APP cleavage in live cells guided by C99 / 中华神经医学杂志

Objective To construct recombinant eukaryotic expression plasmid encoding Swedish and Flemish mutations of amyloid precursor protein (APP) fused with fluorescent protein and to investigate the APP cleavage progress. Methods The last 300 bases of APP (named as C99 containing Flemish mutation), together with cyan and yellow fluorescence sequence (named as CFP and YFP,respectively) were obtained by polymerase chain reaction (PCR). The 54 bases in the middle of APP sequence were synthesized (named as 54 bp containing Swedish mutation). The 4 fragments mentioned above (CFP, YFP, C99 as well as 54 bp) were inserted into the vector pcDNA3.0. By genetic engineering, the recombinant plasmid pcDNA3.0-CFP-54bp-YFP-C99 was constructed and identified by enzyme digestion, PCR and sequencing. Then the plasmid was transfected into SH-SY5Y cells. Its expression was examined by fluorescence confocal microscopy and the fluorescence resonance energy transfer (FRET) signal was collected. The amyloid beta (A) deposition was detected by immunocytochemistry. Results (1) DNA sequencing showed the sequence of the constructed recombinant plasmid was correct. (2) FRET and two types of fluorescence could be seen by the spectrum confocal fluorescence microscopy. (3) The expression product of fusion gene was correct and cleaved by and secretases. (4) The A deposition was detected in the cell membrane, cytoplasma and intercellular space. Conclusion (1) The fusion protein can generate A by and γproteolytic processing. (2) It is for the first time to observe the APP cleavage by FRET. (3) It is also the first time to find that APP may be cleaved during its transportation from cell plasma to cell membrane. (4) C99 is very important for the correct cleavage of APP. Our test data strongly suggest that C99 may function as the signal peptide. It might guide and direct the APP to the right location for the cleavage.

Regionspecific survival and differentiation of mouse embryonic stem cell-derived implants in the adult rat brain / 生理学报

Totipotent and regionally non-specified embryonic stem (ES) cells provide a powerful tool to understand mechanisms controlling stem cell differentiation in different regions of the adult brain. As the development capacity of ES cells in the adult brain is still largely unknown, we grafted small amounts of mouse ES (mES) cells into adult rat brains to explore the survival and differentiation of implanted mES cells in different rat brain regions. We transplanted the green fluorescent protein (GFP)-positive mES cells into the hippocampus, septal area, cortex and caudate nucleus in rat brains. Then the rats were sacrificed 5, 14 and 28 d later. Of all the brain regions, the survival rate of the transplanted cells and their progeny were the highest in the hippocampus and the lowest in the septal area (P<0.01). The grafted ES cells could differentiate into nestin-positive neural stem cells. Nestin-positive/GFP-positive cells were observed in all brain regions with the highest frequency of nestin-positive cells in the hippocampus and the lowest in the medial septal area (P<0.01). mES cells differentiated into end cells such as neurons and glial cells in all transplantation sites in recipient brains. In the hippocampus, the ES cells differentiated into neurons in large amounts. These results demonstrate that only some brain regions permit survival of mES cells and their progeny, and form instructive environments for neuronal differentiation of mES cells. Thus, because of region specific presence of microenvironmental cues and their environmental fields, the characteristics of the recipient tissue were considerably important in formulating cell replacement strategies for neural disorders.

Differentiation of human embryonic stem cells into neural stem cells in vitro / 中华神经科杂志

Objective To explore a kind of simple and high efficient approach to differentiate human embryonic stem cells(hESCs)into neural stem cells(NSCs).Methods hESCs were cultured in bacterial culture dish filled with serum free medium to gain embryoid bodies.Then the mature embryoid bodies grew in the special medium including B27 and noggin by adherent culture to differentiate into NSCs. Results The hESCs kept floating in the bacterial culture dish and growing all the time and then formed mature embryoid bodies 7 to 10 days later.The embryoid bodies could be differentiated into highly pure (96.4%)nestin positive cells.And these cells were differentiated into all kinds of neural cells if cultured further.Conclusions This kind of method is less time-consuming,cheaper,and more efficient than those of the results in literatures reported.It affords very good source of seed cells for cell transplantation therapy in the future.

The study on the immunogenicity of hESCs and the derived NSCs / 中华神经科杂志

Objective To study the immunogenicity of human embryonic stem cells(hESCs)and the derived neural stem cells(NSCs)in vitro.Methods The constitutive expression of human leucocyte antigen(HLA)Ⅰ and Ⅱ in hESCs and the NSCs derived from these hESCs were detected by flow cytometry (FCM), as well as the expression of HLA-Ⅰ,Ⅱin NSCs induced by 30 ng/ml recombination human interferon-?(IFN-?).Meanwhile, the NSCs before and after induction of IFN-? were co-cultured with peripheral blood lymphocyte obtained from healthy person.Lymphocyte proliferation standing for the immunoreactivity of NSCs was then investigated.Results The hESCs slightly expressed HLA-Ⅰ(6.18%) and hardly any HLA-Ⅱ before differentiation.However, the NSCs expressed more HLA-Ⅰ(23.56%)as well as HLA-Ⅱ(1.28%, 1.73%)than the hESCs did.Both HLA-Ⅰ(46.43%)and HLA-Ⅱ(8.73%, 10.57%)expressed by the NSCs after they were induced by IFN-? were up-regulated.Conclusions hESCs express certain level of HLA-Ⅰ molecules but do not constitutively express HLA-Ⅱ molecules.The derived NSCs express heavy HLA-Ⅰ and a little HLA-Ⅱ, when treated by IFN-? they can inducibly up- regulated both molecules.The NSCs derived from HESCs are of immunogenicity, which induce rejection aiming at HLA-Ⅰ molecules or even at HLA-Ⅱ molecules when the host is inflammative or under stress, which can result in a failure of cellular transplantation.