BACKGROUND: The transplantation of limbal epithelial cells cultivated on amniotic membrane is a newly developed treatment for limbal stem cell deficiency. The purpose of our study was to investigate the biological cha...BACKGROUND: The transplantation of limbal epithelial cells cultivated on amniotic membrane is a newly developed treatment for limbal stem cell deficiency. The purpose of our study was to investigate the biological characteristics of limbal epithelial cells and evaluate the effect of transplantation of cultivated human limbal epithelial cells on ocular surface reconstruction in limbal stem cell deficiency rat model. METHODS: Human limbal cells were isolated and cultivated in vitro. Cytokeratins 3, 12, and 19 (K3, K12 and K19) and p63 were detected by immunofluorescent staining or RT-PCR. BrdU labelling test was used to identify the slow cycling cells in the cultures. Limbal stem cell deficiency was established in rat cornea by alkali burn. Two weeks after injury, the rats received transplants of human limbal stem cells cultivated on amniotic membrane carrier. The therapeutic effect was evaluated by slit lamp observation, Hemotoxin and Eosin (HE) staining and immunofluorescent staining. RESULTS: On day 7 in primary culture, p63 and K19 were strongly expressed by most cells but only a few cells expressed K3. On days 14 and 21, p63 and K19 were still expressed by a majority of cells, but the expressive intensity of p63 decreased in a number of cells, while the proportion of K3 positive cells increased slightly and some cells coexpressed p63 and K3. RT-PCR showed that gene expression of both p63 and K12 were positive in cultivated limbal cells, but in mature superficial epithelial cells, only K12 was detected. BrdU labelling test showed that most cells were labelled with BrdU after 7 days' labelling and BrdU label retaining cells were observed after chasing for 21 days with BrdU free medium. For in vivo test, slit lamp observation, HE staining and immunofluorescent staining showed that the rats receiving transplant of human limbal stem cells cultivated on amniotic membrane grew reconstructed corneas with intact epithelium, improved transparency and slight or no neovascularization. A majority of epithelial cells of the展开更多
Manipulation of mouse genome has merged as one of the most important approaches for studying genefunction and establishing the disease model because of the high homology between human genome and mouse genome. In this ...Manipulation of mouse genome has merged as one of the most important approaches for studying genefunction and establishing the disease model because of the high homology between human genome and mouse genome. In this study, the chemical mutagen ethylnitrosourea (ENU) was employed for inducing germ cell mutations in maleC57BL/6J mice. The first generation (G1) of the backcrossof these mutated mice, totally 3172, was screened for abnor-mal phenotypes on gross morphology, behavior, learning and memory, auditory brainstem response (ABR), electrocardio-gram (ECG), electroretinogram (ERG), flash-visual evoked potential (F-VEP), bone mineral density, and blood sugarlevel. 595 mice have been identified with specific dominantabnormalities. Fur color changes, eye defects and hearing loss occurred at the highest frequency. Abnormalities related to metabolism alteration are least frequent. Interestingly, eye defects displayed significant left-right asymmetry and sexpreference. Sex preference is also observed in mice with ab-normal bone mineral density. Among 104 G1 generation mutant mice examined for inheritability, 14 of them have been confirmed for passing abnormal phenotypes to their progenies. However, we did not observe behavior abnormali-ties of G1 mice to be inheritable, suggesting multi-gene con-trol for these complicated functions in mice. In conclusion, the generation of these mutants paves the way for under-standing molecular and cellular mechanisms of these ab-normal phenotypes, and accelerates the cloning of disease-related genes.展开更多
Mutagenized populations have provided important materials for introducing variation and identifying gene function in plants. In this study, an ethyl methanesulfonate（EMS）-induced soybean（Glycine max） population,co...Mutagenized populations have provided important materials for introducing variation and identifying gene function in plants. In this study, an ethyl methanesulfonate（EMS）-induced soybean（Glycine max） population,consisting of 21,600 independent M_2 lines, was developed.Over 1,000 M_（4（5））families, with diverse abnormal phenotypes for seed composition, seed shape, plant morphology and maturity that are stably expressed across different environments and generations were identified. Phenotypic analysis of the population led to the identification of a yellow pigmentation mutant, gyl, that displayed significantly decreased chlorophyll（Chl） content and abnormal chloroplast development. Sequence analysis showed that gyl is allelic to Minn Gold, where a different single nucleotide polymorphism variation in the Mg-chelatase subunit gene（ChlI1a） results in golden yellow leaves. A cleaved amplified polymorphic sequence marker was developed and may be applied to marker-assisted selection for the golden yellow phenotype in soybean breeding. We show that the newly developed soybean EMS mutant population has potential for functional genomics research and genetic improvement in soybean.展开更多
文摘BACKGROUND: The transplantation of limbal epithelial cells cultivated on amniotic membrane is a newly developed treatment for limbal stem cell deficiency. The purpose of our study was to investigate the biological characteristics of limbal epithelial cells and evaluate the effect of transplantation of cultivated human limbal epithelial cells on ocular surface reconstruction in limbal stem cell deficiency rat model. METHODS: Human limbal cells were isolated and cultivated in vitro. Cytokeratins 3, 12, and 19 (K3, K12 and K19) and p63 were detected by immunofluorescent staining or RT-PCR. BrdU labelling test was used to identify the slow cycling cells in the cultures. Limbal stem cell deficiency was established in rat cornea by alkali burn. Two weeks after injury, the rats received transplants of human limbal stem cells cultivated on amniotic membrane carrier. The therapeutic effect was evaluated by slit lamp observation, Hemotoxin and Eosin (HE) staining and immunofluorescent staining. RESULTS: On day 7 in primary culture, p63 and K19 were strongly expressed by most cells but only a few cells expressed K3. On days 14 and 21, p63 and K19 were still expressed by a majority of cells, but the expressive intensity of p63 decreased in a number of cells, while the proportion of K3 positive cells increased slightly and some cells coexpressed p63 and K3. RT-PCR showed that gene expression of both p63 and K12 were positive in cultivated limbal cells, but in mature superficial epithelial cells, only K12 was detected. BrdU labelling test showed that most cells were labelled with BrdU after 7 days' labelling and BrdU label retaining cells were observed after chasing for 21 days with BrdU free medium. For in vivo test, slit lamp observation, HE staining and immunofluorescent staining showed that the rats receiving transplant of human limbal stem cells cultivated on amniotic membrane grew reconstructed corneas with intact epithelium, improved transparency and slight or no neovascularization. A majority of epithelial cells of the
基金supported by the State“863”High-Tech Project and the National Gongguan Project
文摘Manipulation of mouse genome has merged as one of the most important approaches for studying genefunction and establishing the disease model because of the high homology between human genome and mouse genome. In this study, the chemical mutagen ethylnitrosourea (ENU) was employed for inducing germ cell mutations in maleC57BL/6J mice. The first generation (G1) of the backcrossof these mutated mice, totally 3172, was screened for abnor-mal phenotypes on gross morphology, behavior, learning and memory, auditory brainstem response (ABR), electrocardio-gram (ECG), electroretinogram (ERG), flash-visual evoked potential (F-VEP), bone mineral density, and blood sugarlevel. 595 mice have been identified with specific dominantabnormalities. Fur color changes, eye defects and hearing loss occurred at the highest frequency. Abnormalities related to metabolism alteration are least frequent. Interestingly, eye defects displayed significant left-right asymmetry and sexpreference. Sex preference is also observed in mice with ab-normal bone mineral density. Among 104 G1 generation mutant mice examined for inheritability, 14 of them have been confirmed for passing abnormal phenotypes to their progenies. However, we did not observe behavior abnormali-ties of G1 mice to be inheritable, suggesting multi-gene con-trol for these complicated functions in mice. In conclusion, the generation of these mutants paves the way for under-standing molecular and cellular mechanisms of these ab-normal phenotypes, and accelerates the cloning of disease-related genes.
基金supported by National Key R&D Program for Crop Breeding (2016YFD0100201)the Crop Germplasm Resources Protection (2014NWB030, 2015NWB030-05)+1 种基金Platform of National Crop Germplasm Resources of China (2014-004, 2015-004)The Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences (CAAS)
文摘Mutagenized populations have provided important materials for introducing variation and identifying gene function in plants. In this study, an ethyl methanesulfonate（EMS）-induced soybean（Glycine max） population,consisting of 21,600 independent M_2 lines, was developed.Over 1,000 M_（4（5））families, with diverse abnormal phenotypes for seed composition, seed shape, plant morphology and maturity that are stably expressed across different environments and generations were identified. Phenotypic analysis of the population led to the identification of a yellow pigmentation mutant, gyl, that displayed significantly decreased chlorophyll（Chl） content and abnormal chloroplast development. Sequence analysis showed that gyl is allelic to Minn Gold, where a different single nucleotide polymorphism variation in the Mg-chelatase subunit gene（ChlI1a） results in golden yellow leaves. A cleaved amplified polymorphic sequence marker was developed and may be applied to marker-assisted selection for the golden yellow phenotype in soybean breeding. We show that the newly developed soybean EMS mutant population has potential for functional genomics research and genetic improvement in soybean.
