過表達CXCR4的骨髓間充質干細胞歸巢治療潰瘍性結腸炎及復方苦參湯的協(xié)同作用研究
發(fā)布時間：2018-10-14 11:28
【摘要】：第一部分CXCR4基因過表達慢病毒載體的構建及鑒定 目的：探討構建大鼠趨化因子受體4(chemokine receptor4, CXCR4)基因過表達慢病毒載體的技術方法并檢測其體外表達目的基因的水平。 方法：設計CXCR4基因引物,采用聚合酶鏈反應(PCR)擴增CXCR4基因片段；用Age I酶切GV208載體；通過連接酶將CXCR4基因片段連接至線性化的GV208載體上；運用PCR方法鑒定陽性克隆的CXCR4-GV208載體；按GV208慢病毒包裝試劑盒說明書包裝慢病毒并運用實時定量聚合酶鏈反應(RT-qPCR)法行滴度檢測；然后用重組慢病毒轉染人胚腎細胞(293T細胞),觀察GFP表達情況。 結果：通過PCR成功地擴增了CXCR4基因片段并連接到了GV208病毒載體上,通過PCR及DNA測序鑒定,證明CXCR4-GV208質粒構建正確,重組慢病毒轉染293T細胞后可觀察到熒光及蛋白表達。包裝過表達慢病毒并測其濃縮滴度為2.0×108TU/mL。 結論：成功構建CXCR4-GV208慢病毒載體,為CXCR4基因的后期研究提供了實驗基礎。 第二部分SDF-1α/CXCR4軸促進間充質干細胞歸巢于實驗性結腸炎受損結腸及復方苦參湯的協(xié)同效應 目的：調查基質細胞衍生因子(SDF-1α)／趨化因子受體4(CXCR4)軸在骨髓間充質干細胞(BMSCs)治療2,4,6-三硝基苯磺酸(TNBS)誘導的結腸炎中的作用及復方苦參湯的協(xié)同效應。 方法：從SD大鼠骨髓中分離BMSCs并使用流式細胞術鑒定。通過慢病毒技術使BMSCs表達GFP (Ad-GFP-BMSCs)或共表達CXCR4和GFP (Ad-CXCR4-BMSCs), Western blot檢鋇BMSCs轉染前后CXCR4蛋白表達。40只大鼠被隨機分成5組(n=8)：空白組、模型組、Ad-GFP-BMSCs組、Ad-CXCR4-BMSCs組和二聯組。采用TNBS誘導實驗性結腸炎,尾靜脈注射Ad-CXCR4-BMSCs或Ad-GFP-BMSCs,二聯組予復方苦參湯灌胃治療。細胞和中藥治療1wk后收集結腸組織,免疫熒光或western blot檢測結腸部位GFP、CXCR4和SDF-1α表達。 結果：慢病毒轉染48h后BMSCs的存活率大約為90%,80%的BMSCs能夠穩(wěn)定表達GFP。相對于空白組,結腸炎大鼠結腸部位SDF-1α蛋白表達明顯上升。第12d,Ad-GFP-BMSCs組結腸只能發(fā)現少量的綠色熒光表達。相對于Ad-GFP-BMSCs組,Ad-CXCR4-BMSCs組和二聯組結腸部位則能發(fā)現綠色熒光表達顯著增多。類似的是,Western blot顯示Ad-GFP-BMSCs組結腸部位僅表達少量的GFP蛋白；相對于Ad-GFP-BMSCs組,Ad-CXCR4-BMSCs組結腸部位GFP蛋白表達量則明顯增多(P0.05)；相對于Ad-CXCR4-BMSCs組,二聯組GFP蛋白表達顯著提高(P0.05)。 結論：SDF-1α/CXCR4軸在BMSCs歸巢于受損的結腸組織中發(fā)揮重要的作用,復方苦參湯可能協(xié)同改善BMSCs的歸巢效率。這可能為潰瘍性結腸炎的細胞治療提供潛在的方法和理論依據。 第三部分過表達CXCR4的骨髓間充質干細胞治療實驗性結腸炎的免疫機制研究 目的：調查過表達CXCR4的骨髓間充質干細胞治療2,4,6-三硝基苯磺酸誘導的實驗性結腸炎的效果及其潛在的免疫作用機制。 方法：從雌性SD大鼠骨髓中分離BMSCs并使用流式細胞術鑒定。通過慢病毒技術使BMSCs表達GFP(Ad-GFP-BMSCs)或共表達CXCR4和GFP(Ad-CXCR4-BMSCs),40只大鼠被隨機分成5組(n=8)：空白組、模型組、Ad-GFP-BMSCs組、Ad-CXCR4-BMSCs組和二聯組。采用TNBS誘導實驗性結腸炎,尾靜脈注射Ad-CXCR4-BMSCs或Ad-GFP-BMSCs,二聯組予復方苦參湯灌胃治療。在整個實驗過程中,每天記錄大鼠的DAI。治療1wk后,收集結腸組織進行HE染色和病理學分析。PCR檢測結腸部位IFN-γ、L-6和11-10的mRNA表達,Western blot檢測STAT-3和磷酸化STAT-3蛋白表達。 結果：相對于空白組,模型組結腸部位的IFN-γ、IL-6和IL-10的mRNA表達顯著上升,STAT-3及磷酸化STAT-3的蛋白表達明顯上升(P0.05)。系統(tǒng)治療一周后,相對于模型組,Ad-GFP-BMSCs組大鼠的臨床癥狀及結腸病理損害并沒有得到有效的緩解。相對于Ad-GFP-BMSCs組,Ad-CXCR4-BMSCs組和二聯組大鼠的DAI及病理炎癥分數顯著降低,結腸長度明顯恢復。大鼠結腸組織的IFN-γ、IL-6的mRNA表達顯著下降,IL-10的mRNA表達顯著上升(P0.05); STAT3及磷酸化STAT-3的蛋白表達顯著下降(P0.05)。 結論：過表達CXCR4的BMSCs可能通過抗炎及免疫調節(jié)機制來緩解實驗性結腸炎,這可能為BMSCs治療UC提供可靠的理論依據。
[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).
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R574.62
本文編號：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).
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R574.62
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