CXCR-4基因转染的BMMSCs对实验性IBD小鼠肠黏膜修复机制研究
发布时间：2019-06-01 11:28
【摘要】：研究背景：炎症性肠病(inflammatory bowel disease,IBD)作为一种病因及发病机制尚未完全明确的,以累及肠道为主的消化道慢性炎症性疾病,在临床上一般包括克罗恩病(Crohn's Disease,CD)和溃疡性结肠炎(Ulcerative Colitis,UC)。骨髓间充质干细胞(Bone Marrow Mesenchymal Stem Cells,BMMSCs)作为最早被人工分离出的成体干细胞,具有许多生物学特点和优势,如：自身低免疫原性、多向分化潜能、免疫调节作用、取材方便等,因此成为临床上移植疗法的推荐选择之一,对IBD而言,因其可通过促进病变肠黏膜修复并纠正机体及局部的免疫功能紊乱,而成为治疗本病的首选细胞。然而,经静脉移植的BMMSCs最终定植于病变肠黏膜的数量有限,限制了其治疗作用的发挥。由于干细胞的归巢行为涉及多种趋化因子及其受体的参与,其中趋化因子CXC亚组受体-4/基质细胞衍生因子-1(Chemokine Receptor-4/Stromal cell-derived Factor-1,CXCR4/SDF-1)的作用已经被证实在介导干细胞归巢中发挥着十分重要的地位。故利用CXCR-4基因转染BMMSCs,能够使其在体内的靶向性定位增强,从而提高疗效。实验目的：通过移植未经修饰的BMMSCs和CXCR-4基因修饰的BMMSCs来比较两者在治疗实验性IBD小鼠模型中肠道黏膜局部定植率及疗效,并探讨CXCR-4基因修饰的BMMSCs促进受损肠黏膜愈合的机制,从而为临床上确定一种选择靶向性更强的干细胞用于治疗IBD提供理论依据。实验内容：1、构建并鉴定BMMSCs。2、构建并鉴定经CXCR-4基因修饰的靶向性BMMSCs及评估其体外迁移率。3、观察经CXCR-4基因修饰的BMMSCs能否向IBD小鼠损伤肠黏膜定向迁移并加快其愈合。4、探讨靶向性BMMSCs通过何种机制在肠道局部大量定植并发挥修复作用。实验方法：1、采用细胞贴壁分离法从雄性BALB/c小鼠(4-5周龄)的骨髓中分离并体外培养BMMSCs;并对BMMSCs进行荧光染料(CM-Dil)标记。2、通过形态学观察、流式细胞学技术检测细胞表面标志物、成骨／成脂肪样细胞诱导分化实验对所培养的BMMSCs进行鉴定。3、将携带CXCR-4的慢病毒转染入BMMSCs构建成CXCR4-BMMSCs;进行嘌呤霉素筛选实验,从中提纯CXCR-BMMSCs;采用Real time-PCR检测并比较转染组和未转染组的CXCR-4的表达量。4、通过形态学观察、流式细胞技术检测细胞表面标志物、成骨／成脂样细胞诱导分化实验、台盼蓝拒染实验间接测定细胞存活率、流式细胞学测定细胞周期,来比较两组生物学特性的差异。5、通过Transwell体外迁移实验,比较两组细胞向SDF-1的迁移率。6、选择雌性BALB/c小鼠(6-8周龄),分别随机分为6组;①对照组(标记为Control, Ctr组)：生理盐水灌肠(0.1m1/只),灌肠后第2天,给予尾静脉注射不含BMMSCs的PBS 0.1ml, n=20; ②TNBS组(T组)：TNBS-乙醇灌肠剂(0.1m1/只),并于造模后第2天,给予尾静脉注射不含BMMSCs的PBS 0.1ml,n=20;③ BMMSC移植组(MT组)：TNBS-乙醇灌肠剂(0.1ml/只),并于造模后第2天,尾静脉注射含BMMSCs(1×106/只)的PBS 0.1ml,n=20;④CXCR-BMMSC移植组(CMT组)：TNBS-乙醇灌肠剂(0.1ml/只),并于造模后第2天,给予尾注射含CXCR4-BMMSCs(1×106/只)的PBS 0.1ml,n=20。移植当天标记为D0,移植后第1-13天,分别记为D1-13。7、观察各组小鼠一般情况、体重变化及存活率、进行临床症状评分、肉眼及放大镜下观察结肠大体外观、黏膜状况,进行组织病理学评分。8、分别于D0、D1、D3、D5、D7、D9、D11、D13天采用颈椎脱臼法处死小鼠3只/组,并取材血液、肠道组织、肠系膜淋巴结及脾脏,一部分进行石蜡包埋,一部分经液氮速冻后,-80。C冰箱保存。9、荧光显微镜下观察各组细胞在肠道内的定植情况,应用Real time PCR检测并比较Y染色体的性别决定区段(SRY)基因的肠道局部的相对量。10、采用免疫组织化学检测肠道干细胞标志物Lgr-5、肠道上皮间的紧密连接蛋白Occludin、血管生成指标VEGF的表达情况。11、采用Western Blot技术检测P13K的表达情况。实验结果：1、采用贴壁分离法培养的BMMSCs随着不断纯化其细胞形态呈长梭状且按束状排列,细胞表型鉴定显示为：CD90.2+;CD105+;CD45-;CDllb-,成骨诱导可见明显的骨结节形成,成脂肪诱导可见脂滴。经荧光染料染色后对细胞的生物学特性无明显影响。2、将CXCR-4基因导入BMSCs后,与BMMSCs组比较可检测到CXCR-4基因的高表达(p0.05)：3、CXCR-BMMSCs组细胞均呈长梭形并束状排列；细胞表型鉴定未发生改变(CD90.2+;CD105+;CD45-;CD11b-);成骨诱导可见明显的骨结节形成,成脂肪诱导可见脂滴；活细胞比率均在90%以上;以上结果与未经修饰的BMMSCs相比无明显差异(p0.05);在细胞周期检测中发现过表达CXCR-4基因的BMMSCs处于复制期的细胞增多(p0.05)。4、体外迁移实验证实：在SDF-1的诱导下，CXCR-BMMSCs组的迁移率BMMSCs组(p0.05)。5、TNBS-乙醇灌肠法诱导后的实验性IBD小鼠模型的存活率明显下降、小鼠精神状态差且体重明显下降,临床症状及组织病理学评分上升,肉眼观察可见结肠皱缩,局部肠段狭窄,肠黏膜伴有充血水肿、糜烂形成,黏膜及黏膜下层有大量中性粒细胞浸润,与正常组比较均有明显差异(p0.05)；MT组、CMT组移植后第2天,与T组相比,存活率开始上升,疾病症状减轻、体重回升,临床症状及组织病理学评分下降(p0.05), CMT组诱导缓解的效果更好(p0.05)。6、荧光显微镜下可见MT组、CMT组的肠道组织均存在红色荧光;治疗组及雄性对照组的肠道黏膜组织中均能检测到SRY基因；体内定植率的比较显示CMT组MT组。(p0.01)7、免疫组织化学染色结果显示：IBD模型小鼠接受BMSCs移植后,肠道干细胞标志物Lgr-5、肠道上皮间的紧密连接蛋白Occludin及VEGF的表达量较造模未治疗组有所升高。8、Western Blot的结果显示：与T组相比,MT组、CMT组P13K的蛋白表达量均升高,其中CMT组升高最为明显(p0.01)。实验结论：1、利用慢病毒载体转染CXCR-4基因至BMMSCs,几乎不会改变BMMSCs原有的生物学特性。2、体内外实验均证实了CXCR-4基因的过表达可增加BMMSCs的迁移率。3、通过干细胞移植治疗实验性IBD小鼠模型时,其治疗效果与细胞在肠道的定植率呈正相关,并且可通过促进肠道干细胞分化、重构肠上皮紧密连接、激活P13K信号通路来促进肠黏膜再生、修复、上皮屏障重建及血管生成。
[Abstract]:Background: The etiology and pathogenesis of inflammatory bowel disease (IBD), as a cause and pathogenesis, are not well defined, and in the clinical practice, Crohn's disease (CD) and ulcerative colitis (UC) are generally involved in the treatment of chronic inflammatory diseases of the digestive tract. Bone marrow mesenchymal stem cells (BMMSCs), as the earliest adult stem cells, have many biological characteristics and advantages, such as low immunogenicity, multi-directional differentiation potential, immunoregulation, and convenient materials. Therefore, it is one of the recommended options for clinical transplantation therapy. For IBD, it is the preferred cell to treat the disease by promoting the repair of the intestinal mucosa of the lesion and correcting the body and local immune function disorder. However, the limited number of BMMSCs that have been transplanted through the vein in the intestinal mucosa of the lesion is limited, and the therapeutic effect of the BMMSCs is limited. The role of the chemokine CXC subgroup receptor-4/ matrix cell-derived factor-1 (CXCR4/ SDF-1) has been demonstrated to play a very important role in mediating stem cell homing, as the homing behavior of stem cells involves a variety of chemokines and their receptors. Therefore, using the CXCR-4 gene to transfect the BMMSCs, the targeted localization of the BMMSCs in the body can be enhanced, and the curative effect can be improved. Objective: To compare the local colonization rate and efficacy of the intestinal mucosa in the experimental IBD mouse model by transplantation of the unmodified BMMSCs and the modified BMMSCs of the CXCR-4, and to explore the mechanism of the BMMSCs modified by the CXCR-4 to promote the healing of the damaged intestinal mucosa. So as to provide a theoretical basis for the clinical determination of a stem cell with stronger targeting property for the treatment of IBD. 1. Construct and identify the BMMSCs.2, construct and identify the targeted BMMSCs modified by the CXCR-4 gene and evaluate their in vitro mobility.3. To observe whether the BMMSCs modified by the CXCR-4 can damage the intestinal mucosa and accelerate the healing of the intestinal mucosa in the IBD mice. To explore the mechanism of targeting BMMSCs to colonize the intestinal tract and to play a repair role. Methods:1. BMMSCs were isolated from bone marrow of male BALB/ c mice (4-5 weeks old) by cell wall-wall separation and BMMSCs were cultured in vitro. The cultured BMMSCs were identified by an osteogenic/ fat-like cell-induced differentiation experiment.3. The lentivirus carrying the CXCR-4 was transfected into the BMMSCs to construct the CXCR4-BMMSCs, and the CXCR-BMMSCs were purified from the BMMSCs using a real time-PCR method and the expression of the CXCR-4 in the transfected and untransfected groups was compared by the Real time-PCR. The cell cycle was determined by flow cytometry, the cell cycle was determined by flow cytometry, and the difference between the two groups of biological characteristics was compared. The mobility of two groups of cells to SDF-1 was compared by a Transwell in vitro migration experiment.6. The female BALB/ c mice (6-8 weeks old) were randomly divided into 6 groups, and the control group (labeled Control, Ctr group): normal saline enema (0.1ml/ min), and the second day after the enema. The tail vein was given 0.1 ml of PBS without BMMSCs, n = 20; TNBS group (T group): TNBS-ethanol enema (0.1 ml/ min), and the second day after the model, the tail vein was given a PBS 0.1 ml without BMMSCs, n = 20; and the BMMSC transplantation group (MT group): TNBS-ethanol enema (0.1 ml/ min), In the second day of the model, 0.1 ml of PBS containing BMMSCs (1-106/ s) was injected intravenously, and n = 20; the CCXCR-BMMSC transplantation group (CMT group): TNBS-ethanol enema (0.1 ml/ min), and the tail was given 0.1 ml of PBS containing CXCR4-BMMSCs (1-106/ only) after the second day of the model, and n = 20. On the day of transplantation, the marker was D0, and the first to 13 days after the transplantation, respectively, were recorded as D1-13.7, and the general condition, weight change and survival rate of each group of mice were observed, and the general appearance and the mucous membrane status of the colon were observed under the naked eye and the magnifying glass, and the pathological scores of the tissues were observed. D3, D5, D7, D9, D11 and D13 were treated with cervical dislocation to kill 3 mice/ group, and the blood, intestinal tissue, mesenteric lymph node and spleen were obtained. and the relative amount of the intestinal part of the sex determination section (SRY) gene of the Y chromosome is detected by using the Real time PCR and the relative amount of the intestinal part of the sex determination section (SRY) gene of the Y chromosome is detected under the fluorescence microscope,10, the intestinal stem cell marker Lgr-5 is detected by immunohistochemistry, The expression of the close-connexin Occidin, the angiogenesis index and the expression of VEGF in the intestinal epithelium was detected in 11, and the expression of P13K was detected by Western Blot technique. The results of the experiment were as follows:1. The BMMSCs cultured by the method of wall-wall separation were in the form of long-shuttle and bundle-like, and the cell phenotype was shown as: CD90.2 +; CD105 +; CD45-; CDllb-; osteogenic induction of the visible bone nodule formation; and the fat-induced visible lipid droplets. After the expression of the CXCR-4 gene into BMSCs, the high expression of the CXCR-4 gene (p0.05):3, CXCR-BMMSCs, and the cell phenotype identification were not changed (CD90.2 +, CD105 +). CD45-; CD11b-); osteogenic induction of visible bone nodule formation, and fat-induced fat drop; the ratio of living cells was above 90%; the above results did not differ significantly from the unmodified BMMSCs (p0.05); In the cell cycle test, it was found that the BMMSCs expressing the CXCR-4 gene were in the replication stage (p0.05).4. In vitro migration experiments confirmed that the survival rate of the experimental IBD mouse model induced by the TNBS-ethanol enema method was significantly decreased under the induction of SDF-1, the mobility of the CXCR-BMMSCs group (p0.05). The mouse's mental state is poor and the body weight is obviously reduced, the clinical symptom and the histopathological score are increased, the visible colon is observed with the naked eye, the local intestinal segment is narrow, the intestinal mucosa is accompanied with hyperemia and edema, the erosion is formed, and the lower layer of the mucosa and the mucous membrane has a large number of neutrophil infiltration, Compared with the normal group, there was a significant difference (p0.05); in the MT group, the second day after the transplantation of the CMT group, the survival rate began to increase, the symptoms of the disease were relieved, the weight recovered, the clinical symptoms and the histopathological score decreased (p0.05), and the effect of the CMT group induction response was better (p0.05).6, The SRY gene was detected in the intestinal tissues of the MT group and the CMT group under the fluorescence microscope. The SRY gene can be detected in the intestinal mucosa tissues of the treatment group and the male control group, and the MT group of the CMT group is displayed in comparison with the in vivo colonization rate. (p0.01)7. The results of the immunohistochemical staining showed that the expression of the close-linked protein Occidin and VEGF in the intestinal stem cell marker Lgr-5 and the intestinal epithelium was higher than that of the untreated group. The results of the Western Blot showed that the MT group, The expression of P13K in the CMT group was increased, with the most significant increase in the CMT group (p0.01). Conclusion:1. Transfection of the CXCR-4 gene to the BMMSCs with the lentiviral vector can hardly change the original biological characteristics of the BMMSCs. In vivo and in vivo, the overexpression of the CXCR-4 gene can increase the mobility of the BMMSCs. The treatment effect is positively related to the colonization rate of the cells in the intestinal tract, and the intestinal mucosa regeneration, repair, epithelial barrier reconstruction and blood vessel generation can be promoted by promoting the differentiation of the intestinal stem cells, reconstructing the intestinal epithelial connection, and activating the P13K signal path.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R574
本文编号：2490237
[Abstract]:Background: The etiology and pathogenesis of inflammatory bowel disease (IBD), as a cause and pathogenesis, are not well defined, and in the clinical practice, Crohn's disease (CD) and ulcerative colitis (UC) are generally involved in the treatment of chronic inflammatory diseases of the digestive tract. Bone marrow mesenchymal stem cells (BMMSCs), as the earliest adult stem cells, have many biological characteristics and advantages, such as low immunogenicity, multi-directional differentiation potential, immunoregulation, and convenient materials. Therefore, it is one of the recommended options for clinical transplantation therapy. For IBD, it is the preferred cell to treat the disease by promoting the repair of the intestinal mucosa of the lesion and correcting the body and local immune function disorder. However, the limited number of BMMSCs that have been transplanted through the vein in the intestinal mucosa of the lesion is limited, and the therapeutic effect of the BMMSCs is limited. The role of the chemokine CXC subgroup receptor-4/ matrix cell-derived factor-1 (CXCR4/ SDF-1) has been demonstrated to play a very important role in mediating stem cell homing, as the homing behavior of stem cells involves a variety of chemokines and their receptors. Therefore, using the CXCR-4 gene to transfect the BMMSCs, the targeted localization of the BMMSCs in the body can be enhanced, and the curative effect can be improved. Objective: To compare the local colonization rate and efficacy of the intestinal mucosa in the experimental IBD mouse model by transplantation of the unmodified BMMSCs and the modified BMMSCs of the CXCR-4, and to explore the mechanism of the BMMSCs modified by the CXCR-4 to promote the healing of the damaged intestinal mucosa. So as to provide a theoretical basis for the clinical determination of a stem cell with stronger targeting property for the treatment of IBD. 1. Construct and identify the BMMSCs.2, construct and identify the targeted BMMSCs modified by the CXCR-4 gene and evaluate their in vitro mobility.3. To observe whether the BMMSCs modified by the CXCR-4 can damage the intestinal mucosa and accelerate the healing of the intestinal mucosa in the IBD mice. To explore the mechanism of targeting BMMSCs to colonize the intestinal tract and to play a repair role. Methods:1. BMMSCs were isolated from bone marrow of male BALB/ c mice (4-5 weeks old) by cell wall-wall separation and BMMSCs were cultured in vitro. The cultured BMMSCs were identified by an osteogenic/ fat-like cell-induced differentiation experiment.3. The lentivirus carrying the CXCR-4 was transfected into the BMMSCs to construct the CXCR4-BMMSCs, and the CXCR-BMMSCs were purified from the BMMSCs using a real time-PCR method and the expression of the CXCR-4 in the transfected and untransfected groups was compared by the Real time-PCR. The cell cycle was determined by flow cytometry, the cell cycle was determined by flow cytometry, and the difference between the two groups of biological characteristics was compared. The mobility of two groups of cells to SDF-1 was compared by a Transwell in vitro migration experiment.6. The female BALB/ c mice (6-8 weeks old) were randomly divided into 6 groups, and the control group (labeled Control, Ctr group): normal saline enema (0.1ml/ min), and the second day after the enema. The tail vein was given 0.1 ml of PBS without BMMSCs, n = 20; TNBS group (T group): TNBS-ethanol enema (0.1 ml/ min), and the second day after the model, the tail vein was given a PBS 0.1 ml without BMMSCs, n = 20; and the BMMSC transplantation group (MT group): TNBS-ethanol enema (0.1 ml/ min), In the second day of the model, 0.1 ml of PBS containing BMMSCs (1-106/ s) was injected intravenously, and n = 20; the CCXCR-BMMSC transplantation group (CMT group): TNBS-ethanol enema (0.1 ml/ min), and the tail was given 0.1 ml of PBS containing CXCR4-BMMSCs (1-106/ only) after the second day of the model, and n = 20. On the day of transplantation, the marker was D0, and the first to 13 days after the transplantation, respectively, were recorded as D1-13.7, and the general condition, weight change and survival rate of each group of mice were observed, and the general appearance and the mucous membrane status of the colon were observed under the naked eye and the magnifying glass, and the pathological scores of the tissues were observed. D3, D5, D7, D9, D11 and D13 were treated with cervical dislocation to kill 3 mice/ group, and the blood, intestinal tissue, mesenteric lymph node and spleen were obtained. and the relative amount of the intestinal part of the sex determination section (SRY) gene of the Y chromosome is detected by using the Real time PCR and the relative amount of the intestinal part of the sex determination section (SRY) gene of the Y chromosome is detected under the fluorescence microscope,10, the intestinal stem cell marker Lgr-5 is detected by immunohistochemistry, The expression of the close-connexin Occidin, the angiogenesis index and the expression of VEGF in the intestinal epithelium was detected in 11, and the expression of P13K was detected by Western Blot technique. The results of the experiment were as follows:1. The BMMSCs cultured by the method of wall-wall separation were in the form of long-shuttle and bundle-like, and the cell phenotype was shown as: CD90.2 +; CD105 +; CD45-; CDllb-; osteogenic induction of the visible bone nodule formation; and the fat-induced visible lipid droplets. After the expression of the CXCR-4 gene into BMSCs, the high expression of the CXCR-4 gene (p0.05):3, CXCR-BMMSCs, and the cell phenotype identification were not changed (CD90.2 +, CD105 +). CD45-; CD11b-); osteogenic induction of visible bone nodule formation, and fat-induced fat drop; the ratio of living cells was above 90%; the above results did not differ significantly from the unmodified BMMSCs (p0.05); In the cell cycle test, it was found that the BMMSCs expressing the CXCR-4 gene were in the replication stage (p0.05).4. In vitro migration experiments confirmed that the survival rate of the experimental IBD mouse model induced by the TNBS-ethanol enema method was significantly decreased under the induction of SDF-1, the mobility of the CXCR-BMMSCs group (p0.05). The mouse's mental state is poor and the body weight is obviously reduced, the clinical symptom and the histopathological score are increased, the visible colon is observed with the naked eye, the local intestinal segment is narrow, the intestinal mucosa is accompanied with hyperemia and edema, the erosion is formed, and the lower layer of the mucosa and the mucous membrane has a large number of neutrophil infiltration, Compared with the normal group, there was a significant difference (p0.05); in the MT group, the second day after the transplantation of the CMT group, the survival rate began to increase, the symptoms of the disease were relieved, the weight recovered, the clinical symptoms and the histopathological score decreased (p0.05), and the effect of the CMT group induction response was better (p0.05).6, The SRY gene was detected in the intestinal tissues of the MT group and the CMT group under the fluorescence microscope. The SRY gene can be detected in the intestinal mucosa tissues of the treatment group and the male control group, and the MT group of the CMT group is displayed in comparison with the in vivo colonization rate. (p0.01)7. The results of the immunohistochemical staining showed that the expression of the close-linked protein Occidin and VEGF in the intestinal stem cell marker Lgr-5 and the intestinal epithelium was higher than that of the untreated group. The results of the Western Blot showed that the MT group, The expression of P13K in the CMT group was increased, with the most significant increase in the CMT group (p0.01). Conclusion:1. Transfection of the CXCR-4 gene to the BMMSCs with the lentiviral vector can hardly change the original biological characteristics of the BMMSCs. In vivo and in vivo, the overexpression of the CXCR-4 gene can increase the mobility of the BMMSCs. The treatment effect is positively related to the colonization rate of the cells in the intestinal tract, and the intestinal mucosa regeneration, repair, epithelial barrier reconstruction and blood vessel generation can be promoted by promoting the differentiation of the intestinal stem cells, reconstructing the intestinal epithelial connection, and activating the P13K signal path.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R574
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