| Objective(s):To study the effect and mechanism of allogeneic murine mesenchymal stem cell transplantation on the structure and function of thymus in aged e57 mice,and provide a new method and theoretical basis for the treatment of senile thymic atrophy.Methods:1.Screening and evaluation of aging model:A total of 50 female C57BL/6 mice,aged 18 months and weighing 38±5g,were selected from the SPF animal laboratory of the 920th Hospital of the People's Liberation Army Joint Service Support Unit for screening aging models.Screen the mouse population.Evaluation method:Appearance to observe hair growth,color,spirit,and activity;Three C57BL/6 mice were randomly selected and sacrificed.The thymus size was observed by gross anatomical observation.The thymus/body mass index was calculated by weighing method.Conventional thymus tissue sections,HE staining,thymus tissue structure under light microscope;The changes of thymus skin with mass volume and lymphocyte ratio were analyzed by immunofluorescence.The expression levels of aging and autophagy-related proteins were analyzed by immunohistochemical staining.2.Preparation of mUCMSC:The C57 pregnant rats,which were raised in the SPF animal laboratory of the 920th Hospital of the People's Liberation Army Joint Service Support Unit and pregnant for 21 days,were sacrificed by cervical dislocation,the umbilical cord was aseptically collected,and the tissue block was cut into 1 mm3 tissue by ophthalmology.The blocks were inoculated on the bottom wall of a 10 cm2 culture dish.After the mesenchymal stem cells climbed out and grew at a fusion rate of 80%,the primary mUCMSCs were digested with trypsin and further expanded and cultured to the third generation(p3).For p4 generation mUCMSC,the growth morphology during culture was observed under an inverted microscope.The positive expression rates of CD29,CD90 and CD34 in mUCMSC were analyzed by flow cytometry.The induced differentiation assay kit was used to perform adipogenesis and formation according to the instructions,Bone and chondrogenesis induce differentiation and identify differentiation potential.3.mUCMSC transplantation therapy:The C57 mice obtained in the previous screening and meeting the requirements of thymic aging were randomly divided into the treatment group and the model control group.At the same time,the C57 mice of 2 months old were used as the youth control group,with 15 rats in each group.For the experimental group,mUCMSC was infused through the tail vein at a dose of 1 x 107 cells/kg twice a week(Monday,Thursday)for 3 weeks;The remaining 2 groups were injected with an equal volume of normal saline.The animals were routinely kept,and the activity of the mice was recorded daily.After the last transplantation of 6W,the mice in each group were sacrificed,and the efficacy and mechanism were evaluated.4.Evaluation method of mUCMSC treatment:After 6 weeks of the last transplantation,the hair growth and color changes of the mice were observed and compared.After weighing,the cervical spine was sacrificed and the size and shape of the thymus were dissected.The thymus was weighed and the body weight ratio was calculated.The thymus tissue was collected,embedded in paraffin,sectioned,and stained with HE.The changes of tissue structure were observed under light microscope.The changes of thymus skin volune and lymphocyte ratio were analyzed by immunofluorescence.The intact thymus of 3 mice in each group was digested with type ? collagenase into a single cell suspension,and then the proportion of T lymphocyte subsets was analyzed by flow cytometry.5.Mechanism study:For thymus tissue sections,chemically labeled antibodies were used for immunohistochemical staining of p16,p53,SOD1,beclinel,LC3b,sirtl,and sirt3,and the expression levels of related protein molecules were analyzed.CK5,CK8,CD4 and CD8 staining were performed using fluorescently labeled antibodies,and changes in expression levels were observed.Results:1.Evaluation of aging model:The appearance of 18-month-old C57 mice showed white hair loss,shedding,slow movement,poor mental function,reduced activity,and body weight in the range of 38±5g.The thymus index of 18-month-old aged rats was(0.39±0.21)×10.3,and the thymus index of young rats at 2 months old was(6.4±0.21)×10-3.The thymus index of young C57 mice/young C57 mouse thymus Index=6.0%.The structure observation showed that the thymus gland atrophy and the medullary gland-like structure disappeared in the 18-month-old mice.Immunohistochemical staining analysis showed an increase in the expression of senescent proteins.2.Preparation of mUCMSC:Inoculated in the umbilical cord tissue block of the culture dish,a small amount of mUCMSC was observed to climb out from the tissue block on the 2nd day after culture,the cells were long spindle-shaped,and the cell fusion rate reached 70%on the 3rd day,and the digestion was collected.After mUCMSCs were subcultured to the third generation(P3),the growth morphology of mUCMSCs was long fusiform and arranged in a spiral shape.Flow cytometry analysis of P4 generation mUCMSC showed that CD29 positive rate was 99.6%,CD90 positive rate was 99.7%,CD34 positive rate was 0.109%,oil red O staining was positive after adipogenic induction culture,and alizarin red staining after osteogenic induction culture Ace blue staining was positive after positive and chondrogenic induction culture.3.the efficacy of mUCMSC:(1)After the treatment of mUCMSC,part of the hair of the mice turned black,and the hair that had fallen off the back grew again.The average gray value of the hair of the head,back and hip was statistically analyzed.The gray value of the treatment group was increased compared with the model control group(p<0.01);(2)The thymus volume of the treatment group was larger than that of the model control group(p<0.05).(3)Increased mean thymus index:treatment group/model control group=664%(p<0.01);(4)Thymus tissue structure changes:The thymus gland of the treatment group was intact and had a glandular structure,while the thymus gland-like structure of the model control group disappeared.(5)The expression levels of CK5 and CK8 in the thymus of the model control group and the treatment group were higher than those of the young group(p<0.01);The expression of CK5 in the thymus tissue of the treatment group was higher than that of the model control group(p<0.01),while the expression level of CK8 was increased,but the difference was not significant(p>0.05).(6)Changes in the proportion of CD4+and CD8+T cells:The proportion of CD8+T cells in the thymus tissue of the treatment group increased(p<0.001),indicating that mUCMSC treatment may increase the CD8+T cell output fimction of mouse thymus.The expression of CD4 and CD8 in the thymus of the treatment group was enhanced compared with the model control group,and the increase of CD8 was higher.In the young control group,the thymus was mainly CD4+cells,and the number of CD8+cells was small.4.Mechanism study:(1)Changes in aging-related protein expression:Comparing the immunohistochemical results of thymus tissue,we found that the expression of p53 and P16 protein in the young control group,the treatment group,and the model control group showed an upward trend,but the difference was not significant(p>0.05).The activity of SOD1,Sirtl and Sirt3 protein was the lowest in the young control group,the highest in the mUCMSC treatment group,and the model control group was in the middle position(p>0.05).(2)Autophagy-related protein expression changes:LC3b activity was lowest in the young control group,the treatment group was the highest,and the model control group was in the middle position.Young control group/treatment group?15.4%(p<0.01);young control group/model control group=34.3%(P<0.05);model control group/treatment group=45.1%(p>0.05);Beclinel activity was lowest in the young control group,the treatment group was the highest,and the model control group was in the middle position.Youth control group/treatment group=24.0%(p<0.05);The young control group/model control group=34.4%(p<0.05);the model control group/treatment group=69.8%(p>0.05);the P62 activity was the lowest in the model control group,the treatment group was the highest,and the young control group was in the middle position.The young control group/treatment group=87.0%(p>0.05);the young control group/model control group=150.6%(p>0.05);the model control group/treatment group=57.7%(p>0.05).Conclusion(s):1.The color,spirit and activity of 18-month-old C57 mice were aging,thymus atrophy,structural disorder,and decreased T cell output function,which was consistent with the thymic aging model;2.C57 mouse mUCMSCs with growth characteristics,immunophenotype and differentiation potential in accordance with UCMSC standards were prepared.3.The treatment of mUCMSC can promote the hair regeneration of aging mice,promote the partial blackening of hair,partially regenerate the structure of thymus and the immune output function mainly by CD8+T cells.4.mUCMSC inhibits the process of thymic aging and promotes the structural and functional regeneration of thymus by down-regulating the expression of p53,p1 6 and up-regulated SOD1,Sirtl and Sirt3 genes,but promotes the expression of autophagy-related genes LC3b,beclinel and P62,but The mechanism needs further research. |
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