稳定过表达IL-9的小鼠CT-26肿瘤细胞株及BALB/C小鼠皮下移植瘤模型的建立
|
摘要
目的建立稳定过表达IL-9的小鼠CT-26肿瘤细胞株及BALB/C小鼠皮下移植瘤模型,为后续研究IL-9在结肠癌肿瘤微环境中的作用及其机制提供实验基础。方法将目的基因IL-9片段插入带有绿色荧光蛋白(GFP)的慢病毒载体GV492,构建重组过表达IL-9-GV492质粒,转染293T细胞后,用倒置荧光显微镜观察GFP的表达强度,用蛋白免疫印迹法检测IL-9的表达;包装病毒后检测病毒滴度。将过表达IL-9的慢病毒转染小鼠结肠癌CT-26细胞后,用倒置荧光显微镜观察GFP的表达强度,用流式细胞仪检测GFP及IL-9的表达,用qRT-PCR法检测IL-9的mRNA表达。过表达IL-9的CT-26细胞皮下接种BALB/C小鼠建立皮下移植瘤模型后,用免疫组化法检测肿瘤中IL-9的蛋白表达,用qRT-PCR法检测IL-9的mRNA表达。结果阳性重组质粒PCR扩增产物大小约617 bp,DNA测序显示重组质粒的编码序列与目标序列完全一致。293T细胞自身不表达IL-9,重组质粒转染293T细胞后表达IL-9;慢病毒滴度为2×109 TU/ml。慢病毒转染小鼠结肠癌CT-26细胞后,Control组、LV-NC组、LV-IL-9组GFP表达阳性率分别为0、96.4%、91.2%;LV-IL-9组IL-9的MFI及mRNA的表达均高于LV-NC组(P均<0.05)。BALB/C小鼠皮下移植瘤模型建立后,实验组小鼠肿瘤IL-9的阳性表达率及mRNA的表达均高于阴性对照组(P均<0.05)。结论稳定过表达IL-9的小鼠结肠癌CT-26细胞株及BALB/C小鼠皮下移植瘤模型建立成功。
Objective To construct the CT-26 cell lines stably over-expressing IL-9 gene and establish the BALB/C mouse models with subcutaneous xenograft, aiming to provide experimental basis for further study of the function and mechanism of IL-9 in the tumor microenvironment of colon cancer. Methods The IL-9 gene fragment was inserted into the lentiviral plasmid GV492 which carried green fluorescent protein( GFP) to establish the recombinant IL-9-GV492. After the 293 T cells were transfected, the expression intensity of GFP was observed under an inverted fluorescence microscope and the expression level of IL-9 was measured by Western blot. The virus titer was detected after packaging the lentiviral vectors. After the lentivirus over-expressing IL-9 were transfected with the CT-26 cells, the expression intensity of GFP was observed under an inverted fluorescence microscope. The expression levels of GFP and IL-9 were measured by flow cytometry. The expression level of IL-9 m RNA was measured by q RT-PCR. The BALB/C mouse models with subcutaneous xenograft were established by subcutaneous inoculation of IL-9-CT-26 cells. The expression level of IL-9 protein in the tumors was detected immunohistochemistry, and the expression level of IL-9 m RNA was measured by q RT-PCR. Results The PCR amplified product of positive recombinant plasmid was approximately 617 bp in size. DNA sequencing demonstrated that the coding sequence of the recombinant plasmid was completely consistent with the designed sequence. The 293 T cells didn't express IL-9, whereas expressed IL-9 after the cells were transfected with the recombinant plasmid. The virus titer of the packaged lentivirus was 2×109 TU/ml. After the CT-26 cells were transfected with the lentivirus over-expressing IL-9, the positive expression rates of GFP in the control, LV-NC and LV-IL-9 groups were 0, 96.4% and 91.2%, respectively. The IL-9 expression( MFI) and IL-9 m RNA in the LV-IL-9 group were significantly higher than those in the LV-NC group(all P < 0.05). After the BALB/C mouse models with subcutaneous xenograft model were established, the expression rate of IL-9 and the expression level of IL-9 m RNA in the experimental group were significantly higher compared with those in the control group(all P < 0.05). Conclusions The CT-26 cell lines stably over-expressing IL-9 gene and the BALB/C mouse models with subcutaneous xenograft model are successfully established.
Objective To construct the CT-26 cell lines stably over-expressing IL-9 gene and establish the BALB/C mouse models with subcutaneous xenograft, aiming to provide experimental basis for further study of the function and mechanism of IL-9 in the tumor microenvironment of colon cancer. Methods The IL-9 gene fragment was inserted into the lentiviral plasmid GV492 which carried green fluorescent protein( GFP) to establish the recombinant IL-9-GV492. After the 293 T cells were transfected, the expression intensity of GFP was observed under an inverted fluorescence microscope and the expression level of IL-9 was measured by Western blot. The virus titer was detected after packaging the lentiviral vectors. After the lentivirus over-expressing IL-9 were transfected with the CT-26 cells, the expression intensity of GFP was observed under an inverted fluorescence microscope. The expression levels of GFP and IL-9 were measured by flow cytometry. The expression level of IL-9 m RNA was measured by q RT-PCR. The BALB/C mouse models with subcutaneous xenograft were established by subcutaneous inoculation of IL-9-CT-26 cells. The expression level of IL-9 protein in the tumors was detected immunohistochemistry, and the expression level of IL-9 m RNA was measured by q RT-PCR. Results The PCR amplified product of positive recombinant plasmid was approximately 617 bp in size. DNA sequencing demonstrated that the coding sequence of the recombinant plasmid was completely consistent with the designed sequence. The 293 T cells didn't express IL-9, whereas expressed IL-9 after the cells were transfected with the recombinant plasmid. The virus titer of the packaged lentivirus was 2×109 TU/ml. After the CT-26 cells were transfected with the lentivirus over-expressing IL-9, the positive expression rates of GFP in the control, LV-NC and LV-IL-9 groups were 0, 96.4% and 91.2%, respectively. The IL-9 expression( MFI) and IL-9 m RNA in the LV-IL-9 group were significantly higher than those in the LV-NC group(all P < 0.05). After the BALB/C mouse models with subcutaneous xenograft model were established, the expression rate of IL-9 and the expression level of IL-9 m RNA in the experimental group were significantly higher compared with those in the control group(all P < 0.05). Conclusions The CT-26 cell lines stably over-expressing IL-9 gene and the BALB/C mouse models with subcutaneous xenograft model are successfully established.
引文
[1]曾一文,黎洁瑶,田旷怡,于涛,许稷豪,陈其奎.通过AOM及AOM联合DSS建立C57BL/6J小鼠结肠癌诱导模型的对比研究.新医学,2018,49(10):720-725.
[2]Chen W,Zheng R,Baade PD,Zhang S,Zeng H,Bray F,Jemal A,Yu XQ,He J.Cancer statistics in China,2015.CACancer J Clin,2016,66(2):115-132.
[3]Naldini L.Gene therapy returns to centre stage.Nature,2015,526(7573):351-360.
[4]Yang Y.Cancer immunotherapy:harnessing the immune system to battle cancer.J Clin Invest,2015,125(9):3335-3337.
[5]Uyttenhove C,Simpson RJ,Van Snick J.Functional and structural characterization of P40,a mouse glycoprotein with T-cell growth factor activity.Proc Natl Acad Sci U S A,1988,85(18):6934-6938.
[6]Nakatsukasa H,Zhang D,Maruyama T,Chen H,Cui K,Ishikawa M,Deng L,Zanvit P,Tu E,Jin W,Abbatiello B,Goldberg N,Chen Q,Sun L,Zhao K,Chen W.The DNA-binding inhibitor Id3 regulates IL-9 production in CD4(+)Tcells.Nat Immunol,2015,16(10):1077-1084.
[7]Purwar R,Schlapbach C,Xiao S,Kang HS,Elyaman W,Jiang X,Jetten AM,Khoury SJ,Fuhlbrigge RC,Kuchroo VK,Clark RA,Kupper TS.Robust tumor immunity to melanoma mediated by interleukin-9-producing T cells.Nat Med,2012,18(8):1248-1253.
[8]Lu Y,Hong S,Li H,Park J,Hong B,Wang L,Zheng Y,Liu Z,Xu J,He J,Yang J,Qian J,Yi Q.Th9 cells promote antitumor immune responses in vivo.J Clin Invest,2012,122(11):4160-4171.
[9]Miao BP,Zhang RS,Sun HJ,Yu YP,Chen T,Li LJ,Liu JQ,Liu J,Yu HQ,Zhang M,Liu ZG,Yang PC.Inhibition of squamous cancer growth in a mouse model by Staphylococcal enterotoxin B-triggered Th9 cell expansion.Cell Mol Immunol,2017,14(4):371-379.
[10]Abdul-Wahid A,Cydzik M,Prodeus A,Alwash M,Stanojcic M,Thompson M,Huang EH,Shively JE,Gray-Owen SD,Gariépy J.Induction of antigen-specific TH 9 immunity accompanied by mast cell activation blocks tumor cell engraftment.Int J Cancer,2016,139(4):841-853.
[11]Huang Y,Cao Y,Zhang S,Gao F.Association between low expression levels of interleukin-9 and colon cancer progression.Exp Ther Med,2015,10(3):942-946.
[12]Touzot F,Hacein-Bey-Abina S,Fischer A,Cavazzana M.Gene therapy for inherited immunodeficiency.Expert Opin Biol Ther,2014,14(6):789-798.
[13]Misra S.Human gene therapy:a brief overview of the genetic revolution.J Assoc Physicians India,2013,61(2):127-133.
[14]Kaufmann KB,Büning H,Galy A,Schambach A,Grez M.Gene therapy on the move.EMBO Mol Med,2013,5(11):1642-1661.
[15]Tang H,Qiao J,Fu YX.Immunotherapy and tumor microenvironment.Cancer Lett,2016,370(1):85-90.
[16]Luo M,Liang X,Luo ST,Wei XW,Liu T,Ren J,Ma CC,Yang YH,Wang BL,Liu L,Song XR,He ZY,Wei YQ.Folate-modified lipoplexes delivering the interleukin-12 gene for targeting colon cancer immunogene therapy.J Biomed Nanotechnol,2015,11(11):2011-2023.
[2]Chen W,Zheng R,Baade PD,Zhang S,Zeng H,Bray F,Jemal A,Yu XQ,He J.Cancer statistics in China,2015.CACancer J Clin,2016,66(2):115-132.
[3]Naldini L.Gene therapy returns to centre stage.Nature,2015,526(7573):351-360.
[4]Yang Y.Cancer immunotherapy:harnessing the immune system to battle cancer.J Clin Invest,2015,125(9):3335-3337.
[5]Uyttenhove C,Simpson RJ,Van Snick J.Functional and structural characterization of P40,a mouse glycoprotein with T-cell growth factor activity.Proc Natl Acad Sci U S A,1988,85(18):6934-6938.
[6]Nakatsukasa H,Zhang D,Maruyama T,Chen H,Cui K,Ishikawa M,Deng L,Zanvit P,Tu E,Jin W,Abbatiello B,Goldberg N,Chen Q,Sun L,Zhao K,Chen W.The DNA-binding inhibitor Id3 regulates IL-9 production in CD4(+)Tcells.Nat Immunol,2015,16(10):1077-1084.
[7]Purwar R,Schlapbach C,Xiao S,Kang HS,Elyaman W,Jiang X,Jetten AM,Khoury SJ,Fuhlbrigge RC,Kuchroo VK,Clark RA,Kupper TS.Robust tumor immunity to melanoma mediated by interleukin-9-producing T cells.Nat Med,2012,18(8):1248-1253.
[8]Lu Y,Hong S,Li H,Park J,Hong B,Wang L,Zheng Y,Liu Z,Xu J,He J,Yang J,Qian J,Yi Q.Th9 cells promote antitumor immune responses in vivo.J Clin Invest,2012,122(11):4160-4171.
[9]Miao BP,Zhang RS,Sun HJ,Yu YP,Chen T,Li LJ,Liu JQ,Liu J,Yu HQ,Zhang M,Liu ZG,Yang PC.Inhibition of squamous cancer growth in a mouse model by Staphylococcal enterotoxin B-triggered Th9 cell expansion.Cell Mol Immunol,2017,14(4):371-379.
[10]Abdul-Wahid A,Cydzik M,Prodeus A,Alwash M,Stanojcic M,Thompson M,Huang EH,Shively JE,Gray-Owen SD,Gariépy J.Induction of antigen-specific TH 9 immunity accompanied by mast cell activation blocks tumor cell engraftment.Int J Cancer,2016,139(4):841-853.
[11]Huang Y,Cao Y,Zhang S,Gao F.Association between low expression levels of interleukin-9 and colon cancer progression.Exp Ther Med,2015,10(3):942-946.
[12]Touzot F,Hacein-Bey-Abina S,Fischer A,Cavazzana M.Gene therapy for inherited immunodeficiency.Expert Opin Biol Ther,2014,14(6):789-798.
[13]Misra S.Human gene therapy:a brief overview of the genetic revolution.J Assoc Physicians India,2013,61(2):127-133.
[14]Kaufmann KB,Büning H,Galy A,Schambach A,Grez M.Gene therapy on the move.EMBO Mol Med,2013,5(11):1642-1661.
[15]Tang H,Qiao J,Fu YX.Immunotherapy and tumor microenvironment.Cancer Lett,2016,370(1):85-90.
[16]Luo M,Liang X,Luo ST,Wei XW,Liu T,Ren J,Ma CC,Yang YH,Wang BL,Liu L,Song XR,He ZY,Wei YQ.Folate-modified lipoplexes delivering the interleukin-12 gene for targeting colon cancer immunogene therapy.J Biomed Nanotechnol,2015,11(11):2011-2023.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |