王进 董晓强 赵鑫 朱新国 赵华 张江磊

【摘要】目的 建立穩定过表达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。

【关键词】慢病毒转染;IL-9基因;CT-26肿瘤细胞;BALB/C小鼠;异种移植瘤模型

【Abstract】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 293T 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 mRNA was measured by qRT-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 mRNA was measured by qRT-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 293T cells didnt 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 mRNA 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 mRNA 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.

【Key words】Lentiviral transfection;IL-9 gene;CT-26 cell;BALB/C mouse;Xenograft model

结肠癌是消化道常见的恶性肿瘤之一，其全球发病率居于第3位[1]。在我国，结肠癌的发病率及病死率呈现逐年上升趋势，每年因结肠癌死亡人数在恶性肿瘤中居于第5位[2]。结肠癌除传统的手术、化学治疗及放射治疗等治疗方式，免疫基因治疗逐渐呈现其优越性，因此寻找免疫治疗的新策略对于结肠癌的治疗至关重要[3-4]。

IL-9最早于1988年由Uyttenhove等在建立的多个Th2细胞株中发现，主要来源于活化的Th9细胞，近年来逐渐发现IL-9具有抗肿瘤免疫作用[5-6]。……