New Methods For Detection Of Tumor Cells Apoptosis And Tumor Cells

Cancer, which is also being referred to as malignant tumor, is very dangerousfor human beings. Once the malignant tumor occurs, the normal programmed celldeath mechanism would be broken, and the tumor cell hyperplasia would occur.When the unlimited growing tumor cells invaded to other parts of body, the effectswere destructive. Many people die of cancer every year. However, if the cancercould be diagnosed in the early stage, early treatment could be given, and survivalrate could be improved. On the other hand, a cure method for cancer is to induceapoptosis of tumor cells, and measurement of apoptotic tumor cells could be used toevaluate therapeutic effects and prognosis. In this paper, several new methods fordetection of tumor cells and apoptosis were proposed, and some valuable resultswere achieved. The main research contents of this thesis are as follows:1. The caspases family of proteases has been proved to play a crucial role inapoptosis. A simple and quick colorimetric method for detection of apoptosis wasproposed using unmodified gold nanoparticles (AuNPs) based on caspase–3activityassay. An octa peptide, Ac–Gly–Asp–Glu–Val–Asp–Cys–Cys–Arg–NH2(GR–8),was designed as the substrate of caspase–3. Once GR–8was treated by caspase–3, apeptide fragment containing two–SH and two–NH3+(+3HN–Cys–Cys–Arg–NH3+,CR–3) was released from GR–8, that would promote the combination of CR–3toAuNPs as well as lower the stability of AuNPs, and then cause aggregation of AuNPsin a short time. The color change of AuNPs was related to the caspase–3activity thatreflected the process of apoptosis. UV–Vis absorption spectrometry was used toquantitative analysis of caspase–3activity. Then, the proposed colorimetric assaywas applied to the detection of apoptosis of Jurkat cells. In the test, we can tell thedifference between apoptotic Jurkat cell and the normal set by naked eyes.2. A novel strategy for sensitive and visible detection of early stage apoptosiswas proposed based on silver–enhanced gold nanoparticle label method. Annexin–Vmodified substrate was constructed by layer–by–layer (LBL) method for specificcapture of early stage apoptotic Jurkat cells. A new kind of aminophenylboronic acidmodified gold nanoparticles (APBA–GNPs) was synthesized and utilized forlabeling cells and as the substrate of silver enhancement. Anodic strippingvoltammetry (ASV) was applied to sensitive detection of Ag+which reflected the number of apoptotic cells, and a good linear range from1×102to3.5×103cells wasachieved, with the detection limit of38apoptotic cells. Moreover, the gray color ofsilver particles deposited on the gold nanoparticles could be used for colorimetricdetection of apoptosis. This method is not only sensitive and visible, but also simple,rapid and cheap. It has the potential to be used as diagnosis of apoptosis which is ofgreat significance to the early detection of therapy efficiency and the evaluation ofdisease progression and prognosis.3. Poly(o–phenylenediamine)(Po PD) memberane was modified on quarzcrystal microbalance (QCM) through electro–polymerization. The membrane isinsoluble in both aqueous phase and oil phase. Streptavidin was combined to themembrane through glutaraldehyde crosslinking, and then biotin–Annexin–V wasimmobilized to the membrane through the interaction between streptavidin andbiotin. Thus a novel Annexin–V modified QCM sensor was constructed for detectionof early stage apoptotic cells. The sensor could be used to distinguish apoptotic cellsfrom normal cells, because apoptotic cells would arouse bigger frequence changethan normal cells. The detection limit was52apoptotic cells. As a result, theAnnexin–V modified QCM sensor could be applied for specific detection ofapoptotic cells.4. An electrochemical sensor for specific detection of leukemia CCRF–CEMcells was constructed. Aptamer sgc8c modified gold electrode was used for specificcapture of CCRF–CEM cells. Then APBA GNPs were used for cells labeling. Inorder to get sensitive results, silver enhancement was employed based on the goldlabeling. At last, the silver particles deposited on the gold labeling were dissolvedand detected by ASV technique. The quantity of silver ions reflected the quantity ofcells. The method was very sensitive, and the detection limit was15cells. It has thepotential to be used in the detection of other leukemia cells, or even some othertumor cells.