| Background:Chronic glomerulonephritis(chronic glomerulonephritis,CGN)patients have variable onset times and early symptoms are not obvious.The glomeruli in the patient's kidney continue to accumulate slowly and eventually develop into uremia.Three-dimensional genomics(Three-Dimensional Genomics,3D Genomics)is the study of biological processes in three-dimensional spatial structure.For example,genomic sequences with different three-dimensional structures play a role in gene transcription,replication,repair and regulation.Peripheral blood mononuclear cells(PBMCs)contain a large number of immune cells and are often used in the study of the human immune system.There is a difference in gene expression between immune cells of patients with glomerulonephritis and normal human immune cells,but we do not understand the process of changes in gene regulation of immune cells that lead to glomerular damage.In order to explore the relationship between changes in three-dimensional genomics and disease,we performed Hi-C(High-throughput chromosome conformation capture,Hi-C)analysis on immune cells of patients with glomerulonephritis and normal human immune cells.Objective:In this study,high-throughput sequencing was used,and peripheral blood mononuclear cells from patients were used as research objects to observe changes in three-dimensional genomics.At the same time,the pathogenesis of peripheral blood mononuclear cells in uremia and the effect of related pathways are explored,which provides important help for further research on the molecular mechanism of peripheral blood monocytes and disease.Methods:The experimental group of this study consisted of 20 patients with chronic glomerulonephritis and uremia,and the control group consisted of 20 healthy people who had undergone physical examination without uremia-related symptoms.First,the peripheral blood mononuclear cells of the experimental group and the control group were separated by density gradient centrifugation,and formaldehyde cross-linking was performed.Second,use restriction enzymes to cut chromatin into different fragments.At the same time,these fragments are labeled with biotin.After labeling,blunt ends of the fragments are ligated with ligase and the ligation product is purified to prepare Hi-C samples.And sampling for the first quality control point detection.After end repair,add adapter A,biotin retrieval,add sequencing adapter,and screening and amplification of PCR conditions.The amplified product of the library is sampled and tested for a second quality control point.When the test is qualified,it indicates that the preparation of the library has been completed.After the standard library is constructed,the sequencing analysis is performed.Results:(1)Two samples of Hi-C standard libraries were constructed,and the samples were tested from blood cell counting method to agarose gel electrophoresis method.The results of both samples met the Hi-C library requirements.(2)Patient groups and experimental control groups obtained 867,096,347 and953,779,734 pairs of filtered Reads,respectively.The filtered sequences accounted for95.43% and 96.28% of the original reads.(3)In the uremia group,the effective Reads pair was 284,574,670.The cis-interacting Reads pair is 107,006,632;the cis-interacting Reads pair accounts for 37.60% of the effective Reads pair.The trans-interacting Reads pair was 177,568,038;the trans-interacting Reads pair accounted for 62.40% of the effective Reads pair.In the normal control group,the effective Reads pair was 439,473,459.The cis-interactive Reads pairs are 372,249,382;the cis-interactive Reads pairs account for 84.70% of the effective Reads pairs.The trans-interacting Reads pair was 67,224,077;the trans-interacting Reads pair accounted for 15.30% of the effective Reads pair.(4)We normalized the intensity of the interaction between different chromosomes according to the Hi-C data processing standard to obtain the interaction matrix diagram of the different chromosomes.It was found that the closer the chromosome fragments are,the higher the interaction value is.(5)The interaction value of the change in the intensity of the action between the patient group and the control group is basically the same.The interaction value of the same chromosome is higher than the interaction value with other chromosomes.Enhanced.(6)According to statistics,in 23 pairs of chromosomes,most of the two groups of cells have no significant changes in area A and area B.The highest proportion of conversion from area A to area B is 7.11% of chromosome 9,and the highest proportion of conversion from area B to area A The proportion of chromosome 22 is7.39%.(7)Both groups of cells have some unique TAD boundaries.The number of TAD boundaries in the patient group is 1001,the number of boundary in the control group is 1166,and the number of common boundaries is 780.The TAD boundaries of the chromosomal fragments of the two groups of immune cells are different,but about70% of the TAD boundaries of the two groups of cells are the same.Conclusion:(1)The original Hi-C animal specimens were cancer cell tissues or conventional cell specimens.In this experiment,human peripheral blood specimens were used directly to build the library.Obtaining human disease or healthy tissues and organs for Hi-C database construction is relatively difficult,and the peripheral blood specimens obtained in this experiment are relatively easy.(2)In this experiment,some steps in the Hi-C experiment process were improved and further optimized.The sample quality control was qualified.The Hi-C molecule was successfully prepared and a standard library was constructed.Mononuclear cells extracted from peripheral blood have established a set of standard operating procedures for human immune cells Hi-C,which can provide reference for the future establishment of peripheral blood Hi-C libraries.(3)Most of the interactions in this sequencing will fall into the same region of the same chromosome,and few interactions will fall to other regions of the same chromosome,indicating that the interactions are not random.Most interactions are caused by the same chromosome,and rarely occur between different chromosomes,which indicates that the chromosomes are not randomly distributed in the nucleus.(4)Longer chromosomes tend to approach and act on longer chromosomes,while smaller chromosomes tend to act and approach smaller chromosomes.(5)The change in the intensity of the action between the patient group and the control group is basically the same.The interaction between the same chromosome is stronger than the effect with other chromosomes.(6)The genomes of N20 and U20 showed similar distribution of open and closed compartments.Only some regions have A to B or B to A transformation,and many genes in these transformed regions are related to disease pathways.Changes in A / B compartments suggest that there are more changes in gene expression regulation in chromosome 9 and 22 in the patient group and the control group.(7)The TAD sliding boundary is highly conservative.Changes in the boundaries of TAD may promote changes in gene expression.The TAD boundary is basically the same between the two groups of cells,but there are also differences in TAD boundaries.These difference boundaries may be related to the occurrence of uremia. |
PDF Full Text Request