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The Expression and Significance of Indoleamine 2, 3-dioxygenase in 4T1 Cells after Transfection

Author: ZhengLei
Tutor: LiuJunTian;YuJinPu
School: Tianjin Medical University
Course: Oncology
Keywords: Indoleamine 2,3-Dioxygenase(IDO) Metastasis Transfection 4T1 cells Apoptosis
CLC: R737.9
Type: Master's thesis
Year: 2010
Downloads: 70
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Abstract


Objective:1. Through the establishment of mouse breast cancer metastasis model, investigating the effects and possible mechanisms of indoleamine 2,3-dioxygenase (IDO) during the metastasis of breast cancer.2. Detection of the expression and significance of IDO in 4T1 cells after transfection. To detect the inhibitory action of trasfected 4T1 cells to the immune system, and exploring the possible mechanisms of metastasis by trasfected 4T1 cells.Methods:1. After the establishment of 4T1 mouse breast cancer metastasis model, RT-PCR, Western blot and Immunohistochemistry were used to detect the expression of IDO in mouse cancer tissues both not having and having metastasis.2. After the establishment of 4T1 cell line which was high expression of IDO gene by transfection,RT-PCR and Western blot were used to detect the expression of IDO in transfected 4T1 cells.The transfected 4T1 cells co-cultured with T cells, then Flow cytometer was used to detect the percentage of apoptosis of T cells, and Western blot was used to detect the expression of Fas/FasL and Bcl-2/Bax of co-cultured T cells.Results:1. RT-PCR results showed that the expression of IDO was negative in 4T1 cells, and it can not be up-regulated by IFN-y.2. Immunohistochemistry results showed that the percentage of IDO+cells in both primary cancer[(12.40±5.15)%] and metastatic carcinoma[(14.28±6.46)%] of mouse breast cancer having metastasis was higher than that in cancer tissues of mouse breast cancer not having metastasis[(3.16±1.63)%](P<0.05). The expression of IDO in metastatic carcinoma of mouse breast cancer having metastasis was higher than primary cancer of it, but having no statistic difference(P>0.05). RT-PCR and Western blot results were consistent with Immunohistochemistry results.3. Taking of IDO gene exon(CDS) region, then cloning it into the pMD19-T simple vector and being sequenced.Joining it into the eukaryotic expression vector pIRES2-EGFP, and verifying the connection successfully. 4. The eukaryotic expression vector pIRES2-EGFP-IDO transfected 4T1 cells, and making the control group of pIRES2-EGFP transfected 4T1 cells. RT-PCR and Western blot showed that the expression of IDO in pIRES2-EGFP-IDO transfected 4T1 cells was positive, but negative in both control group and common 4T1 cells.5. pIRES2-EGFP-IDO transfected 4T1 cells, pIRES2-EGFP transfected 4T1 cells and common 4T1 cells respectively co-cultured with T cells, and taking of the T cells which didn’t co-culture with 4T1 cells as control group. Flow cytometer was used to detect the percentage of apoptosis of T cells. After 48 hours, Flow cytometer results showed that the percentage of apoptosis of CD3+T cells which co-cultured with pIRES2-EGFP-IDO transfected 4T1 cells was (47.07±6.13)%, T cells which co-cultured with pIRES2-EGFP-IDO transfected 4T1 cells had obviously apoptosis.6. Taking of the T cells which co-cultured with pIRES2-EGFP-IDO transfected 4T1 cells, pIRES2-EGFP transfected 4T1 cells, common 4T1 cells and the T cells which didn’t co-culture with 4T1 cells. To detect the expression of Fas/FasL and Bcl-2/Bax proteins among them. Western blot results showed that the expression of Fas,FasL and Bax proteins in T cells which co-cultured with pIRES2-EGFP-IDO transfected 4T1 cells was higher than others(P<0.05). On the contrary, the expression of Bcl-2 protein in T cells which co-cultured with them was lower than others(P<0.05).Conclusion:1. The expression of IDO in primary cancer and metastatic carcinoma of breast cancer having metastasis was both higher than that in cancer tissues of breast cancer not having metastasis. It shows that IDO may play a role in the process of breast cancer metastasis.2. After the transfected 4T1 cells co-cultured with T cells, the percentage of apoptosis of T cells rised. Western blot results showed that the expression of Fas,FasL and Bax proteins increased, but the expression of Bcl-2 protein decreased. It means that IDO may induce the apoptosis of T cells by inside and outside the two channels, then Suppress the immune system function and accelerate the tumor metastasis.

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