發(fā)布時間：2018-12-12 13:26

【摘要】：目的：觀察粒細(xì)胞集落刺激因子（granulocyte colony-stimulating factor，G-CSF）對D-半乳糖胺/脂多糖(D-GalN/LPS)所致小鼠急性肝衰竭的保護(hù)作用，探討G-CSF在肝衰竭中治療作用及其抗凋亡可能機(jī)制。 方法： 1、采取腹腔注射D-GalN/LPS制造小鼠急性肝衰竭模型。選取三個可能影響造模成功率的指標(biāo)為實(shí)驗(yàn)因素:D-GalN劑量、LPS劑量、稀釋倍數(shù)，每個實(shí)驗(yàn)因素選取四個水平，利用L16(43)正交表安排試驗(yàn)，以小鼠24h病死率為考察指標(biāo)。觀察小鼠血清ALT、肝組織學(xué)改變，肝臟細(xì)胞凋亡驗(yàn)證造模效果，確定D-GalN、LPS致小鼠肝衰竭的理想劑量搭配。 2、腹腔注射D-GalN（350mg/kg）/LPS（30μg/kg）制造小鼠急性肝衰竭模型。將雄性C57/BL6小鼠隨機(jī)分為對照組、治療組及正常對照組。治療組在肝衰竭造模前96h、72h、48、24H和0H皮下注射重組人G-CSF（250mg/kg體重），對照組在相應(yīng)時間點(diǎn)皮下注射無菌生理鹽水0.2ml/只。通過檢測比較各組小鼠生存率、不同時間點(diǎn)血清ALT水平、肝組織損傷程度證明G-CSF對急性肝衰竭小鼠保護(hù)作用；采用TUNEL染色方法檢測小鼠肝組織中細(xì)胞凋亡，明確G-CSF具有減少細(xì)胞凋亡作用。使用Real-time PCR方法觀察肝組織中Bcl-2、Bcl-xlmRNA表達(dá)變化。蛋白免疫印跡（Western blot）檢測小鼠肝組織Bcl-2表達(dá)，進(jìn)一步探討G-CSF抗凋亡作用機(jī)制。 結(jié)果: 1、經(jīng)方差分析造模中各因素作用主次為: D-GalN劑量LPS劑量稀釋倍數(shù)，D-GalN劑量為350mg/kg、LPS劑量為30μg/kg、原液稀釋三倍為理想造模條件。經(jīng)檢測小鼠ALT值、造模后肝組織HE染色以及TUNEL染色證明上述優(yōu)化條件造模后符合急性肝衰竭模型。 2、G-CSF對D-GalN/LPS所致小鼠急性肝衰竭具有治療作用：G-CSF治療可提高D-GalN/LPS所致小鼠急性肝衰竭生存率，治療組小鼠24小時生存率為40%，高于對照組15%（χ~2=3.989, P=0.046）；造模后6h，治療組小鼠血清ALT水平顯著低于對照組（681.0±113.2VS1371±210.9t=2.882,P=0.044）；HE染色結(jié)果示對照組小鼠肝小葉結(jié)構(gòu)破壞，肝索解離，肝細(xì)胞崩解、彌漫性大片壞死，殘存肝細(xì)胞腫脹，大量炎性細(xì)胞浸潤。G-CSF治療組小鼠肝組織小葉結(jié)構(gòu)紊亂但仍可辨，可見大量細(xì)胞腫脹，炎細(xì)胞浸潤，無彌漫性大片壞死，未見肝索解離，未見大量小葉內(nèi)出血。G-CSF抑制D-GalN/LPS致急性肝衰竭造模小鼠肝細(xì)胞凋亡：造模后6h，肝組織中TUNEL染色陽性細(xì)胞計(jì)數(shù)，治療組亦低于對照組（654±64.29VS1155±192.6t=2.467P=0.033）；造模后6H經(jīng)G-CSF治療組抗凋亡基因Bcl-2表達(dá)高于對照組（4.793±0.900VS2.093±0.733；P=0.017），，抗凋亡基因Bcl-xl表達(dá)同為治療組高于對照組（0.412±0.095VS0.162±0.0737P=0.027）。 結(jié)論: G-CSF可通過抑制肝組織細(xì)胞凋亡的方式對小鼠急性肝衰竭起到治療作用，其抗凋亡作用可能通過上調(diào)肝組織內(nèi)Bcl-2、Bcl-xl實(shí)現(xiàn)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 鹵210.9t 鹵210.9t 鹵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 鹵64.29VS1155 鹵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 鹵0.900VS2.093 鹵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 鹵0.095VS0.162 鹵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.
【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】：2013
【分類號】：R575.3

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;肝衰竭診療指南[J];中華肝臟病雜志;2006年09期

本文編號：2374650