| Hepatocellular carcinoma(HCC)is a common and severe liver malignant tumor,which mainly comes from liver epithelial cells infected with the hepatitis B virus(HB V)and hepatitis C virus(HCV).Generally speaking,the main manifestations of HCC are chronic inflammation,fibrosis,abnormal regeneration,liver function damage,and so on.Because of the liver’s physiological and structural characteristics,HCC mainly presents with insidious onset,delayed disease extension,and invasion&metastasis;However,although many treatments have been developed,HCC still exhibits problems such as drug-treatment resistance,post-operative recurrence,new metastasis formation,becoming a significant cause of organ failure and death in patients with HCC.With the interactive development of current diagnosis and treatment technology and nanomedicine,a novel disease judgment model using "nanoparticle " as a carrier has progressed in the early diagnosis,efficacy determination,disease detection,and prognosis evaluation of diseases.However,bottleneck problems remain,such as high treatment costs and toxic side effects.Therefore,a new and intelligent nano platform with low cost,easy synthesis,and high biocompatibility,which has both diagnostic and therapeutic roles,should be developed to provide a theoretical and experimental basis to realize the "integration of diagnosis and treatment" of hepatocellular carcinoma and so on.Semiconductor polymer dots(Pdots),an emerging class of nano dye particles,have been applied in fluorescence imaging,target molecular labeling,and other fields with an excellent spatiotemporal resolution,low cost,radiation hazard-free,high sensitivity,and detection specificity.Pdots can play different roles in various areas through optimal design,preparation process optimization,and subsequent functionalization modification.For example,a.biomedical imaging,early diagnosis,disease monitoring,and efficacy determination can be achieved through modification with specific antibodies or molecules;b.Using the particular adsorption effect between antigen and antibody to realize drug carriers’ targeting ability and specificity distribution in living organisms;c.Intratumoral irradiation or photodynamic therapy can be achieved by controlling the synthetic path,utilizing a variety of substances with different physicochemical attributes for doping;d.Using the optical properties of nanoparticles,real-time tracing or long-term label imaging is performed to achieve precision and individualization of therapy;e.And the combination of multiple treatments means realizing the multi-level,multi-dimensional therapy of tumors and so on.However,whether it can be applied in the liver is unknown.Although the large-scale culture of exogenous MSCs and the in vitro artificial cultivation of organoids hold a pivotal position within the field,many cognitive gaps remain regarding the process of directed differentiation,self-development,cell migration,and spatial assembly of organoid cells within organoid clusters,and the potential of organoids to be used in disease model construction and specific diagnosis and treatment.Directed induced differentiation of mesenchymal stem cells(MSCs),induced pluripotent stem cells(iPSCs),and Embryonic stem cells(ESCs)at the twodimensional(2D)cell level are difficult to recapitulate the developmental process of the embryonic organ,ultimately dramatically limiting their clinical use.In contrast,the culture of three-dimensional(3D)organoids could offer such potential applications.Therefore,the second part of this research will use Pdots that may have the characteristics of recruiting functional cells to establish a new cell culture system and make the induced differentiation of cells more closely resemble the biological function and molecular phenotype of embryonic viscera(liver).Based on the different uses of multifunctional nanoparticles,this study mainly revolves around the specific application of Pdots in the preoperative diagnosis and postoperative liver injury treatment of HCC patients,a clinical diagnosis and treatment idea.First,complementary antisense sequences of pathological grade-specific miRNAs were used to functionalize Pdots.Then,a multi-channel compound imaging modality was used to make the interpretation and prognosis evaluation of the pathological grade of HCC.Subsequently,we utilized an in vitro three-dimensional self-assembled cell cluster nurturing system of liver organoids,performed long-term labeling with cellpenetrating peptide R8 to synthesize R8-Pdots,and then explored the value of organoids for the treatment value of the post-operative liver injury.To provide exogenous functionalized cells and artificial organs to extend the life span and improve patients’quality of life with HBV and HCC.Purpose:Based on the multifunctional properties of semiconductor polymer dots(Pdots),this study mainly explores the specific application value of this nanoparticle in the preoperative diagnosis and the treatment of postoperative liver injury in HCC patients,and provides a new thinking direction for precision medicine and personalized therapy.Experiment content:(1)Investigate the correlation between 4 specific miRNAs and the clinical characteristics of patients was investigated,and explore and improve the functionalized modification scheme between Pdots and specific antisense complementary sequences of short single-stranded miRNA,based on the public database information.(2)Investigate the interpretation scheme of multiple miRNAs sensitive Pdots-anti miRNA labeled tissue staining slides,explore the significance of the interpretation results for the early diagnosis or prognosis evaluation of HCC patients,and establish a new basis for pathological grading diagnosis.(3)Explore the protocol for in vitro induction of cell differentiation from MSCs to HLC s/MHs,and create a method with low cost,low manipulation complexity,and high reproducibility.(4)Probe the processes of cell differentiation,interaction,and migration within organoids,by deconstructing the structural features of organoid cell clusters using digital imaging,thereby providing additional insights into human regenerative medicine,liver developmental physiology,and liver disease modeling.Materials and methods:Ⅰ:Based on the database information,the specific miRNAs of HCC pathological grading were screened.1.The clinical gene expression data of the samples of hepatocellular liver carcinoma(LIHC)patients were collected from TCGA/UCSC database,aiming at exploring the correlation between miRNA(has-miRNA-1292-5p,has-miRNA-13013p,has-miRNA-3614-5p,has-miRNA-5589-3p)expression and clinical tumor shape with the bioinformatics methods(such as R language,GraphPad,and SPSS).Ⅱ:Preparation of microRNA-specific labeled nanoprobe Pdots-anti miRNA and its application value in preoperative pathological grading diagnosis of HCC.2.Precipitation method was adopted to prepare four kinds of Pdots host cores with different excitation and emission bands,namely PFO Pdots,PFBT Pdots,PFDTBT Pdots,and NIR Pdots.Also,the precipitation method could modify miRNA-specific antisense complementary sequences on the outer surface of core particles by EDC(1ethyl-(3-dimethylaminopropyl)carbodiimide)reaction,thus characterizing the material-related attributes of staining medium Pdots polymers with super-resolution imaging ability.3.Liver normal cell line L-02,liver cancer cell lines SMMC 7721,HepG2,and HuH-6 cell lines were selected and cultured in high glucose DMEM,and RPMI 1640 mediums,and then fixed and stained,thus exploring the feasibility of the novel probe or novel multiplex miRNAs in situ labeling strategy as for these analyses.4.By using two-photon confocal microscopy and digitalization software,the compound staining imaging was applied to the ultrathin frozen sections from in vitro cell staining models and ex vivo tissues of HCC,thereby analyzing the collected data,obtaining the corresponding colorimetric plate information,and further accumulating technical experience for the subsequent pathological grading interpretation or probe commercialization.5.Based on the reference sequence of hsa-miR-1292-5p,a mutation site was designed every five bases,and Cy3dt was modified on the outside of the T base in the nucleotide chain of the reference sequence and the mutant sequence.As a subsequent detection,the Cy3dt tag could achieve energy transfer(namely Forster/fluorescence resonance energy transfer,FRET)between Cy3dt and nanoparticles(PFO Pdots),thereby exploring the specificity and sensitivity of the probe for specific miRNA detection.Ⅲ:Isolation and culture of primary cells and preparation of long-term labeling probe R8-Pdots.6.MSCs derived from the fresh umbilical cord(≤24 hours ex vivo)were collected,thus characterizing the biological features,including the gross morphology,differentiation potential,cell membrane molecular phenotype,cell stemness of MSCs,and potential for inducing differentiation,by adopting light microscope,specific staining,and flow cytometry.7.By utilizing the as-prepared Pdots nanoprobe(PFBT Pdots&PFDTBT Pdots)in Item 2,the cell-penetrating peptide(R8)could be modified on the outer surface of the Pdots by virtue of electrostatic adsorption.As for the characterization of the related attributes of R8-Pdots before and after modification,transmission electron microscopy(TEM),fluorescence spectroscopic detection,and dynamic light scattering(DLS)were used.Ⅳ:Directional differentiation of MSCs in the 2D plane and cultivation of 3D selfassembled liver-like organs in vitro.8.To explore the protocol of induced culture and differentiation of MSCs into MHs in 2D planar ordinary culture medium in vitro,some experimental techniques,such as PCR,agarose gel electrophoresis,Western blot,and immunofluorescence labeling,were adopted,aiming at detecting the amount of mRNA expression of specific molecules,the molecular phenotype of the extracellular membrane,and the expression of specific proteins inside the cells during the induced transformation process.9.MSCs cells were pretreated with different endocytosis pathway inhibitors,thereby exploring the endocytosis pathway of R8-Pdots crossing the lipid bilayer into MSCs before and after R8 modification.Also,the fluorescence intensity of MSCs cell lysates was detected by adopting flow cytometry,thus determining the endocytosis pathway screened by specific endocytosis inhibitors.10.MHs described in Item 8 were co-cultured with HUVECs and Wharton Jelly,aiming at thoroughly investigating the 3D organoid self-assembly procession and cultivation scheme in vitro,by adopting HE staining,two-photon confocal microscopy,digital image processing,and other experimental technologies.Besides,tracking imaging,tracking capture,and data analysis were given to cell migration trajectories,self-assembly processes,and interactions between cells in the organoid cell cluster selfassembly process.Ⅴ:Virtual imaging of organoid space cell mass.11.Finally,a co-culture organoid space cell mass,with the shape similar to a tuber,was simulated successfully by adopting computer digitalization software.Moreover,the spatially disordered structural elements of liver organoid tissue cell clusters were digitally modeled and spatially reconstructed,by tracing the R8-Pdots with high resolution and excellent biocompatibility.Results:1.The clinical gene expression data of the samples of LIHC patients in the TCGA/UCSC database were collected and processed.The change of miRNA expression(has-miR-1292-5p,has-miR-1301-3p,has-miR-3614-5p,has-miR-5589-3p)was obviously related to the pathological grade,TNM stage,proliferation,metastasis,and independent prognostic diagnosis of the tumor.2.Pdots,functionalized with miRNA-specific antisense complementary sequence,showed the regular spheroid shape under TEM.The particle sizes of the four labeling probes were mainly 30 ± 5 nm.Furthermore,the surface potentials of modified nanoprobes were concentrated at-20～-40 mV,indicating the excellent ability of target miRNA labeling in the cell labeling tests.Moreover,the results exhibited that the Pdotsanti miRNA probes designed in this work had excellent detection sensitivity and specificity and the detection accuracy was down to the fM level.3.The miRNA-sensitive Pdots probe presented noticeable regular changes in the independent and composite imaging of the frozen ultrathin sections of HCC;as the cell source tended to vary from almost normal tissue to malignant hepatocellular carcinoma,the color palette after the calculation and stacking ranged progressively from bright orange for the normal tissue to dark blue for the cancerous tissue.The result illustrated that the labeling probe designed in this work had an excellent binding ability to certain ribonucleotide chains in the tissue.4.MSCs,extracted from the umbilical cord in vitro,grew in the shape of a spindle under a microscope,showing excellent membrane molecular phenotypes and potential biological functions.During the differentiation of MSCs into Hepatocyte-like Cells(HLCs),the cell shape gradually changed from long spindle to polygonal hepatocytelike cells.The characteristic liver proteins,such as HepPAR1&ALB,were exhibited according to the immunofluorescence results.The proportion of cells with positive expression was no less than 83%.The changing trend of marker molecules indicating liver cell function and the results of PCR quantitative analysis determined the final fate of MSCs differentiating into Hepatocyte-like Cells(HLCs)and Mature Hepatocytes(MHs).5.3D liver organ clusters could be self-assembled in vitro.Three kinds of primitive cells were paved into a 2D plane.After 72 hours of culture,the cell structure in the selfassembled organoid gradually changed from disorder to order.As indicated by the fluorescent pictures,HUVECs formed an endothelial biological skeleton network,and the MHs induced by MSCs attached to the biological skeleton formed by HUVECs.Also,they,together with MSCs,were distributed in the gaps of the grid.6.Based on the results of a light microscope,PCR,specific protein immunofluorescence labeling,and metabonomic analysis,the cultivation of 3D liverlike organoid clusters could realize large-scale target cell expansion under the unit surface area.Simultaneously,the differentiated liver-like cells had biological functions and molecular phenotypes closer to the embryonic organs(e.g.,the liver).7.By adopting computer technology and digital processing software,the digital model of an organ-like spatial cell cluster similar to a truffle shape was successfully simulated,after 96 hours of organ-like co-cultivation.This method was expected to provide new solutions and directions,for the organoid research,target cell labeling,and long-term tracking in regenerative medicine.Conclusions:1.After the screening,the results indicated that the altered expression levels of microRNAs(has-miR-1292-5p,has-miR-1301-3p,has-miR-3614-5p,has-miR-55893p)were significantly correlated with the pathological grade,TNM stage,proliferation,metastasis,and independent prognostic diagnosis of HCC patients.2.Pdots,modified by functionalization with miRNA-specific antisense complementary sequences,had excellent labeling ability for target miRNA.Moreover,Pdots could image HCC frozen ultrathin sections independently or give composite imaging,and exhibit noticeable regular changes,indicating that the labeling probe designed in this work had good binding labeling and imaging ability for single ribonucleotide chains in tissues.3.The spatial culture of target cells and the cultivation of 3D liver organoid clusters could enable large-scale target cell expansion per unit surface area.Moreover,the induced differentiation of spatial organoids had biological functions and molecular phenotypes,that were more closely resembling those of the embryonic viscera(liver).4.By using high-resolution fluorescent Pdots as bioimaging labeling sites,the selfassembly process of 3D liver organoid tissues could be traced,thereby deconstructing the structural features during the development of liver bud organoid-like tissues,such as the reconstruction of organoid cell clusters with tuber-like appearance,the bioskeletal function of HUVECs,and the adhesion of MHS to this specific bone.Moreover,some problems in the bioimaging of organics have been solved to some extent.For example,target cells were labeled in cell clusters by using nanoprobes with low tissue scattering properties,and stereo imaging of specific disordered structures was achieved with the aid of multimedia computer technology(e.g.,AR,VR,capturing point cloud,digital model reconstruction.). |
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