Adenovirus-mediated delivery of bcl-2 gene attenuates cisplatin-induced degeneration of cultured spiral ganglion cells / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To assess the protection against cisplatin-induced ototoxicity by adenovirus-mediated overexpression of the bcl-2 gene in cultured spiral ganglion cells (SGC).</p><p><b>METHODS</b>SGC from P3 rats were cultured in vitro and exposed to adenovirus vector carrying green fluorescent protein gene (Ad-GFP), followed by immunocytochemical analysis for expression of the neuron-specific marker Neurofilament 200 (NF200) and detection under laser scanning confocal fluorescence microscope. Then, SGC were transduced by Ad-bcl-2 and the expression of human bcl-2 protein was evaluated by Western Blot. Finally, the cultures of SGC were divided into 4 groups: the group of Ad-bcl-2 transfection followed by cisplatin treatment, the group of Ad-GFP transfection followed by cisplatin treatment, the group of cisplatin treatment only and the untreated group. Cisplatin worked for 48 hours at a concentration of 2 microg/ml. Outcome measures included survival number of SGC and longest neurite length by using ImageJ software.</p><p><b>RESULTS</b>SGC were cultured successfully in vitro and transfected by adenovirus vector safely and efficiently. By Western Blot, human bcl-2 protein was expressed in the group after exposure to Ad-bcl-2, but not in the Ad-GFP transfected SGC. Cisplatin exposure resulted in shrinking of neuritis and pyknosis of cell body, even cell death. Expression of bcl-2 in the SGC provided a significant level of protection against cisplatin-induced SGC degeneration.</p><p><b>CONCLUSIONS</b>Our results suggest that SGC can be transduced by adenovirus vector safely and efficiently in vitro. Adenovirus-mediated delivery of the bcl-2 gene attenuates cisplatin-induced SGC degeneration.</p>

Gene transfer into primary cultures of fetal neural stem cells by a recombinant adenovirus carrying the gene for green fluorescent protein / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

<p><b>OBJECTIVE</b>To evaluate the transduction efficiency of a recombinant adenovirus carrying the gene for green fluorescent protein (Ad-GFP) into the primary cultures of fetal neural stem cells (NSCs) by the expression of GFP.</p><p><b>METHODS</b>The Ad-GFP was constructed by homologous recombination in bacteria with the AdEasy system; NSCs were isolated from rat fetal hippocampus and cultured as neurosphere suspensions. After infection with the recombinant Ad-GFP, NSCs were examined with a fluorescent microscopy and a flow cytometry for their expression of GFP.</p><p><b>RESULTS</b>After the viral infection, flow cytometry analysis revealed that the percentage of GFP-positive cells was as high as 97.05%. The infected NSCs sustained the GFP expression for above 4 weeks. After differentiated into astrocytes or neurons, they continued to express GFP efficiently.</p><p><b>CONCLUSION</b>We have successfully constructed a viral vector Ad-GFP that can efficiently infect the primary NSCs. The reporter gene was showed fully and sustained expression in the infected cells as well as their differentiated progenies.</p>

The effect of vitamin A on the conjunctival goblet cells of rat after corneal transplantation / 国际眼科杂志(Guoji Yanke Zazhi)

·AIM: To investigate the effect of vitamin A on the conjunctival goblet cells of rat after corneal transplantation.·METHODS: Rat graft rejection models of corneal transplantation were established. SD rats were receptor and Wistar rats were donors. After corneal allografts were performed, 48 SD rats were randomly divided into three groups, 16 rats in each group. Group A was blank control group; group B was treated by oculotect gel (containing vitamin A); group C was treated by 1g/L dexamethasone eyedrops. Besides, group D was normal unoperated eyes.Slit-lamp microscope was employed to record and compare rejection index (RI) of corneal transplantation. Through HE,PAS staining of conjunctival histological sections and image analysis system, the number and morphology of conjunctival goblet cells were observed and analyzed between operation group and normal group.·RESULTS: The HE, PAS staining detection showed that the number of conjunctival goblet cells in oculotect gel group,1g/L dexamethasone eyedrops group and control group is lower than that in normal group after surgery (P＜0.01). The number of conjunctival goblet cells in oculotect gel group and 1g/L dexamethasone eyedrops group is higher than that in control group (P＜0.05). The number of conjunctival goblet cells in 1g/L dexamethasone eyedrops group is higher than that in oculotect gel group (P＜0.05).·CONCLUSION: The results indicate that vitamin A may inhibit the decrease of conjunctival goblet cells after corneal allograft rejection in rats.

Inhibitory effects of small interfering RNA specific to protein kinase CK2a on the growth of laryngeal carcinoma cells / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To assess the effect of small interfering RNA (siRNA) specific to protein kinase CK2a on proliferation and apoptosis of Hep-2 cell line.</p><p><b>METHODS</b>siRNA expression plasmid psiRNA-hH1neo-CK2 specific to protein kinase CK2a and non-specific siRNA expression plasmid psiRNA-hH1neo-cont were constructed respectively, and then were transfected into Hep-2 cells by lipofectamine methods. Protein kinase CK2a mRNA and protein of the transfected cells were detected by reverse transcription polymerase chain reaction (RT-PCR) and Western Blot, respectively. Proliferation and apoptosis of the transfected cells were observed by methyl thiazolyl tetrazolium (MTT) method and flow cytometry (FCM), respectively.</p><p><b>RESULTS</b>Protein kinase CK2a mRNA and protein expressions were significantly decreased in the cells transfected with psiRNA-hH1neo-CK2 (P < 0.05). The Hep-2 cells grew slowly after transfected with psiRNA-hH1neo-CK2(P < 0.05). Obvious subdiploid peaks were found in the cells transfected with psiRNA-hH1neo-CK2 (P < 0.05).</p><p><b>CONCLUSIONS</b>siRNA expression plasmid specific to protein kinase CK2a suppressed the protein kinase CK2a expression and the proliferation of Hep-2, and induced apoptosis of Hep-2 cells.</p>

Construction of recombinant adenovirus containing human Bcl-2 and its expression in the spiral ganglion cells / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To construct the adenoviral vector containing human Bcl-2 gene and to study the expression of the gene in the spiral ganglion cells (SGC) in vitro.</p><p><b>METHODS</b>Human Bcl-2 cDNA obtained from the plasmid pUC-CAGGS/Bcl-2 was cloned into the plasmid pAdTrack-CMV. Then, pAdTrack/Bcl-2 was cotransferred with adenoviral backbone vector into E. coli strain BJ5183. The recombinant adenoviral plasmid was identified by restriction analysis with Pac I and transfected into HEK293 cells to package and amplify recombinant adenovirus particles which would be identified by Electron microscope. After the adenovirus infected the rat spiral ganglion cells, the expression of Bcl-2 gene was detected by Western Blot and RT-PCR.</p><p><b>RESULTS</b>The recombinant AdEGFP/Bcl-2 plasmid was correctly constructed and confirmed by restriction endonuclease analysis. The viral particles in HEK293 cells were identified by Electron microscope. RT-PCR and Western blot showed that Bcl-2 gene was exactly transcripted and expressed in transgene SGC.</p><p><b>CONCLUSIONS</b>The method based on homologous recombination in bacteria is simple and high efficient. The recombinant adenoviral vector containing human Bcl-2 cDNA was constructed and the transgene SGC expressed human Bcl-2 gene in vitro successfully. It provided foundation for the further study of protection for the impaired SGC by hBcl-2 gene.</p>