Establishment of goat limbal stem cell strain expressing Venus fluorescent protein and construction of limbal epithelial sheets / 生物工程学报

The integrity and transparency of cornea plays a key role in vision. Limbal Stem Cells (LSCs) are precursors of cornea, which are responsible for self-renewal and replenishing corneal epithelium. Though it is successful to cell replacement therapy for impairing ocular surface by Limbal Stem Cell Transplantation (LSCT), the mechanism of renew is unclear after LSCT. To real time follow-up the migration and differentiation of corneal transplanted epithelial cells after transplanting, we transfected venus (a fluorescent protein gene) into goat LSCs, selected with G418 and established a stable transfected cell line, named GLSC-V. These cells showed green fluorescence, and which could maintain for at least 3 months. GLSC-V also were positive for anti-P63 and anti-Integrinbeta1 antibody by immunofluorescent staining. We founded neither GLSC-V nor GLSCs expressed keratin3 (k3) and keratinl2 (k12). However, GLSC-V had higher levels in expression of p63, pcna and venus compared with GLSCs. Further, we cultivated the cells on denude amniotic membrane to construct tissue engineered fluorescent corneal epithelial sheets. Histology and HE staining showed that the constructed fluorescent corneal epithelial sheets consisted of 5-6 layers of epithelium. Only the lowest basal cells of fluorescent corneal epithelial sheets expressed P63 analyzed by immunofluorescence, but not superficial epithelial cells. These results showed that our constructed fluorescent corneal epithelial sheets were similar to the normal corneal epithelium in structure and morphology. This demonstrated that they could be transplanted for patents with corneal impair, also may provide a foundation for the study on the mechanisms of corneal epithelial cell regeneration after LSCT.

Program optimization for bovine somatic cells nuclear transfer / 生物工程学报

To optimize program of bovine somatic nuclear transfer, we used two different enucleation procedures (by Spindle-view system & Hoechst 33342 staining), two different procedures to introduce donor nuclei (by ooplasm microinjection & electrofusion), and three different group electrofusion parameters (group 1: 1.9 kV/cm, 10 micros, two; group 2: 1.5 kV/cm, 25 micros, two; group 3: 0.6 kV/cm, 100 micros, one) to reconstruct bovine cloned embryos. The cleavation rates and blastocyst development rates of cloned embryos were used to assess the efficiency of different operational procedure. Finally, the best combination of operational procedure, that the spindle-viewer system was used for oocytes enucleating, and donor cell was electrofused into ooplasm by electrical pulse (1.9 kV/cm, 10 micros, two) to reconstruct bovine cloned embryos. Then the excellent blastocysts were transferred to fosters for producing cloned cattle 80 high-quality cloned blastocysts were transferred into 33 fosters, two cloned calves were produced. According to the results, the optimized program could be used to produce cloned cattle.

Isolation and cultivation of goat embryo stem cells / 生物工程学报

Morulaes and blastocysts obtained from Guanzhong dairy goats 6-7 days after mating were treated with whole embryo cultivaton, enzymatic digestion and immunosurgery separately. The goat embryonic stem cells (ESC) were isolated and cultured on a feeder layer of mitomycin-inactivated mouse embryo fibroblasts (MEF). The characteristics of goat ESCs were analyzed by immunohistochemisty, RT-PCR and inducing differentiation in vitro. The results indicated that the embryos were easier to attach the culture dish and form primary colonies with whole embryo method. There were colonies that maintained undifferentiated for 18 passages. The ESCs expressed the protein of Nanog, Oct4 and SSEA-3, whereas the protein of SSEA-4 was absent and the protein of SSEA-1 was weakly expressed. In addition, the genes of Nanog, Oct4, TERT and CD117 were expressed in goat ESCs. The cells also could differentiate to myocardial cells when induced in vitro by DMSO. These results suggest that the goat ESCs have characteristics of ESCs.