| Aim:1. To develop and master the operation methods of heterotopic cervical heart transplantation in mice.2.Select C57BL/6 , Balb/c and treditional gene-knockout mice as the object,and heterotopic transverse part cardiac transplantation was performed to investigate the effect of the transcription factor RBP-J in the Notch/ RBP-J pathway on subgroup T cells after allogene heart transplantation in mice.Mathods:1.Under microscopy , the right external jugular vein and the common carotid artery of the recipient were liberated . After heparinization all over the body of the donor ,the heart was obtained in low temperature. The innominate artery of the donor heart and the right common carotid artery of the recipient were anastomosed using the end-to-end interrupted suture technique.The same method was used in the connection between the pulmonary artery of the donor heart and the right external jugular vein of the recipient.2. Generated Lck-Cre x RBP-J flox/flox mouse in which RBP-J, the key transcription factor of Notch pathway,can be specifically inactivated in T cells.Identified lck-cre mouse lines in which RBP-J can be completely inactivated in T cells by PCR.And select gene-knockou mice as the object.3. A total of 32mice of 3 inbred lines(C57BL/6, Balb/c and conditional knockout C57BL/6) were randomly divided into 2 groups. Group A(n=8)had aniso-strain operation(BALB/c→C57BL/6); group B(n=8)got the same operation (BALB/c→conditional knockout RBP-J C57BL/6). Samples of grafts were taken 6 days after transplantation, then allograft were detected by pathology.4. Peripheral blood was stringed blood routine examination. Lymphoid nodes and spleens were taken after graft stopped work,and the T cells were isolated from spleens,then marked with anti-mouse CD4-PE,anti-mouse CD8-APC, anti-mouse CD25-FITC ,and other colored immune fluorescence antibodies to detect the quantity of sub-T cell with flow cytometerResult:1. We developed the operation methods,the result showed that the the total ischemic time of the graft was 40 min, the rate of the heart re-beat was increased to 90%.2.The mice of conditional knockout transcriptional factor RBP-J is got and identified successfully,the mice is available which lays the foundation for the study of the effect of the transcription factor RBP-J in the Notch/ RBP-J pathway on sub-T cells after allogene heart transplantation in mice.3. Conditional Knockout RBP-J in mice shorted survival time to 4±1.8 days compared to 6±1.21 days in group C57 (p<0.01).Acute rejection pathologically was observed in group A and B.The proliferation of RBP-J deficient T cells,epecially in CD8+ T cells, increased a lot in response to the allograft compared with group C57 (P<0.01),but the Treg cell decreased.Conclusions:1. The advantage of this model lies in easy exposition, short duration of the operation, easy treatment of the blood vessel, low probability of contaminate and high rate of success,so deserves to use in experimental study widely.2. We get the mice in which conditional knockout transcriptional factor RBP-J in T cell, We identified thelck-cre mouse line in which RBP-J can be completely inactivated in T cells by PCR.3.Through established the heterotopic cervical heart transplantation,we found that the the transcription factor RBP-J influenced the acute rejection. The proliferation of RBP-J deficient T cells,epecially in CD8+ T cells, increased a lot,and the Treg cell decreased.These results convinced us to make a deep research on the relationship between the Notch pathway and the transplantation rejection. |
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