[Development of human embryonic stem cell model for toxicity evaluation].

The current international standard for toxicity screening of biomedical devices and materials recommend the use of immortalized cell lines because of their homogeneous morphologies and infinite proliferation which provide good reproducibility for in vitro cytotoxicity screening. However, most of the widely used immortalized cell lines are derived from animals and may not be representative of normal human cell behavior in vivo, in particular in terms of the cytotoxic and genotoxic response. Therefore, It is vital to develop a model for toxicity evaluation. In our studies, two Chinese human embryonic stem cell (hESC) lines as toxicity model were established. hESC derived tissue/organ cell model for tissue/organ specific toxicity evaluation were developed. The efficiency and accuracy of using hESC model for cytoxicity, embryotoxicity and genotoxicity evaluation were confirmed. The results indicated that hESCs might be good tools for toxicity testing and biosafety evaluation in vitro.

[Effects of Epimedium on proliferation, function and apoptosis of mouse osteoblasts in vitro].

OBJECTIVE: To investigate the effects of Epimedium on proliferation, function and apoptosis of mouse osteoblasts in vitro. METHODS: Primary osteoblasts were obtained by sequential digestion of mouse calvaria with collagenase and hyaluronidase. The identification of derived cells was done by histochemical staining of alkaline phosphatase (ALPase) and immunohistochemical staining of type I collagen, bone sialoprotein and osteopontin. MTT assay was employed to examine the proliferation of osteoblasts after treatment with Epimedium. The alkaline phosphatase activity level of mouse osteoblasts was also determined through an enzyme dynamical method. Apoptosis of osteoblasts was induced by dexamethasone and flow cytometry was utilized to examine the effects of Epimedium on the dexamethasone-induced apoptosis of osteoblasts. RESULTS: Five populations of bone cells were obtained by sequential digestion. Osteoblasts were purely obtained by discarding the first two populations and identified by the positive staining of ALPase, type I collagen, bone sialoprotein, and osteopontin. The alkaline phosphatase activity level of osteoblasts was significantly increased by the addition of Epimedium at 0.1 - 10 g/L, with the most significant increase at 1 g/L. On the other hand, the proliferation of osteoblasts was not affected after different doses of Epimedium added into the culture medium. Determined by flow cytometry, apoptosis of osteoblasts were induced by treatment with dexamethasone for 72 h. However, simultaneous administration of 1 g/L Epimedium had no effects on dexamethasone-induced apoptosis in osteoblasts. CONCLUSION: Epimedium did not affect the cell proliferation and cell survival of mouse osteoblasts, but could significantly increase alkaline phosphatase activity of the cells. The increase of alkaline phosphatase activity by Epimedium in osteoblasts may be one of the important mechanisms by which Epimedium can effectively prevent osteoporosis.

Sublingual delivery of insulin: effects of enhancers on the mucosal lipid fluidity and protein conformation, transport, and in vivo hypoglycemic activity.

The purposes of this study were to evaluate effects of enhancers for sublingual delivering insulin on the mucosal lipid fluidity and protein conformation, transport, and in vivo hypoglycemic activity in normal rats. The effects on sublingual mucosa, and aggregation states of insulin were estimated using fluorescence polarization, and circular dichroism method, respectively. The human immortalized oral epithelial cell monolayer was used for evaluating transport of insulin. Hydroxylpropyl-beta-cyclodextrin (HP-beta-CD), chitosan, polyethylene-polypropylene glycol, polyoxyethylene lauryl ether, polysorbate 80, egg lecithin, or oleic acid, was used as a penetration enhancer, respectively. The fluidity of sublingual mucosal lipid was markedly reduced by these enhancers excluding polysorbate 80, and the secondary structure of the mucosal proteins was also influenced by these enhancers. The hexamers of insulin were dissociated to monomers only by chitosan, polyoxyethylene lauryl ether, and egg lecithin. Nonetheless, plasma glucose levels in normal rats were significantly lowered after sublingual administration of insulin with an enhancer compared with those without an enhancer at the same time-point. The enhancing effects may be due to one or multiple factors: increasing the mucosal lipid fluidity, directly loosing the tight junction of epithelia, and dissociating the hexamers of insulin to monomers. Among these, the opened tight junction may correlate most with the enhancing effect in the mucosal permeability. Because the aggregates of insulin exist, the dissociation of the aggregates by an enhancer would benefit the permeability.