1.Role of epidermal growth factor in ocular diseases
Huixin SONG ; Jianfeng WU ; Hongsheng BI
Recent Advances in Ophthalmology 2017;37(5):484-487
Epidermal growth factor (EGF) is a multifunctional growth factor,which is widely expressed in most ocular tissues including cornea,retina and sclera.It may regulate the growth and proliferation of various ocular cells with the interaction of epidermal growth factor receptor in vitro or in vivo.This article reviews the role of EGF in pathological and physiological processes of ocular diseases,including corneal injury,retinal proliferative diseases,myopia,pterygium,glaucoma,cataract,and eye tumors.
2.Effect of heat shock protein 47 on epithelial-mesenchymal transdifferentiation induced by transforming growth factor β1 in the renal tubular epithelial cells
Huixin BI ; Ruihong LIU ; Lin SUN ; Hong LIU ; Shuguang YUAN ; Guxiang HUANG ; Nanlan CHEN
Chinese Journal of Nephrology 2013;29(10):775-781
Objective To detect the expression of heat shock protein 47(HSP47) in renal proximal epithelial cell lines (HK-2) and to investigate the role of HSP47 in the progress of transforming growth factor β1 (TGF-β1) induced epithelial-mesenchymal transdifferentiation (EMT) in HK-2 cells.Methods HK-2 cells were exposed to TGF-β1 (0,2.5,5,10 μg/L) for different time (0,12,24,48 h).The expression of HSP47 was examined by Western blotting.Then HK-2 cells were exposed to 10 μg/L TGF-β1,the expressions of vimentin,zona occludens-1 (ZO-1) were examined by Western blotting and real-time PCR.Furthermore,the expressions of p-Smad3 and Smad3 were examined by Western blotting.HK-2 cells were transfected with HSP47 siRNA and siRNA negative control before exposing to TGF-β1.Then the expressions of vimentin,ZO-1 were detected by Western blotting and real-time PCR,meanwhile Western blotting for HSP47,p-Smad3 and Smad3.Results Stimulating HK-2 with TGF-β1 resulted in a significant increased expression of HSP47 in time-and concentration-dependent manner (P < 0.05).Meanwhile,TGF-β1 up-regulated the protein and mRNA expression of vimentin (P < 0.05),and down-regulated the protein and mRNA expression of ZO-1 (P < 0.05),all in time-dependent manner.Stimulating HK-2 with TGF-β1 resulted in phosphorylation of Smad3,which was peaked at 30 min,slightly decreased at 1 h,and then increased again between 24 and 48 h (P < 0.05).Compared to the TGF-β1 group,inhibition of HSP4.7 expression in HK-2up-regulated the protein and mRNA expression of ZO-1,down-regulated the protein and mRNA expression of vimentin (P < 0.05) and down-regulated the ratio of p-Smad3/Smad3.HSP47 siRNA negative control had no significant effect on the expressions of ZO-1,vimentin and p-Smad3/Smad3 (P > 0.05).Conclusion HSP47 can promote the EMT of renal tubular epithelial cell which is possibly via the TGF-β1-Smad3 pathway.
3.Construction of a new patient-derived xenograft model of human liver cancer in mice with normal immunity
Huixin TANG ; Shanshan LI ; Feng HONG ; Yanzhen BI ; Quanyi WANG ; Xiaobei ZHANG ; Shumin CHENG ; Zhongping DUAN ; Zhenfeng SHU ; Yu CHEN
Journal of Clinical Hepatology 2021;37(11):2584-2588
Objective To establish a new patient-derived xenograft (PDX) model of human liver cancer by inoculating the complex of human primary liver cancer cells and a novel microcarrier (microcarrier 6) into mice with normal immune function. Methods Primary liver cancer cells were isolated and extracted from the fresh human liver cancer tissue of five patients and were then co-cultured with microcarrier 6 to construct a three-dimensional tumor cell culture model in vitro . According to the type of graft, 75 male C57BL/6 mice were divided into cell control group, microcarrier control group, and experimental group (each sample corresponded to three groups, with 15 groups in total and 5 mice in each group). The liver cancer cell-microcarrier complex was implanted into the mice by subcutaneous inoculation, and tumor formation time, tumor formation rate, and histopathological manifestations were observed. The Fisher's exact test was used for comparison of categorical data between two groups. Results As for the liver cancer cells from the five patients, tumor formation was observed in the mice corresponding to three patients. In these three experiments, tumor formation was not observed in the control groups and was only observed in the experimental groups, and 12 of the 15 mice in the experimental groups had successful tumor formation, with a tumor formation rate as high as 80%, which was significantly different from that in the cell control groups and the microcarrier control groups (all P < 0.05). The tumor formation time was 5-7 days; the xenograft tumor grew rapidly, and HE staining showed nested or flaky cells with obvious heteromorphism, with the presence of pathological mitosis; immunohistochemical staining showed positive CK8/18, Hep, and Gpc-3, which was in accordance with the characteristics of human liver cancer cells. Conclusion This experiment successfully establishes a new PDX model of human liver cancer based on the complex of microcarrier 6 and human primary liver cancer cells in mice with normal immunity. This model can be used to better elucidate the mechanism of the development and progression of liver cancer in the body with normal immunity, and besides, it also provides a new animal model with higher value for the precise treatment of liver cancer.
4.Effect of exosomes from adult human liver-derived stem cells on concanavalin A-induced acute liver injury in mice
Luxiang HAN ; Huixin TANG ; Zhenfeng ZHAO ; Shanshan LI ; Quanyi WANG ; Lingbin KONG ; Huiying BI ; Zhenfeng SHU ; Zhongping DUAN ; Yu CHEN ; Feng HONG
Journal of Clinical Hepatology 2022;38(5):1101-1105
Objective To investigate the protective effect of adult human liver-derived stem cell exosomes (HLSC-exo) intravenously injected at different time points against acute liver injury induced by concanavalin A (ConA) in mice. Methods HLSC-exo was extracted by differential centrifugation. Western blot was used to measure the expression of the marker proteins CD9 and CD63, and nanoparticle tracking analysis was used to investigate particle size distribution. A total of 56 male C57BL/6 mice were randomly divided into blank control group, ConA model group, and HLSC-exo treatment group. The ConA model group and the HLSC-exo treatment group were further divided into 3-, 6-, and 12-hour subgroups according to the interval between phosphate buffer or HLSC-exo injection and ConA injection. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), tumor necrosis factor-α (TNF-α), and interleukin-10 (IL-10) were measured, and the gross morphology and histopathology of the liver were compared between groups. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t -test was used for further comparison between two groups. Results HLSC-exo was a membranous vesicle with a diameter of 90-110 nm, with a clear saucer-like structure under an electron microscope and marked expression of its specific marker proteins CD9 and CD63. In the blank control group, the levels of ALT and AST were 31.81±6.74 U/L and 69.75±8.30 U/L, respectively. Compared with the blank control group, the 3-, 6-, and 12-hour ConA model groups had significant increases in the levels of ALT and AST (all P < 0.001); compared with the 3-and 6-hour ConA model groups, the 3-and 6-hour HLSC-exo treatment groups had significant reductions in the levels of ALT and AST (225.58±115.59 U/L vs 1989.32±347.67 U/L, 1174.71±203.30 U/L vs 2208.33±349.96 U/L, 303.53±126.68 U/L vs 2534.27±644.72 U/L, 1340.70±262.56 U/L vs 2437.13±288.13 U/L, all P < 0.001); compared with the 6-hour HLSC-exo treatment group, the 3-hour HLSC-exo treatment group had significantly greater reductions ( P < 0.001). In the blank group, the levels of IL-10 and TNF-α were 313.51±10.97 pg/ml and 476.05±7.31 pg/ml, respectively. Compared with the blank control group, the 3-, 6-, and 12-hour ConA model groups had a significant reduction in the level of IL-10 (all P < 0.001); compared with the 3-and 6-hour ConA model groups, the 3-and 6-hour HLSC-exo treatment groups had a significant increase in the level of IL-10(331.61±10.46 pg/ml vs 266.20±8.15 pg/ml, 288.13±10.74 pg/ml vs 264.41±9.12 pg/ml, both P < 0.001); compared with the 6-hour HLSC-exo treatment group, the 3-hour HLSC-exo treatment group had a significantly greater increase ( P < 0.001). Compared with the blank control group, the 3-, 6-, and 12-hour ConA model groups had a significant increase in the level of TNF-α (all P < 0.001); compared with the 3-and 6-hour ConA model groups, the 3-and 6-hour HLSC-exo treatment groups had a significant reduction in the level of TNF-α (478.26±12.99 pg/ml vs 551.31±17.70 pg/ml, 515.58±7.18 pg/ml vs 556.21±11.15 pg/ml, both P < 0.001); compared with the 6-hour HLSC-exo treatment group, the 3-hour HLSC-exo treatment group had a significantly greater reduction ( P < 0.001). Compared with the 3-and 6-hour ConA model groups in terms of the gross morphology and histopathology of the liver, the 3-and 6-hour HLSC-exo treatment groups had a significant reduction in the degree of hepatocyte necrosis, and the 3-hour HLSC-exo treatment group had a basically complete lobular structure, with sporadic spotty necrosis; the 12-hour HLSC-exo treatment group had no significant improvement in hepatocyte necrosis compared with the 12-hour ConA model group. Conclusion Intravenous injection of adult HLSC-exo can alleviate acute liver injury induced by ConA in mice, and injection at 3 hours in advance has the most significant protective effect. Regulation of cytokines is one of the important mechanisms for HLSC-exo to alleviate liver injury.