BACKGROUND: The investigation of culturing, passaging and establishing human limbal stem cells can strengthen the recognition of the stem cells and provide the enough cellular reserve for the basic and clinical research of limbal stem cell transplantation.OBJECTIVE: To explore a method of pessaging and establishing cell line of human limbal stem cells cultured in vitro.DESIGN: Randomized controlled observation.SETTING: Gannan Medical College.MATERIALS: The experiment was performed at Scientific Center of Gannan Medical College and the National Key Laboratory of Ophthalmology Hospital Affiliated to Sun Yat-Sen University from June 2003 to April 2004. Fresh human limbus corneae were isolated from two healthy donors. Procedures were performed according to the informed consent of the donors. Main reagents contained RPMI-1640 (Sigma R8755, containing L-glutamine) and 200 g/L fetal calf serum (FCS) (Gibco 16140-071). DMEM medium, chondroitin sulfatase and human epidermal growth factor (hEGF) were purchased from Sigma Co. USA; HEPES and DMSO were bought from Gibco, USA; 100% glycerinum was purchased from Yunjia Huangpu Pharmaceutical Product Limited Company, PRC; glutaraldehyde was bought from E.Merk, Germany; Alcohol, chlorhydric acid, acetone and methyl aldehyde were purchased from Beijing Chemical Agent Company, PRC; 0.25% parenzyme was bought from Shanghai Xinhua Pharmaceutical Factory, PRC.Above-mentioned reagents were analytical pure grade.METHODS: After digestion, human limbal tissues in limbal basilar part with an abundant pigment were cultured in the culture flask containing RPMI-1640 and 200 g/L FCS and in culture dish containing amniotic extracellular matrix (AECM) as the cultural supporter. Primary and passage cells were observed under light microscope and scanning electron microscope (SEM). The revival ratio of stem cell refrigeration of every generation was calculated by the trypanblau exclusion experiment.MAIN OUTCOME MEASURES: ① Observational results of limbal stem cells during the primary culture and serial subcultivation in vitro, and ② revival ratio of stem cell refrigeration.RESULTS: ①Findings of primary culture: Most limbal stem cells in the culture flask had the adherence and were arrayed uniformly sparsely to form monolayer and adhered to the bottom of culture flask under the inverted phase contrast microscope after 1-day culture. ② Findings of serial subcultivation: After human epidermal growth factor (hEGF) was added into the second passage, cells were scattered into the monolayer and adhered to grow quickly.Morphological variability of all the cells increased obviously when passage the 30th generation. The cellular volume was obviously increasing, and the round or irregular round cells gathered together. The 33rd generation human limbal stem cells still could vigorously differentiate, proliferate and grow in ACEM. ③ The revival ratio of stem cell refrigeration was 82.2%.CONCLUSION: The human limbal stem cell lines were preliminarily established by culturing and freezing the cells of 33 generations in vitro. The human limbal stem cell lines preferred to grow in the culture dish containing AECM as the cultural supporter.

The hydroponic culture test method was used to study the physiological and biochemical responses of Paulownia fortunei seedlings under Zn stress, Cd stress, and combined Zn and Cd stress as well as changes in the enrichment and transfer characteristics of heavy metals. Under single and combined heavy metal stress, the biomass, plant height, and peroxidase (POD) activity of P. fortunei decreased as the treatment concentration increased. Combined Zn and Cd affected adversely plant height and biomass. As the concentration of Zn increased when applied alone, the chlorophyll content and catalase (CAT) activity of P. fortunei first increased and then decreased, the superoxide dismutase (SOD) activity increased, and the aboveground malondialdehyde (MDA) content first decreased and then increased. As the concentration of Cd increased when applied alone, chlorophyll content and CAT activity increased, and SOD activity and aboveground MDA content first increased and then decreased. Under both Cd and Zn, the physiological response was more complex. Cd in the seedlings of P. fortunei was concentrated in the root. In contrast, Zn was concentrated in the upper part of the ground, and its transfer coefficient was greater than 1.00. Thus, the addition of Zn promotes the transfer of heavy metals to the above-ground portions of plants. Generally, P. fortunei can effectively promote ecological restoration under complex forms of heavy metal pollution.
Cadmium ; Chlorophyll ; Metals, Heavy ; Plant Roots/chemistry* ; Seedlings ; Soil Pollutants ; Stress, Physiological ; Superoxide Dismutase ; Zinc