Effects Of Lipofectemine Mediated BFGF-Math1 Gene Transfer To UEC-4 Cell On Reduction And Differentiation

The mammalian inner ear contains the cochlea and vestibular organs , which are responsible for hearing and balance, respectively. The epithelia of these sensory organs contain hair cells that function as mechanoreceptors to transduce sound and head motion. Loss of hair cells leads to permanent hearing loss and imbalance. Therefore, how to induce the hair cells to regenerate is the research focus all alone.Studies over the past 20 years indicate that many growth factors can promote hair cell regeneration, including epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), brain derived neurotrophic factor (BDNF), and insulin growth factor (IGF). bFGF has broad range of biological effect, such as promoting the growth and differentiation of embryonic nerve cell cultures in vitro. And it has been demonstrated that bFGF may participate in the initiation mechanism of hair cell regeneration.Hair cell regeneration is considered as the reappearance of embryonic hair cell development. It has been universal accepted that basic helix-loop-helix (bHLH) transcription factor genes may play an important role during the development of inner ear hair cells. Many scientists suppose that hair cell injury can induce regeneration through progenitor or stem cell transdifferentiation. Math1 is an essential gene for the generation of inner ear hair cells. Embryonic Math1-null mice failed to generate cochlear and vestibular hair cells andoverexpression of Math1 induces robust production of ectopic hair cells in postnatal rat inner ears. The latest demonstration shows that Mathl induces regeneration of hair cells and substantially improves hearing thresholds in the mature deaf inner ear after delivery to nonsensory cells using adenovectors.The UEC-4 (utricular epithelial cell line-4) cells derive from rat utricular supporting epithelial cells and are regarded as one of the progenitor cells of vestibule hair cells. The UEC-4 cells express early hair cell maker such as calretimn and calmodulin but not hair cell specific maker such as myosinâ…¦a.To investigate the mechanism of hair cell regeneration and to make a further study about the biological effect of bFGF -Mathl fusion gene expression vector, bFGF-Math1 gene was transfered into the UEC-4 cells. The main results are as follows.1. The selection and optimization of gene transfection methods Enhanced green fluorescent protein (EGFP) was used as the report gene tocompare the transfection efficiency of several common gene transfection methods involving calcium phosphate, Fugene 6 and liposome mediated way. The observation showed that liposome mediated way is effective for the study of gene transfection in vitro in terms of its high efficiency, good stability and reproducibility, and the transfection efficiency can reach 40-56% after optimization.2. The expression of bFGF-Mathl gene in UEC-4 cellsTo detect the expression of bFGF-Mathl, We performed RT-PCR analysis with total RNA extracted from UEC-4 cells prepared respectively from 24h, 48h, 72h and 96h after gene transfection. The results indicated that bFGF-Mathl was expressed from 24h. Its expression became elevated at 48h, 72h and 96h and was maintained stably in this several stages.3. The differentiation inducing effect of bFGF-Mathl gene in UEC-4...