The Expression And Research Of Dystrophin Gene In Dermal Fibroblast Cells

Objective:To study the expression of dystrophin mRNA and dystrophin in dermal fibroblasts.Investigating the application of dermal fibroblasts for donor's cells for the research of transformation of muscular cells with genetic deficiencyMethods:1,Culture the dermal fibroblasts of normal mice on the age of 0～1 by the tissue pact outside.Obliterating the tissue when the cells full filled the plate and digesting the cells by 0.25% tryplar and profating them by the proportion of one to three.Then,respectively detect the expression of dystrophin mRNA and dystrophin in the cultured parental and the first to fourth generation of dermal fibroblasts of the mouse by RT-PCR and Western blot.2,Cutting the skeletal muscle,heart,liver,kidney and skin tissue of the mouse into pieces,transfer to liquid nitrogen and homogenize them with lysis solution,in the end, respectively detect the expression of dystrophin mRNA and dystrophin in these tissues of the mouse by RT-PCR and Western blot.3,Cutting the skeletal muscle and skin tissues of the mouse into pieces,transfer to liquid nitrogen and homogenize them with lysis solution untill the tissues are disappeared,in the end,confirm the DNA sequences of the dermal fibroblasts of the mouse contain dystrophin gene by PCR.Results:1,The result of RT-PCR:(1) detection based on cells:One about 573bp specific DNA strip is found by amplifying in the skeletal muscle of the mouse(positive control ) ,but,there is no any DNA strip by amplifying in the cultured parental and the first to fourth generation of dermal fibroblasts of the mouse.(2) detection based on tissues: One about 573bp specific DNA strip is found by amplifying in the skeletal muscle and heart of the mouse, but,there is no any DNA strip by amplifying in the skin,liver and kidney tissues of the mouse.The experiment result states that there is no expression of dystrophin mRNA in dermal fibroblasts.2,The result of Western blot:(1) detection based on cells:There is one about 427 KD specific strip in the skeletal muscle of the mouse,but,there is no any reaction strip in the cultured parental and the first to fourth genenation of dermal fibroblasts of the mouse.(2) detection based on tissues: One about 427 KD specific strip appears in the skeletal muscle and heart of the mouse,but there is no any reaction strip in the skin,liver and kidney tissues of the mouse.The experiment result states that there is no expression of dystrophin in dermal fibroblasts.3,The result of PCR:There is one about 331bp specific DNA strip by amplifying in the skeletal muscle and skin tissues of the mouse.The result shows the DNA sequences of the dermal fibroblasts contain dystrophin gene.Conclusion:In the process of experiment on discussion genetic material shift using cell integration , dermal fibroblast cells could be one of the ideal donor's cells for the research of transformation of muscular cells with genetic deficiency...