| Objective1. To explore the effects of aging on the expression of splenic stem cell-related genes and aging-related genes.2. To observe the age-related dynamic changes of T cell subsets using flow cytometry.3. To establish a efficient isolation culture system of bone marrow mesenchymal stem cells and observe the age-related changes of bone marrow mesenchymal stem cells. To provide sufficient cells with good quality which can be used in the next cell therapy.4. To assess the effects of transplanted mesenchymal stem cells on dynamic changes of T cell subsets in spleen and the expression of splenic stem cell-related genes and aging-related genes.Methods1. BALB/c mice were divided into three groups according age:Young adult (2mos, n=5), mature (8mos, n=5) and old (18mos, n=5). Mice were executed and the following tests were made.(1) Histology characteristics of spleen were examined using hematoxylin-eosin staining.(2) The expressions of splenic stem cell-related genes and aging-related genes(AUF1、P16INK4、 Oct3/4、Sox2、KLF4、c-MYC、NANOG、HOX11、ADAM12、GIL3、Wnt) of three groups were tested with real-time PCR. Finally, the data were analyzed with software lightcycler480and statistical software spss16.0.2. BALB/c mice were divided into three groups according age:Young adult (2mos, n=5), mature (8mos, n=5) and old (18mos, n=5). Harvest mouse spleen and prepare a single cell suspension with Red Blood Cell Lysis Buffer. Count cells、just density (2×107/ml), and proceed with cell staining procedures. Dynamic changes of splenic T cell subsets (CD3+、CD+、CD8+、CD8+CD44lowCD62Lhigh CD8+CD44lowCD62Lhigh Sca-1+) was detected by flow cytometry (FCM). Finally, the data were analyzed with software Kaluza and statistical software spss16.0. 3. Bone marrow stem cell(BMSCs) were isolated from C57BL/6mouse (3weeks,22months)compact bone. The procedure included flushing bone marrow out of the long bones, digesting the bone chips with collagenase typeⅡ. The cells were culture in MesenCult Proliferation Kit(Mouse). The morphology of the cells was observed under inverted microscope. The surface markers of6th passage BMSCs were detected by flow cytometer. The multilineage differentiation capacity of the6th passage BMSCs were in investigated by differentiating the cells into osteoblasts and adipocytes.4. BALB/c mice were divided into three groups according age:Young adult (2mos, n=5), mature (8mos, n=5) and old (18mos, n=5).The sixth passage C57BL/6(3wk) bone marrow stem cell (BMSCs) were labled by5μM fluorescent dye(CM-Dil), suspended in200ul Dulbecco’s Phosphate Buffered Saline (DPBS).The1×107labled cells were injected through the tail vein into the three groups mice. A month after the injection, mice were executed and the following tests were made.(1) Dynamic changes of splenic T cell subsets (CD3+、CD4+、CD8+、CD8+CD28+、 CD8+CD44lowCD62LhighCD8+CD44low CD62Lhigh Sca-1+) was detected by flow cytometry(FCM).(2)The expressions of splenic stem cell-related genes and aging-related genes(AUF1、P16INK4、Oct3/4、Sox2、 KLF4、c-MYC、NANOG、HOX11、ADAM12、GIL3、Wnt) of three groups were tested with real-time PCR. Finally, the data were compared with experiment part I、 II and analyzed by statistical software spss16.0.ResultsPart IThe strutural changes of spleen in BALB/c mice during aging:compared with young adult mice, structural of old mice were disorder with more macrophage and hemosiderin. The mRNA relative egression of P16INK4in three group mice was6.20(young adult)、11.56(mature)、18.48(old)respectively. The RNA relative egression of AUF1was0.154(young adult)、0.130(mature)0.09(old) respectively. With aging, the expression of P16INK4showed tendency to ascend and AUF1showed tendency to descend (P<0.05). With aging, the mRNA relative egression of genes related with splenic stem cell showed tendency to descend(P<0.05), These genes include Oct3/4、Sox2、KLF4、c-MYC、NANOG、 HOX11、ADAM12、GIL3、Wnt. Part II(1)The percentage of CD3+T was47.8%(Young adult)、50.1%(mature)'36.3%(old) respectively. There were significant differences betweenyoung adult mice and old mice, and between mature mice and old mice.(2)The percentage of CD4+T was24.2%(Young adult)、22.7%(mature) and21.0%(old) respectively. There were no significant differences between groups.(3)The percentage of CD8+T was17.8%(Young adult)、15.0%(mature)'9.80%(old) respectively. There were significant differences between young adult mice and old mice, and between mature mice and old mice.(4)The percentage of CD8+CD28+was13.1%(Young adult)、7.3%(mature)'6.0%(old) respectively. There were no significant differences between groups.(5)The percentage of CD8+CD44lowCD62Lhigh of total splenic cells was4.5%(Young adult)、2.2%(mature)'3.7%(old)respectively. The percentage of CD8+CD44lowCD62Lhigh of CD8+cells was24.5%、(Young adult)、12.7%(mature) and16.5%(old) respectively. There were no significant differences between groups.(6)The percentage of CD8+CD44lowCD62LhighSca-1+of total splenic cells was1.4%(Young adult)、1.8%(mature)'3.2%(old) respectively. The percentage of CD8+CD44lowCD62Lhigh Sca-1+of CD8+was14.8%(Young adult).12.6%(mature) and26.8%(old) respectively. There were no significant differences between groups.PartⅢBone marrow stem cell(BMSCs) were isolated from mouse (C57BL/6) compact bone after10days during primamy culture. After passage, cell morphology became uniform. BMSCs at the sixth passage isolated were positive on the expression of CD29(3wk,94.1%;2mos,94.7%)、CD11b(3wk,6.6%;2mos,8.1%) and CD45(3wk,2.68%;2mos,2.24%). The multilineage differentiation capacity of the6th passage BMSCs were successfully induced into osteoblasts and adipocytes.PartrⅣA month after the injection, the percentage of CD3+and CD8+T showed tendency to descend in Young adult and mature mice (P<0.005). the percentage of CD4+also showed tendency to descend in mature mice (P<0.01). There were no significant differences of T cell subsets in old mice. There were no significant differences of CD8+CD44lowCD62Lhigh and CD8+CD44lowCD62LhighSca-1+in the all three group. Injection of allogeneic bone marrow stem cell can affect the mRNA relative egression of genes related with splenic stem cell and age-related genes.Conclusion1. Bone marrow stem cell (BMSCs) can be obtained by mouse (C57BL/6) compact bone. Sufficient cells with good quality can be provided. There were age-related changes on multilineage differentiation capacity between3weeks BMSCs and22months BMSCs.2. There were age-related changes on mice spleen and splenic T cell subset. After the injection of allogenic BMSCs, mice spleen and splenic T cell subset can be affected. These effects are not the same in different age group. |
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