Han Baoqin

Establishment of a novel corneal endothelial cell line from domestic rabbit, Oryctolagus curiculus

To develop a rabbit corneal endothelial (RCE) cell line, in vitro culture of RCE cells was initiated from Oryctolagus curiculus corneas and a novel RCE cell line was established in this study. To initiate the primary culture of RCE cells, corneas from rabbit eyes were sliced and attached into glutin-coated wells with endothelial cell surface down. After being cultured at a time-gradient interval from 48 to 6 h, the corneal slices were detached and reattached into new wells, respectively. Cells in the wells containing only a pure population of RCE cells were collected and cultured in 20% FBS-DMEM/F12 medium containing chondroitin sulfate, ocular extract, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), carboxymethyl-chitosan, N-acetylglucosamine hydrochloride, glucosamine hydrochloride, culture medium of rabbit corneal stromal cells and oxidation-degradation products of chondroitin sulfate at 37°C, 5% CO2. The cultured RCE cells, in quadrangle and polygonal shapes, proliferated to confluence 3 weeks later. During the subsequent subculture, the shape of RCE cells changed gradually from polygonal to more fibroblastic. A novel RCE cell line, growing at a steady rate, with a population doubling time of 53.8 h, has been established and subcultured to passage 67. Chromosome analysis showed that the RCE cells exhibited chromosomal aneuploidy with the modal chromosome number of 44. The results of immuno-cytochemical staining with neuron specific enolase (NSE) confirmed that the RCE cells were in neuroectodermal origin. Combined with the results of vascular endothelial growth factor (VEGF) treatment and endothelial cell morphology recovery, it can be concluded that the cell line established here is an RCE cell line. This RCE cell line may serve as a useful tool in theoretical researches of mammalian corneal endothelial cells, and may also have potential application in artificial corneal endothelium development.

Purification and inhibition fungal growth of chitinases fromVibrio pacini

Vibrio pacini synthesizes multiple chitinases, of which three have been purified in this study by ammonium sulphate fractionation, chitin affinity chromatography and gel chromatography. Molecular weights of the three chitinases, Chi1, Chi2 and Chi3 are 27×103, 39×103 and 46×103 respectively, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified enzymes have optimal activity at pH 7–8, and retain 50% enzymatic activity pH 4–9. The activities of chitinases are inhibited by Pb2+, Fe3+ and Cu2+, and increased by Ca2+, Mg2+ and Mn2+. Chi3 is found to inhibit the growth of six species of fungi. Such characters of chitinase are different from those of any other chitinase that were reported before.

Evaluation on biological compatibility of carboxymethyl chitosan as biomaterials for antitumor drug delivery

Carboxymethyl-chitosan, a water-soluble derivative of chitosan, has emerged as a promising candidate for biomedical applications due to its excellent water solubility, biodegradation, biocompatibility, hydrating, antimicrobial, and nontoxicity. In this paper, the antitumor proliferation and metastasis was studied in vitro and in vivo to evaluate biocompatibility of carboxymethyl-chitosan as biomaterials for antitumor drug delivery. The results showed that carboxymethyl-chitosan could significantly reduce the clone formation and tumor migration of human cancer cells including kidney cancer cell line OS-RC-2, gastric cancer cell line SGC-7901, colon cancer cell line HT-29, and nonsmall cell lung cancer cell line NCI-H1650 in vitro. Through Lewis tumor-bearing C57BL/6 mouse model, carboxymethyl-chitosan was proved to be able to inhibit solid tumor growth and tumor metastasis to the liver and lung, meanwhile increase the level of tissue inhibitor of metalloproteinase 1 and E-cadherin, and decrease the level of mice blood serum matrix metalloproteinase 9. This study suggested that carboxymethyl-chitosan had certain antimetastasis effect and good biocompatibility and may have a potential application as a synergic antitumor reagent.

Activities of the deriviatives of chitin on the osbeoblast proliferation and the effect on bone strength in ovariectomized rats

This study intends to examine the effects of different concentrations of four kinds of degradations of chitin: glucosamine (GLC), N-acetyl-D-glucosamine (NAG), chitooligosaccharide (COS), CM-chitooligosaccharide (CM-COS)—on the proliferation of MC3T3-E1 cell line cultured in vitro. Results suggest that all of the glucoses mentioned above promoted the proliferation of osteoblast, and various concentrations have different effects: the proliferation was remarkable when the concentration of GLC*. NAG, COS, CM-COS was 100, 100, 500, 500 μg/mL, respectively. Furthermore we choose the glucosamine as the material and study the effect on the bone strength in ovariectomized rats. The results showed that the middle does of glucosamine can significantly increase the bone strength of femur in ovariectomized rats.