Visual Typing Of Glioma Gene Mutations Based On In Situ Rolling Circle Amplification

Gliomas are the most prevalent primary intracranial tumors with high histological and genetical heterogeneity.This extremely high heterogeneity is accompanied by alterations in complex molecular genetic information that can lead to differences in tumor proliferative capacity,invasive capacity,and drug sensitivity,which ultimately affect the diagnosis and prognosis of tumor patients.The classification of glioma diseases by classical histopathology often ignores the high heterogeneity of tumors,and it is difficult to achieve accurate diagnosis,treatment and clinical prognosis of patients.In 2016,the World Health Organization added molecular diagnosis to the framework of histopathology to classify glioma into different pathological subtypes.Genotype-based glioma typing can guide individualized diagnosis and treatment of patients more objectively and reasonably.In situ imaging and precise quantification of genes and mutated loci at the single-cell level are the most powerful tools for precise cell typing,clinical diagnosis,prognosis,and pathological mechanisms study of glioma.In this thesis,we constructed a strategy of padlock probe-coupling in situ coding reaction,for in situ imaging of multiple RNA mutations and RNA-related markers in glioma cells,achieving visual typing of different pathological grades of gliomas at the single-cell and tissue levels.The visual classification of gliomas provides a new analysis method for glioma heterogeneity study,pathological diagnosis and prognosis.The specific content and research results are as follows:1.RNA mutation detection requires methods to identify RNA sequences with high base resolution.By constructing a modular padlock probe and coupling in situ rolling circle amplification reaction,in situ and single-molecule resolution detection of single base mutation sites is achieved at the single-cell level.According to the principle that ligation reaction requires complementary pairing of the 5’ ligation site,with the help of the designability of padlock probe,the 5’ end of the padlock probe was designed as the complementary base of the mutation site.When the mutation site exists,the padlock probe can be circularized by ligase and trigger the downstream amplification reaction,so that the padlock probe can specifically recognize the single-base mutation site.By optimizing different module sequences of the padlock probe,the influence of secondary structure,steric hindrance and potential binding proteins on sequence hybridization were reduced,and the recognition efficiency and accuracy of the target were improved.The feasibility of constructing probes for rolling circle amplification reactions and the targeting accuracy of RNA mutations were verified through a series of experiments.2.Glioma typing often relies on the expression status of multiple markers,and multi-target detection can improve the accuracy of molecular diagnosis.To address the current challenges of low detection throughput faced by in situ imaging,a fluorescent coding strategy was constructed to achieve simultaneous detection and quantitation of multiple glioma molecular marker genes at the single-molecule level.Taking advantage of the designability of nucleic acid sequences,two nucleic acid modules were designed for the padlock probe,coupled with four fluorescent labeling motifs to achieve simultaneous labeling of eight molecular markers.This strategy enables the analysis and quantification of IDH1,ATRX,and TERT gene mutation status combined with deletion status of chromosome 1p/19 q at the single-molecule level.Through fluorescence confocal imaging and image decoding analysis,the feasibility of the fluorescence encoding strategy and the accuracy of multi-target detection were verified.3.Visual typing of glioma cells at the single-cell level and the tissue level for different pathological levels was achieved.In this study,specific types of glioma cells were characterized according to different expression patterns of eight specific target genes.Oligodendroglioma cells were characterized by gene IDH mutation,TERT mutation,ATRX wild and co-deletion of 1p and 19 q.Astroglioma cells were characterized by gene IDH mutation,intact expression of 1p and 19 q accompanied by TERT mutation or ATRX mutation.Primary glioma cells were characterized by the absence of IDH mutations and the intact expression of 1p and 19 q accompanied by ATRX mutations or TERT mutations.The constructed method was first used to analyze the detection of molecular markers for glioma cells in the polytrophic cells,achieving accurate differentiation of glioma cells at the cellular level.After pretreatment of brain tissue slice samples,in situ rolling circle amplification technology was used to achieve in situ imaging of molecular markers of glioma cells in brain tissue of different pathological grades.A visual typing method for the status and location of glioma cells in brain tissues was constructed,which provides a new analytical tool for pathological research and prognosis judgment of glioma.