粒细胞集落刺激因子（G-CSF）对小鼠急性肝衰竭的治疗作用及抗凋亡机制研究
发布时间：2018-12-12 13:26
【摘要】：目的：观察粒细胞集落刺激因子（granulocyte colony-stimulating factor，G-CSF）对D-半乳糖胺/脂多糖(D-GalN/LPS)所致小鼠急性肝衰竭的保护作用，探讨G-CSF在肝衰竭中治疗作用及其抗凋亡可能机制。 方法： 1、采取腹腔注射D-GalN/LPS制造小鼠急性肝衰竭模型。选取三个可能影响造模成功率的指标为实验因素:D-GalN剂量、LPS剂量、稀释倍数，每个实验因素选取四个水平，利用L16(43)正交表安排试验，以小鼠24h病死率为考察指标。观察小鼠血清ALT、肝组织学改变，肝脏细胞凋亡验证造模效果，确定D-GalN、LPS致小鼠肝衰竭的理想剂量搭配。 2、腹腔注射D-GalN（350mg/kg）/LPS（30μg/kg）制造小鼠急性肝衰竭模型。将雄性C57/BL6小鼠随机分为对照组、治疗组及正常对照组。治疗组在肝衰竭造模前96h、72h、48、24H和0H皮下注射重组人G-CSF（250mg/kg体重），对照组在相应时间点皮下注射无菌生理盐水0.2ml/只。通过检测比较各组小鼠生存率、不同时间点血清ALT水平、肝组织损伤程度证明G-CSF对急性肝衰竭小鼠保护作用；采用TUNEL染色方法检测小鼠肝组织中细胞凋亡，明确G-CSF具有减少细胞凋亡作用。使用Real-time PCR方法观察肝组织中Bcl-2、Bcl-xlmRNA表达变化。蛋白免疫印迹（Western blot）检测小鼠肝组织Bcl-2表达，进一步探讨G-CSF抗凋亡作用机制。 结果: 1、经方差分析造模中各因素作用主次为: D-GalN剂量LPS剂量稀释倍数，D-GalN剂量为350mg/kg、LPS剂量为30μg/kg、原液稀释三倍为理想造模条件。经检测小鼠ALT值、造模后肝组织HE染色以及TUNEL染色证明上述优化条件造模后符合急性肝衰竭模型。 2、G-CSF对D-GalN/LPS所致小鼠急性肝衰竭具有治疗作用：G-CSF治疗可提高D-GalN/LPS所致小鼠急性肝衰竭生存率，治疗组小鼠24小时生存率为40%，高于对照组15%（χ~2=3.989, P=0.046）；造模后6h，治疗组小鼠血清ALT水平显著低于对照组（681.0±113.2VS1371±210.9t=2.882,P=0.044）；HE染色结果示对照组小鼠肝小叶结构破坏，肝索解离，肝细胞崩解、弥漫性大片坏死，残存肝细胞肿胀，大量炎性细胞浸润。G-CSF治疗组小鼠肝组织小叶结构紊乱但仍可辨，可见大量细胞肿胀，炎细胞浸润，无弥漫性大片坏死，未见肝索解离，未见大量小叶内出血。G-CSF抑制D-GalN/LPS致急性肝衰竭造模小鼠肝细胞凋亡：造模后6h，肝组织中TUNEL染色阳性细胞计数，治疗组亦低于对照组（654±64.29VS1155±192.6t=2.467P=0.033）；造模后6H经G-CSF治疗组抗凋亡基因Bcl-2表达高于对照组（4.793±0.900VS2.093±0.733；P=0.017），，抗凋亡基因Bcl-xl表达同为治疗组高于对照组（0.412±0.095VS0.162±0.0737P=0.027）。 结论: G-CSF可通过抑制肝组织细胞凋亡的方式对小鼠急性肝衰竭起到治疗作用，其抗凋亡作用可能通过上调肝组织内Bcl-2、Bcl-xl实现的。
[Abstract]:Aim: to observe the protective effect of granulocyte colony stimulating factor (granulocyte colony-stimulating factor,G-CSF) on acute hepatic failure induced by D-galactosamine / lipopolysaccharide (D-GalN/LPS) in mice. To investigate the therapeutic effect of G-CSF in liver failure and its mechanism of anti-apoptosis. Methods: 1. Acute liver failure in mice was induced by intraperitoneal injection of D-GalN/LPS. Three indexes which may affect the success rate of model making were selected as experimental factors: D-GalN dose, LPS dose, dilution multiple, four levels of each experimental factor. The test was arranged by L16 (43) orthogonal table, and the 24 h mortality of mice was taken as the index. The liver histological changes of serum ALT, in mice were observed and the effect of liver cell apoptosis was verified. The ideal dose of D-GalNLPS-induced liver failure in mice was determined. 2. D-GalN (350mg/kg) / LPS (30 渭 g/kg) was injected intraperitoneally to induce acute liver failure in mice. Male C57/BL6 mice were randomly divided into control group, treatment group and normal control group. In the treatment group, recombinant human G-CSF (250mg/kg body weight) was injected subcutaneously 96 hours before the liver failure model was made. The control group was subcutaneously injected with aseptic saline 0.2ml/ at the corresponding time point. The survival rate, serum ALT level at different time points and the degree of liver tissue injury in each group were measured and compared. The protective effect of G-CSF on acute liver failure mice was proved. TUNEL staining was used to detect apoptosis in mouse liver tissue. It was confirmed that G-CSF could reduce apoptosis. Real-time PCR method was used to observe the expression of Bcl-2,Bcl-xlmRNA in liver tissue. Western blot (Western blot) was used to detect the expression of Bcl-2 in liver tissue of mice, and to further explore the mechanism of anti-apoptosis of G-CSF. Results: 1. By ANOVA, the main factors were as follows: D-GalN dose, LPS dose dilution multiple, D-GalN dose was 350 mg / kg, D-GalN dosage was 30 渭 g / kg, and the dilution of the original solution was the ideal condition. The ALT value of mice, the HE staining of liver tissue and TUNEL staining showed that the above optimized conditions were in accord with the model of acute liver failure. 2G-CSF has therapeutic effect on acute liver failure induced by D-GalN/LPS in mice: G-CSF treatment can improve the survival rate of mice with acute liver failure induced by D-GalN/LPS, and the 24-hour survival rate of mice in the treatment group is 40%. It was higher than the control group by 15% (蠂 ~ 2 = 3.989, P = 0.046). The serum ALT level in the treatment group was significantly lower than that in the control group (681.0 卤210.9t 卤210.9t 卤2.882P0. 044). The results of HE staining showed that the hepatic lobule structure was destroyed, the hepatic cord dissociated, the liver cells disintegrated, the diffuse necrosis, the residual hepatocytes swelling, and the inflammatory cells infiltrated in the control group. In the G-CSF treatment group, the hepatic lobule structure was disordered but still recognizable. The results showed that a large number of cells were swollen, inflammatory cells infiltrated, no diffuse necrosis, no dissociation of hepatic cord, no massive intralobular hemorrhage. G-CSF inhibited the apoptosis of hepatocytes induced by D-GalN/LPS in mice with acute liver failure. The number of TUNEL positive cells in the liver tissue in the treatment group was also lower than that in the control group (654 卤64.29VS1155 卤192.6t=2.467P=0.033). The Bcl-2 expression of anti-apoptotic gene in G-CSF treated group was higher than that in control group (4.793 卤0.900VS2.093 卤0.733 P0.017), and the Bcl-xl expression of anti-apoptotic gene in treatment group was higher than that in control group (0.412 卤0.095VS0.162 卤0.0737P=0.027). Conclusion: G-CSF can treat acute liver failure by inhibiting apoptosis of liver tissue, and its anti-apoptotic effect may be achieved by up-regulating Bcl-2,Bcl-xl in liver tissue.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R575.3
本文编号：2374650
[Abstract]:Aim: to observe the protective effect of granulocyte colony stimulating factor (granulocyte colony-stimulating factor,G-CSF) on acute hepatic failure induced by D-galactosamine / lipopolysaccharide (D-GalN/LPS) in mice. To investigate the therapeutic effect of G-CSF in liver failure and its mechanism of anti-apoptosis. Methods: 1. Acute liver failure in mice was induced by intraperitoneal injection of D-GalN/LPS. Three indexes which may affect the success rate of model making were selected as experimental factors: D-GalN dose, LPS dose, dilution multiple, four levels of each experimental factor. The test was arranged by L16 (43) orthogonal table, and the 24 h mortality of mice was taken as the index. The liver histological changes of serum ALT, in mice were observed and the effect of liver cell apoptosis was verified. The ideal dose of D-GalNLPS-induced liver failure in mice was determined. 2. D-GalN (350mg/kg) / LPS (30 渭 g/kg) was injected intraperitoneally to induce acute liver failure in mice. Male C57/BL6 mice were randomly divided into control group, treatment group and normal control group. In the treatment group, recombinant human G-CSF (250mg/kg body weight) was injected subcutaneously 96 hours before the liver failure model was made. The control group was subcutaneously injected with aseptic saline 0.2ml/ at the corresponding time point. The survival rate, serum ALT level at different time points and the degree of liver tissue injury in each group were measured and compared. The protective effect of G-CSF on acute liver failure mice was proved. TUNEL staining was used to detect apoptosis in mouse liver tissue. It was confirmed that G-CSF could reduce apoptosis. Real-time PCR method was used to observe the expression of Bcl-2,Bcl-xlmRNA in liver tissue. Western blot (Western blot) was used to detect the expression of Bcl-2 in liver tissue of mice, and to further explore the mechanism of anti-apoptosis of G-CSF. Results: 1. By ANOVA, the main factors were as follows: D-GalN dose, LPS dose dilution multiple, D-GalN dose was 350 mg / kg, D-GalN dosage was 30 渭 g / kg, and the dilution of the original solution was the ideal condition. The ALT value of mice, the HE staining of liver tissue and TUNEL staining showed that the above optimized conditions were in accord with the model of acute liver failure. 2G-CSF has therapeutic effect on acute liver failure induced by D-GalN/LPS in mice: G-CSF treatment can improve the survival rate of mice with acute liver failure induced by D-GalN/LPS, and the 24-hour survival rate of mice in the treatment group is 40%. It was higher than the control group by 15% (蠂 ~ 2 = 3.989, P = 0.046). The serum ALT level in the treatment group was significantly lower than that in the control group (681.0 卤210.9t 卤210.9t 卤2.882P0. 044). The results of HE staining showed that the hepatic lobule structure was destroyed, the hepatic cord dissociated, the liver cells disintegrated, the diffuse necrosis, the residual hepatocytes swelling, and the inflammatory cells infiltrated in the control group. In the G-CSF treatment group, the hepatic lobule structure was disordered but still recognizable. The results showed that a large number of cells were swollen, inflammatory cells infiltrated, no diffuse necrosis, no dissociation of hepatic cord, no massive intralobular hemorrhage. G-CSF inhibited the apoptosis of hepatocytes induced by D-GalN/LPS in mice with acute liver failure. The number of TUNEL positive cells in the liver tissue in the treatment group was also lower than that in the control group (654 卤64.29VS1155 卤192.6t=2.467P=0.033). The Bcl-2 expression of anti-apoptotic gene in G-CSF treated group was higher than that in control group (4.793 卤0.900VS2.093 卤0.733 P0.017), and the Bcl-xl expression of anti-apoptotic gene in treatment group was higher than that in control group (0.412 卤0.095VS0.162 卤0.0737P=0.027). Conclusion: G-CSF can treat acute liver failure by inhibiting apoptosis of liver tissue, and its anti-apoptotic effect may be achieved by up-regulating Bcl-2,Bcl-xl in liver tissue.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R575.3
【参考文献】
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1 ;肝衰竭诊疗指南[J];中华肝脏病杂志;2006年09期
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