| The pathology change of sensorineural hearing loss is mainly the trauma of hair cells, spiral ganglion, support cells and neural terminal. The trauma of hair cells caused by strong sound, drug, aging, injury and various diseases in vitro and in vivo is sensorineural deafness and equilibrium problems' pathological base. The study in recent years indicates that the hair cells in cochlea and vesticular of non-mammalian and the hair cells in vestivular of mammalian have regenerate ability in anatomy and function. Whereas, the study of mammalian cochlear hair cells regeneration, include human, has not broken through. The treatment of sensorineural hearing loss is a key question in clinic.Stem cells are a kind of cells with self-renewal ability in one's whole life. They have the potential in differentiation into many kinds of mature cells with special configuration and function, such as cardiac muscle cells, epidermal cells and neuron under proper conditions or being given proper signals. Bone marrow mesenchymal stem cells (BM-MSCs) are adult stem cells in bone marrow stroma. It is found that BM-MSCs can differentiate into some kinds of cells, for instance, mesenchymal cells, fibroblast, bone cells, cartilage cells, adipocytes , neuron and epidermal cells.Neurotrophin is a protein family having close relationship with neural structure and function. It includes nerve growth factor (NGF) , brain-derived neurotrophic factor (BDNF) , glial cell line-derived neurotrophic factor (GDNF), neurotrophin-3 ( NT3 ) , Neurotrophin-4/5(NT4) and neurotrophin-6(NT6). They are the most important endogenesis protein in peripheral and central nervous system that adjust neuron's growth,-5-differentiation and living. BDNF and NT3 are the most significant nourish factors for inner ear. The former is especial for vestibule and the latter for cochlea.In this research, we explored the method of isolating, culturing and expanding human bone marrow mesenchymal stem cells (BM-MSC) in vitro and study their phenotypical properties. NT3 gene was transfected into Human BM-MSC by liposome mediated method, genetic engineering cells were constructed to produce NT3 in vitro. Detecting the expression of NT3 by PCR and observing culture medium's effect on guinea pig cochlea hair cells' survival.Method1.Cells culturing: using the methods of cell culture to isolate and expandhuman bone marrow mesenchymal stem cells (BM-MSCs) in vitro.Observing the cells configuration and growth characteristic under opticsmicroscope.2.Detecting BM-MSCs phenotypes: the third generation BM-MSCs thatalmost confluent were detached by 0.25% trypsin-EDTA, centrifuged,washed by PBS, than detected phenotypes with FCM.3.Drawing growth curve by MTT: the third, sixth, ninth generationBM-MSCs were made as single cell liquid, detected OD value (X=570nm) .The growth curves was described.4.Transfecting BM-MSCs with pEGFP-Nl: the fourth and seventh generationBM-MSCs were transferred with pEGFP-N 1 and count the transfection rate.5. Construction of the eukaryon expressing vector pcDNA3.1(+)/NT3 :pUC 18/NT-3 and pcDNA3.1 (+) were digested by Hind III and EcoR I. NT3gene and pcDNA3.1(+) segment were collected and connect with T4 DNAligase.6.NT3 gene transfection and the screening of steady clone:pcDNA3.1(+)/NT3 and pcDNA3.1(+) were transferred into humanBM-MSCs in vitro by liposome mediated method . The genetic engineeringBM-MSCs were screened several times with 300ug/ml G418 for three-6-weeks and cultured continually in 200g/ml G418 for another two weeks in order to get steady clone.T.Culturing guinea pig cochlear hair cells in vitro: the guinea's temporal was taken out. The basilar membrane was separated from modiolus then putted into culturing medium containing Hanks' and supernatant of BM-MSCs transfected pcDNA3.1(+)/NT3 while the control group containing Hanks' and supernatant of BM-MSCs transfected pcDNA3.1(+). Both were incubated under the condi...
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