IL-35基因修饰间充质干细胞治疗小鼠溃疡性结肠炎的实验研究
发布时间：2018-11-14 13:24
【摘要】：本课题研究白介素35(interleukin35, IL-35)基因修饰间充质干细胞对小鼠溃疡性结肠炎的治疗作用,并探讨其作用机制。通过建立稳定的小鼠溃疡性结肠炎模型,经尾静脉注射IL-35基因修饰的间充质干细胞,观察其对小鼠急性溃疡性结肠炎的治疗效果,探索出治疗溃疡性结肠炎新途径。 一、IL-35基因修饰C57BL/6小鼠间充质干细胞制备 目的：构建IL-35过表达慢病毒载体,以其感染小鼠间充质干细胞,从而获得IL-35基因修饰的间充质干细胞。方法：采用I型胶原酶消化法获得小鼠脂肪来源间充质干细胞,并以流式细胞术(FCM)和定向诱导分化实验加以鉴定；应用DNA聚合酶将IL-35基因连接入慢病毒工作质粒,测序鉴定无误后与包装质粒共转染包装细胞获得过表达IL-35慢病毒颗粒；以过表达IL-35慢病毒颗粒感染小鼠间充质干细胞,采用酶联免疫吸附法(ELISA)法检测不同感染条件下细胞上清中IL-35的表达情况,从而获得IL-35基因修饰的间充质干细胞。结果：脂肪来源间充质干细胞获取成功；过表达IL-35慢病毒载体可以成功感染间充质干细胞,感染后细胞上清中IL-35蛋白浓度可达329.5±16.5pg/ml. 二、C57BL/6小鼠急性溃疡性结肠炎模型构建 目的：建立C57BL/6小鼠急性溃疡性模型。方法：应用不同浓度葡聚糖硫酸钠(DSS)喂养小鼠7日,每日记录体重变化及一般情况改变,第8日处死小鼠,观察结肠大体标本；将结肠标本石蜡包埋后切片检查,HE染色评估病理改变。结果：2%DSS可成功、稳定诱导小鼠急性溃疡性结肠炎,模型组小鼠结肠长度为4.10±0.03cm,较对照组6.17±0.10cm明显缩短(P0.001),第4日后,模型组较对照组体重明显减轻(P0.05)。 三、 IL-35基因修饰间充质干细胞对小鼠急性溃疡性结肠炎保护作用的研究 目的：观察IL-35基因修饰间充质干细胞(IL-35-MSCs)对小鼠急性溃疡性结肠炎的保护作用,并探讨其机制。方法：在DSS喂养第2、4、6天尾静脉注射IL-35基因修饰间充质干细胞,并与注射间充质干细胞(MSCs)和生理盐水(对照)的小鼠进行对比；观察不同组间小鼠一般情况及病理改变；采用实时定量聚合酶链式反应(RealTime PCR)及流式细胞术(FCM)对不同组小鼠结肠标本进行分析,测定结肠标本中IL-17、TNF-а、IFNγ的表达情况和结肠固有层淋巴细胞(LPL)中CD4+CD25+FoxP3+调节性T细胞(Treg)分布情况。结果：①IL-35-MSCs注射组小鼠体重下降趋势较对照组变缓,但与MSCs注射组无明显差异。②IL-35-MSCs注射组小鼠结肠标本长度(4.75±0.05cm)较MSCs注射组(4.06±0.04cm)及对照组(4.03±0.07cm)显著变长(P0.001),病理评分显著降低(P0.001)。③IL-35-MSCs注射组LPL中Treg分布(7.3±0.2%)较另外两组显著升高(P0.001)。IL-35-MSCs注射组小鼠结肠mRNA水平IL-17表达量较另外两组升高(P0.001),而TNF-α和IFN-γ表达降低(P0.001)。 结论：IL-35基因修饰的间充质干细胞对DSS诱导的小鼠急性溃疡性结肠炎有保护作用。其机制可能是通过刺激CD4+CD25+FoxP3+调节性T细胞的生成、上调结肠中IL-17的表达、下调TNF-α和IFN-γ表达,从而抑制局部免疫反应实现的。IL-35基因修饰的间充质干细胞可能成为治疗溃疡性结肠炎的新途径。
[Abstract]:Objective To study the therapeutic effect of interleukin35 (IL-35) gene-modified mesenchymal stem cells on ulcerative colitis in mice, and to explore its mechanism of action. The effect of IL-35 gene-modified mesenchymal stem cells (IL-35) on the treatment of acute ulcerative colitis in mice was observed through the establishment of a stable model of mouse ulcerative colitis, and a new approach to the treatment of ulcerative colitis was explored. I. IL-35 gene-modified C57BL/ 6 mice The purpose of the cell preparation is to construct an IL-35 overexpressing lentiviral vector to infect mouse mesenchymal stem cells to obtain an IL-35 gene-modified mesenchymal stem cell. Methods: The adipose-derived mesenchymal stem cells of mice were obtained by type I collagenase digestion, and were identified by flow cytometry (FCM) and directional induction differentiation. The IL-35 gene was ligated into the lentis by using the DNA polymerase. The IL-35 lentivirus particles were obtained by co-transfecting the packed cells with the packaging plasmid after the identification of the virus working plasmid and the sequencing and identification. The IL-35 lentiviral particles were overexpressed to infect the mesenchymal stem cells in the mice, and the IL-35 in the supernatant of the cells under different infection conditions was detected by the enzyme-linked immunosorbent assay (ELISA) method. The expression of IL-35 gene is obtained by the expression of IL-35 gene. The results showed that the accumulation of mesenchymal stem cells was successful. The expression of IL-35 lentiviral vector could successfully infect the mesenchymal stem cells, and the concentration of IL-35 in the supernatant of the cells after infection could reach 32.9. 5-16. 5. pg/ ml. 2, acute collapse of C57BL/ 6 mice Objective of the establishment of a model of enterocolitis: the establishment of C57BL Methods: The mice were fed with different concentration of dextran sodium sulfate (DSS) for 7 days, the body weight was recorded daily and the general condition was changed. The mice were sacrificed on the 8th day, and the colon was observed. The pathological changes were assessed by HE staining. The results showed that 2% DSS could successfully and stably induce the acute ulcerative colitis of mice. The length of colon in the model group was 4.10-0. 03cm, and that of the control group was 6.17-0. 10cm (P. 001). After the 4th day, the weight of the model group was higher than that of the control group. Obvious reduction (P0.05). Three-and IL-35 gene-modified mesenchymal stem cells were used in mice Objective: To study the protective effects of IL-35 gene-modified mesenchymal stem cells (IL-35-MSCs) on acute collapse of mice. Methods: IL-35 gene-modified mesenchymal stem cells were injected into the tail of DSS feeding group 2, 4 and 6, and compared with those of injected mesenchymal stem cells (MSCs) and normal saline (control). A real-time quantitative polymerase chain reaction (real time PCR) and flow cytometry (FCM) were used to analyze and measure the colon of different groups of mice. The expression of IL-17, TNF-1 and IFN in the colon specimens and the expression of CD4 + CD25 + FoxP3 in the colonic lamina lymphocytes (LPL) + regulatory T cell (Treg) distribution. Results: The decrease of body weight in the mice injected with IL-35-MSCs was higher than that in the control group. There was no significant difference with the group of MSCs in the injection group. The length of the colon specimen (4.75-0.05cm) in the mice injected with IL-35-MSCs was significantly longer than that of the control group (4.06-0. 04cm) and the control group (4.03-0. 07cm) (P 0.001). The expression of IL-17 in the colon of the injection group of IL-35-MSCs was higher than that of the other two groups (P 0.001), while the level of IL-17 in the IL-35-MSCs injection group was higher than that of the other two groups (P 0.001), while the TNF-1 and IL-35-MSCs were significantly higher than those in the other two groups (P 0.001). Conclusion: The IL-35 gene-modified mesenchymal stem cell pair D The mechanism of the regulation of the expression of IL-17 in the colon by stimulating the production of CD4 + CD25 + FoxP3 + regulatory T cells and the down-regulation of TNF-1 and I The expression of FN-35 in order to inhibit the realization of local immune response. Inter-charging of IL-35 gene modification
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R574.62
本文编号：2331278
[Abstract]:Objective To study the therapeutic effect of interleukin35 (IL-35) gene-modified mesenchymal stem cells on ulcerative colitis in mice, and to explore its mechanism of action. The effect of IL-35 gene-modified mesenchymal stem cells (IL-35) on the treatment of acute ulcerative colitis in mice was observed through the establishment of a stable model of mouse ulcerative colitis, and a new approach to the treatment of ulcerative colitis was explored. I. IL-35 gene-modified C57BL/ 6 mice The purpose of the cell preparation is to construct an IL-35 overexpressing lentiviral vector to infect mouse mesenchymal stem cells to obtain an IL-35 gene-modified mesenchymal stem cell. Methods: The adipose-derived mesenchymal stem cells of mice were obtained by type I collagenase digestion, and were identified by flow cytometry (FCM) and directional induction differentiation. The IL-35 gene was ligated into the lentis by using the DNA polymerase. The IL-35 lentivirus particles were obtained by co-transfecting the packed cells with the packaging plasmid after the identification of the virus working plasmid and the sequencing and identification. The IL-35 lentiviral particles were overexpressed to infect the mesenchymal stem cells in the mice, and the IL-35 in the supernatant of the cells under different infection conditions was detected by the enzyme-linked immunosorbent assay (ELISA) method. The expression of IL-35 gene is obtained by the expression of IL-35 gene. The results showed that the accumulation of mesenchymal stem cells was successful. The expression of IL-35 lentiviral vector could successfully infect the mesenchymal stem cells, and the concentration of IL-35 in the supernatant of the cells after infection could reach 32.9. 5-16. 5. pg/ ml. 2, acute collapse of C57BL/ 6 mice Objective of the establishment of a model of enterocolitis: the establishment of C57BL Methods: The mice were fed with different concentration of dextran sodium sulfate (DSS) for 7 days, the body weight was recorded daily and the general condition was changed. The mice were sacrificed on the 8th day, and the colon was observed. The pathological changes were assessed by HE staining. The results showed that 2% DSS could successfully and stably induce the acute ulcerative colitis of mice. The length of colon in the model group was 4.10-0. 03cm, and that of the control group was 6.17-0. 10cm (P. 001). After the 4th day, the weight of the model group was higher than that of the control group. Obvious reduction (P0.05). Three-and IL-35 gene-modified mesenchymal stem cells were used in mice Objective: To study the protective effects of IL-35 gene-modified mesenchymal stem cells (IL-35-MSCs) on acute collapse of mice. Methods: IL-35 gene-modified mesenchymal stem cells were injected into the tail of DSS feeding group 2, 4 and 6, and compared with those of injected mesenchymal stem cells (MSCs) and normal saline (control). A real-time quantitative polymerase chain reaction (real time PCR) and flow cytometry (FCM) were used to analyze and measure the colon of different groups of mice. The expression of IL-17, TNF-1 and IFN in the colon specimens and the expression of CD4 + CD25 + FoxP3 in the colonic lamina lymphocytes (LPL) + regulatory T cell (Treg) distribution. Results: The decrease of body weight in the mice injected with IL-35-MSCs was higher than that in the control group. There was no significant difference with the group of MSCs in the injection group. The length of the colon specimen (4.75-0.05cm) in the mice injected with IL-35-MSCs was significantly longer than that of the control group (4.06-0. 04cm) and the control group (4.03-0. 07cm) (P 0.001). The expression of IL-17 in the colon of the injection group of IL-35-MSCs was higher than that of the other two groups (P 0.001), while the level of IL-17 in the IL-35-MSCs injection group was higher than that of the other two groups (P 0.001), while the TNF-1 and IL-35-MSCs were significantly higher than those in the other two groups (P 0.001). Conclusion: The IL-35 gene-modified mesenchymal stem cell pair D The mechanism of the regulation of the expression of IL-17 in the colon by stimulating the production of CD4 + CD25 + FoxP3 + regulatory T cells and the down-regulation of TNF-1 and I The expression of FN-35 in order to inhibit the realization of local immune response. Inter-charging of IL-35 gene modification
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R574.62
【参考文献】
相关期刊论文 前2条
1 杨春练;方青枝;林俊玲;王光华;;小鼠尾静脉作为新护士静脉穿刺训练模型[J];医药世界;2006年05期
2 段征;徐艳华;陈小云;汪维伟;;间充质干细胞移植后在溃疡性结肠炎大鼠体内的迁移[J];重庆医科大学学报;2010年08期
，本文编号：2331278
本文链接：https://www.wllwen.com/yixuelunwen/xiaohjib/2331278.html
最近更新
教材专著
