发酵虫草菌粉对人肝癌细胞增殖、凋亡及VEGF,MMP-2表达的影响

Effects of Paecilomyces hepiali on Proliferation,Apoptosis and Expression of VEGF,MMP-2 of SMMC 7721 Cells

目的:探讨从天然冬虫夏草中分离出的虫草拟青霉Cs-4菌株,经人工发酵所得发酵虫草菌粉对SMMC-7721人肝癌细胞的生长抑制作用。方法:20只体重(200±20)g雄性SD大鼠随机分为4组(每组5只):空白组及Cs-4低、中、高剂量组,空白组ig予蒸馏水,Cs-4低、中、高剂量组(0.77,1.54,3.08 g·kg-1,ig);给药3 d后,制备含药血清。肝癌细胞悬液调整至4×104/m L,96孔板每孔加入100μL,培养24 h后加入大鼠含药血清及顺铂注射液;干预48 h MTT法测各组细胞增殖抑制率。将SMMC-7721细胞悬液调整至2.8×104/m L,接种于6孔板培养24 h,加入大鼠含药血清及顺铂,干预培养48 h后Annexin V-FITC/PI染色,流式细胞仪检测各组细胞凋亡率;ELISA检测各组细胞上清液中血管内皮生长因子(VEGF),金属基质蛋白酶-2(MMP-2)表达量。结果:SMMC-7721细胞增殖被Cs-4含药血清明显抑制(与空白组比较P<0.05),低、中、高剂量组抑制率分别为12.2%,23.2%,38.1%,呈剂量依赖性(Cs-4不同剂量组间比较P<0.05)。Cs-4含药血清低、中、高剂量组细胞凋亡率分别为13.9%,23.5%,24.9%,均高于空白组(P<0.05)。Cs-4含药血清低、中、高剂量组细胞上清液中VEGF分别为443.5,436.9,411.0 ng·L-1,低于空白组(P<0.05);Cs-4低、中、高剂量组细胞上清液中MMP-2浓度与空白组比无显著差异。结论:Cs-4含药血清能抑制SMMC-7721细胞增殖,提高体外培养的SMMC-7721细胞凋亡率,抑制SMMC-7721细胞VEGF表达,提示Cs-4有抗肝癌作用,对SMMC-7721细胞MMP-2表达无显著影响。

Objective： To explore the effect of the Chinese caterpillar fungus isolated from natural Paecilomyces hepiali strain （Cs-4） inhibited by artificial fermentation, the Fermented-4 powder on SMMC-7721 human hepatocellular carcinoma cell growth. Method： Twenty male SD rats were randomly divided into four groups （n = 5）： control group and Cs-4 low, medium and high dose groups, control group was treated with distilled water, Cs4 low, middle and high dose groups were treated with distilled water to prepare the Cs-4 （0.77, 1.54, 3.08 g·kg^-1, ig） suspension, according to the 10 mL .kg-1 body weight orallyorally, 2 times twice a daily ; after intragastric administrationfor 3 days, the serum containing drug was prepared. The liver cell suspension was adjusted to 4 ×10^4 eells/mL, each hole of 96 hole plate was added with 100 μL cell suspension culture. After 24 h, the holes were respectively added with 160 μL cells culture fluid and 40 μL blank serum and containing Cs-4. After 48 h MTT detection was carried out, the inhibition rate of proliferation of cells was calculated. The SMMC-7721 cell suspension was adjusted to 2.8 ×10^4 cells/mL, were cultured in 6 well plates （each hole 2 mL inoculation） for 24 h, the holes were added to 1.6 mL cells suspension and 0.4 mL black serum and containingdrug serum were added after 48 h, Annexin V-FITC/PI staining by flow cytometry was used to detect of apoptosis ; expression of vascular endothelial growth factor （VEGF） , matrix metalloproteinases-2 （MMP-2） were detected with ELISA method. Result： MTT showed the proliferation of SMMC-7721 cells was significantly inhibited by containing Cs-4 serum compared with the blank control group （P 〈 0.05） , the different dose inhibition rates were 12.2% , 23.2% , 38.1% in a dose-dependent manner. Apoptosis detection showed that in containing Cs-4 serum groups, the apoptosis rates were 13.9% , 23.5% , 24.9% , which was higher than those in the blank group （P 〈 0.05）. In containing Cs4 serum groups, the concentration of VEGF in supernatant was respectively 443.5, 436.9, 411.0 ng ·L^-1, which was lower than that in control group （P 〈 0.05 ）. Conclusion ： The containing Cs-4 serum can inhibit the proliferation and VEGF expression of SMMC-7721 cells, and increase the apoptosis of SMMC-7721 suggesting Cs-4 had anti-tumor metastasis action.