【摘要】：目的：研究狗肝菜多糖通過抑制肝星狀細胞(HSC)活化抗肝纖維化(HF)的機制 方法： 1建立二甲基亞硝胺致肝纖維化體內動物模型，組織切片HE染色、Masson三色特染評價肝纖維化程度。 2不同劑量狗肝菜多糖干預，免疫組化檢測α-SMA的表達；免疫組化檢測TGF-β1及其下游的效應分子pSmad2/3的表達。 3體外培養(yǎng)HSC細胞株，1ng/mL的轉化生長因子-β1（TGF-β1）激活HSC,采用MTT比色法檢測用不同濃度狗肝菜多糖干預對活化HSC的生長增殖的抑制情況以及對未經TGF-β1刺激的HSC細胞的影響。 4在倒置相差顯微鏡下觀察經TGF-β1刺激后活化的HSC細胞形態(tài)變化。 5用Western Blotting檢測不同濃度的狗肝菜多糖作用于活化的HSC中α-SMA、TGF-β1下游效應分子pSmad2/3蛋白表達水平。 結果： 1HE染色和Masson染色結果顯示：與模型組相比，狗肝菜多糖(300mg/kg,100mg/kg)劑量組中肝組織炎癥壞死和纖維化程度明顯減輕，尤以狗肝菜多糖（300mg/kg）劑量組更明顯。 2免疫組化結果顯示：與模型組相比，狗肝菜多糖(300mg/kg,100mg/kg)劑量組中α-SMA、TGF-β1及其下游的效應分子pSmad2/3的表達均有明顯減輕，尤以狗肝菜多糖（300mg/kg）劑量組更明顯。 3狗肝菜多糖抑制HSC細胞體外增殖與活化，不同濃度（25mg/L,50mg/L,100mg/L,200mg/L,400mg/L）狗肝菜多糖能抑制HSC細胞增殖，，且呈劑量依賴性（P＜0.05），而對未經TGF-β1激活的HSC細胞增殖無明顯影響。 4在倒置相差顯微鏡下觀察：活化的HSC細胞呈星狀，纖維樣細胞形狀，多樹突樣突起，表面有較多微絨毛。 5Western Blotting檢測結果顯示：狗肝菜多糖可以明顯抑制活化的HSC細胞中α-SMA、pSmad2/3蛋白的表達，且呈劑量依賴性（P＜0.05）。 結論：狗肝菜多糖可能通過抑制HSC細胞增殖與活化，下調α-SMA、pSmad2/3的表達，從而發(fā)揮其抗肝纖維化的作用。
[Abstract]:Objective: To study the mechanism of dog liver polysaccharide in inhibiting hepatic fibrosis (HF) by activating hepatic stellate cells (HSC).
Method:
1 Establish the animal model of dimethylnitrosamine-induced liver fibrosis in vivo, and evaluate the degree of liver fibrosis by HE staining and Masson trichrome staining.
The expression of alpha-SMA was detected by immunohistochemistry and the expression of TGF-beta 1 and its downstream effector molecule pSmad2/3 was detected by immunohistochemistry.
3. HSC cell lines were cultured in vitro and 1 ng/mL transforming growth factor-beta 1 (TGF-beta 1) activated HSC. MTT colorimetric assay was used to detect the inhibitory effect of different concentrations of Polysaccharide on the growth and proliferation of activated HSC cells and the effect on HSC cells without TGF-beta 1 stimulation.
4 the morphological changes of HSC cells stimulated by TGF- beta 1 were observed under inverted phase contrast microscope.
5 Western Blotting was used to detect the expression of pSmad2/3 protein, a downstream effector molecule of TGF-beta 1, in activated HSC treated with different concentrations of CAP.
Result:
The results of 1HE staining and Mason staining showed that compared with the model group, the degree of inflammation, necrosis and fibrosis of liver tissue in the dosage group of CGP (300mg/kg, 100mg/kg) was significantly reduced, especially in the dosage group of CGP (300mg/kg).
The results of immunohistochemistry showed that compared with the model group, the expression of alpha-SMA, TGF-beta 1 and its downstream effector pSmad 2/3 were significantly decreased in the dose group of CGP (300mg/kg, 100mg/kg), especially in the dose group of CGP (300mg/kg).
3. Polysaccharide of C. coli inhibited the proliferation and activation of HSC cells in vitro. Different concentrations (25 mg/L, 50 mg/L, 100 mg/L, 200 mg/L, 400 mg/L) of C. coli polysaccharide could inhibit the proliferation of HSC cells in a dose-dependent manner (P < 0.05), but had no significant effect on the proliferation of HSC cells not activated by TGF-beta 1.
4. Under inverted phase contrast microscope, the activated HSC cells were stellate, fibrous and dendritic with many microvilli on the surface.
5Western Blotting assay showed that the polysaccharides could significantly inhibit the expression of alpha-SMA and pSmad2/3 protein in activated HSC cells in a dose-dependent manner (P<0.05).
CONCLUSION: Polysaccharide from Lysimachia cordata Thunb. may inhibit the proliferation and activation of HSC cells and down-regulate the expression of alpha-SMA and pSmad2/3, thus exerting its anti-hepatic fibrosis effect.
【學位授予單位】：桂林醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R575

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