狗肝菜多糖抑制肝星状细胞活化抗肝纤维化的机制
发布时间：2018-08-26 16:52
【摘要】：目的：研究狗肝菜多糖通过抑制肝星状细胞(HSC)活化抗肝纤维化(HF)的机制 方法： 1建立二甲基亚硝胺致肝纤维化体内动物模型，组织切片HE染色、Masson三色特染评价肝纤维化程度。 2不同剂量狗肝菜多糖干预，免疫组化检测α-SMA的表达；免疫组化检测TGF-β1及其下游的效应分子pSmad2/3的表达。 3体外培养HSC细胞株，1ng/mL的转化生长因子-β1（TGF-β1）激活HSC,采用MTT比色法检测用不同浓度狗肝菜多糖干预对活化HSC的生长增殖的抑制情况以及对未经TGF-β1刺激的HSC细胞的影响。 4在倒置相差显微镜下观察经TGF-β1刺激后活化的HSC细胞形态变化。 5用Western Blotting检测不同浓度的狗肝菜多糖作用于活化的HSC中α-SMA、TGF-β1下游效应分子pSmad2/3蛋白表达水平。 结果： 1HE染色和Masson染色结果显示：与模型组相比，狗肝菜多糖(300mg/kg,100mg/kg)剂量组中肝组织炎症坏死和纤维化程度明显减轻，尤以狗肝菜多糖（300mg/kg）剂量组更明显。 2免疫组化结果显示：与模型组相比，狗肝菜多糖(300mg/kg,100mg/kg)剂量组中α-SMA、TGF-β1及其下游的效应分子pSmad2/3的表达均有明显减轻，尤以狗肝菜多糖（300mg/kg）剂量组更明显。 3狗肝菜多糖抑制HSC细胞体外增殖与活化，不同浓度（25mg/L,50mg/L,100mg/L,200mg/L,400mg/L）狗肝菜多糖能抑制HSC细胞增殖，，且呈剂量依赖性（P＜0.05），而对未经TGF-β1激活的HSC细胞增殖无明显影响。 4在倒置相差显微镜下观察：活化的HSC细胞呈星状，纤维样细胞形状，多树突样突起，表面有较多微绒毛。 5Western Blotting检测结果显示：狗肝菜多糖可以明显抑制活化的HSC细胞中α-SMA、pSmad2/3蛋白的表达，且呈剂量依赖性（P＜0.05）。 结论：狗肝菜多糖可能通过抑制HSC细胞增殖与活化，下调α-SMA、pSmad2/3的表达，从而发挥其抗肝纤维化的作用。
[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.
【学位授予单位】：桂林医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575
本文编号：2205550
[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.
【学位授予单位】：桂林医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575
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