IL-35基因修飾間充質(zhì)干細(xì)胞治療小鼠潰瘍性結(jié)腸炎的實(shí)驗(yàn)研究
發(fā)布時(shí)間：2018-11-14 13:24
【摘要】：本課題研究白介素35(interleukin35, IL-35)基因修飾間充質(zhì)干細(xì)胞對小鼠潰瘍性結(jié)腸炎的治療作用,并探討其作用機(jī)制。通過建立穩(wěn)定的小鼠潰瘍性結(jié)腸炎模型,經(jīng)尾靜脈注射IL-35基因修飾的間充質(zhì)干細(xì)胞,觀察其對小鼠急性潰瘍性結(jié)腸炎的治療效果,探索出治療潰瘍性結(jié)腸炎新途徑。 一、IL-35基因修飾C57BL/6小鼠間充質(zhì)干細(xì)胞制備 目的：構(gòu)建IL-35過表達(dá)慢病毒載體,以其感染小鼠間充質(zhì)干細(xì)胞,從而獲得IL-35基因修飾的間充質(zhì)干細(xì)胞。方法：采用I型膠原酶消化法獲得小鼠脂肪來源間充質(zhì)干細(xì)胞,并以流式細(xì)胞術(shù)(FCM)和定向誘導(dǎo)分化實(shí)驗(yàn)加以鑒定；應(yīng)用DNA聚合酶將IL-35基因連接入慢病毒工作質(zhì)粒,測序鑒定無誤后與包裝質(zhì)粒共轉(zhuǎn)染包裝細(xì)胞獲得過表達(dá)IL-35慢病毒顆粒；以過表達(dá)IL-35慢病毒顆粒感染小鼠間充質(zhì)干細(xì)胞,采用酶聯(lián)免疫吸附法(ELISA)法檢測不同感染條件下細(xì)胞上清中IL-35的表達(dá)情況,從而獲得IL-35基因修飾的間充質(zhì)干細(xì)胞。結(jié)果：脂肪來源間充質(zhì)干細(xì)胞獲取成功；過表達(dá)IL-35慢病毒載體可以成功感染間充質(zhì)干細(xì)胞,感染后細(xì)胞上清中IL-35蛋白濃度可達(dá)329.5±16.5pg/ml. 二、C57BL/6小鼠急性潰瘍性結(jié)腸炎模型構(gòu)建 目的：建立C57BL/6小鼠急性潰瘍性模型。方法：應(yīng)用不同濃度葡聚糖硫酸鈉(DSS)喂養(yǎng)小鼠7日,每日記錄體重變化及一般情況改變,第8日處死小鼠,觀察結(jié)腸大體標(biāo)本；將結(jié)腸標(biāo)本石蠟包埋后切片檢查,HE染色評估病理改變。結(jié)果：2%DSS可成功、穩(wěn)定誘導(dǎo)小鼠急性潰瘍性結(jié)腸炎,模型組小鼠結(jié)腸長度為4.10±0.03cm,較對照組6.17±0.10cm明顯縮短(P0.001),第4日后,模型組較對照組體重明顯減輕(P0.05)。 三、 IL-35基因修飾間充質(zhì)干細(xì)胞對小鼠急性潰瘍性結(jié)腸炎保護(hù)作用的研究 目的：觀察IL-35基因修飾間充質(zhì)干細(xì)胞(IL-35-MSCs)對小鼠急性潰瘍性結(jié)腸炎的保護(hù)作用,并探討其機(jī)制。方法：在DSS喂養(yǎng)第2、4、6天尾靜脈注射IL-35基因修飾間充質(zhì)干細(xì)胞,并與注射間充質(zhì)干細(xì)胞(MSCs)和生理鹽水(對照)的小鼠進(jìn)行對比；觀察不同組間小鼠一般情況及病理改變；采用實(shí)時(shí)定量聚合酶鏈?zhǔn)椒磻?yīng)(RealTime PCR)及流式細(xì)胞術(shù)(FCM)對不同組小鼠結(jié)腸標(biāo)本進(jìn)行分析,測定結(jié)腸標(biāo)本中IL-17、TNF-а、IFNγ的表達(dá)情況和結(jié)腸固有層淋巴細(xì)胞(LPL)中CD4+CD25+FoxP3+調(diào)節(jié)性T細(xì)胞(Treg)分布情況。結(jié)果：①IL-35-MSCs注射組小鼠體重下降趨勢較對照組變緩,但與MSCs注射組無明顯差異。②IL-35-MSCs注射組小鼠結(jié)腸標(biāo)本長度(4.75±0.05cm)較MSCs注射組(4.06±0.04cm)及對照組(4.03±0.07cm)顯著變長(P0.001),病理評分顯著降低(P0.001)。③IL-35-MSCs注射組LPL中Treg分布(7.3±0.2%)較另外兩組顯著升高(P0.001)。IL-35-MSCs注射組小鼠結(jié)腸mRNA水平IL-17表達(dá)量較另外兩組升高(P0.001),而TNF-α和IFN-γ表達(dá)降低(P0.001)。 結(jié)論：IL-35基因修飾的間充質(zhì)干細(xì)胞對DSS誘導(dǎo)的小鼠急性潰瘍性結(jié)腸炎有保護(hù)作用。其機(jī)制可能是通過刺激CD4+CD25+FoxP3+調(diào)節(jié)性T細(xì)胞的生成、上調(diào)結(jié)腸中IL-17的表達(dá)、下調(diào)TNF-α和IFN-γ表達(dá),從而抑制局部免疫反應(yīng)實(shí)現(xiàn)的。IL-35基因修飾的間充質(zhì)干細(xì)胞可能成為治療潰瘍性結(jié)腸炎的新途徑。
[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
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R574.62
本文編號：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
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R574.62
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相關(guān)期刊論文 前2條
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2 段征;徐艷華;陳小云;汪維偉;;間充質(zhì)干細(xì)胞移植后在潰瘍性結(jié)腸炎大鼠體內(nèi)的遷移[J];重慶醫(yī)科大學(xué)學(xué)報(bào);2010年08期
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