肠上皮是机体内最大的免疫系统之一，一般生物体内肠上皮细胞的寿命为4~5天。本研究旨在从树鼩小肠分离小肠上皮细胞，探索分离培养及永生化方法和条件，建立永生化树鼩小肠上皮细胞系，补充树鼩这一新资源的基础实验材料，为应用树鼩开展药物开发和病原感染机制研究提供体外细胞模型。

首先，采用胶原酶Ⅺ和中性蛋白酶Ⅰ及DTT联合消化树鼩小肠组织块，得到的细胞经完全培养液培养，用相差显微镜观察细胞形态，并采用细胞角蛋白荧光染色等方法鉴定细胞。经优化分离法得到的肠上皮隐窝细胞团在24h后贴壁，6d汇合成片，在显微镜下观察发现细胞团延辐射状向外长出铺路石样和多角状的细胞；第二代后，每隔2~3d可传代1次；细胞接种3~6d为对数生长期；细胞角蛋白18进行免疫荧光鉴定后确定为小肠上皮细胞。因此成功地建立了树鼩小肠上皮细胞体外培养的方法。

 端粒酶是由端粒逆转录酶催化亚基和端粒酶相关RNA组成的多蛋白复合体。端粒酶可以端粒重复序列加在染色体末端，保护端粒不在染色体复制过程中受到损耗。所以，我们将人的端粒酶基因（hTERT）转入到树鼩肠上皮细胞中来突破细胞有丝分裂的Hayflick极限。把hTERT DNA序列连接pHBLV-CMVIE-ZsGreen-Puro慢病毒载体，在体外包装慢病毒；用稀释计数法检测病毒滴度；以感染复数MOI=10加入病毒原液，72h后荧光显微镜观察转染效果并计算转染效率；转染15d后加入筛选剂puromycin，连续筛选3代；将阳性转染的细胞接种于96孔板，稀释至1个细胞/孔，获得单克隆。经过体外包装的慢病毒感染树鼩肠上皮细胞72h后在荧光显微镜下观察，转染率为6.3%；经过15d的筛选和纯化，最终有4个孔获得了单克隆细胞，分别命名为TIEC1、TIEC2、TICE3和TIEC4。

对转染后的细胞进行生物学特性的研究。用RT-PCR的方法检测外源性hTERT mRNA以及免疫组化法检测hTERT蛋白的表达；CK18、pan-cytokeratin、occludin抗体对TIEC1细胞进行免疫荧光染色；Western blot 检测；生长曲线分析以及确定最佳血清浓度； TIEC1细胞的染色体分析；沙门菌感染TIEC1细胞和LPS刺激TIEC1细胞后，用real-time PCR方法检测相关免疫因子的表达。结果发现TIEC1细胞相比原代树鼩肠上皮细胞外源性hTERT mRNA表达为阳性。同时免疫组化TIEC1细胞被染成深褐色，而原代细胞染成浅褐色；CK18、pan-cytokeratin、occludin抗体对TIEC1细胞进行免疫荧光染色均为阳性，说明TIEC1细胞是上皮来源的细胞；TIEC1细胞较树鼩原代肠上皮细胞有更高的增殖活性，生长曲线显示TIEC1细胞提前进入稳定期；TIEC1细胞在5%血清浓度下增殖情况较好，染色体分析结果显示为62条染色体，与正常生理条件下的树鼩细胞染色体数目一致；TIEC1细胞对沙门菌感染和LPS刺激后相关免疫因子均上调，说明TIEC1能够保持原有生物学功能，对病原产生免疫应答。

总之，本研究通过慢病毒介导的hTERT过表达载体感染树鼩肠上皮细胞后，获得了阳性转染的细胞，经过鉴定后，确定该细胞为永生化的树鼩肠上皮细胞TIEC1，并且保持了原代细胞的特性，能够对病原刺激产生免疫应答。该细胞系的建立，为今后树鼩肠道病原感染、细胞信号转导、药物研究等方面提供了可备选的细胞模型。

Intestinal epithelium is one of the largest immune systems in the body. the general life  time of intestinal epithelial cells is 4~5 days. The small intestinal epithelial cells(IECs) were  isolated from the small intestine of tree shrews to explore the methods and conditions of the cells isolation, to provide an in vitro cell model for drug development and pathogen infection mechanism and to establish small intestinal epithelial cell lines of tree shrews.

First, the small intestine tissue of tree shrews was digested with collagenase XI and neutral protease I and DTT. The cells were cultured in complete culture medium. Cell morphology was observed by phase contrast microscope. Fluorescence staining and other methods were using to identify the cells. The attachment of intestinal epithelial crypt cells obtained by optimized separation performed in 24 hours, Epithelial outgrowth from isolated crypts were formed with confluent monolayers cobblestone and multi-angular morphology. After the second generation, cells can be passaged every 2~3 days; IECs can be identified by cytokeratin 18 immunofluorescence. Therefore, the method of isolating IECs from tree shrews was successfully established.

   Telomerase is a polyprotein complex consisting of telomeric reverse transcriptase catalytic subunit and telomerase-related RNA. Telomerase add telomeric repeats to the end of chromosome , protecting telomeres from loss of chromosome replication. Therefore, we transfected the human telomerase gene (hTERT) into the tree shrew’s IECs to break the cell mitotic Hayflick limit. The hTERT DNA sequence was ligated with pHBLV-CMVIE-ZsGreen-Puro lentivirus vector, and the virus titer was detected by dilution counting method. The transfection efficiency was observed by fluorescence microscope after 72 hours of infection with MOI = 10. The transfection rate was calculated. After 15 days of transfection, the agent puromycin was added into the 3rd generation of cells. The positive transfected cells were inoculated into 96-well plates and diluted to 1 cell / well to obtain monoclonal. The transfection rate was 6.3%. After 15 days of screening and purification, four wells of monoclonal cells were obtained named TIEC1, TIEC2, TICE3 and TIEC4 respectively.

The biological characteristics of the transfected cells were studied. The expression of hTERT mRNA level was detected by RT-PCR. The expression of hTERT protein was detected by immunohistochemistry. Anti-CK18, pan-cytokeratin and occludin were used to fluorescent staining of the TIEC1 cells. The growth curve was analyzed and the optimal serum concentration was determined. The expression of the TIEC1 cells’ relevant immunological factors after stimulated by S.T. and LPS was detected by real-time PCR. The results showed TIEC1 cells positive by hTERT mRNA expression compared to the IECs of primary tree shrews. At the same time, the immunohistochemical staining of TIEC1 cells were dark brown compared with light brown of the primary cells . Anti-CK18, pan-cytokeratin and occludin fluorescent staining showed TIEC1 cells positive, indicating that TIEC1 cells were epithelial cells; The cell growth rate of TIEC1 cells was higher than the primary intestinal epithelial cells, which showed that the proliferation rate of TIEC1 cells was higher than that of the primary intestinal epithelial cells. The number of chromosomes in tree shrews was the same with the primary intestinal epithelial cells under physiological conditions. TIEC1 cells’ Immune - related factors were up-regulated by Salmonella infection and LPS stimulation, which indicated that TIEC1 cells could perform immune response to pathogen.

In conclusion, the positive transfected cells of the tree shrew were obtained by lentiviral-mediated hTERT overexpressing vector. After identification, it shows the cells were immortalized tree shrew intestinal epithelial cell line--TIEC1, and the TIEC1 cells maintained The characteristics of primary cells, which can perform immune response to pathogen stimulation. For intestinal infection, cell Signaling, intestinal drug research and other aspects ,TIEC1 can be considered alternative cell model.