Research Of Liver Tumor Model In Vitro Based On Three-dimensional Bio-printing Technique And RNA-Sequencing Technique

Background:Liver cancer is one of the most common malignant tumors in China,and the malignant degree of liver cancer is second only to pancreatic cancer in digestive system.The curative resection is the standard treatment of liver cancer,but the chemotherapy and targeted drug therapy are still in an important position for postoperatively or advanced patients.Clinical drug test is the most effective method to screen anti-tumor drugs,however,due to safety and ethical factors,it can't be widely applied.Two-dimensional culture in vitro and animal models are the most commonly used models in cancer research.Nevertheless,there are many spatial structural defects and species differences in models,so it can't really simulate the microenvironment of human tumor growth.In recent years,the rapid development of three-dimensional bio-printing technology makes it possible to construct an three-dimensional cell model in a fast and economic way.However,there is no report focus on constructing liver cancer models by using three-dimensional bio-printing and evaluating the physiological function of the model by using the transcriptional sequencing technology.Objectives:1.We constrnucted two-dimensional and three-dimensional liver cancer models by using three-dimensional bio-printing technology,then the cell survival rate of 3D groups was calculated and proliferation of two groups were compared;2.We compared the function of two-dimensional and three-dimensional liver cancer models by using transcriptional sequencing technology.Methods:1.HepG2 cells,gelatin and sodium alginate were located in vitro to form 3D liver cancer model by using 3D bio-printing technology.Simultaneously,we constructed 2D models as well.We used immunofluorescence staining to calculate the survival rate of HepG2 cells in 3D structure.Then,the CCK-8 kit was used to analysis the proliferation of cells in 3D and 2D models at 1?3?5 and 7 days.2.Transcriptome analysis of HepG2 cells in 2D and 3D liver cancer models was carried out by using RNA-seq technology.Results:1.The survival rate of cells in the 3D culture structure was 95.4±4.6%;The proliferation activity of cells in 3D and 2D culture were compared:the cell proliferation rate of cells in 2D models was faster than that in the 3D models at 1,3,5 days.But the proliferation rate of cells in 3D culture was faster than that in 2D culture s after 5 days,and the proliferation rate of cells in 2D samples was decreasing.2.RNA-Seq results showed that there were 617 differentially expressed genes in 3D and 2D models,among which 235 genes were up-regulated and 382 genes were down regulated.After functional enrichment analysis of differentially expressed genes,it is speculated that there are differences in cellular function between 3D and 2D structural models.The study found that the mRNA levels of 40 transcription factors have significant differences between 3D and 2D cultured cells,and the differences in these transcription factors may be involved in the regulation of biological processes such as cell proliferation and differentiation.Conclusions:1.The survival rate and proliferative activity of cells in 3D bioprinting models were at a higher level.It is proved that the cells in the 3D culture have advantages than the 2D culture in space structure and extracellular matrix microenvironment.The survival conditions of cells in 3D models are similar with the body environment.2.There are differentially expressed genes and different characteristic gene expression profiles in 3D and 2D models.Through the functional analysis of differential genes?it can be concluded that there are differences in metabolic function and immune function between the two different culture structures.