CXCR-4基因轉(zhuǎn)染的BMMSCs對實驗性IBD小鼠腸黏膜修復(fù)機制研究
發(fā)布時間：2019-06-01 11:28
【摘要】：研究背景：炎癥性腸病(inflammatory bowel disease,IBD)作為一種病因及發(fā)病機制尚未完全明確的,以累及腸道為主的消化道慢性炎癥性疾病,在臨床上一般包括克羅恩病(Crohn's Disease,CD)和潰瘍性結(jié)腸炎(Ulcerative Colitis,UC)。骨髓間充質(zhì)干細胞(Bone Marrow Mesenchymal Stem Cells,BMMSCs)作為最早被人工分離出的成體干細胞,具有許多生物學(xué)特點和優(yōu)勢,如：自身低免疫原性、多向分化潛能、免疫調(diào)節(jié)作用、取材方便等,因此成為臨床上移植療法的推薦選擇之一,對IBD而言,因其可通過促進病變腸黏膜修復(fù)并糾正機體及局部的免疫功能紊亂,而成為治療本病的首選細胞。然而,經(jīng)靜脈移植的BMMSCs最終定植于病變腸黏膜的數(shù)量有限,限制了其治療作用的發(fā)揮。由于干細胞的歸巢行為涉及多種趨化因子及其受體的參與,其中趨化因子CXC亞組受體-4/基質(zhì)細胞衍生因子-1(Chemokine Receptor-4/Stromal cell-derived Factor-1,CXCR4/SDF-1)的作用已經(jīng)被證實在介導(dǎo)干細胞歸巢中發(fā)揮著十分重要的地位。故利用CXCR-4基因轉(zhuǎn)染BMMSCs,能夠使其在體內(nèi)的靶向性定位增強,從而提高療效。實驗?zāi)康模和ㄟ^移植未經(jīng)修飾的BMMSCs和CXCR-4基因修飾的BMMSCs來比較兩者在治療實驗性IBD小鼠模型中腸道黏膜局部定植率及療效,并探討CXCR-4基因修飾的BMMSCs促進受損腸黏膜愈合的機制,從而為臨床上確定一種選擇靶向性更強的干細胞用于治療IBD提供理論依據(jù)。實驗內(nèi)容：1、構(gòu)建并鑒定BMMSCs。2、構(gòu)建并鑒定經(jīng)CXCR-4基因修飾的靶向性BMMSCs及評估其體外遷移率。3、觀察經(jīng)CXCR-4基因修飾的BMMSCs能否向IBD小鼠損傷腸黏膜定向遷移并加快其愈合。4、探討靶向性BMMSCs通過何種機制在腸道局部大量定植并發(fā)揮修復(fù)作用。實驗方法：1、采用細胞貼壁分離法從雄性BALB/c小鼠(4-5周齡)的骨髓中分離并體外培養(yǎng)BMMSCs;并對BMMSCs進行熒光染料(CM-Dil)標(biāo)記。2、通過形態(tài)學(xué)觀察、流式細胞學(xué)技術(shù)檢測細胞表面標(biāo)志物、成骨／成脂肪樣細胞誘導(dǎo)分化實驗對所培養(yǎng)的BMMSCs進行鑒定。3、將攜帶CXCR-4的慢病毒轉(zhuǎn)染入BMMSCs構(gòu)建成CXCR4-BMMSCs;進行嘌呤霉素篩選實驗,從中提純CXCR-BMMSCs;采用Real time-PCR檢測并比較轉(zhuǎn)染組和未轉(zhuǎn)染組的CXCR-4的表達量。4、通過形態(tài)學(xué)觀察、流式細胞技術(shù)檢測細胞表面標(biāo)志物、成骨／成脂樣細胞誘導(dǎo)分化實驗、臺盼藍拒染實驗間接測定細胞存活率、流式細胞學(xué)測定細胞周期,來比較兩組生物學(xué)特性的差異。5、通過Transwell體外遷移實驗,比較兩組細胞向SDF-1的遷移率。6、選擇雌性BALB/c小鼠(6-8周齡),分別隨機分為6組;①對照組(標(biāo)記為Control, Ctr組)：生理鹽水灌腸(0.1m1/只),灌腸后第2天,給予尾靜脈注射不含BMMSCs的PBS 0.1ml, n=20; ②TNBS組(T組)：TNBS-乙醇灌腸劑(0.1m1/只),并于造模后第2天,給予尾靜脈注射不含BMMSCs的PBS 0.1ml,n=20;③ BMMSC移植組(MT組)：TNBS-乙醇灌腸劑(0.1ml/只),并于造模后第2天,尾靜脈注射含BMMSCs(1×106/只)的PBS 0.1ml,n=20;④CXCR-BMMSC移植組(CMT組)：TNBS-乙醇灌腸劑(0.1ml/只),并于造模后第2天,給予尾注射含CXCR4-BMMSCs(1×106/只)的PBS 0.1ml,n=20。移植當(dāng)天標(biāo)記為D0,移植后第1-13天,分別記為D1-13。7、觀察各組小鼠一般情況、體重變化及存活率、進行臨床癥狀評分、肉眼及放大鏡下觀察結(jié)腸大體外觀、黏膜狀況,進行組織病理學(xué)評分。8、分別于D0、D1、D3、D5、D7、D9、D11、D13天采用頸椎脫臼法處死小鼠3只/組,并取材血液、腸道組織、腸系膜淋巴結(jié)及脾臟,一部分進行石蠟包埋,一部分經(jīng)液氮速凍后,-80。C冰箱保存。9、熒光顯微鏡下觀察各組細胞在腸道內(nèi)的定植情況,應(yīng)用Real time PCR檢測并比較Y染色體的性別決定區(qū)段(SRY)基因的腸道局部的相對量。10、采用免疫組織化學(xué)檢測腸道干細胞標(biāo)志物L(fēng)gr-5、腸道上皮間的緊密連接蛋白Occludin、血管生成指標(biāo)VEGF的表達情況。11、采用Western Blot技術(shù)檢測P13K的表達情況。實驗結(jié)果：1、采用貼壁分離法培養(yǎng)的BMMSCs隨著不斷純化其細胞形態(tài)呈長梭狀且按束狀排列,細胞表型鑒定顯示為：CD90.2+;CD105+;CD45-;CDllb-,成骨誘導(dǎo)可見明顯的骨結(jié)節(jié)形成,成脂肪誘導(dǎo)可見脂滴。經(jīng)熒光染料染色后對細胞的生物學(xué)特性無明顯影響。2、將CXCR-4基因?qū)隑MSCs后,與BMMSCs組比較可檢測到CXCR-4基因的高表達(p0.05)：3、CXCR-BMMSCs組細胞均呈長梭形并束狀排列；細胞表型鑒定未發(fā)生改變(CD90.2+;CD105+;CD45-;CD11b-);成骨誘導(dǎo)可見明顯的骨結(jié)節(jié)形成,成脂肪誘導(dǎo)可見脂滴；活細胞比率均在90%以上;以上結(jié)果與未經(jīng)修飾的BMMSCs相比無明顯差異(p0.05);在細胞周期檢測中發(fā)現(xiàn)過表達CXCR-4基因的BMMSCs處于復(fù)制期的細胞增多(p0.05)。4、體外遷移實驗證實：在SDF-1的誘導(dǎo)下，CXCR-BMMSCs組的遷移率BMMSCs組(p0.05)。5、TNBS-乙醇灌腸法誘導(dǎo)后的實驗性IBD小鼠模型的存活率明顯下降、小鼠精神狀態(tài)差且體重明顯下降,臨床癥狀及組織病理學(xué)評分上升,肉眼觀察可見結(jié)腸皺縮,局部腸段狹窄,腸黏膜伴有充血水腫、糜爛形成,黏膜及黏膜下層有大量中性粒細胞浸潤,與正常組比較均有明顯差異(p0.05)；MT組、CMT組移植后第2天,與T組相比,存活率開始上升,疾病癥狀減輕、體重回升,臨床癥狀及組織病理學(xué)評分下降(p0.05), CMT組誘導(dǎo)緩解的效果更好(p0.05)。6、熒光顯微鏡下可見MT組、CMT組的腸道組織均存在紅色熒光;治療組及雄性對照組的腸道黏膜組織中均能檢測到SRY基因；體內(nèi)定植率的比較顯示CMT組MT組。(p0.01)7、免疫組織化學(xué)染色結(jié)果顯示：IBD模型小鼠接受BMSCs移植后,腸道干細胞標(biāo)志物L(fēng)gr-5、腸道上皮間的緊密連接蛋白Occludin及VEGF的表達量較造模未治療組有所升高。8、Western Blot的結(jié)果顯示：與T組相比,MT組、CMT組P13K的蛋白表達量均升高,其中CMT組升高最為明顯(p0.01)。實驗結(jié)論：1、利用慢病毒載體轉(zhuǎn)染CXCR-4基因至BMMSCs,幾乎不會改變BMMSCs原有的生物學(xué)特性。2、體內(nèi)外實驗均證實了CXCR-4基因的過表達可增加BMMSCs的遷移率。3、通過干細胞移植治療實驗性IBD小鼠模型時,其治療效果與細胞在腸道的定植率呈正相關(guān),并且可通過促進腸道干細胞分化、重構(gòu)腸上皮緊密連接、激活P13K信號通路來促進腸黏膜再生、修復(fù)、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.
【學(xué)位授予單位】：中國人民解放軍醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R574
本文編號：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.
【學(xué)位授予單位】：中國人民解放軍醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R574
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