Method for evaluating functions of effective cells in thymus tissue based on multi-factor combination

Abstract

The invention provides a method for evaluating effective cell functions in thymus tissue based on multi-factor combination, which comprises the following steps: detecting the content of molecular markers in thymus tissue slice culture supernatant, the molecular markers being one or a combination of more of IL16, L-Selectin, CCL19, CCL21, CXCL16 and CXCL12, and the content of the molecular markers being one or more of IL16, L-Selectin, CCL19, CCL21, CXCL16 and CXCL12. The combination of one or more of IL16 and L-Selectin is used for evaluating the effective cell function of the thymus tissue raw material, and the combination of one or more of CCL19, CCL21, CXCL16 and CXCL12 is used for evaluating the effective cell function of the thymus tissue slice finished product. The thymus tissue slice does not need to be lost, the consumed time is short, and the functions of effective cells in the thymus tissue slice can be accurately reflected.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and particularly relates to a method for jointly evaluating the functions of effective cells in thymus tissue based on multiple factors. Background Art

[0002] The thymus is an important central immune organ of the body, a key site for the development, maturation, and functional output of T cells, and plays an important role in the body's anti-infection, anti-tumor, and maintenance of immune tolerance. Although the thymus is the foundation for T cell generation, it is the most vulnerable organ in the body. Aging, pathogen infection, clinical radiotherapy and chemotherapy, and bone marrow transplantation can all cause irreversible damage to it. Age-related thymic involution leads to a gradual reduction in thymocyte structure and damage to the thymic stromal microenvironment, including the loss of the clear cortex-medulla connection, a decrease in cortical thymic epithelial cells and medullary thymic epithelial cells, fibroblast expansion, and an increase in the perivascular space, which is also the reason for the reduced resistance of the elderly to pathogens, the increased incidence of autoimmunity, and the weakened tumor immune surveillance.

[0003] In the 1990s, Professor M. Louise Markert of Duke University in the United States prepared tissue sections with a thickness of 0.5-1 mm from discarded thymus tissue in vitro. Studies have shown that after in vitro culture, the T lymphocytes in the thymus tissue sections will gradually apoptose, and finally, thymic stromal cells and tissue architecture will be retained. Further transplanting the in vitro-cultured thymus tissue sections into the thigh muscles of children with congenital thymic defects (complete DiGeorge syndrome), clinical data show that thymus transplantation therapy can effectively reconstruct the thymus function of patients with congenital thymic defects. This technology was approved by the US FDA in 2021. Since the thymus tissue sections need to undergo 14-21 days of in vitro culture before transplantation, the quality assessment of the finished thymus tissue sections is a key link before transplantation. Previous quality assessment schemes mainly relied on histological detection, evaluating the content of residual lymphocytes, the integrity of the tissue skeleton, and the number of thymic corpuscles in the thymus tissue sections by hematoxylin-eosin (H&E), and evaluating the content of CD3+ T cells and CK14+ epithelial cells by immunohistochemistry. However, this technology has three problems: the evaluation time of H&E and immunohistochemistry is relatively long, affecting subsequent surgical arrangements; each detection requires the consumption of tissue sections; relying solely on H&E and immunohistochemistry cannot accurately reflect the functions of the effective cells in the tissue sections. Therefore, there is a clinical need to develop a scheme for quickly evaluating the effective functions of thymus tissue sections. Summary of the Invention

[0004] To solve the problems in the prior art, the present invention provides a method for jointly evaluating the functions of effective cells in thymic tissue based on multiple factors. Based on thymic epithelial cell molecular markers (CCL19, CCL21, CXCL16), fibroblast molecular markers (CXCL12), and T cell function molecular markers (IL-16, L-selectin), the functions of effective cells in thymic tissue sections are jointly evaluated, without consuming thymic tissue sections, with short time consumption, and can accurately reflect the functions of effective cells in thymic tissue sections.

[0005] The present invention realizes the solution of its technical problems by adopting the following technical solutions:

[0006] The first object of the present invention is to provide a method for jointly evaluating the functions of effective cells in thymic tissue based on multiple factors, including the following steps: detecting the content of molecular markers in the culture supernatant of thymic tissue sections, where the molecular markers are one or more combinations of IL16 and L-selectin and CCL19, CCL21, CXCL16, CXCL12. One or more combinations of IL16 and L-selectin are used to evaluate the functions of effective cells in thymic tissue raw materials, and one or more combinations of CCL19, CCL21, CXCL16, CXCL12 are used to evaluate the functions of effective cells in finished thymic tissue sections.

[0007] Further, CCL19, CCL21, and CXCL16 are specifically expressed in epithelial cells, CXCL12 is specifically expressed in fibroblasts, and IL16 and L-selectin are specifically expressed in T cells.

[0008] Further, for the functions of effective cells in thymic tissue raw materials, the supernatant of thymic tissue sections cultured in vitro for 1 day is used as a sample. The content standards of the molecular markers are that the content of CCL19 is not less than 2000 pg / mL, the content of CCL21 is not less than 200 pg / mL, the content of CXCL16 is not less than 10 pg / mL, the content of CXCL12 is not less than 100 pg / mL, the content of IL16 is not less than 3000 pg / mL, and the content of L-selectin is not less than 8000 pg / mL.

[0009] Furthermore, the effective cell function of the finished thymus tissue section uses the supernatant of the thymus tissue section cultured in vitro for 14 to 21 days as a sample. The content standards of the molecular markers are that the content of CCL19 is not less than 10,000 pg / mL, the content of CCL21 is not less than 2,000 pg / mL, the content of CXCL16 is not less than 500 pg / mL, the content of CXCL12 is not less than 2,000 pg / mL, the content of IL16 is less than 500 pg / mL, and the content of L-selectin is less than 2,000 pg / mL.

[0010] Furthermore, the method for detecting the content of the molecular marker is enzyme-linked immunosorbent assay.

[0011] Furthermore, IL16 and L-selectin are jointly used for evaluating the effective cell function of thymus tissue raw materials, and CCL19, CCL21, CXCL16 and CXCL12 are jointly used for evaluating the effective cell function of finished thymus tissue sections.

[0012] Furthermore, the molecular markers are jointly used to construct a model for evaluating the effective cell function in thymus tissue, and the model is a Logistics regression model.

[0013] Furthermore, when used for evaluating the effective cell function of thymus tissue raw materials, the construction method of the Logistics regression model is to use whether the content detection of the molecular marker is qualified as the dependent variable, and the contents of IL16 and L-Selectin as the covariates to construct the Logistics regression model.

[0014] Furthermore, the evaluation value of the model is logit(P) = -3.891 + 0.001[IL16 content] + 0.000[L-Selectin content], and the optimal cut-off value is 0.667.

[0015] Furthermore, when the predicted value of the regression model is higher than the cut-off value, the evaluation of the effective cell function of thymus tissue raw materials is qualified; when the predicted value of the regression model is lower than the cut-off value, the evaluation of the effective cell function of thymus tissue raw materials is unqualified.

[0016] Furthermore, when used for evaluating the effective cell function of finished thymus tissue sections, the construction method of the Logistics regression model is to use whether the content detection of the molecular marker is qualified as the dependent variable, and the contents of CCL19 and CCL21, CXCL12 and CXCL16 as the covariates to construct the Logistics regression model.

[0017] Furthermore, the evaluation value of the model is logit(P) = -42.803 + 0.001 [CCL19 content] + 0.004 [CCL21 content] + -0.001 [CXCL12 content] + 0.003 [CXCL16 content], and the optimal cut-off value is 0.491.

[0018] Furthermore, when the predicted value of the regression model is higher than the cut-off value, the evaluation of the effective cell function of the finished thymus tissue section is qualified; when the predicted value of the regression model is lower than the cut-off value, the evaluation of the effective cell function of the finished thymus tissue section is unqualified.

[0019] The second object of the present invention is to provide an application of the above evaluation method in a thymus tissue section transplantation model.

[0020] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0021] Based on thymic epithelial cell molecular markers (CCL19, CCL21, CXCL16), fibroblast molecular markers (CXCL12), and T cell function molecular markers (IL-16, L-selectin), the present invention does not require the consumption of thymus section tissues for the evaluation of effective cells in thymus raw materials and finished tissue sections, with short time consumption, simple operation, and high evaluation accuracy. In particular, the combination of IL16 and L-selectin is used for the evaluation of the effective cell function of thymus tissue raw materials, and the combination of CCL19, CCL21, CXCL16, and CXCL12 is used for the evaluation of the effective cell function of finished thymus tissue sections, with better evaluation result accuracy. It avoids problems such as sample destruction, consumption of thymus sections, and sampling errors existing in traditional histological diagnosis, and is of great significance for rapid and accurate pre-transplant quality assessment in future clinical practice.

[0022] The above description is only an overview of the technical solution of the present invention. In order to understand the technical means of the present invention more clearly, it can be implemented according to the content of the specification. In order to make the above and other objects, features, and advantages of the present invention more obvious and understandable, the following specific embodiments of the present invention are specifically described. Description of the Drawings

[0023] Figure 1 It is the distribution of cell subsets in the thymus tissue section of the present invention.

[0024] Figure 2 It is the screening of thymic epithelial cells, fibroblasts, and T cell molecular markers of the present invention.

[0025] Figure 3 It is the dynamic change diagram of the secretion levels of molecular markers in the supernatant of the thymus tissue section of the present invention on the 1st, 5th, 9th, and 14th days of in vitro culture.

[0026] Figure 4 This is the overall view and partial view of the hematoxylin-eosin (H&E) staining of the thymus tissue section of the present invention on the 14th day of in vitro culture.

[0027] Figure 5 This is the level of molecular markers in the supernatant of the thymus tissue section of the present invention on the 1st day and the 14th day of in vitro culture.

[0028] Figure 6 This is the ROC curve for the single evaluation and combined evaluation of the effective function of the thymus tissue section by the molecular markers IL-16 and L-selectin in the thymus tissue section of the present invention on the 1st day of in vitro culture.

[0029] Figure 7 This is the ROC curve for the single evaluation and combined evaluation of the effective function of the thymus tissue section by the molecular markers CCL19, CCL21, CXCL12, and CXCL16 in the thymus tissue section of the present invention on the 14th day of in vitro culture.

[0030] Figure 8 This is the peripheral blood flow diagram and the proportions of mature CD3+ TCR-β+ T lymphocytes, CD4+ T lymphocytes, and CD8+ T lymphocytes in the quality qualified group, quality unqualified group, and sham operation control group of the present invention at the 16th week after surgery.

[0031] Figure 9 This is the hematoxylin-eosin (H&E) staining and immunohistochemical staining diagrams of the transplantation site in the quality qualified group and quality unqualified group of the present invention at the 16th week after surgery. Detailed implementation manners

[0032] The technical solution of the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following embodiments are only used to illustrate and explain the present invention exemplarily, and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0033] In addition, unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchase or prepared by existing methods.

[0034] Example 1 Analysis of the main effector cell types in thymus tissue sections

[0035] Collect the thymus tissues of clinical children with congenital heart disease. After preparing thymus tissue sections in vitro and culturing for 14 days, the thymus tissue sections cultured for 14 days are dissociated into single-cell suspensions, and then cell type analysis and pathway enrichment analysis are carried out.

[0036] The dissociation of the thymus tissue section refers to placing the thymus tissue section in 5 mL of 0.25% Trypsin-EDTA, using surgical scissors to cut the tissue section into tissue homogenate to release cells, then passing it through a 70-μm cell strainer, centrifuging at 200 rcf for 5 minutes to collect the cell pellet, and finally resuspending it in FACS solution (PBS containing 2% fetal bovine serum) to obtain a single-cell suspension.

[0037] The analysis of cell types mainly includes the following steps: constructing a single-cell transcriptome sequencing library using the Chip G Single Cell Kit (10×Genomics, PN-1000146), performing single-cell transcriptome sequencing, processing the sequencing data using CellRanger software for sequencing library splitting, barcode identification and assignment, and UMI counting. Further data processing uses the Seurat R package (V.4.3.0) to remove cells with less than 200 gene expressions and more than 20% mitochondrial gene expressions. Then, the data from different samples are integrated and batch-corrected, and then the standardization, normalization, clustering, dimensionality reduction, and visualization of all sample cells are completed, and the marker genes of cell clusters are listed.

[0038] The pathway enrichment analysis refers to using the findMarkers function to calculate the differential genes between different subpopulations, using the scibet software to determine cell clusters, and then using the clusterProfiler package to perform GO and KEGG pathway annotation and enrichment analysis on the differentially expressed genes to understand the biological functions of cells.

[0039] As Figure 1 shown, the thymus tissue section is mainly composed of 11 cell populations, including T cells (T), B cells (B), NKT cells (NKT), endothelial cells (Endo), dendritic cells (DC), monocytes (Mono), macrophages (Mac), epithelial cells (Epi), fibroblasts (Fib), smooth muscle cells (SMC), and vascular smooth muscle cells (VSMC). Among them, T cells account for 59.95%, epithelial cells account for 7.20%, and fibroblasts account for 16%. Although there are some T lymphocytes in the thymus tissue section, epithelial cells and fibroblasts are the main stromal cell populations.

[0040] Example 2 Screening and Evaluating Molecular Markers of Effective Cell Functions in Thymus Tissue Sections

[0041] In view of the fact that the evaluation of effective cells in traditional thymic tissue sections mainly relies on histological detection to evaluate the content of effective cells, but this method has the disadvantages of consuming thymic tissue sections, taking a long time, and not being able to accurately reflect the functions of effective cells. Therefore, the expression of secretion-related genes that can reflect the functions of the main effector cells (thymic epithelial cells, fibroblasts, and T cells) in thymic tissue sections was further explored.

[0042] As Figure 2 shown, first, the pathways related to cytokine production were anchored, mainly including chemotaxis pathway, cellular response to chemokines, epithelial cell migration, positive regulation of cytokine production, T cell receptor signaling, etc., and 50 genes were screened from them. Among them, 18 genes such as CCL19, CXCL16, CCL20, CCL21, and MIF were mainly expressed in epithelial cells; 18 genes such as MMP1, IL6, FGF7, IGFBP5, FN1, IL24, and CXCL12 were mainly expressed in fibroblasts; 14 genes such as IL16, TNFSF8, ITGB2, and SELL were mainly expressed in T cells. Further, 6 characteristic genes: CCL19, CCL21, CXCL16, CXCL12, IL16, and SELL were screened out as molecular markers of thymic functional cells. The UMAP plot showed that CCL19, CCL21, and CXCL16 were specifically expressed in epithelial cells, CXCL12 was specifically expressed in fibroblasts, and IL16 and SELL were specifically expressed in T cells.

[0043] CCL19: This molecular marker is specifically expressed in thymic epithelial cells and transfers CD4+CD8+ T lymphocytes to the thymic medulla through chemotaxis to undergo negative selection.

[0044] CCL21: This molecular marker is specifically expressed in thymic epithelial cells and is crucial for the migration of thymocyte precursors and the transfer of CD4+CD8+ T lymphocytes to the thymic medulla to undergo negative selection.

[0045] CXCL12: This molecular marker has been shown to be produced by thymic cortical and medullary epithelial cells, and can also be produced by thymic fibroblasts and endothelial cells, retaining immature CD4+CD8+ T lymphocytes in the cortical region and participating in the positive selection process. At the same time, there are relevant reports indicating that high expression of CXCL12 promotes thymopoiesis in in vitro human thymic organ cultures.

[0046] CXCL16: This molecular marker is only expressed in thymic medullary epithelium, and its role in thymocyte development is not yet clear.

[0047] IL-16: This molecular marker is secreted by T lymphocytes and can activate the proliferation of T lymphocytes.

[0048] SELL: Expresses L-Selectin, a molecular marker that is highly expressed on developing and naive T cells. It is not a secreted factor, but it is constitutively shed during cell adhesion and migration and is rapidly re-expressed. A decrease in the shedding level reflects the gradual loss of thymocyte viability.

[0049] Example 3 Defines the Secretion Level and Content Standard of Molecular Markers

[0050] To evaluate the dynamic changes in the secretion level of molecular markers, supernatants from 20 batches of thymic tissue sections cultured in vitro for 1, 5, 9, and 14 days were collected. The absorbance values of the molecular markers in the culture supernatants at a wavelength of 450 nm were detected using enzyme-linked immunosorbent assay (ELISA). The content of the molecular markers was calculated according to the standard curve, and the content standard of the molecular markers during the culture period was statistically analyzed.

[0051] The enzyme-linked immunosorbent assay in this example refers to using the IL16 human ELISA reagent (D1600), L-selectin human uncoated ELISA kit (DY728), CCL19 human uncoated ELISA kit (DY361), CCL21 human uncoated ELISA kit (DY366), CXCL12 human ELISA kit (DSA00), and CXCL16 human ELISA kit (DCX160) from R&D system company and operating according to the kit instructions.

[0052] As Figure 3 shown, the contents of CCL19, CCL21, CXCL12, and CXCL16 in the culture supernatants gradually increased during in vitro culture, indicating that the number and function of epithelial cells and fibroblasts were effectively retained; while the contents of IL-16 and L-selectin (expressed by the SELL gene) gradually decreased during in vitro culture, indicating that the number and viability of thymocytes, including T cells, gradually decreased. The above results show that the secretion levels of CCL19, CCL21, CXCL12, CXCL16, IL-16, and L-selectin during the culture period can fully reflect the effective cell function in thymic tissue sections. The content standards of each molecular marker in the evaluation of effective cell function in thymic tissue sections are shown in Table 1.

[0053] Table 1 Content Standards of Molecular Markers

[0054]

[0055] Example 4 Verifies the Reliability of Molecular Markers in Evaluating Effective Cell Function in Thymic Tissue Sections

[0056] Thymus tissue sections with qualified and unqualified quality inspections were continuously cultured in vitro for 21 days. The thymus tissue sections on the 14th day of culture were stained with hematoxylin-eosin (H&E). See Figure 4 . It can be seen that the thymus tissue sections with qualified quality inspection retain the normal thymic lobule structure, and the cortical area and medullary area can be clearly distinguished; the reticular thymic epithelial cells are dense, and the lymphocyte clearance rate reaches more than 90%; there are at least 2 clearly structured thymic corpuscles in a single field of view. The thymus tissue sections with unqualified quality inspection showed phenomena such as large-area tissue damage, tissue necrosis, tissue ablation, and karyolysis.

[0057] The supernatants on the 1st and 14th days of culture were taken, and the contents of molecular markers in the culture supernatants, including CCL19, CCL21, CXCL12, CXCL16, IL-16, and L-selectin, were detected by enzyme-linked immunosorbent assay (ELISA). See Figure 5 . It can be seen that during the in vitro culture of thymus tissue sections with qualified and unqualified quality inspections, the levels of molecular markers CCL19, CCL21, CXCL12, and CXCL16 in their culture supernatants all increased, while the levels of molecular markers IL-16 and L-selectin both decreased. Compared with the thymus tissue sections with unqualified quality inspection, on the 1st day of culture, the levels of IL-16 and L-selectin in the culture supernatant of the thymus tissue sections with qualified quality inspection were higher, but there was no significant difference on the 14th day of culture; on the 14th day of culture, the levels of CCL19, CCL21, CXCL12, and CXCL16 in the culture supernatant of the thymus tissue sections with qualified quality inspection were higher, but there was no significant difference on the 1st day of culture. This shows that the molecular markers CCL19, CCL21, CXCL12, CXCL16, IL-16, and L-selectin can be used as detection indicators for evaluating the effective cell function of thymus tissue sections.

[0058] Example 5 Use the ROC curve to evaluate the value of molecular markers in predicting the effective cell function of thymus tissue sections

[0059] The Receiver Operating Characteristic Curve (ROC curve) is commonly used in clinical medicine and epidemiological research to study the prediction accuracy of the test variable X for the status variable Y. X and Y in the ROC curve can be of any data type (both categorical and quantitative data are acceptable), but there can be many test variables X, and the status variable Y has only two categories of results (such as positive or negative, yes or no, diseased or non-diseased, etc.). When drawing the ROC curve, generally, the False Positive Rate (FPR), which is 1 - specificity, is used as the ordinate to reflect the proportion of all prediction errors (FP), and the True Positive Rate (TPR), also known as sensitivity or recall rate, is used as the abscissa to reflect the proportion of all correct predictions (TP), to draw a two-dimensional plane curve. AUC is defined as the Area Under the Curve (AUC) of the ROC curve, and this area value ranges from 0 to 1, indicating the probability that the sample is correctly classified. When AUC is less than 0.5, it means that the detection model does not match the actual situation; when AUC is equal to 0.5, it means that the detection model has no predictive evaluation value at all; when AUC is greater than 0.5 and less than 1, it means that the detection model has predictive evaluation value. Further, when AUC is between 0.5 and 0.7, it means that the detection model has relatively low predictive evaluation value; when it is between 0.7 and 0.9, it means that the detection model has certain predictive evaluation value; when it is above 0.9, it means that the detection model has high predictive evaluation value. At the same time, the prediction effect can be measured by the p-value. In ROC analysis, if the p-value is less than 0.05, it means that this indicator is effective for the prediction result.

[0060] Using SPSS 27.0 software, with 11 batches of thymus tissue sections with qualified quality inspections (positive) and 7 batches with unqualified quality inspections (negative) as the status variable Y, set as the grouping, named 1 and 0 respectively, and taking the content of a single molecular marker (CCL19 or CCL21 or CXCL12 or CXCL16 or IL-16 or L-selectin) in the supernatant on the 1st day or the 14th day of culture as the test variable, upload the data to the SPSSAU system, select the ROC curve analysis method in classification, draw the curves of single molecular markers of CCL19, CCL21, CXCL12, CXCL16, IL-16, and L-selectin, calculate the area under the curve, and judge the evaluation value of the single molecular marker.

[0061] As shown in Table 2, on the first day of in vitro culture of thymus tissue sections, the areas under the curves of IL-16 and L-selectin were 0.792 and 0.805 respectively, and the p-values were 0.042 and 0.033 respectively, indicating that the molecular markers IL-16 and L-selectin were effective in predicting the effective function of thymus tissue sections and had high evaluation value (AUC > 0.7, p < 0.05); while the AUCs of the molecular markers CCL19, CCL21, CXCL12, and CXCL16 were all less than 0.7, and the p-values were all greater than 0.05, indicating that their accuracy in evaluating the effective function of thymus tissue sections was low and they had no evaluation value. On the 14th day of in vitro culture of thymus tissue sections, the areas under the curves of CCL19, CCL21, CXCL12, and CXCL16 were 0.818, 0.870, 0.792, and 0.805 respectively, and the p-values were 0.026, 0.010, 0.042, and 0.033 respectively, indicating that the molecular markers CCL19, CCL21, CXCL12, and CXCL16 were effective in predicting the effective function of thymus tissue sections and had high evaluation value (AUC > 0.7, p < 0.05); while the AUC value of IL-16 was greater than 0.7, but the p-value was greater than 0.05, and the AUC of L-selectin was less than 0.7, and the p-value was greater than 0.05, indicating that the evaluation value of these two molecular markers was low.

[0062] Table 2 Area under the ROC curve (AUC), p-value, and confidence interval

[0063]

[0064] Example 6 Construction of a combined molecular marker to evaluate the effective function of thymus tissue sections using a Logistics regression model

[0065] In view of the high evaluation value of the molecular markers IL-16 and L-Selectin for the effective function of thymus tissue sections on the first day of culture, and CCL19, CCL21, CXCL12, and CXCL16 for thymus tissue sections on the 14th day of culture, the optimal combination of molecular markers for evaluating the effective function of thymus tissue sections was further explored by establishing a regression model. The regression model is to study one dependent variable (Y) or multiple dependent variables (Y 1 , Y 2 , …, Y n ) with respect to another covariate (X) or a set of covariates (X 1 , X 2 , …, X nStatistical analysis method of the dependency relationship. In common linear regression models, the dependent variable belongs to quantitative data and is used to predict continuous numerical data. While in the logistic regression model, the dependent variable belongs to categorical data, and the linear combination (value) of covariates is mapped to a probability range from 0 to 1 through the sigmoid function to predict the classification category. In medical statistics and epidemiopathology, it is often used to examine the effects of multiple factors on a disease (the outcome variable).

[0066] Using SPSS 27.0 software, with 11 batches of thymus tissue sections with qualified quality inspection (positive) and 7 batches with unqualified quality inspection (negative) as the status variable Y, set as the grouping, and named 1 and 0 respectively; and import the contents of IL-16 and L-selectin in the supernatant on the 1st day of culture into the SPSSAU system as the detection variables; then select the Logistic regression model in regression classification, use the grouping (status variable) as the dependent variable, and the contents of IL16 and L-Selectin (detection variables) as the covariates to generate a Logistic regression. The final expression of the model is: logit(P) = -3.891 + 0.001[IL16 content] + 0.000[L-Selectin content]; fit the original variables [IL16 content] and [L-Selectin content] into the combined predictor Pre 1; finally, with the grouping as the status variable and Pre 1 as the detection variable, select the ROC curve analysis method in classification, draw the combined ROC curve of IL-16 and L-selectin, calculate the area under the curve, judge the value of the combined evaluation of molecular markers, and calculate the AUC and p values, etc.

[0067] Using SPSS 27.0 software, with 11 batches of thymus tissue sections with qualified quality inspection (positive) and 7 batches with unqualified quality inspection (negative) as the status variable Y, set as the grouping, and named 1 and 0 respectively; and import the contents of CCL19, CCL21, CXCL12 and CXCL16 in the supernatant on the 14th day of culture into the SPSSAU system as the detection variables; then select the Logistic regression model in regression classification, use the grouping (status variable) as the dependent variable, and the contents of CCL19 and CCL21, CXCL12 and CXCL16 (detection variables) as the covariates to generate a Logistic regression. The final expression of the model is:

[0068] logit(P) = -42.803 + 0.001[CCL19 content] + 0.004[CCL21 content] + -0.001[CXCL12 content] + 0.003[CXCL16 content]; The original variables [CCL19 content], [CCL21 content], [CXCL12 content], and [CXCL16 content] were fitted into a combined prediction factor Pre 1; Finally, with the grouping as the state variable and Pre 1 as the detection variable, the ROC curve analysis method was selected in the classification to plot the combined ROC curve of CCL19, CCL21, CXCL12, and CXCL16, calculate the area under the curve, judge the value of the combined evaluation of the molecular markers, and calculate the AUC and p values, etc.

[0069] As Figure 6 and Figure 7 shown, on the 1st day of in vitro culture of thymus tissue sections, for the prediction factor Pre 1 evaluated jointly by IL-16 and L-selectin, the corresponding AUC was 0.883 (p < 0.05, 95% CI: 0.724 - 1.000), which was much higher than the AUC of single evaluation. Pre 1 was greater than the cut-off value of 0.667. Being greater than the cut-off value indicated that the thymus tissue raw material was qualified, with a sensitivity of 81.8% and a specificity of 85.7%; On the 14th day of in vitro culture of thymus tissue sections, for the prediction factor Pre 1 evaluated jointly by CCL19, CCL21, CXCL12, and CXCL16, the corresponding AUC was 0.948 (p < 0.05, 95% CI: 0.850 - 1.000), which was close to 1 and also much higher than the AUC of single evaluation. Pre 1 was greater than the cut-off value of 0.491. Being greater than the cut-off value indicated that the finished product of thymus tissue sections was qualified, with a sensitivity of 90.9% and a specificity of 85.7%. This result shows that the evaluation value and judgment accuracy of the combined molecular markers constructed using the Logistic regression model are superior to using only a single indicator, and the optimal cut-off value for jointly evaluating thymus tissue raw materials by IL-16 and L-selectin is 0.667; The optimal cut-off value for jointly evaluating the finished product of thymus tissue sections by CCL19, CCL21, CXCL12, and CXCL16 is 0.491. Being greater than this cut-off value can be judged as qualified for thymus tissue raw materials / finished products of thymus tissue sections, and being less than this cut-off value will be judged as unqualified for thymus tissue raw materials / finished products of thymus tissue sections.

[0070] Example 7 Verifying the Accuracy of Joint Evaluation Using a Mouse Transplantation Model

[0071] The finished products of thymus tissue sections with qualified quality and unqualified quality obtained by combined evaluation based on thymic epithelial cell molecular markers (CCL19, CCL21, CXCL16), fibroblast molecular markers (CXCL12), and T cell function molecular markers (IL-16, L-selectin) were transplanted subcutaneously into the back of 6-8-week-old Balb / c-Nude athymic nude mice at a transplantation dose of 1-2 mg / mouse, respectively.

[0072] The qualified quality of thymus tissue sections means that on the 1st day of culturing thymus tissue sections, the predicted values jointly evaluated by IL-16 and L-selectin are higher than the cut-off value; on the 14th day of culturing, the predicted values jointly evaluated by CCL19, CCL21, CXCL12, and CXCL16 are higher than the cut-off value.

[0073] The unqualified quality of thymus tissue sections means that on the 1st day of culturing thymus tissue sections, the predicted values jointly evaluated by IL-16 and L-selectin are lower than the cut-off value; on the 14th day of culturing, the predicted values jointly evaluated by CCL19, CCL21, CXCL12, and CXCL16 are lower than the cut-off value.

[0074] In this example, for the qualified thymus tissue sections obtained, on the 1st day of culturing, the predicted value jointly evaluated by IL-16 and L-selectin was 0.796, and on the 14th day of culturing, the predicted value jointly evaluated by CCL19, CCL21, CXCL12, and CXCL16 was 0.640. For the unqualified thymus tissue sections, on the 1st day of culturing thymus tissue sections, the predicted value jointly evaluated by IL-16 and L-selectin was 0.543, and on the 14th day of culturing, the predicted value jointly evaluated by CCL19, CCL21, CXCL12, and CXCL16 was 0.379.

[0075] At the 16th week after transplantation surgery, the tail vein blood of athymic Balb / c-Nude nude mice transplanted with qualified thymus tissue sections (qualified quality group) and unqualified thymus tissue sections (unqualified quality group) was collected respectively for flow cytometry analysis. At the same time, a sham operation model without transplanted thymus tissue sections was collected as a control group.

[0076] To analyze the newly emigrated lymphocytes and the proportions of CD4 and CD8 T cells, the following combinations were used: anti-mouse CD45 BV510 (Biolegend); anti-mouse CD3 BUV395 (BD); anti-mouse CD4 PE-Cy7 (Biolegend); anti-mouse CD8 Percp Cy5.5 (Biolegend); anti-mouse TCR-β APC (Biolegend); L / D APC-Cy7 (Invitrogen).

[0077] To analyze the reconstruction effect of the thymus at the transplantation site, at the 16th week after transplantation surgery, tissues at the transplantation site were collected for hematoxylin-eosin (H&E) staining and immunohistochemistry (IHC) detection. Anti-mouse CD3 antibody was used to label T lymphocytes in the thymus; anti-mouse CD4 antibody was used to label CD4 T lymphocytes; anti-mouse CD8a antibody was used to label CD8 T lymphocytes; and anti-human CK5 and CK14 antibodies were used to label thymic medullary epithelial cells.

[0078] As Figure 8 and Figure 9 shown, at the 16th week after transplantation surgery, the proportions of mature CD3 + TCR-β + T lymphocytes, CD4 + T lymphocytes, and CD8 + T lymphocytes in the peripheral blood of nude mice transplanted with thymus tissue sections with qualified effective functions were much higher than those in the control group; while the proportions of mature CD3 + TCR-β + T lymphocytes, CD4 + T lymphocytes, and CD8+ T lymphocytes in the peripheral blood of nude mice transplanted with thymus tissue sections with unqualified effective functions had no significant difference from those in the control group. At the same time, histology showed that the thymus tissue sections in the qualified quality group developed normally at the transplantation site (H&E), had a complete thymic structure and a large number of surviving and reticularly distributed epithelial cells (CK5 + , CK14 + ), and attracted lymphocytes of nude mice to form aggregation areas (CD3 + , CD4 + , CD8 + ); while the thymus tissue sections in the unqualified quality group were fatty at the transplantation site, showing a large number of fat vacuoles, no complete thymic structure, no surviving epithelial cells, and no lymphocyte aggregation. The above results indicate that the thymus tissue sections with qualified quality obtained by combined evaluation with molecular markers can develop into thymic structures at the transplantation site of nude mice and reconstruct the central immunity of nude mice, while the thymus sections with unqualified quality transplanted into nude mice will become fatty. It proves the reliability of the combined evaluation model of molecular markers, and at the same time avoids problems such as sample destruction, consumption of thymus sections, and sampling errors existing in traditional histological diagnosis, which has important significance for rapid and accurate pre-transplantation quality assessment in future clinical practice.

[0079] The serial numbers of the above embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.

[0080] The embodiments of the present invention have been described above in conjunction with the accompanying drawings. However, the present invention is not limited to the above specific embodiments. The above specific embodiments are merely illustrative and not restrictive. Under the inspiration of the present invention, those of ordinary skill in the art can also make many forms without departing from the spirit of the present invention and the scope protected by the claims. All of these are within the protection scope of the present invention.

Claims

1. A method for evaluating effective cell function in thymus tissue based on multi-factor combination, characterized in that: The following steps are involved: The content of molecular markers in the culture supernatant of thymic tissue slices is detected, wherein the molecular markers are a combination of IL16 and L-Selectin and one or more of CCL19, CCL21, CXCL16, and CXCL12, wherein the one or more combinations of IL16 and L-Selectin are used to evaluate the effective cell function of thymic tissue raw materials, and the one or more combinations of CCL19, CCL21, CXCL16, and CXCL12 are used to evaluate the effective cell function of thymic tissue slice finished products.

2. A method for evaluating the effective cell function in thymus tissue based on multi-factor combination as claimed in claim 1, characterized in that: The CCL19, CCL21 and CXCL16 are specifically expressed in epithelial cells, CXCL12 is specifically expressed in fibroblasts, and IL16 and L-Selectin are specifically expressed in T cells.

3. A method for evaluating the effective cell function in thymus tissue based on multi-factor combination as claimed in claim 1, characterized in that: The effective cell function of thymus tissue samples takes the supernatant of thymus tissue sections cultured in vitro for 1 day as the sample, and the content standards of the molecular markers are respectively that the content of CCL19 is not less than 2000pg / mL, the content of CCL21 is not less than 200pg / mL, the content of CXCL16 is not less than 10pg / mL, the content of CXCL12 is not less than 100pg / mL, the content of IL16 is not less than 3000pg / mL, and the content of L-Selectin is not less than 8000pg / mL.

4. A method for evaluating the effective cell function in thymus tissue based on multi-factor combination as claimed in claim 1, characterized in that: The effective cell function of the thymus tissue slice finished product uses the supernatant of the thymus tissue slice cultured in vitro for 14 to 21 days as a sample, and the content standards of the molecular markers are respectively CCL19 content not less than 10000pg / mL, CCL21 content not less than 2000pg / mL, CXCL16 content not less than 500pg / mL, CXCL12 content not less than 2000pg / mL, IL16 content less than 500pg / mL, and L-Selectin content less than 2000pg / mL.

5. A method for evaluating the effective cell function in thymus tissue based on multi-factor combination as claimed in claim 1, characterized in that: The IL16 and L-Selectin are used in combination for evaluating the effective cell function of thymus tissue raw materials, and the CCL19, CCL21, CXCL16 and CXCL12 are used in combination for evaluating the effective cell function of thymus tissue slice finished products.

6. A method for evaluating the effective cell function in thymus tissue based on multi-factor combination as claimed in claim 5, characterized in that: The molecular markers are combined to construct a model for evaluating the effective cell function in thymic tissue. The model is a Logistics regression model. When used for evaluating the effective cell function of thymic tissue raw materials, the Logistics regression model is constructed by using whether the content of the molecular marker is qualified as the dependent variable and the content of IL16 and L-Selectin as covariates to construct a Logistics regression model.

7. A method for evaluating the effective cell function in thymus tissue based on multi-factor combination as claimed in claim 6, characterized in that: The evaluation value of the model was logit(P)=-3.891+0.001[IL16 content]+0.000[L-Selectin content], and the optimal cutoff value was 0.

667.

8. A method for evaluating the effective cell function in thymus tissue based on multi-factor combination as claimed in claim 5, characterized in that: The molecular markers are combined to construct a model for evaluating the effective cell function in thymic tissue. The model is a Logistics regression model. When used for evaluating the effective cell function of thymic tissue slice finished products, the Logistics regression model is constructed by using whether the content of the molecular marker is qualified as the dependent variable, and using the contents of CCL19 and CCL21, CXCL12 and CXCL16 as covariates to construct a Logistics regression model.

9. A method for evaluating the effective cell function in thymus tissue based on multi-factor combination as claimed in claim 8, characterized in that: The evaluation value of the model was logit(P)=-42.803+0.001[CCL19 content]+0.004[CCL21 content]+-0.001[CXCL12 content]+0.003[CXCL16 content], and the optimal cutoff value was 0.

491.

10. Use of the evaluation method according to any one of claims 1 to 9 in a thymus tissue slice transplantation model.

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