骨髓间充质干细胞对横纹肌溶解致急性肾损伤大鼠肾脏保护作用及机制研究
发布时间：2018-09-03 10:38
【摘要】：目的:对Sprague-Dwley(SD)大鼠骨髓间充质干细胞(Bone Marrow derived Mesenchymal Stem Cells,BMSCs)进行体外抽提,并进行分离、培养、纯化,对生物学特性进行鉴定。用绿色荧光蛋白(Green Fluorenscent Protein,GFP)标记慢病毒载体进行体内示踪。观察BMSCs移植治疗大鼠横纹肌溶解致急性肾损伤(Rhabdomyolysis-induced AKI,RIAKI)的可行性,评价其对RIAKI大鼠肾脏形态结构、功能的影响,并从细胞水平观察BMSCs治疗RIAKI的效果。观察BMSCs移植后RIAKI大鼠Th17细胞及Treg细胞变化及炎症介质水平变化,探讨BMSCs治疗横纹肌溶解致急性肾损伤的机制。方法:1、无菌条件选取健康雄性SD大鼠双后肢的骨髓细胞,采用密度梯度离心法体外分离、培养BMSCs并传代,保存P2~P6代BMSCs,治疗时用P3代BMSCs。流式细胞仪检测细胞表面标志分子及生物学性质,包括生长曲线及细胞周期分析。细胞化学法鉴定BMSCs的多向分化潜能。BMSCs慢病毒载体介导GFP标记和扩增,以感染复数(multiplicity of infection,MOI)(5、10、25、50)进行感染,得到最佳感染效率和细胞活性。2、BMSCs移植治疗RIAKI大鼠效果:⑴成年健康雄性SD大鼠禁水24h后,双后肢肌内注射50%甘油(生理盐水稀释)10ml/Kg制作RIAKI大鼠模型。⑵将SD大鼠随机分为4组:正常对照组(Normal组);假手术组(NS组);模型组(RIAKI组);治疗组(BMSCs组)。分别观察各组大鼠一般情况;血清生化指标:尿素氮(BUN)、肌酐(Scr)、肌酸激酶(CK);形态学与病理学改变:双后肢肌肉(HE染色);肾(HE、PAS染色);肾脏细胞凋亡(TUNEL凋亡分析,Western Blot检测肾组织caspase3,caspase 9,Bcl-2,cytochrome C蛋白表达)。3、BMSCs移植治疗大鼠RIAKI作用机制:⑴细胞水平:流式细胞技术检测各组大鼠脾脏组织中Treg细胞(CD4+CD25+Foxp3+)在CD4+T细胞中的比例及Th17细胞(CD4+、IL-17+代表Th17)比例。⑵基因水平:RT-PCR检测各组大鼠脾脏及肾组织Th17细胞特异性转录因子RORγtm RNA及Treg细胞特异性转录因子Foxp3m RNA水平⑶特异性细胞因子水平:ELISA检测各组大鼠血清及肾组织中Treg细胞释放的特异性细胞因子:TGF-β、IL-10(抗炎);Th17细胞释放IL-17(促炎);相关炎性细胞因子TNF-α、IL-6的水平。结果:1、采用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,胞核位于中央,呈圆形或椭圆形。流式细胞检测结果为:BMSCs均表达CD44,CD90,阳性率分别为89.9%,91.3%;而CD45,呈阴性,阳性率分别为2.8%。从传代细胞生长曲线可见:第1~2d为细胞生长潜伏期,第3~7d为对数生长期,7d后进入平台期。感染复数(multiplicity of infection,MOI)=25时高表达绿色荧光蛋白,在荧光显微镜下绿色荧光明显可见,细胞生长速度快,BMSCs-GFP90%,本实验最佳MOI确定为25。2、生化指标变化:RIAKI组大鼠后肢甘油注射后血BUN、Scr和CK显著升高,第6h、24h、48h、72h、96h及1W(周)后与干预前及Normal组比较差异显著(P0.01),其中血BUN、SCr于第72h达峰值,CK于第24h达峰值,上述生化指标升高水平及变化趋势符合横纹肌溶解并急性肾损伤的实验室诊断标准。BMSCs组血BUN、Scr水平于第24h、48h、72h、96h及1W较RIAKI组相同时间点降低,差异显著,有统计学意义(P0.01)。BMSCs组第24h、48h、72h、96h肌肉组织损伤指标血CK值较RIAKI组相同时间点降低,差异显著,有统计学意义(P0.01)。NS组与Normal组两组间各指标比较无统计学意义(P0.05)。光镜下可见RIAKI组及BMSCs组大鼠双后肢局部肌肉组织呈肌溶解性病理改变,BMSCs组大鼠24h、48h、72h横纹肌溶解损伤程度较RIAKI组明显减轻,BMSCs组大鼠24h、48h、72h肾组织损伤程度较RIAKI组减轻,表现为肾小管内管型减少,上皮细胞坏死程度减轻。TUNEL法检测细胞凋亡情况,荧光显微镜观察BMSCs组较RIAKI组第24h、48h、72h、1w大鼠肾脏凋亡细胞减少,凋亡指数(AI)降低(P0.01)。WB检测结果显示Normal组及NS组caspase 3、caspase 9、cytochrome C蛋白极低水平表达,Bcl-2蛋白表达水平较高。BMSCs组第24h、48h、72h、1w各时间点caspase 3、caspase 9、cytochrome C蛋白含量与均低于RIAKI组表达水平(P0.05),BMSCs组各时间点Bcl-2蛋白表达水平均高于RIAKI组(P0.05)。3、流式技术检测大鼠脾脏Treg细胞比例变化,结果显示BMSCs组与RIAKI组比较第24h、48h、72h Treg细胞比例升高,Th17细胞比例降低,差异显著,有统计学意义(P0.05)。RT-PCR检测RORγt m RNA和Foxp3 m RNA的表达发现BMSCs组与RIAKI组比较脾脏及肾组织RORγt m RNA水平下调,Foxp3m RNA水平上调,差异显著,有统计学意义(P0.05)。ELISA检测血清及肾组织细胞因子IL-17、IL-10、TGF-β、IL-6和TNF-α的含量发现:BMSCs组与RIAKI组比较IL-17水平下,L-10水平升高,TGF-β水平升高,IL-6、TNF-α水平降低,差异显著,有统计学意义(P0.05)。结论:1、通过密度梯度离心法联合BMSCs贴壁筛选法可获得足量纯化的BMSCs,GFP慢病毒载体成功转染BMSCs,并高效的表达GFP,不影响BMSCs生长、分化等生物学特性及功能,可作为移植BMSCs细胞标记示踪的手段。2、双后肢肌肉注射50%甘油溶液可成功诱导了RIAKI大鼠模型,BMSCs治疗降低RIAKI大鼠的血BUN、SCr和CK水平,在一定程度上改善了急性损伤肾脏的结构与功能,证实了BMSCs对RIAKI治疗效果;进一步从细胞水平观察发现BMSCs治疗可减少RIAKI大鼠肾脏凋亡细胞数量,降低凋亡指数。BMSCs移植上调RIAKI大鼠肾组织抗凋亡蛋白Bcl-2的表达,抑制促凋亡因子cyt C释放,下调凋亡效应因子caspase-9、caspase-3表达,BMSCs抑制肾脏细胞凋亡可能是通过使细胞凋亡通路中线粒体途径受抑,从而发挥肾保护作用。未发现BMSCs在肾脏的定植,推测BMSCs可能通过“非分化修复”的旁分泌及免疫调节等减轻RIAKI。3、在第二部分实验基础上进一步研究BMSCs改善RIAKI的“非分化修复”机制,结果证实RIAKI大鼠机体存在Thl7/Treg细胞免疫失衡及其特异性、炎性细胞因子水变化,机体存在促炎-抗炎反应失衡。BMSCs治疗可上调Treg细胞比例,在基因水平下调Th17细胞特异RORγtm RNA表达,促进Treg细胞特异性转录因子Foxp3m RNA上调;BMSCs抑制Thl7细胞特异性促炎性细胞因子IL-17的表达,促进Treg细胞因子抗炎性细胞因子IL-10和TGF-β的表达,同时抑制炎性效应因子IL-6、TNF-α表达,从而验证了BMSCs可以调节RIAKI大鼠体内Thl7/Treg细胞免疫失衡、抑制炎症反应,这可能是BMSCs减轻横纹肌溶解致急性肾损伤程度,发挥肾保护作用的重要机制。
[Abstract]:AIM: To isolate, culture, purify and identify the biological characteristics of bone marrow mesenchymal stem cells (BMSCs) from Sprague-Dwley (SD) rats in vitro. The lentiviral vectors were labeled with green fluorescent protein (GFP). The feasibility of treating acute renal injury (RIAKI) induced by rhabdomyolysis in rats was evaluated. The effects of BMSCs on renal morphology, structure and function in RIAKI rats were evaluated. The effects of BMSCs on RIAKI were observed at cellular level. The changes of Th17 cells and Treg cells and inflammatory mediators in RIAKI rats after BMSCs transplantation were observed. Methods: 1. Bone marrow cells from both hind limbs of healthy male SD rats were isolated by density gradient centrifugation in vitro. BMSCs were cultured and passaged. P2-P6 generation BMSCs were preserved. P3 generation BMSCs were used for treatment. Flow cytometry was used to detect cell surface markers and biological properties, including biological properties. Long curve and cell cycle analysis. Multidifferentiation potential of BMSCs was identified by cytochemical method. GFP marking and amplification mediated by BMSCs lentiviral vector were carried out. The optimal infection efficiency and cell viability were obtained by multiple infection (MOI) (5,10,25,50). 2. Effect of BMSCs transplantation on RIAKI rats: _Adult healthy male SD was large. Rats were randomly divided into 4 groups: normal control group (Normal group); sham operation group (NS group); model group (RIAKI group); treatment group (BMSCs group). The general conditions of rats in each group were observed; serum biochemical indexes: BUN, Scr, and so on. Creatine kinase (CK); morphological and pathological changes: muscle of both hind limbs (HE staining); kidney (HE, PAS staining); renal cell apoptosis (TUNEL apoptosis analysis, Western Blot detection of caspase 3, caspase 9, Bcl-2, cytochrome C protein expression) 3, BMSCs transplantation treatment of RIAKI mechanism: _Cell level: flow cytometry detection of rats in each group The proportion of Treg cells (CD4+CD25+Foxp3+) in CD4+T cells and the proportion of Th17 cells (CD4+, IL-17+ representing Th17) in spleen tissue were measured. _Gene level: RT-PCR was used to detect the levels of RORgamtmRNA and Foxp3m RNA in spleen and kidney tissue of rats. The levels of specific cytokines (TGF-beta, IL-10, anti-inflammation), IL-17, TNF-alpha and IL-6) released by Treg cells in serum and kidney of rats were detected. The positive rate of BMSCs was 89.9% and 91.3% respectively, while that of CD45 was negative with a positive rate of 2.8%. From the growth curve of passaged cells, it was found that the cell growth incubation period was 1-2 days, the logarithmic growth period was 3-7 days, and the plateau period was 7 days later. High expression of green fluorescent protein was observed under fluorescence microscope. The growth rate of BMSCs-GFP was 90%. The optimum MOI was 25.2. The biochemical indexes of RIAKI group increased significantly after glycerol injection in hind limbs. The BUN, Scr and CK of RIAKI group increased significantly after 6 h, 24 h, 48 h, 72 h, 96 h and 1 W (week) compared with that before intervention and Normal group. Blood BUN, SCr reached the peak at 72h, CK reached the peak at 24h. The above biochemical indexes were in accordance with the laboratory diagnostic criteria of rhabdomyolysis and acute renal injury. The levels of BUN, Scr in BMSCs group were lower at 24h, 48h, 72h, 96h and 1W than RIAKI group at the same time point, the difference was significant (P 0.01). Compared with RIAKI group at the same time point, the level of serum CK in MSCs group at 24h, 48h, 72h and 96h decreased significantly (P 0.01). There was no significant difference between NS group and Normal group (P 0.05). The degree of rhabdomyolysis injury in rats at 24h, 48h, 72h was significantly less than that in RIAKI group. The degree of renal tissue injury in BMSCs group was less than that in RIAKI group at 24h, 48h, 72h. The renal tubular type was reduced and the degree of epithelial cell necrosis was reduced. The expression of caspase 3, caspase 9 and cytochrome C protein was very low and the expression of Bcl-2 protein was high in Normal group and NS group. The expression of caspase 3, caspase 9 and cytochrome C protein in BMSCs group was lower than that in RIAKI group at 24, 48, 72, and 1 W time points (P 0.05). The expression of Bcl-2 protein in BMSCs group was higher than that in RIAKI group (P 0.05). The percentage of Treg cells in spleen was detected by flow cytometry. The percentage of Treg cells in BMSCs group was higher than that in RIAKI group at 24h, 48h, 72h, and the percentage of Th17 cells was lower than that in RIAKI group (P 0.05). Compared with RIAKI group, the levels of ROR-gamma t RNA in spleen and kidney were down-regulated and Foxp3m RNA was up-regulated in BMSCs group (P 0.05). The levels of IL-17, IL-10, TGF-beta, IL-6 and TNF-alpha in serum and kidney tissue were detected by ELISA. The results showed that the levels of L-10 and TGF-beta were up-regulated in BMSCs group compared with RIAKI group. Conclusion: 1. Full purified BMSCs can be obtained by density gradient centrifugation combined with BMSCs adherence screening method. GFP lentiviral vector can successfully transfect BMSCs and efficiently express GFP, which does not affect the biological characteristics and functions of BMSCs, such as growth and differentiation, and can be used as fine BMSCs transplantation. 2. Intramuscular injection of 50% glycerol solution into both hind limbs could induce RIAKI rat model successfully. BMSCs treatment reduced the levels of BUN, SCr and CK in RIAKI rats, and improved the structure and function of acute injured kidney to a certain extent, which confirmed the therapeutic effect of BMSCs on RIAKI. BMSCs transplantation can up-regulate the expression of anti-apoptotic protein Bcl-2, inhibit the release of pro-apoptotic factor cyt C, and down-regulate the expression of apoptotic effector caspase-9 and Caspase-3 in RIAKI rats kidney. BMSCs may inhibit the apoptosis of renal cells through the mitochondrial pathway of apoptosis pathway. No BMSCs were found to colonize the kidneys. It is speculated that BMSCs may alleviate RIAKI.3 by "undifferentiated repair" paracrine and immunoregulation. On the basis of the second part of the experiment, the mechanism of BMSCs improving RIAKI by "undifferentiated repair" was further studied. The results confirm the existence of Thl7/Treg cells in RIAKI rats. BMSCs can up-regulate the proportion of Treg cells, down-regulate the expression of specific ROR gamma TM RNA and up-regulate the expression of Treg cell-specific transcription factor Foxp3m RNA at the gene level. BMSCs inhibit the up-regulation of Thl7 cell-specific pro-inflammatory cytokine IL-17. BMSCs can regulate the immune imbalance of Thl7/Treg cells in RIAKI rats and inhibit inflammation, which may be due to BMSCs alleviating acute renal injury induced by rhabdomyolysis and exerting renal protection. Important mechanisms used.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R685.5;R692.5
[Abstract]:AIM: To isolate, culture, purify and identify the biological characteristics of bone marrow mesenchymal stem cells (BMSCs) from Sprague-Dwley (SD) rats in vitro. The lentiviral vectors were labeled with green fluorescent protein (GFP). The feasibility of treating acute renal injury (RIAKI) induced by rhabdomyolysis in rats was evaluated. The effects of BMSCs on renal morphology, structure and function in RIAKI rats were evaluated. The effects of BMSCs on RIAKI were observed at cellular level. The changes of Th17 cells and Treg cells and inflammatory mediators in RIAKI rats after BMSCs transplantation were observed. Methods: 1. Bone marrow cells from both hind limbs of healthy male SD rats were isolated by density gradient centrifugation in vitro. BMSCs were cultured and passaged. P2-P6 generation BMSCs were preserved. P3 generation BMSCs were used for treatment. Flow cytometry was used to detect cell surface markers and biological properties, including biological properties. Long curve and cell cycle analysis. Multidifferentiation potential of BMSCs was identified by cytochemical method. GFP marking and amplification mediated by BMSCs lentiviral vector were carried out. The optimal infection efficiency and cell viability were obtained by multiple infection (MOI) (5,10,25,50). 2. Effect of BMSCs transplantation on RIAKI rats: _Adult healthy male SD was large. Rats were randomly divided into 4 groups: normal control group (Normal group); sham operation group (NS group); model group (RIAKI group); treatment group (BMSCs group). The general conditions of rats in each group were observed; serum biochemical indexes: BUN, Scr, and so on. Creatine kinase (CK); morphological and pathological changes: muscle of both hind limbs (HE staining); kidney (HE, PAS staining); renal cell apoptosis (TUNEL apoptosis analysis, Western Blot detection of caspase 3, caspase 9, Bcl-2, cytochrome C protein expression) 3, BMSCs transplantation treatment of RIAKI mechanism: _Cell level: flow cytometry detection of rats in each group The proportion of Treg cells (CD4+CD25+Foxp3+) in CD4+T cells and the proportion of Th17 cells (CD4+, IL-17+ representing Th17) in spleen tissue were measured. _Gene level: RT-PCR was used to detect the levels of RORgamtmRNA and Foxp3m RNA in spleen and kidney tissue of rats. The levels of specific cytokines (TGF-beta, IL-10, anti-inflammation), IL-17, TNF-alpha and IL-6) released by Treg cells in serum and kidney of rats were detected. The positive rate of BMSCs was 89.9% and 91.3% respectively, while that of CD45 was negative with a positive rate of 2.8%. From the growth curve of passaged cells, it was found that the cell growth incubation period was 1-2 days, the logarithmic growth period was 3-7 days, and the plateau period was 7 days later. High expression of green fluorescent protein was observed under fluorescence microscope. The growth rate of BMSCs-GFP was 90%. The optimum MOI was 25.2. The biochemical indexes of RIAKI group increased significantly after glycerol injection in hind limbs. The BUN, Scr and CK of RIAKI group increased significantly after 6 h, 24 h, 48 h, 72 h, 96 h and 1 W (week) compared with that before intervention and Normal group. Blood BUN, SCr reached the peak at 72h, CK reached the peak at 24h. The above biochemical indexes were in accordance with the laboratory diagnostic criteria of rhabdomyolysis and acute renal injury. The levels of BUN, Scr in BMSCs group were lower at 24h, 48h, 72h, 96h and 1W than RIAKI group at the same time point, the difference was significant (P 0.01). Compared with RIAKI group at the same time point, the level of serum CK in MSCs group at 24h, 48h, 72h and 96h decreased significantly (P 0.01). There was no significant difference between NS group and Normal group (P 0.05). The degree of rhabdomyolysis injury in rats at 24h, 48h, 72h was significantly less than that in RIAKI group. The degree of renal tissue injury in BMSCs group was less than that in RIAKI group at 24h, 48h, 72h. The renal tubular type was reduced and the degree of epithelial cell necrosis was reduced. The expression of caspase 3, caspase 9 and cytochrome C protein was very low and the expression of Bcl-2 protein was high in Normal group and NS group. The expression of caspase 3, caspase 9 and cytochrome C protein in BMSCs group was lower than that in RIAKI group at 24, 48, 72, and 1 W time points (P 0.05). The expression of Bcl-2 protein in BMSCs group was higher than that in RIAKI group (P 0.05). The percentage of Treg cells in spleen was detected by flow cytometry. The percentage of Treg cells in BMSCs group was higher than that in RIAKI group at 24h, 48h, 72h, and the percentage of Th17 cells was lower than that in RIAKI group (P 0.05). Compared with RIAKI group, the levels of ROR-gamma t RNA in spleen and kidney were down-regulated and Foxp3m RNA was up-regulated in BMSCs group (P 0.05). The levels of IL-17, IL-10, TGF-beta, IL-6 and TNF-alpha in serum and kidney tissue were detected by ELISA. The results showed that the levels of L-10 and TGF-beta were up-regulated in BMSCs group compared with RIAKI group. Conclusion: 1. Full purified BMSCs can be obtained by density gradient centrifugation combined with BMSCs adherence screening method. GFP lentiviral vector can successfully transfect BMSCs and efficiently express GFP, which does not affect the biological characteristics and functions of BMSCs, such as growth and differentiation, and can be used as fine BMSCs transplantation. 2. Intramuscular injection of 50% glycerol solution into both hind limbs could induce RIAKI rat model successfully. BMSCs treatment reduced the levels of BUN, SCr and CK in RIAKI rats, and improved the structure and function of acute injured kidney to a certain extent, which confirmed the therapeutic effect of BMSCs on RIAKI. BMSCs transplantation can up-regulate the expression of anti-apoptotic protein Bcl-2, inhibit the release of pro-apoptotic factor cyt C, and down-regulate the expression of apoptotic effector caspase-9 and Caspase-3 in RIAKI rats kidney. BMSCs may inhibit the apoptosis of renal cells through the mitochondrial pathway of apoptosis pathway. No BMSCs were found to colonize the kidneys. It is speculated that BMSCs may alleviate RIAKI.3 by "undifferentiated repair" paracrine and immunoregulation. On the basis of the second part of the experiment, the mechanism of BMSCs improving RIAKI by "undifferentiated repair" was further studied. The results confirm the existence of Thl7/Treg cells in RIAKI rats. BMSCs can up-regulate the proportion of Treg cells, down-regulate the expression of specific ROR gamma TM RNA and up-regulate the expression of Treg cell-specific transcription factor Foxp3m RNA at the gene level. BMSCs inhibit the up-regulation of Thl7 cell-specific pro-inflammatory cytokine IL-17. BMSCs can regulate the immune imbalance of Thl7/Treg cells in RIAKI rats and inhibit inflammation, which may be due to BMSCs alleviating acute renal injury induced by rhabdomyolysis and exerting renal protection. Important mechanisms used.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R685.5;R692.5
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