过表达CXCR4的骨髓间充质干细胞归巢治疗溃疡性结肠炎及复方苦参汤的协同作用研究
发布时间：2018-10-14 11:28
【摘要】：第一部分CXCR4基因过表达慢病毒载体的构建及鉴定 目的：探讨构建大鼠趋化因子受体4(chemokine receptor4, CXCR4)基因过表达慢病毒载体的技术方法并检测其体外表达目的基因的水平。 方法：设计CXCR4基因引物,采用聚合酶链反应(PCR)扩增CXCR4基因片段；用Age I酶切GV208载体；通过连接酶将CXCR4基因片段连接至线性化的GV208载体上；运用PCR方法鉴定阳性克隆的CXCR4-GV208载体；按GV208慢病毒包装试剂盒说明书包装慢病毒并运用实时定量聚合酶链反应(RT-qPCR)法行滴度检测；然后用重组慢病毒转染人胚肾细胞(293T细胞),观察GFP表达情况。 结果：通过PCR成功地扩增了CXCR4基因片段并连接到了GV208病毒载体上,通过PCR及DNA测序鉴定,证明CXCR4-GV208质粒构建正确,重组慢病毒转染293T细胞后可观察到荧光及蛋白表达。包装过表达慢病毒并测其浓缩滴度为2.0×108TU/mL。 结论：成功构建CXCR4-GV208慢病毒载体,为CXCR4基因的后期研究提供了实验基础。 第二部分SDF-1α/CXCR4轴促进间充质干细胞归巢于实验性结肠炎受损结肠及复方苦参汤的协同效应 目的：调查基质细胞衍生因子(SDF-1α)／趋化因子受体4(CXCR4)轴在骨髓间充质干细胞(BMSCs)治疗2,4,6-三硝基苯磺酸(TNBS)诱导的结肠炎中的作用及复方苦参汤的协同效应。 方法：从SD大鼠骨髓中分离BMSCs并使用流式细胞术鉴定。通过慢病毒技术使BMSCs表达GFP (Ad-GFP-BMSCs)或共表达CXCR4和GFP (Ad-CXCR4-BMSCs), Western blot检钡BMSCs转染前后CXCR4蛋白表达。40只大鼠被随机分成5组(n=8)：空白组、模型组、Ad-GFP-BMSCs组、Ad-CXCR4-BMSCs组和二联组。采用TNBS诱导实验性结肠炎,尾静脉注射Ad-CXCR4-BMSCs或Ad-GFP-BMSCs,二联组予复方苦参汤灌胃治疗。细胞和中药治疗1wk后收集结肠组织,免疫荧光或western blot检测结肠部位GFP、CXCR4和SDF-1α表达。 结果：慢病毒转染48h后BMSCs的存活率大约为90%,80%的BMSCs能够稳定表达GFP。相对于空白组,结肠炎大鼠结肠部位SDF-1α蛋白表达明显上升。第12d,Ad-GFP-BMSCs组结肠只能发现少量的绿色荧光表达。相对于Ad-GFP-BMSCs组,Ad-CXCR4-BMSCs组和二联组结肠部位则能发现绿色荧光表达显著增多。类似的是,Western blot显示Ad-GFP-BMSCs组结肠部位仅表达少量的GFP蛋白；相对于Ad-GFP-BMSCs组,Ad-CXCR4-BMSCs组结肠部位GFP蛋白表达量则明显增多(P0.05)；相对于Ad-CXCR4-BMSCs组,二联组GFP蛋白表达显著提高(P0.05)。 结论：SDF-1α/CXCR4轴在BMSCs归巢于受损的结肠组织中发挥重要的作用,复方苦参汤可能协同改善BMSCs的归巢效率。这可能为溃疡性结肠炎的细胞治疗提供潜在的方法和理论依据。 第三部分过表达CXCR4的骨髓间充质干细胞治疗实验性结肠炎的免疫机制研究 目的：调查过表达CXCR4的骨髓间充质干细胞治疗2,4,6-三硝基苯磺酸诱导的实验性结肠炎的效果及其潜在的免疫作用机制。 方法：从雌性SD大鼠骨髓中分离BMSCs并使用流式细胞术鉴定。通过慢病毒技术使BMSCs表达GFP(Ad-GFP-BMSCs)或共表达CXCR4和GFP(Ad-CXCR4-BMSCs),40只大鼠被随机分成5组(n=8)：空白组、模型组、Ad-GFP-BMSCs组、Ad-CXCR4-BMSCs组和二联组。采用TNBS诱导实验性结肠炎,尾静脉注射Ad-CXCR4-BMSCs或Ad-GFP-BMSCs,二联组予复方苦参汤灌胃治疗。在整个实验过程中,每天记录大鼠的DAI。治疗1wk后,收集结肠组织进行HE染色和病理学分析。PCR检测结肠部位IFN-γ、L-6和11-10的mRNA表达,Western blot检测STAT-3和磷酸化STAT-3蛋白表达。 结果：相对于空白组,模型组结肠部位的IFN-γ、IL-6和IL-10的mRNA表达显著上升,STAT-3及磷酸化STAT-3的蛋白表达明显上升(P0.05)。系统治疗一周后,相对于模型组,Ad-GFP-BMSCs组大鼠的临床症状及结肠病理损害并没有得到有效的缓解。相对于Ad-GFP-BMSCs组,Ad-CXCR4-BMSCs组和二联组大鼠的DAI及病理炎症分数显著降低,结肠长度明显恢复。大鼠结肠组织的IFN-γ、IL-6的mRNA表达显著下降,IL-10的mRNA表达显著上升(P0.05); STAT3及磷酸化STAT-3的蛋白表达显著下降(P0.05)。 结论：过表达CXCR4的BMSCs可能通过抗炎及免疫调节机制来缓解实验性结肠炎,这可能为BMSCs治疗UC提供可靠的理论依据。
[Abstract]:Construction and identification of the expression lentiviral vector of the first part of survivin gene Objective: To investigate the technical methods of constructing rat chemokine receptor 4 (CCR5) gene overexpressing lentiviral vector and to detect the expression target gene in vitro Methods: The gene fragment was amplified by polymerase chain reaction (PCR). The gene fragment was digested with Age I enzyme and ligated to the linearized GV208 vector by ligase. The positive clones were identified by PCR. 208 vector; packaging lentivirus according to the specification of GV208 lentivirus packaging kit and using real-time quantitative polymerase chain reaction (RT-qPCR) method to detect droplet size; then transfect human embryonic kidney cells (293T cells) with recombinant lentivirus, and observing GF Results: The gene fragment was amplified by PCR and ligated into GV208 virus vector. It was proved by PCR and DNA sequencing that the plasmid was constructed correctly and the recombinant lentivirus was transfected into 293T cells. Fluorescent and protein expression. Packaging overexpressing lentivirus and measuring its concentration drop of 2.0 kcal 108TU/ mL. Conclusion: We successfully constructed the lentiviral vector of NDV-GV208. An experimental basis was provided for later studies. The second part of SDF-1 A/ T axis promotes the homing of mesenchymal stem cells in experimental colitis damaged knot AIM: To investigate the effect of stromal cell-derived factor (SDF-1)/ chemokine receptor 4 (BMSCs) on the treatment of 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) induced junctions in bone marrow mesenchymal stem cells (BMSCs). The effect of compound sophora flavescens soup and its synergistic effect in enteritis: from sd rats BMSCs were isolated from bone marrow and were identified by flow cytometry. BMSCs expressed GFP (Ad-GFP-BMSCs) or co-expressed GFP (Ad-GFP-BMSCs) and GFP (Ad-GFP-BMSCs) were expressed by lentivirus technique. Western blot analysis of BMSCs was performed before and after transfection. 40 rats were randomly divided into 5 groups (n = 8): blank group, model group, Ad-GFP-BMSCs group. Ad-BMSCs or Ad-GFP-BMSCs or Ad-GFP were induced by TNBS.-BMSCs and two groups were given by gavage for the compound sophora flavescens decoction. The colon tissues, immunofluorescence or western blot were collected for the detection of colon by the cells and traditional Chinese medicine. Results: The survival rate of BMSCs after 48h of lentiviral transfection was similar to that of BMSCs. 90%, 80% BMSCs were able to stably express GFP. Compared with blank group, The expression of SDF-1 protein in colon region of colitis rats increased significantly. Only a small amount of green fluorescent protein was found in the colon of the P-BMSCs group. Ad-GFP-BMSCs were found in Ad-GFP-BMSCs group. Compared with Ad-GFP-BMSCs, the expression of GFP protein in colon of Ad-GFP-BMSCs increased significantly (P0.05). The expression of GFP protein in the two groups increased significantly (P0.05). In order to improve the homing efficiency of BMSCs, the compound sophora flavescens decoction can synergistically improve the homing efficiency of BMSCs. Potential methods and theoretical bases may be provided for the treatment of colonitis, Part 3: Objective: To investigate the immune mechanism of bone marrow mesenchymal stem cells (BMSCs) in the treatment of experimental colitis. Experimental colitis induced by 6-trinitrobenzene sulfonic acid and its potential immune function machine Methods: BMSCs were isolated from the bone marrow of female SD rats and were identified by flow cytometry. BMSCs were expressed GFP (Ad-GFP-BMSCs) or co-expressed GFP (Ad-GFP-BMSCs) by lentivirus technique, 40 rats were randomly divided into 5 groups (n = 8): blank group, model. group, Ad-GFP-BMSCs group, Ad-GFP-BMSCs group and two groups. TNBS was used to induce experimental colitis and tail vein injection Ad-CXC. R4-BMSCs鎴朅d-GFP-BMSC s, the two groups are administered by gavage for the compound flavescens soup, and in the whole experiment, each The results of HE staining and pathological analysis were performed on the colon tissues after 1wk treatment. The mRNA levels of IFN-, L-6 and 11-10 in colon were detected by PCR. The expression of STAT-3 and phosphorylated STAT-3 protein was detected by Western blot. The results showed that the expression of IFN-, IL-6 and IL-10 in the colon of model group compared with blank group. The protein expression of STAT-3 and phosphorylated STAT-3 increased significantly (P0.05). The clinical symptoms and pathological lesions of Ad-GFP-BMSCs were not effectively alleviated. Ad-GFP-BMSCs group, Ad-CXCR The expression of IFN-, IL-6 mRNA and IL-10 mRNA in colon tissues of rats were significantly decreased, and the mRNA expression of IL-10 increased significantly. The expression of STAT3 and phosphorylated STAT-3 was significantly decreased (P0.05).
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R574.62
本文编号：2270324
[Abstract]:Construction and identification of the expression lentiviral vector of the first part of survivin gene Objective: To investigate the technical methods of constructing rat chemokine receptor 4 (CCR5) gene overexpressing lentiviral vector and to detect the expression target gene in vitro Methods: The gene fragment was amplified by polymerase chain reaction (PCR). The gene fragment was digested with Age I enzyme and ligated to the linearized GV208 vector by ligase. The positive clones were identified by PCR. 208 vector; packaging lentivirus according to the specification of GV208 lentivirus packaging kit and using real-time quantitative polymerase chain reaction (RT-qPCR) method to detect droplet size; then transfect human embryonic kidney cells (293T cells) with recombinant lentivirus, and observing GF Results: The gene fragment was amplified by PCR and ligated into GV208 virus vector. It was proved by PCR and DNA sequencing that the plasmid was constructed correctly and the recombinant lentivirus was transfected into 293T cells. Fluorescent and protein expression. Packaging overexpressing lentivirus and measuring its concentration drop of 2.0 kcal 108TU/ mL. Conclusion: We successfully constructed the lentiviral vector of NDV-GV208. An experimental basis was provided for later studies. The second part of SDF-1 A/ T axis promotes the homing of mesenchymal stem cells in experimental colitis damaged knot AIM: To investigate the effect of stromal cell-derived factor (SDF-1)/ chemokine receptor 4 (BMSCs) on the treatment of 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) induced junctions in bone marrow mesenchymal stem cells (BMSCs). The effect of compound sophora flavescens soup and its synergistic effect in enteritis: from sd rats BMSCs were isolated from bone marrow and were identified by flow cytometry. BMSCs expressed GFP (Ad-GFP-BMSCs) or co-expressed GFP (Ad-GFP-BMSCs) and GFP (Ad-GFP-BMSCs) were expressed by lentivirus technique. Western blot analysis of BMSCs was performed before and after transfection. 40 rats were randomly divided into 5 groups (n = 8): blank group, model group, Ad-GFP-BMSCs group. Ad-BMSCs or Ad-GFP-BMSCs or Ad-GFP were induced by TNBS.-BMSCs and two groups were given by gavage for the compound sophora flavescens decoction. The colon tissues, immunofluorescence or western blot were collected for the detection of colon by the cells and traditional Chinese medicine. Results: The survival rate of BMSCs after 48h of lentiviral transfection was similar to that of BMSCs. 90%, 80% BMSCs were able to stably express GFP. Compared with blank group, The expression of SDF-1 protein in colon region of colitis rats increased significantly. Only a small amount of green fluorescent protein was found in the colon of the P-BMSCs group. Ad-GFP-BMSCs were found in Ad-GFP-BMSCs group. Compared with Ad-GFP-BMSCs, the expression of GFP protein in colon of Ad-GFP-BMSCs increased significantly (P0.05). The expression of GFP protein in the two groups increased significantly (P0.05). In order to improve the homing efficiency of BMSCs, the compound sophora flavescens decoction can synergistically improve the homing efficiency of BMSCs. Potential methods and theoretical bases may be provided for the treatment of colonitis, Part 3: Objective: To investigate the immune mechanism of bone marrow mesenchymal stem cells (BMSCs) in the treatment of experimental colitis. Experimental colitis induced by 6-trinitrobenzene sulfonic acid and its potential immune function machine Methods: BMSCs were isolated from the bone marrow of female SD rats and were identified by flow cytometry. BMSCs were expressed GFP (Ad-GFP-BMSCs) or co-expressed GFP (Ad-GFP-BMSCs) by lentivirus technique, 40 rats were randomly divided into 5 groups (n = 8): blank group, model. group, Ad-GFP-BMSCs group, Ad-GFP-BMSCs group and two groups. TNBS was used to induce experimental colitis and tail vein injection Ad-CXC. R4-BMSCs鎴朅d-GFP-BMSC s, the two groups are administered by gavage for the compound flavescens soup, and in the whole experiment, each The results of HE staining and pathological analysis were performed on the colon tissues after 1wk treatment. The mRNA levels of IFN-, L-6 and 11-10 in colon were detected by PCR. The expression of STAT-3 and phosphorylated STAT-3 protein was detected by Western blot. The results showed that the expression of IFN-, IL-6 and IL-10 in the colon of model group compared with blank group. The protein expression of STAT-3 and phosphorylated STAT-3 increased significantly (P0.05). The clinical symptoms and pathological lesions of Ad-GFP-BMSCs were not effectively alleviated. Ad-GFP-BMSCs group, Ad-CXCR The expression of IFN-, IL-6 mRNA and IL-10 mRNA in colon tissues of rats were significantly decreased, and the mRNA expression of IL-10 increased significantly. The expression of STAT3 and phosphorylated STAT-3 was significantly decreased (P0.05).
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R574.62
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