骨髓间充质干细胞预处理对LPS诱导大鼠DIC模型的器官保护作用

发布时间：2018-03-29 19:36

  本文选题：弥漫性血管内凝血　切入点：脂多糖　出处：《山东大学》2013年博士论文

【摘要】：研究目的：研究骨髓间充质干细胞(BMSCs)预处理对脂多糖(LPS)诱导弥漫性血管内凝血(DIC)模型大鼠的器官保护性作用并探讨其机制。 研究方法： 第一部分：取同种异体Wistar大鼠原代骨髓间充质干细胞进行传代培养。对培养出的骨髓干细胞进行形态观察,考察其增殖能力；通过流式细胞仪对其细胞表型进行鉴定；在特定条件下培养BMSCs向内皮细胞,成骨细胞,脂肪细胞的分化,以证实其潜在可塑性,进一步确定其为BMSCS. 第二部分：LPS诱导Wistar大鼠DIC模型的建立。30只成年雄性Wistar大鼠,月龄7周,体重160-170g,分为2组,实验组和对照组各15只,按剂量30mg/kg腹腔内注射异戊巴比妥钠麻醉后,实验组经尾静脉或股静脉缓慢注射2ml LPS (3mg/kg,1小时),而对照组给予等量生理盐水。深麻醉下分别在LPS注射前及注射后4小时和8小时经下腔静脉抽取静脉血,测定以下凝血参数：血小板(PLT)数量,纤维蛋白原(Fib),部分凝血活酶时间(APTT),凝血酶原时间(PT),D-2聚体(D-D)。在LPS注射8小时后处死大鼠。取实验组及对照组大鼠心脏,肝脏,肾脏,肺等主要器官的组织用10%甲醛固定24h,分别行HE染色和Mallory磷钨酸苏木精染色(PTAH染色),显微镜下观察各脏器细胞形态,纤维血栓形成情况,并进行对比；同时计数100个肾小球,观察其中有纤维蛋白微血栓的肾小球所占比例,以判断本组实验LPS能否成功诱导建立DIC模型。 第三部分：慢病毒介导GFP转染骨髓间充质干细胞及体内检测。慢病毒载体(PGCL-GFP、 pHelper1.0、 pHelper2.0)包装后获取病毒的上清液保存。取第3代生长状态良好的大鼠BMSC5×104个,分别与0.25、2.5、5、12.5、25感染复数(MOI)的病毒液,在Polybrene液中(8g/m1),32℃,1000g离心2小时,然后种于12孔板培养,每孔含10%FBS的DMEM1.5ml;12h后换液,此后每3天换液1次并观察BMSCs荧光表达情况；第5天每个孔取5个视野计算平均转染率,了解MOI与转染效率的关系,选择最佳MOI。取月龄7周,体重160-170g成年雄性Wistar大鼠10只,经大鼠尾静脉或股静脉注入1m1培养液(含有GFP标记转染5天后BMSCs数量约1×106)共3次,每次间隔24h,第3次注射24h后处死大鼠,取大鼠心脏组织,用10%甲醛固定24h,然后行HE及抗GFP过氧化物酶染色。观察心肌组织内是否有BMSCs存活。 第四部分：上述实验成功后,取60只成年雄性Wistar大鼠,月龄7周,体重160-170g,按照BMSCs预处理和LPS注射方案不同分为6组,每组各有大鼠10只。处理组1：1ml培养液(不含BMSCs)注射3次,每次间隔24小时,进行预处理。在最后一次预处理结束后24h,注射1ml LPS(剂量,3mg/kg)o处理组2：1ml培养液(含BMSCs1x103)注射3次,每次间隔24小时,进行预处理。在最后一次预处理结束后24h,注射1ml LPS(剂量,3mg/kg).处理组3：1ml培养液(含BMSCs1x104)注射3次每次间隔24小时,进行预处理。在最后一次预处理结束后24h,注射1ml LPS(剂量,3mg/kg).处理组4：1mI培养液(含BMSCs1x105)注射3次每次间隔24小时,进行预处理。在最后一次预处理结束后24h,注射1ml LPS(剂量,3mg/kg).处理组5：1ml培养液(含BMSCs1x106)注射3次,每次间隔24小时,进行预处理。在最后一次预处理结束后24h,注射1ml LPS(剂量,3mg/kg).对照组：1m1培养液,不含BMSCs,注射3次,每次间隔24小时。在最后一次注射后24h,注射生理盐水1m1(不含LPS)。共6组。分别在LPS(或不含LPS)注入前及注入后4小时和8小时各抽取静脉血,测量以下参数：PLT, Fib, D-2聚体(D-D),APTT, PT,肿瘤坏死因子-α(TNF-α),干扰素-γ(IFN-γ),白细胞介素-1β(IL-1β),肌酐(Cr),丙氨酸转氨酶(ALT),磷酸肌酸激酶同工酶(CK-MB),内皮素(ET)。取心脏,肝脏,肾脏及肺部组织进行HE染色及PTAH染色,检测各器官组织细胞形态及微纤维血栓形成情况。为了进一步证明BMSCs预处理对LPS诱导DIC大鼠的保护作用,我们选择另外60只成年雄性Wistar大鼠,月龄7周,体重160-170g,随机分为三组,观察短期内大鼠死亡事件。为更好观察短期内大鼠死亡事件,我们加大LPS注射剂量至10mg/kg,观察24小时内BMSCs预处理对LPS诱导DIC的保护作用。第1组：LPS注射,(LPS10mg/kg);第2组：BMSCs预处理,(1×103/次×3次,每次间隔24小时)+LPS注射(10mg/kg,在BMSCs最后一次预处理结束后24h进行)。第3组：BMSCs预处理,(1×105/次×3次,每次间隔24小时)+LPS注射(10mg/kg,在BMSCs最后一次预处理结束后24h进行)。BMSCs经股静脉注入,LPS行腹腔注射。观察LPS注射后24h内各组大鼠生存情况,统计各组死亡时间,行统计学生存分析,绘制生存曲线。 第五部分：为进一步明确BMSCs预处理的保护机制,我们对不同剂量BMSCs预处理后大鼠外周血淋巴细胞(PBMC)增殖能力的影响进行了测定,并在体外环境下研究了不同剂量同种异体BMSCs对LPS刺激PBMC增殖的影响,以及对炎性细胞因子产生的影响。 结果：1,第1-5天,细胞快速生长,是增殖最快的时期,约第6天BMSCs增值达到顶峰,此后维持小幅波动,总体显示BMSCs生长旺盛。经分化培养,BMSCs细胞可分化成为内皮细胞,成骨细胞,脂肪细胞；经流式细胞学检测,BMSCs CD29, CD44, CD10, CD106表现阳性,而CD34, CD45表现阴性,符合骨髓间充质干细胞分子表型特征；2,LPS注射组和对照组相比,血浆PLT数量明显降低(P＜0.01), Fib含量降低(P0.01),D-2聚体量偏高(P0.01),APTT(P0.01)和PT(P0.01)明显延长,而且肾脏,肺,肝脏,心脏组织切片经HE染色和PTAH染色均提示有微血管血栓形成。LPS注射组发生DIC。3,慢病毒转染293T细胞48小时后可见大量荧光,转染效率良好。病毒转染BMSCs后第5天,可见BMSCs荧光表达,BMSCs生长良好。不同MOI条件下病毒对BMSCs转染的效率不同,当MOI为12.5时,转染效率最高。经静脉注射GFP标记BMSCs三次后(每次间隔24h,剂量为1ml培养液含细胞数1×106),取心肌组织切片,抗GFP过氧化物酶染色呈阳性,提示心肌内有BMSCs细胞存活。4,①和未经BMSCs预处理组相比,BMSCs预处理组血浆PLT(P＜0.01)和Fib(P0.05)含量高,而D-2聚体含量偏低(P0.01),而且,APTT (P＜0.05)和PT(P0.01)延长偏短,凝血状况较好；预处理BMSCs数量为1×105/次和1×106/次时最好。②与未经BMSC预处理组相比,BMSCs预处理组血浆TNF-a (P＜0.01)IFN-γ(P0.01)、 IL-1β(P＜0.05)浓度值均明显偏低,而且组间比较具有统计学意义；在预处理BMSCs数量为1×105/次和1×106/次时,TNF-a, IFN-y和IL-1p值最低。③与未经BMSCs预处理组相比,BMSCs预处理组血浆ALT, Cr, CK-MB (P0.05)和ET(P0.05)浓度明显降低；组间比较发现Cr和ET水平的差异具有统计学意义；预处理BMSCs数量为1×105/次和1×106/次时Cr和ET水平最低。④各组肾脏,肺,肝脏,心脏组织切片经HE染色和PTAH染色均显示BMSCs预处理组器官纤维微血管血栓形成量较少,器官细胞损伤较小。预处理BMSCs数量为1×105/次和1×106/次时,纤维微血管血栓形成量最少,器官细胞损伤最轻。⑤BMSCs预处理对LPS诱导DIC大鼠的死亡事件的观察,结果提示三组大鼠(每组n=20)24小时内总生存率分别为：第一组34.2%,第二组51.3%,第三组64.5%,生存率比较P=0.02,有显著统计学意义；组间生存率比较,第一组VS第二组P=0.011,第一组VS第三组P=0.001,第二组VS第三组P=0.35。5,①和对照组相比,经BMSCs预处理组大鼠PBMC在LPS刺激下的增殖能力明显减弱。②LPS可明显刺激PBMC的增殖,但在BMSCs的影响下,这种刺激作用明显减弱(P0.05)；在Transwell培养下,可达到同样效果(P0.05)。LPS刺激后,培养液炎性因子的浓度明显增加,但和BMSCs混合培养后,炎性因子释放增加的量明显减少,在Transwell培养中也有同样的表现。③和BMSCs混合培养时,LPS刺激PBMC的增殖明显减少,随着BMSCs数量越多,LPS刺激PBMC的增殖数量越来越少。和BMSCs混合培养时,培养液炎性因子的浓度开始减少,随着BMSCs量的增加,炎性因子的浓度越来越低。当BMSCs增加到一定浓度时,PBMC增殖量不再减少,炎性因子浓度也不在降低。 结论：1,用密度梯度法进行BMSCs分离,贴壁法和机械刮除法进行BMSCs培养和体外扩增,可以获得形态较单一的BMSCs样细胞,细胞生长形态和状况良好,经流式细胞表型鉴定和多向分化培养鉴定证实为BMSCs.2,用LPS静脉推注可成功诱导建立Wistar大鼠DIC模型。此法操作简便,成功率高。LPS静脉推注可作为建立大鼠DIC模型的可靠方法。3,用慢病毒载体可成功转染GFP于大鼠骨髓间充质干细胞中,3次静脉注射(每次间隔24h)本实验所培养的GFP标记大鼠骨髓间充质量干细胞后,可在体内发现存活的BMSCS.4, BMSCs预处理可有效减轻LPS诱导大鼠DIC模型的器官损伤,抑制血管内凝血,提高大鼠生存率。这种保护性反应在一定范围内具有BMSCs注射数量依赖性,其机制可能与BMSCs对炎性介质分泌的调节有关。5,在体内和体外实验中,BMSCs均可抑制淋巴细胞增殖,调节炎性因子的释放,这种作用不依赖与细胞与细胞之间的直接接触,而且在一定BMSCs数量范围内,具有数量依赖性。
[Abstract]:Objective : To study the protective effects of bone marrow mesenchymal stem cells ( MSCs ) on lipopolysaccharide ( LPS ) induced diffuse intravascular coagulation ( DIC ) in rats and to explore its mechanism .

Study method :

In the first part , the bone marrow mesenchymal stem cells of Wistar rats were cultured in vitro . The cultured bone marrow stem cells were observed and their proliferative capacity was investigated .
The cell phenotype was identified by flow cytometry .
The differentiation of bone marrow cells into endothelial cells , osteoblasts and adipocytes was cultured under specific conditions to confirm their potential plasticity and to further determine their potential plasticity .

In the second part : LPS - induced DIC model of Wistar rats was established . 30 adult male Wistar rats were randomly divided into 2 groups : platelet ( PLT ) quantity , fibrinogen ( Fib ) , partial thrombase time ( PT ) , prothrombin time ( PT ) and D - 2 mer ( D - D ) .
At the same time , 100 glomeruli were counted , and the proportion of glomeruli with fibrin microthrombus was observed to determine whether LPS could induce the establishment of DIC model successfully .

The third part : lentivirus - mediated GFP - mediated bone marrow mesenchymal stem cells and in vivo detection . The supernatant of the virus was obtained after packaging with slow virus vector ( PGCL - GFP , pHelper1.0 , pHelper2.0 ) .
On the 5th day , the average transfection rate was calculated from 5 visual field of each well , and the relationship between the infection rate and the transfection efficiency was studied . Ten male Wistar rats weighing 160 - 170g were injected into 1 ml culture medium ( about 1 脳 106 ) at intervals of 24 h and 24 h . After 24 h , the rats were sacrificed . After 24 h , the rats were sacrificed . After 24 h of the third injection , the rats were sacrificed and then HE and anti - GFP peroxidase staining were performed .

In the fourth part , 60 adult male Wistar rats , 7 weeks of age , 160 - 170g , were randomly divided into 6 groups according to the pre - treatment and LPS injection protocol . After the last pretreatment , 1 ml of LPS ( dose , 3 mg / kg ) was injected into 2 : 1 ml culture solution ( containing BMSCs1x103 ) for 3 times . After the last pretreatment , 1 ml of LPS ( dose , 3 mg / kg ) was injected . Treatment group 3 : 1 ml of culture solution ( containing BMSCs1x104 ) was injected three times at each interval for 24 hours , and the pretreatment was performed . After the last pretreatment , 1 ml of LPS ( dose , 3 mg / kg ) was injected . Treatment group 4 : 1 mI culture solution ( including BMSCs1x105 ) was injected three times at intervals of 24 hours for pretreatment . After the last pretreatment , 1 ml of LPS ( dose , 3 mg / kg ) was injected . Treatment group 5 : 1 ml culture solution ( containing BMSCs1x106 ) was injected three times , each time interval was 24 hours , pretreatment was performed . After the last pretreatment , 1 ml of LPS ( dose , 3 mg / kg ) was injected . In order to further demonstrate the protective effect of pre - treatment on LPS - induced DIC rats , the following parameters were measured : PLT , Fib , D - dimer ( D - D ) , interferon - 纬 ( IFN - 纬 ) , interleukin - 1尾 ( IL - 1尾 ) , creatinine ( Cr ) , alanine aminotransferase ( ALT ) , interleukin - 1尾 ( IL - 1尾 ) , creatinine ( Cr ) , alanine aminotransferase ( ALT ) , interleukin - 1尾 ( IL - 1尾 ) , creatinine ( Cr ) , alanine aminotransferase ( ALT ) , creatine kinase isoenzyme ( CK - MB ) and endothelin ( ET ) . Group 3 : pre - treatment , ( 1 脳 105 / time 脳 3 times , 24 hours at each interval ) + LPS injection ( 10 mg / kg , 24 h after the last pre - treatment ) . The survival rate of each group was observed in 24 hours after LPS injection , and the survival curves were drawn by statistical survival analysis .

The fifth part : To further clarify the protective mechanism of pre - treatment , we measured the effects of different doses of bone marrow cells on the proliferation of peripheral blood lymphocytes ( PBMC ) , and studied the effects of different doses of allogeneic bone marrow cells on the proliferation of PBMC stimulated by LPS in vitro and the effects on inflammatory cytokines .

Results : 1 , 1 - 5 days , the rapid growth of cells was the fastest growing period , and the value added at the 6th day reached the peak , after which small amplitude fluctuation was maintained , and the growth of bone marrow cells was increased . After differentiation culture , the cells of bone marrow were differentiated into endothelial cells , osteoblasts and fat cells .
The expression of CD29 , CD44 , CD10 and CD106 in bone marrow cells was detected by flow cytometry .
2 銆，

本文编号：1682550