent composition.

Mechanized silica nanoparticles based on pillar[5]arenes for on-command cargo release.

Mechanized silica nanoparticles, equipped with pillar[5]arene-[2]pseudorotaxane nanovalves, operate in biological media to trap cargos within their nanopores, but release them when the pH is lowered or a competitive binding agent is added. Although cargo size plays an important role in cargo loading, cargo charge-type does not appear to have any significant influence on the amount of cargo loading or its release. These findings open up the possibility of using pillar[n]arene and its derivatives for the formation of robust and dynamic nanosystems that are capable of performing useful functions.

Viologen-mediated assembly of and sensing with carboxylatopillar[5]arene-modified gold nanoparticles.

Carboxylatopillar[5]arene (CP[5]A), a new water-soluble macrocyclic synthetic receptor, has been employed as a stabilizing ligand for in situ preparation of gold nanoparticles (AuNPs) to gain new insights into supramolecular host-AuNP interactions. CP[5]A-modified AuNPs with good dispersion and narrow size distributions (3.1 ± 0.5 nm) were successfully produced in aqueous solution, suggesting a green synthetic pathway for the application of AuNPs in biological systems. Supramolecular self-assembly of CP[5]A-modified AuNPs mediated by suitable guest molecules was also investigated, indicating that the new hybrid material is useful for sensing and detection of the herbicide paraquat.

[Investigation on spread and hazards of human parasites in Qushui Village, Suixi County, Zhanjiang City].

OBJECTIVE: To understand the transmission of human parasites in Qushui Village, Yangqing Town, Suixi County, Zhanjing City, Guangdong Province. METHODS: The direct stool smear, floatation, Kato-Katz technique, and hookworm larva culture were used for the parasite infections. The questionnaire survey was applied for the hazards of parasites. The dissections on rats and snails were used for Angiostrongylus cantonensis infection. RESULTS: Five parasites were found and the total infection rate was 10.75%. The infection rates of hookworm (Necator americanus), Ascaris lumbricoides and Trichuris trichiura were 6.07%, 1.87% and 1.87%, respectively, and the infection rates of Enterobius vermicularis and Tyroglyrhus farinae were both 0.47%. The infections were not correlated with the career and age but preferred to males. The densities of infections were slight. The rate of dermatitis caused by hookworm larvae was 69.23%. The infection rates of Angiostrongylus cantonensis were 16.66%, 13.04% and 10.00%, respectively in rats, Achatina fulica and Ampularum crossean. CONCLUSION: The main species of human parasites are nematodes, with hookworm predominately, in Qushui Village, Suixi County. This area is the natural foci of Angiostrongylus cantonensis.

One-pot synthesis of pillar[n]arenes catalyzed by a minimum amount of TfOH and a solution-phase mechanistic study.

A practical and effective trifluoromethanesulfonic acid (TfOH)-catalyzed cyclooligomerization strategy was developed for the synthesis of functionalized pillar[n]arenes and copillar[5]arenes from 1,4-dialkoxybenzenes with paraformaldehyde under mild reaction conditions, and the reaction mechanism of solution-phase catalytic synthesis of pillararenes was investigated by room-temperature X-band ESR spectroscopy, mass spectroscopy, NMR and control experiments, suggesting a free radical process initially and a Friedel-Crafts alkylation process during the consequent coupling and ring-closure stage.

[Investigation on snails Achatina fulica and Pomacea canaliculata infected with Angiostrongylus cantonensis in Panyu region of Guangzhou City].

OBJECTIVE: To understand the natural infection status of Angiostrongylus cantonensis in snails Achatina fulica and Pomacea canaliculata from Panyu region of Guangzhou City. METHODS: The snails Achatina fulica and Pomacea canaliculata captured from the field were digested with the artificial stomach fluid. The third-stage larvae of A. cantonensis were examined and counted under a microscope. The collected third-stage larvae were used to infect SD rats. RESULTS: A total of 367 Achatina fulica and 357 Pomacea canaliculata were examined. The infection rate of A. cantonensis in Achatina fulica was 22.62%, with a mean intensity of 57.00 larvae per positive snail. The infection rate of A. cantonensis in Pomacea canaliculata was 3.08%, with a mean intensity of 1.64 larvae per positive snail. The infection rates of A. cantonensis in Achatina fulica from Dagang, Shiqi, Hualong, and Lanhe towns and Nansha District, were 13.33%, 15.00%, 20.93%, 73.68% and 8.41%, respectively. Those in Pomacea canaliculata were 5.88%, 2.88%, 1.89%, 0% and 3.96%, respectively. CONCLUSIONS: A. cantonensis infection exists in Achatina fulica and Pomacea canaliculata from Panyu region of Guangzhou City, and the infection in Achatina fulica is more serious than that in Pomacea canaliculata. The infection rates of the snails among five sites are different.

Jiangxienone, a new compound with potent cytotoxicity against tumor cells from traditional Chinese medicinal mushroom Cordyceps jiangxiensis.

A new compound, named jiangxienone, has been isolated from a culture of the traditional Chinese medicinal mushroom Cordyceps jiangxiensis, and its chemical structure was established on the basis of spectroscopic and chemical techniques. Jiangxienone showed potent cytotoxic effects against human gastric adenocarcinoma SGC-7901 cell and human lung carcinoma A549 cell with IC(50) values ranging from 1.38 to 2.93 µM, i.e., with at least approximately six-fold stronger cytotoxicity than cisplatin, a first-line chemotherapy drug for cancer patients.

[Study on inducing differentiation of human amniotic epithelial cells into insulin secreting cells in vitro].

OBJECTIVE: To investigate the differentiation of human amniotic epithelial cells (hAECs) into insulin secreting cells (ISCs) in vitro. METHODS: The hAECs were isolated from human amnion by trypsin digestion, and the phenotype of the isolated cells were identified by flow cytometry and immunocytochemical staining. The hAECs at passage 3 were treated with nicotinamide and N2 supplement to investigate their differentiation into ISCs. At different times after differentiation, the expression of insulin and beta2 microglobulin (beta2-MG) was determined by immunocytochemical staining, while the content of insulin in supernatant from cultured hAECs was detected by radioimmunoassay and the expressions of insulin, pancreatic and duodenal homeobox factor-1 (PDX-1) mRNA were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: (1) hAECs expressed high percent of CD29, CD73, CD166 and CK19. (2) At 7, 14 and 21 days, the percentages of insulin-positive cells in induced groups were 74.00% +/- 1.73%, 75.33% +/- 1.15% (see symbol) 75.67% +/- 0.58% respectively, which were negative in control groups. (3) At 7, 14 and 21 days, contents of insulin in supernatant from induced groups were (328.47 +/- 3.22) microIU/ml, (332.26 +/- 1.22) microIU/ml and (329.68 +/- 2.57) microIU/ml respectively, they were significantly higher than those in control groups (All P < 0.01). (4) PDX-1 mRNA and beta2-MG were expressed before and after the induction of hAECs, but insulin mRNA was expressed only in the induced groups. CONCLUSION: hAECs can differentiate into ISCs, having the potential application for therapy of type I diabetes.

Establishment of a cell-based drug screening model for identifying agonists of human peroxisome proliferator-activated receptor gamma (PPARγ).

OBJECTIVES: Peroxisome proliferator-activated receptor gamma (PPARγ) plays a critical role in regulation of diverse biological processes, including lipid metabolism and adipogenesis, cell division and apoptosis, and is involved in variety of disease conditions, such as obesity, atherosclerosis, inflammation and tumour. Developing a cell-based reporter gene model targeting PPARγ would be useful to screen human PPARγ agonists that could be beneficial to patients with these diseases. METHODS: We stably co-transfected human embryonic kidney (HEK) cell line 293T cells with phPPARγ-IRES2-EGFP vector to express human PPARγ (hPPARγ), a reporter vector pPPRE×3-TK-LUC, and control vector pRL-CMV. The efficiency of the co-transfection was evaluated with flow cytometry of hPPARγ expressing cells. Specificity of hPPARγ activity was determined by dual luciferase reporter assay of co-transfected cells exposed to PPARγ agonist rosiglitazone, PPARα agonist WY14643 and retinoic acid receptor alpha (RARα) agonist all-trans-retinoic acid (ATRA). KEY FINDINGS: The phPPARγ-IRES2-EGFP co-transfected HEK293T cells showed concentration- and time-dependent luciferase induction upon exposure to the rosiglitazone, while WY14643 and ATRA were unable to activate the co-transfected HEK293T cells. CONCLUSIONS: These data indicated that the HEK293T cells could be stably transfected with hPPARγ. This cell-based drug screening platform could be used targeting specific nuclear receptor of hPPARγ with effectiveness and specificity for hPPARγ agonists discovery.

Polysaccharides from the Medicinal Mushroom Cordyceps taii Show Antioxidant and Immunoenhancing Activities in a D-Galactose-Induced Aging Mouse Model.

Cordyceps taii, an edible medicinal mushroom native to south China, is recognized as an unparalleled resource of healthy foods and drug discovery. In the present study, the antioxidant pharmacological properties of C. taii were systematically investigated. In vitro assays revealed the scavenging activities of the aqueous extract and polysaccharides of C. taii against various free radicals, that is, 1,1-diphenyl-2-picrylhydrazyl radical, hydroxyl radical, and superoxide anion radical. The EC(50) values for superoxide anion-free radical ranged from 2.04 mg/mL to 2.49 mg/mL, which was at least 2.6-fold stronger than that of antioxidant thiourea. The polysaccharides also significantly enhanced the antioxidant enzyme activities (superoxide dismutase, catalase, and glutathione peroxidase) and markedly decreased the malondialdehyde production of lipid peroxidation in a D-galactose-induced aging mouse model. Interestingly, the immune function of the administration group was significantly boosted compared with the D-galactose-induced aging model group. Therefore, the C. taii polysaccharides possessed potent antioxidant activity closely associated with immune function enhancement and free radical scavenging. These findings suggest that the polysaccharides are a promising source of natural antioxidants and antiaging drugs. Consequently, a preliminary chemical investigation was performed using gas chromatography-mass spectroscopy and revealed that the polysaccharides studied were mainly composed of glucose, mannose, and galactose. Fourier-transform infrared spectra also showed characteristic polysaccharide absorption bands.

[CD133(+) cells derived from human placenta identified as initiating cells for LTC-IC colony formation].

The aim of this study was to evaluate whether human placenta CD133(+) cells have an ability to reconstitute long-term hematopoiesis. Magnetic-activated cell sorting (MACS) was applied to enrich human placental CD133(+) cells. The isolated human placental CD133(+)cells of four different densities were established by limiting-dilution assay and primary fetal bone marrow stromal cells separated from bone marrow as feeder layer cells were co-cultured in long-term culture system so as to observe the incidence of long-term culture initiating-cells (LTC-IC) and their ability of proliferation and differentiation.The results showed that human placenta derived CD133(+) cells contained LTC-IC with frequency of 1/645 which have an ability to proliferate and differentiate into granulocyte/macrophage colony-forming units (CFU-GM) and mixed colony-forming units (CFU-Mix). In all LTC-IC positive wells, 71.43% form only CFU-GM and 28.57% display both CFU-GM and CFU-Mix formation. It is concluded that human placental CD133(+) cells possess LTC-IC with colony-forming capacity of hematopoietic early progenitor cells.

[Investigation on antitumor mechanism of polysaccharide from medicinal fungus Penicillium jiangxiense].

OBJECTIVE: To investigate the possible antitumor mechanism of polysaccharide from medicinal fungus Penicillium jiangxiense. METHODS: The cytotoxic effect was measured by MTT assay, and the cell cycle was analyzed by flow cytometry with Propidium iodide (PI) staining. To apoptotic detections, Hoechst 33258 staining for chromatin, annexin-V FITC/PI double staining for early phase cell apoptosis and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) for late phase cell apoptosis. RESULTS: Both MPPJ4 and MPPJ5, the fine polysaccharide fractions from P. jiangxiense, showed slight cytotoxic effects to inhibit human gastric adenocarcinoma SGC-7901 cells proliferation, but significantly caused cell cycle arrest at the S phase, and the rate of cell population at the subG1 phase was evidently increased. In apoptotic assays, MPPJ5 was more potent than MMPJ4 in inducing tumor cells in a time-dependent manner at the range from 0 to 72 hours, comparable to the negative control. CONCLUSION: The antitumor acting mechanism of P. jiangxiense polysaccharide is associated with the induction of apoptotic cell death and cell cycle arrest.

[Construction of gtfB/CAT eukaryotic expression plasmid of Streptococcus mutans in mammalian cells].

PURPOSE: The study aimed to evaluate the expression of recombinant plasmid pVAX1- gtfB/CAT in mammalian COS-7 cells. METHODS: The eukaryotic plasmid carrying encoding gene of gtfB/CAT of Streptococcus mutans was constructed and introduced into COS-7 cells by lipofectamine reagent. The transient protein expression was detected by immunochemistry technique in COS-7 cells. RESULTS: The positive expression of gefB/CAT was detected in plasma of the cells which were transfected with recombinant plasmid pVAX1- gtfB/CAT. The cells which were transfected with pVAX1 were negative for gtfB/CAT expression. CONCLUSIONS: GtfB/CAT can be translated and expressed in COS-7 cells after transfected with recombinant plasmid pVAX1- gtfB/CAT. The expressed protein is located in the plasma and the protein is able to combine with anti- gtfB/CAT antibody. The expressed protein has the antigenicity and recombinant plasmid pVAX1- gtfB/CAT is a candidate vaccine.Supported by Key Research Project of Bureau of Education of Guizhou Province (Grant No.2004119).