Study On Effects Of BMP7 On Lens Cells In Transgenic Mice

Cataract is a kind of very important blindness diseases in ophthalmology, which is related to the behavior of lens cells. Learning the effects of development signals to lens cells is useful in getting to know the regularity of lens development, and even the mechanism of cataract formation.Mammalian lens provides an ideal system for studying the embryonic development; it was almost isolated from the surrounding tissues. The mature lens is a polarized structure consisting of a mitotic epithelial layer that covers the anterior surface and terminally differentiated lens fiber cells that occupy the interior volume and the posterior surface. Lens epithelial cells and fiber cells are from the same cell spectrum, epithelia are the primary cells and fiber cells are the terminated ones. Only the lens epithelia contains mitotic cells in the lens, once cells move into the equatorial region and differentiate, they become postmitotic. The cyclin dependent kinases have a well-established role in regulating the cell cycle and apoptosis.The major proteins found in the developing and mature lens are the crystallins, a diverse group of soluble proteins, which make possible the refraction of light. Many crystallins genes are expressed almost exclusively in the lens. This has made it possible to use the transcriptional promoters of these genes to target specific gene expression to lens cells in transgenic mice, and thus study the effects of gene perturbation in isolation, without disrupting development in other parts of the embryo. Thus, a unique opportunity is created to use the lens as a general in vivo model for studies of cell cycle dynamics and differentiation without other effects from in vitro experiments.Bone morphogenetic protein 7 (BMP7), also called osteogenic protein-1 (OP-1), a 35kDa homodimer protein, belongs to the transforming growth factor-P (TGF-P) superfamily. BMP7 was first isolated from extracellular bone matrix, based on its ability to induce new bone formation in vivo. During the embryonicdevelopment of the mice, BMP7 was expressed in many embryonic tissues, especially in which reciprocal reactions of epithelial cells and mesenchymal cells are of importance for the morphogenesis.Many complex mechanisms involved in the eye development, eye tissues derived from neuroectoderm, ectoderm and mesoderm. BMPs played very important roles in almost all these ocular tissues and cells, especially for the lens and retina, which attributed to the different signals and reciprocal reactions. The effects of BMPs are one of the hot topics in recent research.Several studies had shown that BMP7 was the signal from the optic vesicle for the induction of lens placode and corneal epithelial cells. Some scholars summarizes the relationship among retina, lens and BMP7 during the early stage of eye development, they supposed that early staged retina produced BMP7 and BMPs binding proteins, which were all secreted to the vitreous cavity, the induction of lens depended on them.Since BMP7 involved in the eye, especially the lens development, we used lens-specific aA-crystallin promoter and human BMP7 to generate transgenic mice, in order to examine the effects of BMP7 in lens cells. The present research focused on the early stage of embryonic eyes, we selected aA-crystallin promoter, which acts from the Embryo 12.5, just after the induction of placode, which occurred on Embryo 9.5. In this study, we paid our attention to the stages of postnatal 20 and 30 when the mouse lenses already develop.In this study, we constructed the transgenic aA-BMP7 gene, which were confirmed by enzyming and sequencing. After amplifying, enzyming, isolating and purifying, injecting DNA segment was got. Microinjection method was used to generate the transgenic mice, PCR and dot blotting for integration, in situ hybridization for expression of transgene, TUNEL technique for apoptosis of lens cells, and ABC method of immunohistochemistry for the detection of Caspase 3, Cyclin Bl, Cyclin Dl and E-cadherin. The results are as follows:1. aA-BMP7 gene was successfully constructed, which was confirmed by enzyme digest and sequencing.2. Microinjection was used to inject aA-BMP7 gene into the pronuclears and...