| Thymus is a primary lymphoid organ, which spatially, orderly educates immature lymphocyte precursors to be mature T cells and provides the main fighters to the peripheral immune response. However, thymus begins to shrink at puberty and its function declines step by step, which reducing the output of naive T cells and limiting the diversity of peripheral T cells and increasing the risk of infection and autoimmunity. Previous studies have shown the change of thymic microenvironment itself rather than the reduced function/number of hematopoietic stem cells is the main inducement of age-related thymic involution. Thymic epithelial cells（TECs） are the most important cellular components in the thymic microenvironment. However, due to their rarity and isolating difficulty, TEC-related studies have made little progress. This study takes murine thymus as research object to analyze the relation between the histological changes of thymus and the gene expression changes of TEC-specific genes during age-related thymic involution, while optimizes the isolation/enrichment methods of adult murine TECs. Hopefully, we can provide the basis for exploring the machenism of age-related thymic involution, setting TEC lines and regenerating thymus. Object:（1） To explore the relation between the histological changes of thymus and the gene expression of TEC-specific genes during age-related thymic involution, and further clarify the influence of thymic microenvironment in age-related thymic involution.（2） To compare the effects among adult murine TECs isolation & enrichment methods, and to find a high-efficiency TECs enrichment method with accurate invivo representation in the hope of providing high-purity TECs for the follow-up studies of molecular profiling analysis and in-vitro TEC cell line establishment. Materials and methods:（1） In involution experiment, BABL/c mice were divided into three groups according to their age, Group A（1-month old）, Group B（4-month old）, Group C（16-month old）. Each group contained two mice, one for HE staining to observe thymic morphologic change, the other for real-time PCR to detect expressions of TEC-specific genes Fox N1, AIRE and Dll4. Each experiment was repeated three times independently（a total of eighteen mice in all）.（2） BABL/c mice（6-8 weeks） were divided into nine groups, and each group contained three mice. Each experiment was repeated three times independently（a total of eighty-one mice in all）. Isolation experiment contained five isolation groups: collagenase V 1.0g·L-1 group and Liberase TM 0.5, 0.7, 1.0, 2.0 U?m L-1 groups. Thymus digestion was performed according to relevant enzyme working concentration. Ⅱ.Enrichment experiment contained four enrichment groups: control, MACS, Percoll and Ficoll enrichment groups; in each group, thymus digestion was performed using the optimal working concentration（1.0 U?m L-1） of Liberase TM and the digests were continued for TECs enrichment by MACS, Percoll, Ficoll respectively（except control group）. For all the nine groups, the cell viability, the percentage of TSCs and the expression of epithelial cell adhesion molecule（Ep CAM） in TSCs were examined by flow cytometry. Furthermore, in both Percoll enrichment group and control group, the percentages of cortical TECs（c TECs） and medullary TECs（m TECs） were examined as well. Results:（1） For involution experiment, thymus in Group A showed intact cortical and medullary regions, and the boundary between them was clear. Thymus in Group B showed adipose hollow space, suggestive of fatty infiltration, but cortimedullary boundary was still identifiable and PVS region had less infiltrated adipose cells. In Group C, large area of adipose hollow space and cyst appeared and even PVS regions were infiltrated as well. Besides, gene expressions of Fox N1, AIRE and Dll4 in Group B were lower than those in Group A（P<0.0001）, but higher than those in Group C（P<0.05）, in other words, three genes all showed an age-related diminishing trend in the three groups.（2） For isolation experiment, the yield of TSCs in Liberase TM 1.0 U?m L-1 group was higher than those in Liberase TM 0.5 and 0.7 U?m L-1 group（P<0.01）; cell viability and digest time in Liberase TM 1.0 U?m L-1 group were superior to collagenase V 1.0g·L-1 group（P<0.01）. The cell viabilities in three enrichment groups were higher, and there were no significant difference between them（P>0.05）. Compared with control group, the percentages of TECs in three enrichment groups were increased（P<0.05 or P<0.01）. And among three enrichment groups, the percentage of TECs in Percoll enrichment group was higher than those in other two groups（P<0.01）. There were no significant differences in the percentages of c TECs and m TECs as well as the ratioes of c TEC to m TEC between control group and Percoll enrichment group（P>0.05）. Conclusion:（1） In the thymic microenvironment, the age-related loss of Fox N1 induces the loss of TECs and further leads to decline in the expression of TEC-specific gene AIRE and Dll4 and then adipose hollow space and cyst appears. Thymopoiesis is hindered by three essential steps——processing（positive selection）, testing（negative selection） & transporting（PVS） and finally, function of aged thymus is gradually weakened.（2） Liberase TM is more suitable for adult murine TECs isolation than collagenase V and its optimal digestion concentration is 1.0 U?m L-1; All the three enrichment groups can increase the percentage of TECs, and they all do little harm to cells; Percoll is the most efficient method for adult murine TECs enrichment among three enrichment methods, and it doesn’t affect the ratio of c TEC to m TEC, thereby making it easier for the following study. |
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