Comparison between mesenchymal stem cells from bone marrow and adipose tissue in the regulatory function of liver fibrosis in rats / 中华消化杂志

ABSTRACT

Objective To compare the difference between bone marrow stomal cell (BMSC) and adipose-derived stem cell (ADSC) of liver fibrosis in rats.Methods BMSC and ADSC of Sprague-Dawley (SD) rats were isolated and purified.The stem cell markers were detected with flow cytometry.The coculture system was set up with 0.4 μm Transwell insert semipermeable membrane.ADSC or BMSC were co-cultured with hepatic stellate cells (HSC).Normal hepatocyte cell line of rat (BRL) was co-cultured with HSC as negative control group and HSC cultured alone was blank control group.After cultured for 72 hours,the proliferation of HSC was determined by cell counting kit-8 (CCK-8) method.The expression of α-smooth muscle actin (α-SMA) of HSC was detected by Western blotting.The apoptosis of HSC was examined by flow cytometry.After BMSC,ADSC and BRL cultured alone for 72 hours,expression level vascular endothelial growth factor (VEGF),interleukin-10 (IL-10),nerve growth factor (NGF) and transforming growth factor-β1 (TGF-β1) in the culture medium were detected by enzymelinked immunosorbent assay (ELISA) method.The rats model of liver fibrosis were established.The rats were divided into BMSC treatment group,ADSC treatment group,BRL group and culture medium group,six rats in each group,which were injected with 1.5 mL BMSC,ADSC and BRL cells suspension (5 × 106) through portal vein,respectively,and same volume of culture medium was injected to the rate of culture medium group,once every two weeks for four weeks.The pathological changes of liver tissue sections were observed and liver fibrosis markers were tested.T test was performed for comparison between two samples and analysis of variance was used for comparison among multiple groups.Results BMSC and ADSC were successfully isolated and cultured.The phenotype of BMSC and ADSC was similar.Compared with blank control group and negative control group,both ADSC and BMSC could inhibit the proliferation of HSC and promote apoptosis (proliferation,2.43±0.27,2.39±0.33,1.92±0.38 and 2.18±0.31,FBMSC =25.61,FADSC =38.63,both P＜0.05 ;apoptosis rate,(5.59 ± 0.40)％,(6.82±0.57)％,(8.31± 1.03) ％ and (9.36 ± 0.54) ％,FBMSC =73.69,FADSC =97.41,both P＜ 0.05).The effects of ADSC were more significant than those of BMSC (t=5.76 and 5.18,both P＜0.05).There was difference in the cytokine levels secreted by ADSC and BMSC (NGF,(7.46 ± 0.54) pg/mL vs (3.95 ± 0.71) pg/mL,t =10.92,P＜0.05; TGF-β1,(8.79 ±0.93) pg/mL vs (6.36±0.85) pg/mL,t=7.58,P＜0.05).The cell transplantation experiment indicated that both BMSC and ADSC had significant inhibitory effect on liver fibrosis.The activity index of inflammation and degree of fibrosis in BMSC treatment group and ADSCs treatment group were 9.87±2.07,4.17 ± 0.94 and 10.13 ± 1.81,3.98 ± 0.82,which were significantly lower than those in blank control group (13.78±2.53 and 5.09±1.15)and negative control group (13.34± 1.89 and 4.95± 1.22,FBMSC=51.26 and 32.29,P＜0.05; FADSC =46.73 and 40.94,P＜0.05).The level of hyaluronic acid ((191.5±33.2) μg/L and (178.8±28.2) μg/L),type Ⅲ collagen ((19.9±5.1) μg/L and (21.7± 3.3) μg/L) and hydroxyproline ((312.6±38.8) μg/g and (325.8±28.2) μg/g) of BMSC treatment group and ADSC treatment group were significantly lower than those of negative control group and blank control group (hyaluronic acid,(282.3 ± 18.7) μg/L and (287.5 ± 26.7) μg/L),F =73.51 ; type Ⅲ collagen,(35.3± 3.3) μg/L and (32.5±4.3) μg/L,F=76.19; hydroxyproline,(458.4 ± 38.1) μg/g and (473.9 ± 63.7) μg/g,F=60.37,all P＜0.05).However,there was no difference between BMSC treatment group and ADSC treatment (all P＜0.05).Conclusions ADSC and BMSC had similar stem cell characteristics.There was difference in inhibiting the activation of HSC between ADSC and BMSC.But there was no significant difference in inhibiting liver fibrosis of rats in vivo.