大鼠骨髓间充质干细胞的生物学特性及其对急性化学性肝损伤的修复作用

发布时间：2018-01-05 10:34

  本文关键词：大鼠骨髓间充质干细胞的生物学特性及其对急性化学性肝损伤的修复作用　出处：《天津医科大学》2009年硕士论文　论文类型：学位论文

  更多相关文章： 骨髓间充质干细胞 慢病毒 增强绿色荧光蛋白 转染 示踪 肝损伤 干细胞移植

【摘要】： 一、大鼠骨髓间充质干细胞的分离、培养、鉴定及EGFP标记 目的:本研究旨在体外分离培养和纯化大鼠骨髓间充质干细胞(Bone marrowmesenchymal stem cells,BMSCs),并初步鉴定BMSCs的生物学特性,为利用骨髓间充质干细胞作细胞治疗提供实验依据;探讨不同浓度慢病毒载体对体外培养的BMSCs的转染效率,为进一步观察体内向肝细胞分化奠定基础。 方法:在无菌条件下取四周龄100～120g健康雄性Wistar大鼠双后肢的骨髓细胞,采用密度梯度离心法与贴壁培养相结合的方法,体外分离BMSCs。采用24小时后贴壁的细胞用含10%～15%胎牛血清(Fetal calf serum,FCS)的DMEM培养基(Dulbecco's Modified Eagle Media,DMEM)进行培养,以后每隔3天换液一次。待细胞80%～90%融合后,消化传代,使之逐渐纯化。1.BMSCs的观察与鉴定:(1)倒置显微镜下观察细胞形态;(2)透射电镜下观察细胞超微结构;(3) MTT法测定细胞生长曲线;(4)流式细胞仪检测CD29,CD34,CD45,CD90的表达。2.BMSCs的增强绿色荧光蛋白(enhanced green fluorescentprotein,EGFP)转染:将分离的BMSCs分为空白对照组和实验组,实验组按照不同感染复数(multiplicity of infection,MOI)(1、10、30、50、70、100)加入携带EGFP的慢病毒液,空白组加入磷酸盐缓冲液(phosphate-buffered saline,PBS)。转染后即刻到14天持续于荧光显微镜蓝光激发下观察绿色荧光蛋白表达情况及细胞形态变化,确定最佳MOI。 结果: 1.采用Percoll密度梯度离心与贴壁培养相结合的方法所培养的BMSCs呈长梭形,胞核圆形或椭圆形,位于中央。流式细胞检测结果为:CD29+/CD34-/CD45-/CD90+; 2.从传代细胞生长曲线可见:第1～2天为细胞生长潜伏期,第3～7天为对数生长期,8天以后进入平台期。 3.透射电镜结果显示:BMSCs有两种不同的形态结构,一种是处于未分化或分化较低状态的小幼稚性细胞;另一种是处于相对活跃期的成熟大细胞。 4.荧光显微镜下观察骨髓间充质干细胞转染后24小时后荧光开始发出,5～7天绿色荧光表达稳定,表达效率最高能达到(79.85±4.92)%(MOI=30,第7天);转染效率随MOI的增加而增加,MOI=30,50,70,100时的转染效率显著高于MOI=1,10时的转染效率(P＜0.05);荧光显微镜蓝光下观察细胞形态与普通显微镜下观察空白组细胞形态一致。 结论:本论文通过体外分离、纯化和鉴定BMSCs,建立了一套较合理的间充质干细胞体外培养的技术方案,成功分离出BMSCs,并对BMSCs的生物学特性有进一步的了解。含EGFP基因的慢病毒载体系统能够成功转染BMSCs,并且能高效的表达EGFP,且不会对细胞的生物学特性产生明显的影响,为进一步观察BMSCs在体内向肝细胞分化奠定基础。 二、EGFP标记的大鼠BMSCs同种异体移植对急性化学性肝损伤的修复作用 目的:观察稳定表达EGFP的BMSCs在肝损伤环境下的定居及对肝损伤的修复作用,了解EGFP标记的BMSCs在肝损伤模型内的变化,为同种异体移植BMSCs为基础的肝损伤治疗提供理论依据。 方法:健康雄性Wistar大鼠30只,体重150～180克,饲养一周后随机分为六组,每组5只,肝损伤组采用2%四氯化碳(carbon tetrachloride,CCL_4)连续灌胃七天诱导急性肝损伤模型,于造模第7天行细胞移植:A组(正常对照组):正常大鼠,不行细胞移植,尾静脉内注入等量PBS;B组(正常+未标记组):正常大鼠,向尾静脉内移植未标记的BMSCs;C组(正常+标记组):正常大鼠,向尾静脉内移植EGFP标记的BMSCs;D组(肝损伤对照组):肝损伤大鼠,不行细胞移植,尾静脉内注入PBS;E组(肝损伤+未标记组):肝损伤大鼠,向尾静脉内移植未标记的BMSCs;F组(肝损伤+标记组):肝损伤大鼠,尾静脉内移植标记的BMSCs。移植后1周处死全部受试动物,血清检测肝功能、HE染色行组织学观察,荧光显微镜下确认EGFP阳性细胞在肝损伤大鼠的定位及对大鼠肝损伤的修复作用。 结果: 1.采用CCL_4制作大鼠急性化学性肝损伤模型1周后,肝损伤组(D、E、F)血清丙氨酸氨基转移酶(alanine aminotransferase,ALT)、天门冬氨酸氨基转移酶(aspartate aminotransferase,AST)、总胆红素(total bilirubin,TBIL)等指标较正常喂养大鼠(A、B、C)相应指标升高,差异有统计学意义(P＜0.05)。肝损伤大鼠肝内出现弥漫性分布的点状坏死、桥接坏死,肝血窦充血明显,弥漫炎性细胞浸润。 2.同种异体移植BMSCs1周后E、F组大鼠血清ALT、AST、TBIL等指标较移植前有所恢复,与未移植BMSCs的肝损伤组(D组)的相应指标比较,差异有统计学意义(P＜0.05)。BMSCs移植组大鼠组织中坏死、充血、炎性细胞浸润情况较未移植组减轻。无论是在正常组大鼠还是在肝损伤大鼠组,EGFP标记的BMSCs移植与未标记的BMSCs移植后,大鼠血清学、肝脏组织病理学情况类似。F组大鼠肝脏冰冻切片在荧光显微镜下可以在肝组织观察到EGFP阳性的细胞。 结论: 1.CCL_4灌胃可以获得比较可靠的肝损伤动物模型。 2.BMSCs移植后可修复急性化学性肝损伤所致的肝功能损害。 3.EGFP标记不影响正常大鼠的肝功能,也不影响BMSCs对急性化学性损伤的修复作用。
[Abstract]:Isolation, culture, identification and EGFP markers of rat bone marrow mesenchymal stem cells
Objective: the purpose of this study is to isolate and purify rat bone marrow mesenchymal stem cells (Bone marrowmesenchymal stem cells, BMSCs), the biological characteristics and preliminary identification of BMSCs, to provide the experimental basis for the use of bone marrow mesenchymal stem cells for cell therapy; to investigate the transfection efficiency of different concentrations of lentiviral vector of BMSCs in vitro that is to lay a foundation for further study of liver cell differentiation in vivo.
Methods: under sterile conditions, take 100 ~ 4 weeks old healthy male 120g Wistar bipedal rat bone marrow cells by density gradient centrifugation combined with adherent culture, in vitro by BMSCs. after 24 hours of adherent cells with 10% ~ 15% fetal bovine serum (Fetal calf, serum, FCS) DMEM medium (Dulbecco's Modified Eagle Media, DMEM) were cultured, after every 3 days. After the cell was changed every 80% to 90% after the fusion of digestive passage, so that gradually purified observation and identification of.1.BMSCs: (1) the cells were observed under inverted microscope; (2) the ultrastructure was observed by transmission electron microscope; (3) the cell growth curve was determined by MTT; (4) the detection of CD29, flow cytometry, CD34, CD45, enhanced green fluorescent protein expression of.2.BMSCs CD90 (enhanced green fluorescentprotein, EGFP) transfection: the isolated BMSCs were divided into control group and experimental group, experimental Group in accordance with different multiplicities of infection (multiplicity of, infection, MOI) (1,10,30,50,70100) with lentivirus carrying EGFP liquid, adding phosphate buffer (phosphate-buffered saline, PBS group). After transfection for 14 days immediately to fluorescent microscopy under blue light to observe the expression of green fluorescent protein and cell morphological changes, determine the best MOI.
Result:
1., the BMSCs cultured by Percoll density gradient centrifugation and adherent culture was spindle shaped, and the nucleus was round or oval. It was located in the center. The result of flow cytometry was CD29+/CD34-/CD45-/CD90+.
2. from the cell growth curve, the incubation period of cell growth was seen from first to 2 days, the logarithmic growth period was third to 7 days, and the platform stage was entered after 8 days.
3. transmission electron microscopy showed that BMSCs has two different morphological structures, one is immature cells in undifferentiated or differentiated state, and the other is mature cells in relatively active stage.
4. observation under fluorescence microscopy of bone marrow mesenchymal stem cells 24 hours after transfection, fluorescence began to produce 5 ~ 7 days, the expression of green fluorescence expression stability, efficiency can reach a maximum (79.85 + 4.92)% (MOI=30, seventh days); transfection efficiency increased with the increase of MOI, MOI=30,50,70100, transfection efficiency was significantly higher than that of MOI=1,10 the transfection efficiency (P < 0.05); control group observation of cell morphology and cell morphology consistent with ordinary microscope fluorescence microscope under blue light.
Conclusion: the in vitro isolation, purification and identification of BMSCs, to establish a set of reasonable technical scheme of mesenchymal stem cells in vitro, BMSCs successfully isolated, and the biological characteristics of BMSCs have further understanding. Lentiviral vector containing EGFP gene can be successfully transfected into BMSCs, and the expression of EGFP efficient, and does not obviously affect on cell biological characteristics, lay the foundation for further study on BMSCs differentiation to hepatocytes in vivo.
Two, EGFP labelled rat BMSCs allograft for the repair of acute chemical liver injury
Objective: To observe the colonization and BMSCs repair of BMSCs with stable expression of EGFP in the liver injury environment, and to understand the change of EGFP labeled BMSCs in the liver injury model, so as to provide a theoretical basis for the treatment of BMSCs based liver injury.
鏂规硶:鍋ュ悍闆勬，

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