Study On The Mechanism Of TERT In Promoting Cochlear Progenitor Cells Proliferation In Vitro

The cochlear hair cells were terminally differentiated cells. They can’t regenerate once they were damaged. The endogenous stem cells were considered as the most potential strategy for replacement of damaged hair cells. Cochlear progenitor cells were successfully isolated from newborn cochlea, which showed features of stem cells. They can be induced to differentiate into hair cell-like cells. The cochlear progenitor cells were not real stem cells and showed limited capability of proliferation. Several genes that relate with cochlear development were proved to promote the proliferation of cochlear progenitor cells. In this study, we examined the expression of TERT in the basilar membrane at different stage of cochlear development and in the cochlear progenitor cells cultured in vitro. The results showed that with the development of cochlea, the expression of TERT decreased. The expression of TERT decreased with the prolonged culture time and passage. We infected the cultured cochlear progenitor cells with adenovirus that carrying TERT gene, and found that overexpression of TERT can promote the proliferation of cochlear progenitor cells. We further confirmedthat Rb/E2 F pass way might involve in TERT of the regulation of cochlear progenitor cells.1 The isolation and culture of cochlear progenitor cells from newborn rats.Objective: To establish a new method to isolate cochlear progenitor cells from epithelium of basilar membrane by combinatorial enzymatic digestion.Methods: Basilar membranes were separated from cochlea of P1-3 neonatal SD rats. The isolated basilar membrane sheets were divided into four groups: Group A, thermolysin digestion; Group B, collagenase type I digestion. Group C, thermolysin and collagenase type I digestion; Group D, thermolysin digestion and mechanical separation. The isolated single cells were collected and cultured under suspension and adherent condition. The number of formed otospheres, the purity of epithelial cells and the proliferation and differentiation of progenitor cells were evaluated.Results: The single cells collected in four groups can give rise to otospheres, which were positive for nestin and Brd U. Under differentiation condition, the cells from epithelium of the basilar membrane can differentiate into supporting cells and hair cells, while the cells from mesenchymal layer of basilar membrane can differentiate into neuron cells. Then most otospheres were harvested from group C that basilar membranes were digested by thermolysin and collagenase type I, and the epithelial origin were confirmed by cytokeratin-18. The purity of epithelial cells among the four groups didn’t show any difference.Conclusions: The combinatorial enzymatic digestion with thermolysin and collagenase type Idigestion can significantly improve the isolation effects of epithelial cells. This method provides a new tool for hair cell regeneration research.2 The expression of TERT in the development of cochlea in vivo and in the cochlear progenitor cells in vitro.Objective: TERT is crucial for maintaining the telomerase activity. It had been proved to promote proliferation of several kinds of cells. But the effects of TERT in the inner ear are still unclear. The aim of this study is to evaluate the expression of TERT in the basilar membrane at different stage of cochlear development and in the cochlear progenitor cells cultured in vitro.Methods: We used RT-PCR and Western blot to evaluate the TERT expression in basilar membrane from P0, P3, P7, P14 and P28 rats. Meanwhile, the cochlear progenitor cells were isolated and cultured in vitro. The expression of TERT at different culture time, generation and different kinds of otospheres were evaluated by RT-PCR.Results: TERT expression was observed in the basilar membrane of P0, P3 and P7 rats and decreased as the cochlear development. The expression of TERT in the progenitor cells cultured in vitro decreased as the culture time became longer and generation passage. The expression of TERT in hollow otospheres was significantly lower than solid and transitional otospheres.Conclusion: The decreasing expression of TERT as the cochlear development indicated that TERT might associate with cochlear development. The stem cell feature and proliferation capability of cochlear progenitor cells decreased as the culture time became longer, along with the decreasing of TERT expression, which indicated that TERT might involve in maintaining the stem cell feature of cochlear progenitor cells.3 Construction and identification of recombinant adenovirus and transfection of the cochlear progenitor cells.Objective: We established the recombinant adenoviral vector of TERT gene. The adenoviruses were used to transfect the cochlear progenitor cells in vitro. Then the efficacy of transfection and suitable MOI were evaluated. Further the expression of TERT m RNA, protein and the activity of telomerase were evaluated.Methods: The recombinant adenoviruses were constructed. The complementary DNA sequence of TERT gene was obtained from Gen Bank. The sequence was subcloned into GV314. The adenovirus generation, amplification and titer process were completed sequentially. The vector containing EGFP were also constructed. The Ad-EGFP was used to infect cochlear progenitor cells at different MOI(250, 200, 150, 100, 50, 25). The expression of EGFP and the morphology of cells were observed under florescent microscopy. The expression of TERT in cochlear progenitor cells after infection was evaluated by RT-PCR and Western blot. The activity of telomerase was evaluated by TRAP-ELISA.Results: The recombinant adenovirus construction was confirmed to carry the TERT gene by the detection of the purpose fragment in PCR and the concentration of virus were identified as 1×1011pfu/ml. A positive dose-dependent relationship was observed between the expressions of EGFP gene ranging from 25-200 MOI. The virus infection didn’t show any effect to the vitality and morphology. However, the cells showed growth arrest and necrosis at a MOI of 250. Accordingly, we chose the MOI of 200 as the suitable infection MOI. Then the vector of Ad-TERT-EGFP was used to infect cochlear progenitor cells at a MOI of 200 and the cells were collected for analysis atday 4 after infection. The expression of TERT m RNA and protein significantly increased in the cochlear progenitor cells infected with Ad-TERT-EGFP. But the activity of telomerase didn’t increase significantly.Conclusion: We can successfully infect cochlear progenitor cells with adenovirus vector. The expression of TERT significantly increased after infection, which may provide powerful tool for the research of TERT on the proliferation of cochlear progenitor cells.4. Over expression of TERT promoting the proliferation of cochlear progenitor cells in vitro.Objective: To evaluate the effect of TERT on the proliferation of cochlear progenitor cells by Ad-TERT-EGFP infection.Methods: The cultured progenitor cells were divided into three groups, including: Ad-TERT-EGFP infection group, Ad-EGFP infection group and control group. The cells were infected with corresponding adenovirus at a MOI of 200. The ratios of three kinds of otospheres were calculated. The cell proliferation was evaluated by MTT test. The Ed U positive ratio was determined, too. The expression of PCNA was tested by RT-PCR and Western blot.Results: The ratio of solid otospheres in the Ad-TERT-EGFP infection group was significantly higher than other two groups at day 7 after infection(P < 0.05). The MTT results suggested that over expression of TERT can promoting the growth of cochlear progenitor cells. The Ed U labeling show that there were significantly more Ed U+ cells in the Ad-TERT-EGFP infection group(P < 0.05). The expression of PCNA in theAd-TERT-EGFP infection group increased significantly(P < 0.05).Conclusion: Over expression of TERT can promote the proliferation of cochlear progenitor cells.5 The mechanism of TERT in promoting cochlear progenitor cell proliferationObjective: To study the mechanism of TERT in promoting cochlear progenitor cell proliferation.Methods: The expressions of P27KIP1, P53, Rb, E2 F, Cyclin D1, Cyclin E1, CDK2, CDK4, β-catenin, P16INK4 a in the Ad-TERT-EGFP group, Ad-EGFP group and Control group were evaluated by RT-PCR. The expression of P27KIP1, P53, Rb, E2 F, Cyclin D1, Cyclin E1, β-catenin protein were evaluated by Western blot.Results: Over expression of TERT decreased the m RNA level of P27KIP1, P53, P16INK4a(P<0.05). The level of CDK2, Cyclin D1 m RNA significantly increased in the Ad-TERT-EGFP infection group(P <0.05). The western blot results showed that over expression of TERT lead to decreasing of P27KIP1, P53, Rb, while the expression of E2 F and Cyclin D1 increased significantly(P <0.05). The expression of β-catenin didn’t show any significant difference among the three groups.Conclusion: Over expression of TERT can promote the proliferation of cochlear progenitor cells, and the Rb/E2 F signaling pathway may be involved.