Quantum Dot-based Fluorescent Signal Amplification System For Detection Of P16^INK4A Expression In Cervical Dysplasia And Neoplasia

Objective:1.Characterize the physical and optical properties of QD550-Streptavidin and biotinylated secondary antibody complexes.2.To achieve the optimum condition of Quantum dot-based fluorescent signal amplification system used in cervical tissues by staining it with HeLa cells.3.To detect the P16^INK4Aprotein expression in cervical dysplasia and neoplasia by the use of Quantum dot-based fluorescent signal amplification system and to explore the correlation between P16^INK4Aprotein and clinical pathological factors of cervical cancer samples.4.To verify the advantages of the Quantum dot-based fluorescent signal amplification system staining method by comparing it with the traditional immun-ohistochemical staining.Methods:Firstly,we characterized the physical features and optical property of QD550-SA and biotinylated secondary antibody compounds by TEM,DLS,UV spectro-photometer and fluorescence spectrometer.Secondly,we subcultured Hela cells and stained HeLa cells with QD550-BAS immunofluorescent method,and then observed the fluorescence labeling effect with inverted fluorescence microscope,in order to analyz its fluorescence intensity and initially validate the signal amplification effect of BAS.Thirdly,we chose different mole ratio of QD550/SA,biotin/secondary antibody as well as different incubation time of primary antibody P16^INK4Ato stain HeLa cells respectively,to obtain optimum reaction conditions by comparing the fluorescence intensity.Finally,we used QD550-BAS to detect the P16^INK4Aexpression in 30 cases of NC,14 cases of CIN Ⅰ,30 cases of CIN Ⅱ,31 cases of CIN Ⅲ,32 cases of CSCC and 25 cases of CAC tissues formalin fixed paraffin section and to explore the correlation between P16^INK4Aprotein expression and clinical pathological factors of cervical cancer samples.We also used immunohistochemical staining the same samples to compare the sensitivity and specificity with QD-BAS staining.Results:1.QD550-SA and biotinylated secondary antibody complexes are successfully coupled.2.There are obvious signal amplification effect by the use of BAS in the QDs immunofluorescent staining with the Hela cells.3.The biotin-secondary antibody ratio 1:50,QD550-SA ratio 1:40,and 2 h incu-bation time are chosen as the optimum value,which were used for cervical tissues immunofluorescent staining.4.The QDs-BAS staining P16^INK4Aexpression positive rate of CIN Ⅰ,CIN Ⅱ,CIN Ⅲ,CSCC and CAC were 0%（0/30）,35.7%（5/14）,70%（21/30）,84.6%（26/31）,96.9%（31/32）and 96%（24/25）,respectively.The QDs-BAS staining exhibited 81.1%sensitivity and 100%specificity which were better than the 67.4%sensitivity and83.3%specificity exhibited by IHC staining using the same antibodies on the same tissues.The P16INK4A expression increased with cervical cancer pathological grade,clinical stage,and the presence of lymph node metastasis and distant metastasis（P<0.05）.Conclusion:1.QDs have small diameter,wonderful optical properties and high degree of biological compatibility,which are appropriate to biomedical research.2.The P16^INK4Aprotein express in cervical lesion tissues,while not express in normal cervical tissues.And the expression of P16^INK4Aprotein increased with cervical cancer infiltration invasion and metastasis,whic can be used as an indicator of cervical cancer detection and targeted therapy.3.QDs-BAS immunofluorescent staining technique have more advantages than raditional IHC,which will have prospects in cancer detection.