| Objectives:1. To construct the recombinant retroviral vector MIGR1-ICAM-1, and examine the effects of ICAM-1transfection on biological characteristics of MSCs. Providing experimental basis for further studies on whether ICAM-1transfected MSCs have effects on the treatment of AIT in vivo.2. C57BL/6mice were immunized by exogenous thyroid globulin to establish AIT animal model (EAT). To provide reliable animal models for further large-scale studies in vivo.Methods:1. The cDNA encoding mouse ICAM-1was construced from mouse spleen cells by reverse transcription polymerase chain reaction (RT-PCR). Then, the mouse ICAM-1cDNA was inserted in pMD19-T vector by restriction enzyme and DNA sequencing, and consequently it was cloned into the retroviral vector MIGR1. Restriction enzyme and DNA sequencing were then used to investigate whether the recombinant vector MIGR1-ICAM-1was correctly built.2. The reconstructed plasmid MIGR1-ICAM-1, empty plasmid MIGR1and packaging plasmid ECOS were transfected into T293cell lines, respectively, and then the supernatant from T293cells were used to infect mouse MSCs cell line C3H10T1/2. Fluorescence microscope, real-time PCR and flow cytometry were used to examine the expression of ICAM-1protein. Besides, cell biological characteristics like cell phenotype, growth characteristics, osteogenic and adipogenic differentiation were examined. In addition, LTT and MLR were introduced to detect the immunological characteristics of these three types of cells.3. Animal model of human AIT was set up in5-week old C57BL/6mice. C57BL/6mice for experimental groups were injected with Porcine Thyroglobulin (PTg) and Freund’s adjuvant (FA) at day0and day14. Peripheral blood was obtained from the retro-orbital sinus at day28. The expression of TgAb、TPOAb、 TMAb in serum were detected. Besides, the thyroid tissue was isolated and hematoxylin and eosin (H&E) was used for histology examination.Results:1. The results showed that1700bp ICAM-1gene fragment was obtained by enzyme digestion and RT-PCR. DNA sequencing further determined the same gene sequence of mouse ICAM-1in gene bank, these data suggest that recombinant retroviral vector MIGR1-ICAM-1was successful construced.2. After virus infection, results showed that the majority of the C3H10T1/2cells expressed green fluorescence under inverted fluorescence microscope, ICAM-1was up-regulated both in mRNA (1625.82±119.42) and protein (90.3%cells expressed ICAM-1) levels. These data suggest that we obtaine the overexpression ICAM-1MSC cell line (C3H10T1/2-MIGR1-ICAM-1/MSC).3. Growth characteristics, cell differentiation and immunological function of C3H10T1/2cells with a high expression of ICAM-1were examined. Results showed that overexpression ICAM-1could induced (29.18±2.47)%cell in S stage and decreased the cell proliferation doubling time. In adipogenic culture system, quantity of adipocytes was significantly decreased in C3H10T1/2-MIGR1-ICAM-1/MSC and lipid droplets were observed smaller. The key transcript factor for adipocyte C/EBPa and PPARγ was significantly down-regulated. Similarly, in ostegenic culture system, activity of alkaline phosphatase and the ability of mineralized bone nodules in overexpression ICAM-1MSCs were obviously downregulating, the mRNA expression levels of Runx2and Osteocalcin were also significant decreased, indicating that ICAM-1overexpression could down regulate the ability of adipogenic and ostegenic induction.4. Furthermore, to investigate the immunoregulatory effect of overexpression ICAM-1in MSCs, we used lymphocyte transformation test (LTT) and mixed allogeneic lymphocytes (MLR) in vitro. We found that in LTT system, when MSCs:T cells was1:40and1:5, the cpm value of C3H10T1/2-MIGR1-ICAM-1/MSC was significant decreased compared with control. The cpm value was (1955.25±292.60)、(2285.33±303.67);(812.00±21.17).(1335.00±209.18). In the MLR system, a higher stimulator cell/effector cell ratio (1:1) could significantly down-regulate T cells proliferation, which cpm value for C3H10T1/2-MIGR1-ICAM-1/MSC and C3H10T1/2-MIGR1/MSC was1598.50±110.74and1859.00±111.25. As the ratio decreased to1:25, cpm value for above groups was1147.50±55.91and1493.00±98.33, respectively.These data implied that overexpression ICAM-1in MSCs was capable of suppressing T cells expansion in a dose-dependent manner. There was a dramatic reduction in the T cells proliferation response in MSCs coculture system according to the MSCs cell number. 5. After C57BL/6mice were immunized subcutaneously exogenous with thyroid globulin, the concentration of TgAb, TPOAb and TMAb in experimental group (60.36±4.53,67.47±6.25, and65.67±5.35) was dramatic increased compared with control group (21.45±4.66,7.68±2.76, and7.59±2.23). Lymphocyte infiltration was observed in follicular stromal by thyroid histological analysis which was typical EAT feature.Conclusions:1. ICAM-1overexpression MSCs cell line (C3H10T1/2-MIGR1-ICAM-1/MSC) was obtained through establishing the recombinant vector of MIGR1-ICAM-1.2. MSCs with an overexpression of ICAM-1showed an enhanced proliferation, decreased osteogenic and adipogenic differentiation ability. The most important is that overexpression of ICAM-1can significantly enhance the immunosuppressive effect of MSCs. Providing experimental basis for further studies on whether ICAM-1transfected MSCs have effects on the treatment of AIT in vivo.3. This study successfully established the EAT model by using exogenous thyroid autoantigen. It was confirmed to be with a high success rate, high feasibility and high repeatability. To provide reliable animal models for further studies in vivo. |
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