| Purpose: Senile Cataract ,which is also called age-related cataract, is the major cause of blindness in the world today. Nearly half of blindness in china was caused by it. Previous studies of cataract mostly focused on the crystalline , while the studies on lens epithelium were rare. Lens epithelial cells, the only cells that have dividing activity in the lens, can divide and differentiate the lens fiber, produce the crystalline and play an important role in maintaining the stabilization of the condition in the lens, retaining the infiltrative pressure of the lens and resisting the attack of harmful factors outside. The study of the changes in gene expressions of cataractous lens epithelium will contribute to find out the specificgene relevant to cataract formation the factors controlling the gene expressions; furthermore, it helps to explain the etiological and developmental mechanism of cataract on the molecular level.Till now, most reserches on gene expressions of catarctous lens epithelium have dealt with the culture cells, and for the defferent treatments(include UV-irradiation , and H2O2 inducing) were employed, the results were conflicting. Some gene's expressions increased, such as the increase of some protein's synthesis and secretion, the increase of apoptosis and the c-fos expression of the lens epithelium, but some others decreased, such as NADH-dehydrogenase subunit 4 and cytochrome-B. Of the sudies on human lens epithelium. Lee reported that the expressions of fibronectin , collagen type I, a-smooth muscle actin, TGF-l, TGF-2 and TGF-3 II receptor in cataractous lens epithelium were different. Kantorow found that there were 3 high-expressed genes and 12 low-expressed genes in age-related cataractous lens epithelium by using the RT-PCR-DD method. Obviously, as the samples, the treatment , and the screen methods were different, so the results were very different until now. The conclusion of gene expression in cataractous lens epithelium remains unclear. The purpose of this study is: using the improved subtractive method , to construct a subtractive cDNA library of cataract, screen the differentially expressed genes of cataractous and normal lens epithelium, and identify some of the differentially expressed genes by collecting the anterior capsule samples of age-related cataract and normal lenses, which may help to find out the specific genes of cataract and provide a new idea for studying the etiology and prevention of cataract.Method: Firstly, a subtractive cDNA library of lens epithelium between age-related cataractous and normal lenses were constructed. The mRNA ofcataractous and normal lens epithelium were isolated. Then the mRNA of cataractous group was reversly transcribed into cDNA by anchor primer method, and the mRNA of normal group was reversly transcribed into cDNA by ordinary method. After hybridizing, infiltrating through the sephacryl S400 column, absorbing by the magnetic beads, and amplifying by PCR method, the fragments were inserted into the plasmid vector and the cDNA library was successfully constructed. As the samples of cataractous and normal lens were in very small quantity, we used the selected PCR method during amplifying the library. The short fragments were cut after the first round of PCR, and the large fragments were amplified by the second round of PCR , so the proportion of large fragments was improved.Secondly, in order to remove the short and nonsense fragments and the false differentially-expressed fragments of this cDNA library, we expended the bacterial germ of the library by shaking them in the LB culture medium , then we isolated the plasmid and identified them by enzyme digestion. The >750bp fragments of this library were saved and hybridized by Southern dot-blotting method .The probe was labeled by isotope with the cDNA of normal group, and it was hybridized with the fragments which had been dotted on the nylon membrane. The negative hybridized dots were sequenced and analyzed by biological imformatics. The sequences were firstly com...
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