The Study On The Cytotoxicity Of Intracellular Non-secretory Expressed Amyloid β Peptide_1-42

The pathogenesis of AD is not completely clear. Although the disease undoubtedlyreflects the interaction of complex multifactorial processes, it is generally accepted that thetoxicity of Aβ is the common path which led to AD. The conventional hypothesisfor the pathophysiology of AD—the so-called "amyloid hypothesis"—states thatAβ plaques are the main cause of neurodegeneration and dementia. Consequently,the majority of studies have focused on the neurotoxic effects of aggregatedextracellular Aβ fragments in AD and in AD animal models. However, in recentyears followed by the development of the molecular biology and the technology ofexperiment, the study showed that before the appearance of senile plaques and NFT inAD there has been an accumulation of intraneuronal Aβ. The detection ofintraneuronal Aβ means that the accumulation of intraneuronal Aβ disruptneural and synaptic function and ultimately lead to neuronal degeneration anddementia. And the detection of intraneuronal Aβ also provoke a generally interestin the field. The study of cytotoxicity of intraneuronal Aβ in the process of AD leadsto the generation of the theory of intracellular toxicity.At present, most of the intracellular toxicity experiment is using microinjectionsof Aβ_1-42 peptide in abroad, and the study of cytotoxicity of intraneuronal Aβ internal isstill in the initial stage. But it is difficult to microinject Aβ_1-42 peptide to the cell, and theoperation will also impact the cell, in addition, the stability of Aβ_1-42 peptide is not very wellthat can not establishment a stable cell line, and the cost of peptide is also very high, all thesefactors restrict the study of the cytotoxicity of intraneuronal Aβ.We suppose if we can packaging a kind of virus which can transfection the cell and non-secretory expressed Aβ1–42 in the cell will solve these problem. Ad hoc, we molecularcloning and sequencing the Aβ1–42 gene, to constructe, and producte the virus of Aβ1–42recombinant retroviral vectors, then transfection the SK-N-SH cell which can non-secretoryexpressed Aβ1–42 in the cell, and approach the cytotoxicity of intraneuronal Aβ1–42. Themethods and the results are as followed:1.Molecular cloning and sequencing the Aβ1–42 gene: By means ofasymmetrical primer/template, double stranded cDNA of Aβ1–42 was cloned byusing PCR and T-vector cloning method. The positive clone was screened andidentified by the restriction enzymes, and then the cloned amplified fragmentswere sequenced and analyzed.Compared the cloned Aβ1–42 gene with the GenBanksequence, we demonstrated that the cloned Aβ1–42 gene sequence was identical to theGenBank sequence. Analyzed the ORF of the cloned Aβ1–42 gene by the DNASIS, wefound that amino acids which encode by the cloned Aβ1–42 gene was not changed.2.Construction and identification of the recombinant viral vector of pL-Aβ1–42 –sn:The resulting gene of Aβ1–42 was inserted into the vector plasmid pLxsn. Identified by therestriction enzymes and agarose gel electrophoresis, we demonstrated that we havesuccessfully construct the recombinant viral vector of pL-Aβ1–42 –sn.3.Packaging the recombinant retroviral vectors which contain Aβ1–42 , the vectorplasmid pLxsn only and the report gene EGFP: The recombinant RV was packaged.80%of confluent PA317 cells were transfected with recombinant viral vector of pL-Aβ1–42 –sn,the vector plasmid pLxsn only and recombinant viral vector of pL-EGFP –sn by calciumphosphate precipitation.The results showed that we had gotten the RV stocks of high titre.4.Recombinant RV transfect the SK-N-SH cell: revival and cultivate SK-N-SH cell,transfect the SK-N-SH cell with the recombinant RV Aβ1–42 , simple RV and recombinant RVEGFP. We detect the expression and the transfect rate of the objective gene in the taget cellby the report gene EGFP and flow cytometry, the result show that the recombinant RVtransfect the SK-N-SH cell effectively. Immunohistochemical stain confirmed that the Aβ1–42can be expressed in the SK-N-SH cell.5.The detection of cytotoxicity of intracellular Aβ1–42 :We prepare the control,simple RV and Aβ1–42 group. By morph observation, we found that Aβ1–42 group showedstronger cytotoxicity, the bulk of the cell obviously diminute, the impairmented cell becameround, the synapse of the cell disappeared and so did the stereo of the cell, the refraction ofthe cell became lower, the multiplication of the cell was not obvious, some of the cell fused,some others shed and dead. MTT showed that the activity of the cell degraded, the growth rateof the cell became lower, can not see the exponential phase of growth and the platform phaseafter that. The flow cytometry showed that the normal cell of the Aβ1–42 group was obviouslydecreased, and the number of early apoptosis cell was increased, and so was the laterapoptosis and necrosis cell, the contrastion is significance compared with the controlgroup(p<0.01).We can draw the follow conclusion by the study:①This study successfully clonedand sequenced the Aβ1–42 gene. ②This study successfully packaged recombinant RVAβ1–42 , and the recombinant RV can transfect SK-N-SH cell effectively. ③ SK-N-SH cellwhich the recombinant RV can non-secretory expressed Aβ1–42 in the cell. ④This studydemonstrated that the intracellular Aβ1–42 showed stronger cytotoxicity.From the above all, we can see that as the finding of intracellular Aβ, the study of Aβ inthe invasion of AD goes to a new stage, the generation of the theory of intracellular toxicityalso challenge the conventional hypothesis for the pathophysiology of AD. We used advancedtechnique of molecular biology, according to the sequence given by the GenBank, by meansof asymmetrical primer/template, cloned the cDNA of Aβ1–42 , and inserted to the RV, thensuccessfully packaged recombinant RV Aβ1–42 which ransfect SK-N-SH cell effectively,though the detection we demonstrated that the intracellular Aβ1–42 showed strongercytotoxicity. The study breakthrough the restrict of the microinjections of Aβ1–42 peptidewhich has been used widely in abroad, and provide a new method and a new idea for the studyof the theory of intracellular toxicity. The result of the study not only demonstratedthat the intracellular Aβ1–42 showed stronger cytotoxicity, but also provide potentevidence for the theory of intracellular toxicity. And at equal pace, the success of theconstruction of recombinant RV which can non-secretory expressed Aβ1–42 in the cell and thecorrect expression in the taget cell is a foundation of the establishment of a cell model whichcan express Aβ1–42 in the cell steadily and lastingly, and a foundation of the establishment of atransgenic animal model which can express Aβ1–42 in vivo as well. And this study can pushabout the study of Aβ1–42 cytotoxicity in the pathophysiology of AD, moreover, it willprovide the theory of the prevention and the therapy of AD, and supply a new method.