Study Of New Methods For Cervical Cancer Biomarker Analysis

Cervical cancer is one of the most frequently diagnosed cancers in women around the world, especially in developing regions. With the support from molecular technology, epidemiological studies have underlined that epithelial cells'persistent HPVs (human papillomaviruses) infection is the cause of lesions ranging from common warts to cervical cancer. Among over 100 different HPV types, HPV16 is the most prevalent type in cervical and other anogenital carcinoma, and is classified as "high-risk" together with HPV 18, HPV31, HPV33, HPV35 and several others. In contrast, HPV types primarily found in verrucas and non-malignant lesions were designated as "low-risk" types. The persistent infection of high-risk types brings to the overexpression of key HPV oncoprotein-producing genes including E6 and E7, and their encoded oncoproteins interacting with important cell cycle regulatory molecules (p33-cyclin dependent kinase 2, E2F transcription factors etc.) or tumor suppressor genes (p53), which result in a malignant transformation. HPV DNA testing has not been recommended as primary screening because of a low clinical specificity and poor positive value. In this work, the integrated MCE methods with NASBA, two-step RT-PCR and one-step RT-PCR strategies respectively were developed to detect HPV16 E6/E7 mRNA. Another cervical cancer biomarker p53 was detected by rolling circle amplification and Hemin/G-quadruplex.The contents of this research are as follows:(1)The relationship between cervical cancer and HPV was reviewed. A brief introduction and comparison of HPV cytological examination, DNA detection and HPV 16 E6/E7 transcripts.(2) Comparative researches towards detection of HPV 16 E6/E7 transcripts were carried out by combination of nucleic acid amplification technologies and microchip electrophoresis (MCE). These studies facilitate the development of high-throughput, high efficient and reliable determination of HPV genotype, and greatly shorten the analysis time, which made an improvement over time-consuming traditional capillary electrophoresis or molecular beacons detection strategy. The approaches of nucleic acid sequence based amplification (NASBA), one-&two-step RT-PCR combined with MCE were successfully developed, among which NASBA only needs a single step isothermal amplification, leaving out the complex and tedious operation. It is believed that with further modification and improvement, this method can be potentially applied for clinical diagnosis and prognosis of cervical and other anogenital carcinoma.(3)p53, a tumor suppressor protein and a transcription factor, plays an important role in cell growth control, DNA repair, and programmed cell death, and was found associated with cervical cancer. In this work, we would achieve excellent sensitivity and high selectivity detection of p53, p53 protein was selectively captured in the microchannels and then was enabled to realize signal amplification by RCA and G-quadruplex catalyzed ABTS-hemin-H2O2 system. As a result, the colorimetric assay was easily achieved.