To prepare a hand-made micropore membrane culture plate insert forco-culture. Methods The plate insert was made using plastic centrifuge tube and micropore membrane.After seeding brain capillary endothelial cells (BCECs) on it (under the effect ofastrocyte-conditioned medium), the plate insert was assessed by analysis of trans-endothelialelectrical resistance (TEER). Results The plate insert has a stability of at least 15 d underculture condition. TEER increased significantly under co-culture condition from (66.1 +- 13.3)Ωcm^2 to (182.2 +- 6.7) Ωcm^2. Conclusion This micropore membrane culture plate insert can beeasily made, on which BCEC culture can be successfully performed. Moreover, it is adjustable andrecyclable. It follows that the plate insert is a useful tool for co-culture and the relatedresearch fields.
Aim To analyze the secondary structure and neurotrophic effect of a specific protein in sensory neurons. Methods Comparison of the proteins expressed in the rat spinal sensory neurons and motor neurons was made by two dimensional electrophoresis. One specific protein in sensory neurons was isolated and purified by DEAE Sephacel ion exchange chromatography and high performance liquid chromatography. A primary analysis of its secondary structure by circular dichroism, and its neurotrophic effects were investigated using the model of dorsal root ganglia(DRG) cultured in vitro . Results The molecular weight and isoelectric point of the protein were 33 1 kDa and 5 52, respectively. Its circular dichroism showed that there were 20 8% α helix, 54 8% β sheet, 7 3% turn, and 17 1% random coil in its secondary structure. Biological experiments showed that the protein could promote the neurite outgrowth of DRG. Conclusion A specific protein in spinal sensory tissue with molecular weight of 33 1 kDa has been purified. There is mainly β sheet in the secondary structure of the protein. And the protein has neurotrophic effects in the model of DRG.
The ability to knockdown the expression of an endogenous gene by RNAi has emerged as a powerful strategy for the rapid identification of specific gene functions. Vector-based constitutive expression of shRNA can result in stable and efficient knockdown of target genes. However, constitutive expression of shRNA imposes major limitations when analyzing the fimction of genes whose expression is vital for the survival of an organism. Inducible RNAi systems can circumvent this limitation by enabling the inhibition of expression of an essential gene only when the inducing agent is present, and the level of knockdown of the essential gene can be controlled and adjusted by the concentration of inducing agent. In this review, we briefly summarize the recent development of various inducible RNAi systems and their potential applications in drug target validation.
