Fluorescence Staining Of SMMC-7721Hepatocarcinoma Tissues Using Aptamers

Cancer is a serious threat to human health and life due to its high mortality.Improving the diagnostic accuracy of cancers holds great significance for theimprovement of its survival rate. Currently, immunohistochemistry has been widelyutilized as a well-established molecular diagnostic method of cancers in the clinicalfield. However, there are some disadvantages such as the time-consuming productionof antibodies and the variation between different batches. Aptamer, as a antibody-likemolecule, possesses a series of inherent advantages, including low variability betweendifferent batches, easy synthesis and modification, excellent binding affinity andspecificity, lack of immunogenicity and fast tissue penetration, these favourablecharacteristics make aptamer an ideal tool for cancer diagnosis. In this work,hepatocellular carcinoma was selected as the object, and its specific aptamer selectedthrough cell-SELEX was constructed as imaging probes for fluorescence staining ofhepatocellular carcinoma tissues. Research contents include:1. The choice of the type of SMMC-7721hepatocarcinoma tissue sectionsTo find the right type of tissue sections, aptamer ZY1was labelled with FAM tosever as a molecular probe and used for fluorescence staining of formalin fixed andparaffin-embedded tissues, acetone-preserved tissues,95%ethanol-preserved tissues,carnoy-preserved tissues, clarke-preserved tissues and fresh-frozen tissues. Theresults indicated that the fresh-frozen SMMC-7721hepatocarcinoma tissues could beeffectively stained by aptamer ZY1.2. Cy5-labeled aptamer for fluorescence staining of fresh-frozen SMMC-7721hepatocarcinoma tissuesTo avoid the interference of tissue autofluorescence, aptamer ZY1was labeledwith cyanine5（Cy5） to serve as a near-infrared aptamer probe (Cy5-ZY1) and usedfor fluorescence staining of fresh-frozen SMMC-7721tissues. The results indicatedthat the Cy5-ZY1not only showed high specificity in identifying SMMC-7721carcinoma, but also exhibited high efficiency in differentiating liver carcinomas ofdifferent subtypes. Moreover, the time requir ed for tissue staining was as short as10min. This work may make a significant contribution to introduce aptamer ZY1intothe diagnosis of hepatocarcinoma in the pathology laboratory.3. Activatable aptamer probe for fluorescence staining of fresh-frozenSMMC-7721hepatocarcinoma tissues In order to overcome the disadvantages of Cy5-labeled aptamer probes, includingcomplicated operation and limted sensitivity, aptamer ZY1was chosen as the modeland an activatable aptamer probe based on target cancer cell s-triggered conformationalteration was successfully constructed. The results demonstrated that AAP couldindicate the presence of target cancer cells with high specificity, and couldefficaciously detect samples containing as low as171SMMC-7721cells. In addition,the AAP showed high specificity and rapidity in identifying SMMC-7721carcinomawith no required washing and separation steps. Therefore, this facile, specific, andwash-free tissue staining strategy may provide a new insight into the diagnosis ofcancers in clinical.