The preparation of supported lipid bilayers and its functional study / 生物医学工程学杂志

Supported lipid bilayers (SLBs) have been widely used in biomedical and bioengineering research because its structure and function are similar to natural cell membrane. A fluorescence recovery after photobleaching (FRAP) technique was used to measure the lateral diffusion of the SLBs composed of 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1, 2-dioleoyl-sn-glycero-3-［(N-(5-amino-1-carboxyp-entyl) iminodiacetic acid)］ (DGS-NTA) on the glass slide, and the effects of the DOPC-to-DGS-NTA ratio, small unilamellar vesicles (SUV) producing method, sizes of bleaching areas and concentrations of loading proteins on the SLBs fluidity and diffusion coefficient were studied systematically in this paper. The results demonstrated that: (1) SUV made by probe sonication exhibited more uniform and smaller size compared with that made by film extrusion, but the whole process of SLBs formation must not be exposed to air. (2) The fluorescence recovery rate and diffusion coefficient of the SLBs decreased with the increasing bleaching area size. With the mole ratio of DOPC to DGS-NTA decreasing from 98∶2 to 84∶16, the fluidity and fluorescence recovery degree decreased gradually, and the SLBs would lose its fluidity if the ratio reached to 82∶18. (3) The average fluorescence intensity of SLBs increased linearly with the loading protein concentration (10-40 nmol·L ), and the protein showed good mobility on the SLBs. The study would provide a good platform of bio-membrane for further research on interactions among cell membrane molecules and subsequent signals response.

Neuronal RNA granule contains ApCPEB1, a novel cytoplasmic polyadenylation element binding protein, in Aplysia sensory neuron

The cytoplasmic polyadenylation element (CPE)-binding protein (CPEB) binds to CPE containing mRNAs on their 3' untranslated regions (3'UTRs). This RNA binding protein comes out many important tasks, especially in learning and memory, by modifying the translational efficiency of target mRNAs via poly (A) tailing. Overexpressed CPEB has been reported to induce the formation of stress granules (SGs), a sort of RNA granule in mammalian cell lines. RNA granule is considered to be a potentially important factor in learning and memory. However, there is no study about RNA granule in Aplysia. To examine whether an Aplysia CPEB, ApCPEB1, forms RNA granules, we overexpressed ApCPEB1-EGFP in Aplysia sensory neurons. Consistent with the localization of mammalian CPEB, overexpressed ApCPEB1 formed granular structures, and was colocalized with RNAs and another RNA binding protein, ApCPEB, showing that ApCPEB1 positive granules are RNA-protein complexes. In addition, ApCPEB1 has a high turnover rate in RNA granules which were mobile structures. Thus, our results indicate that overexpressed ApCPEB1 is incorporated into RNA granule which is a dynamic structure in Aplysia sensory neuron. We propose that ApCPEB1 granule might modulate translation, as other RNA granules do, and furthermore, influence memory.

Visualization of serotonin 1A receptor trafficking in neuron-like PC12 cells / 基础医学与临床

Objective To explore the mechanisms of trafficking and signaling of serotonin 1A receptor(5-HT_(1A))and its spatiotemporal distribution in living cells.Methods The mouse 5-HT_(1A) gene amplified by RT-PCR was recombined into pEGFP-N1 vector and the EGFP coding sequence was located in-frame at the C-terminal end of the 5-HT_(1A) receptor.The 5-HT_(1A)-EGFP was transfected into neuron-like PC12 cells as well as HEK293.The transfected cells were visualized using confocal microscopy,the mobility of 5-HT_(1A)-EGFP was monitored by live measurements and fluorescence recovery after photobleaching.Results The 5-HT_(1A) gene was identitical with the published gene sequence NM_008308.4 and a 5-HT_(1A)-EGFP fusion construct was created.After stable transfection of the plasimd into a PC12 cell line and analysis with a confocal laser scanning microscopy,the EGFP-tagged 5-HT_(1A) was predominantly associated with the plasma membrane,but some intracellular vesicles in the perinuclear region also contained the fusion protein.The predominant localization of 5-HT_(1A)-EGFP at the plasma membrane was confirmed in transiently transfected HEK293 cells.Bleached fluorescence was partialy recovered in 100 seconds,indicating that the 5-HT_(1A)-EGFP was mobiled on the membrane.Conclusion Spatiotemporal distribution and mobility of 5-HT_(1A) tagged with EGFP can be monitored in the 5-HT_(1A)-EGFP stable PC12 cell line,which could be an excellent neuron-like experimental cell model for research of 5-HT_(1A) trafficking and signaling.

Study on Cx43 expression and function of GJIC in ectopic and eutopic endometrial stromal cells in vitro / 重庆医科大学学报

Objective:To investigate Cx43 expression and gap junctional intercellular communication(GJIC) in ectopic and eutopic endometrial stromal cells in endometriosis(EMs),and to explore the influence of aberrant GJIC in stromal cells on pathogenesis of EMs. Methods:The stromal cells were isolated from samples including 24 ectopic endometriotic tissues located in ovaries,41 eutopic endometria with endometriosis and 30 normal endometria. The endometrial stromal cells models were established in vitro by being cultured in manual conditions mimicked with estrogen and progesterone. Laser scanning confocal microscopy(LSCM) was used to determine the expression of Cx43 protein and the function of GJIC in three groups stromal cells. Results:The success rate of isolation and culture of endometriotic stromal cells was 45.8%(11/24);of eutopic endometrial stromal cells with EMs was 92.7(38/41);of normal endometrial stromal cells was 93.3%(28/30). The purities of ectopic and eutopic endometrial stromal cells were 95% and 98% respectively. The level of Cx43 protein and the function of GJIC in stromal cells from ectopic endometrial tissues were much lower than those from the other two groups,the highest level of Cx43 protein and the function of GJIC were observed in normal endometrial stromal cells group,and the differences among these groups were significant (P﹤0.01). Conclusions:(1) It will be helpful to establish models of normal,ectopic and eutopic endometrial stromal cells in vitro simultaneously when investigating the pathgenesis of EMs. (2)Downregulation of Cx43 expression and aberrant function of GJIC are related to pathogenesis of EMs. Regulation of Cx43 or GJIC in endometrial stromal cells is implied to be a potential strategy to treat EMs.

Application of fluorescence recovery after photobleaching in the detection of gap junctional intercellular communication in bladder smooth muscle / 第三军医大学学报

Objective To explore the functional changes of gap junctional intercellular communication(GJIC) in bladder smooth muscle. Methods The functions of GJIC in bladder smooth muscle were detected by fluorescence recovery after photobleaching(FRAP). The mean fluorescence recovery rates of the bladder smooth muscle cells in the experimental group and the control group were compared. Results The mean fluorescence recovery rate of the experimental group was significantly higher than that in the control group( P