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Objective@#To establish a nude mouse model of subcutaneous lung cancer using dual fluorescence reporting genes of luciferase (Luc) and near-infrared fluorescent protein (iRFP).@*Methods@#The Luc and iRFP expressed lentiviral vector was constructed by Gateway method. After verified by sequencing, the lentivirus particle was prepared and infected into lung cancer A549 cells. Successfully infected A549 (mA549) cells were selected by puromycin and amplified. The expression of Luc and iRFP were observed under fluorescence microscope, and the expression of c-Met protein on the cell surface was detected by immunofluorescence. Twelve female nude mice were randomly divided into 2 groups, 6 in each group. A549 and mA549 cells were inoculated subcutaneously into the right forelimb of nude mice. The growth and fluorescence expression of the tumor were observed by in vivo imaging. The tumor formation was evaluated by hematoxylin-eosin (HE) staining and immunohistochemistry.@*Results@#The Luc and iRFP stably expressed mA549 cell line was successfully constructed. The expressions of iRFP and Luc in mA549 cells were observed under fluorescence microscope. The results of immunofluorescence showed that c-Met protein expressed in both A549 cells and mA549 cells. The growth period of mA549 xenograft in nude mice was moderate and the tumorigenesis rate was 100%. The growth trend of mA549 cells in vivo was not significantly different from that of A549 cells (P>0.05). HE staining and immunohistochemistry results showed that the tumor issues displayed typical histopathological features of tumor. Immunohistochemistry results showed that both A549 and mA549 tumors expressed c-Met protein.@*Conclusion@#A stable, real-time monitoring model of iRFP-Luc-A549 lung cancer cell xenograft in nude mice was successfully constructed.
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Objective@#To study the role of Treg cells in the development of mouse experimental autoimmune encephalomyelitis (EAE) through depleting or transplanting Treg cells.@*Methods@#C57BL/6 mice were injected with anti-CD25 monoclonal antibody to deplete natural CD25-expressing Treg cells in vivo, and then treated with MOG35-55/CFA to induce EAE. Their EAE scores were compared with those of the mice without Treg cell deletion (control group). The numbers and percentages of CD4+ Foxp3+ cells in mouse blood samples on 6 d, 10 d, 20 d and 35 d were quantified using flow cytometry. To evaluate the therapeutic effect of Treg cells transplantation on EAE, magnetic activated cell sorting (MACS) combined with fluorescence-activated cell sorting (FACS) was used to isolate Treg cells from spleen and lymph nodes of Foxp3GFP+ transgenic mice on 6 d after EAE induction. Then the cells were injected through tail vein into wild-type mice on 6 d after EAE induction. The EAE scores of both recipient and control mice were recorded and compared.@*Results@#The efficiency of natural Treg cells depletion with anti-CD25 antibody was above 95%. The mice with Treg cell depletion developed significantly more severe EAE than the control mice after MOG35-55/CFA induction. FACS analysis of Treg cells during the development of EAE demonstrated that the lowest Treg cell percentage was detected on 6 d after EAE induction, hence it was the time point for the transplantation of Treg cells. CD4+ GFP+ Treg cells were isolated from Foxp3GFP+ transgenic mice on 6 d after EAE induction and immediately transplanted into wild-type mice on 6 d after EAE induction. The transplantation of isolated Treg cells significantly alleviated the EAE in mice as compared with the control group.@*Conclusions@#Mice with Treg cell depletion developed severer EAE than the control mice after induction, but the EAE score could be significantly reduced with the transplantation of Treg cells. This study showed that the transplanted Treg cells had protective effect on mice during the course of EAE development. Thus, Treg cell transplantation could be used as an effective therapeutic approach for the treatment of multiple sclerosis (MS).
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Objective To study the role of Treg cells in the development of mouse experimental autoimmune encephalomyelitis (EAE) through depleting or transplanting Treg cells. Methods C57BL/ 6 mice were injected with anti-CD25 monoclonal antibody to deplete natural CD25-expressing Treg cells in vi-vo, and then treated with MOG35-55 / CFA to induce EAE. Their EAE scores were compared with those of the mice without Treg cell deletion ( control group). The numbers and percentages of CD4+ Foxp3+ cells in mouse blood samples on 6 d, 10 d, 20 d and 35 d were quantified using flow cytometry. To evaluate the therapeutic effect of Treg cells transplantation on EAE, magnetic activated cell sorting (MACS) combined with fluorescence-activated cell sorting (FACS) was used to isolate Treg cells from spleen and lymph nodes of Foxp3GFP+ transgenic mice on 6 d after EAE induction. Then the cells were injected through tail vein into wild-type mice on 6 d after EAE induction. The EAE scores of both recipient and control mice were recorded and compared. Results The efficiency of natural Treg cells depletion with anti-CD25 antibody was above 95％ . The mice with Treg cell depletion developed significantly more severe EAE than the control mice after MOG35-55 / CFA induction. FACS analysis of Treg cells during the development of EAE demonstrated that the lowest Treg cell percentage was detected on 6 d after EAE induction, hence it was the time point for the transplantation of Treg cells. CD4+GFP+ Treg cells were isolated from Foxp3GFP+ transgenic mice on 6 d after EAE induction and immediately transplanted into wild-type mice on 6 d after EAE induction. The transplan-tation of isolated Treg cells significantly alleviated the EAE in mice as compared with the control group.Conclusions Mice with Treg cell depletion developed severer EAE than the control mice after induction, but the EAE score could be significantly reduced with the transplantation of Treg cells. This study showed that the transplanted Treg cells had protective effect on mice during the course of EAE development. Thus, Treg cell transplantation could be used as an effective therapeutic approach for the treatment of multiple scle-rosis (MS).
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Objective To explore the application value of 256 slice helical multi phase CT scan-ning and three-dimensional reconstruction in the diagnosis of malignant tumor of colorectal obstruction. Methods Using 256 slice spiral CT scanning and three-dimensional reconstruction of multi period in 42 ca-ses of malignant tumor patients with colorectal obstruction, the results and pathological results were analyzed and the postoperative stage.Results Forty-two cases of malignant tumors of colorectal obstruction, 256 slice spiral CT could well reflect the situation of location, range, degree, peripheral intestinal lymph node and distant metastasis, CTA could show the tumor supplying artery and branch sources, on the tumor loca-tion and overall accuracy.Conclusions 256 layer spiral CT scan and three dimensional reconstruction technology is accuracy for clinical diagnosis on malignant tumor of colorectal obstruction.
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Objective To investigate the survival of islet isograft in NOD mice treated with islet antigen-specific regulatory T cells.Methods GAD-65 antigen pulsed immature dendritic cells (imDC) were used to induce naive T cells into islet antigen-specific regulatory T cells.NOD mice which had progressed to type 1 diabetes (T1DM),as the recipients,received islet isografts (500 IEQ) under renal capsule from NOD mice without T1DM.In NOD mice in control group without transplantation,the changes in blood glucose (BG) were observed.NOD mice in simple islet transplantation group were given islet isograft without Treg infusion.In experiment group,NOD mice were infused with 1 × 106 islet antigen-specific regulatory T cells on the 1st day before transplantation,subsequently underwent islet isotransplantation.The survival of the islet isograft was evaluated by BG levels and the pathological changes were observed.Results BG levels were sustained above 11.1 mmol/L in control group.In simple islet transplantation group,BG level was decreased to the normal level in 1 ～2 days after transplantation,and began to rebound in 7～ 17 days posttransplantation and maintained at the preoperative level.The mean survival of the islet isograft in the NOD mice was (12.2 ± 2.6) day;In experiment group,BG level was decreased to the normal level in 1 ～2 days after transplantation,rebounded above 11.1 mmol/L in some mice on the 27th day after transplantation,and rebounded above 11.1 rnmol/L on the 43th day in all mice.The mean survival of the islet isograft in the NOD mice was (35.2 ± 4.3) days,which was significantly prolonged compared to simple islet transplantation group (P＜ 0.01).In simple islet transplantation group,the islet isograft was infiltrated by many lymph cells and damaged severely,and only few residual islet cells secreted insulin without complete islet existing in insulin staining.The islet isograft in experiment group was intact on the 15th day,with little lymph cell infiltration and a great number of islets secreting insulin.Conclusion Infusion of islet antigen-specific regulatory T cells induced by imDC and islet antigen GAD-65 in vitro could delay the destruction of autoimmune system and prolong the islet isograft survival in NOD mice.
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Objective To investigate the effects of donor-specific regulatory T cells (Treg) transfusion on islet allograft survival. Methods Allogeneic fresh islets from Balb/c mice were transplanted to streptozotocin-induced diabetic C57 mice. The survival of islet allografts was observed. The experiment was divided into 3 groups: control group, nothing had been done to the recipients; simple islet transplantation group, the recipients received the islet transplantation only; experimental group, the recipients were given 1 ×106 Treg, then received islet transplantation. Results Blood glucose (BG) was above 16. 7 mmol/L after islet transplantation in control group; In simple islet transplantation group,BG level returned to normal level 1 to 2 days after transplantation, and hyperglycemia appeared 7 to 11 days after transplantation and maintained as the same as that before transplantation; In experimental group, BG level returned to normal level 2 days after transplantation and maintained at a low level,and at the 21st day after transplantation BG level was over 16. 7mmol/L in some recipients. Islet allograft survival in experimental group was significantly prolonged as compared with simple islet transplantation group. Conclusion Donor-specific Treg transfusion could prolong the islet allograft survival,and maybe have positive effect on tolerance induction of islet transplantation.
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Objective:Differential expression analysis and cloning aged related genes of mouse thymus Methods:Different expressions of thymus mRNAs from 1 and 10 month old mouse were analyzed by DDRT PCR and different expression sequence tags (ESTs) were obtained One EST that represented high expressed level in one month mouse thymus was probed to screen mouse thymus cDNA library One 827 bp cDNA fragment was obtained and was extended to 1 406 bp by PCR Results:Homology analysis showed that mt22 1406 contained one 438AA coding region and showed high similarity with human elongation factor1?(EF1?) The Genbank accession number is BE241062 Conclusion:Cloning one gene related with mouse thymus aging