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目的观察甘草酸对胰腺星状细胞(PSC)合成分泌Ⅰ型胶原、基质金属蛋白酶(MMP)及其抑制物(TIMP)的影响,探讨其抗胰腺纤维化的作用机制。方法采用组织块培养法分离培养活化的PSC,培养基中添加0、1、2.5、5和10mg/ml不同剂量的甘草酸,培养24h后分别留取上清和细胞。采用放射免疫法测定上清中Ⅰ型胶原含量;明胶酶谱和反式明胶酶谱测定浓缩上清中MMP-2、MMP-9和TIMP-1、TIMP-2的表达;RT-PCR测定MMP-2、MMP-9、FIMP-1、TIMP-2和Ⅰ型胶原mRNA的表达。结果甘草酸可明显抑制PSC中Ⅰ型胶原含量,以5mg/ml时抑制最明显[从(52±10)ng/ml降至(35±8)ng/ml]。明胶酶谱和RT-PCR显示,PSC有MMP-2、MMP-9和TIMP-2基因和蛋白表达,而TIMP-1仅有基因表达。甘草酸可抑制MMP-2和MMP-9蛋白表达,而MMP-9基因表达未受明显抑制;甘草酸对TIMP-1和TIMP-2基因表达无明显作用,对TIMP-2蛋白的表达仅在10mg/ml浓度时有部分抑制。结论甘草酸可能通过抑制Ⅰ型胶原合成、减少MMP-2和MMP-9表达等途径抑制PSC中细胞外基质的代谢。
Objective To investigate the effect of glycyrrhizin on the synthesis and secretion of type Ⅰ collagen and matrix metalloproteinase (MMP) and its inhibitor (TIMP) in pancreatic stellate cells (PSCs) and to explore the mechanism of anti-pancreatic fibrosis. Methods The activated PSC was isolated and cultured using tissue culture method. Different concentrations of glycyrrhizic acid were added to the culture medium at 0, 1, 2.5, 5 and 10 mg / ml, and the supernatant and cells were collected after culturing for 24 hours. The content of type I collagen in the supernatant was determined by radioimmunoassay. The expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 in the supernatant was determined by gelatin zymogram and transgenetic gelatin zymography. -2, MMP-9, FIMP-1, TIMP-2 and type I collagen mRNA. Results Glycyrrhizin significantly inhibited the type I collagen content in PSC, with the most significant inhibition at 5 mg / ml [from (52 ± 10) ng / ml to (35 ± 8) ng / ml]. Gelatin zymography and RT-PCR showed that PSC had MMP-2, MMP-9 and TIMP-2 gene and protein expression, while TIMP-1 only had gene expression. Glycyrrhizinate could inhibit the expression of MMP-2 and MMP-9, while the expression of MMP-9 was not significantly inhibited. Glycyrrhizinate had no effect on the expression of TIMP-1 and TIMP-2, but the expression of TIMP- Partial inhibition at 10 mg / ml concentration. Conclusion Glycyrrhizin may inhibit the metabolism of extracellular matrix in PSC by inhibiting the synthesis of type Ⅰ collagen and decreasing the expression of MMP-2 and MMP-9.