槲寄生碱对低分化骨肉瘤U2OS的抑制作用

目的：通过体外实验研究槲寄生碱对人低分化骨肉瘤细胞（U2OS）生长、侵袭的影响。方法：以5-氟尿嘧啶(5-FU)作用组为阳性对照组,以不加药组为阴性对照组,槲寄生组为实验组作用U2OS细胞,采用CCK-8试剂盒检测药物对体外培养的U2OS细胞生长抑制率。绘制在不同药物浓度作用下的细胞生长曲线。观察不同药物浓度作用下细胞的形态学变化。应用Boyden小室检测槲寄生碱对U2OS细胞侵袭能力的影响。结果：槲寄生碱作用U2OS细胞的IC50值为7 mg·L^-1;生长曲线显示,加药组细胞生长速度较未加药组明显缓慢,细胞由多角型逐渐变为圆形,排列疏松,附壁性减弱。Boyden小室实验显示,槲寄生碱作用后,迁移至聚碳酸脂膜下的细胞数(47.0±2.4)明显少于未加药组(122.0±12.1)（P<0.05）。结论：槲寄生碱在体外具有抑制人低分化骨肉瘤细胞U2OS细胞生长、侵袭的作用,有望成为治疗骨肉瘤的辅助药物。     

盐酸石蒜碱通过 circASH2L/miR-124-3p 轴调控肝癌 HCCLM3 细胞的 恶性生物学行为

目的：探讨盐酸石蒜碱（lycorine hydrochloride,LH）对肝癌HCCLM3细胞恶性生物学行为的影响及其对circASH2L/ miR-124-3p轴的调控作用。方法：将HCCLM3细胞分为不同浓度LH处理组（LH-L、LH-M、LH-H组）和Con、si-NC、si-circASH2L、 LH+pcDNA、LH+pcDNA-circASH2L组;以 CCK-8法、平板克隆形成实验、流式细胞术、细胞划痕实验和 Transwell小室实验分别 检测HCCLM3细胞的增殖、克隆形成、凋亡、迁移和侵袭;qPCR法检测HCCLM3细胞中circASH2L和miR-124-3p的表达量;双荧 光素酶报告基因实验检测 circASH2L 和 miR-124-3p 的靶向关系;WB 法检测 cleaved-caspase3、cleaved-caspase9、E-cadherin、 N-cadherin蛋白表达量。结果：与Con组比较,LH不同浓度组细胞增殖抑制率升高（P<0.05）,划痕愈合率与N-cadherin蛋白水平降低（P<0.05）,circASH2L的表达水平降低（P<0.05）,细胞增殖抑制率降低（P<0.05）,克隆形成数与侵袭细胞数增多（均P<0.05）,划痕愈合率与N-cadherin蛋白水平升高（均P目的：探讨盐酸石蒜碱（lycorine hydrochloride,LH）对肝癌HCCLM3细胞恶性生物学行为的影响及其对circASH2L/ miR-124-3p轴的调控作用。方法：将HCCLM3细胞分为不同浓度LH处理组（LH-L、LH-M、LH-H组）和Con、si-NC、si-circASH2L、 LH+pcDNA、LH+pcDNA-circASH2L组;以 CCK-8法、平板克隆形成实验、流式细胞术、细胞划痕实验和 Transwell小室实验分别 检测HCCLM3细胞的增殖、克隆形成、凋亡、迁移和侵袭;qPCR法检测HCCLM3细胞中circASH2L和miR-124-3p的表达量;双荧 光素酶报告基因实验检测 circASH2L 和 miR-124-3p 的靶向关系;WB 法检测 cleaved-caspase3、cleaved-caspase9、E-cadherin、 N-cadherin蛋白表达量。结果：与Con组比较,LH不同浓度组细胞增殖抑制率升高（P<0.05）,凋亡率与cleaved-caspase3、cleaved-caspase9、E-cadherin 蛋白水平升高（均P<0.05）,miR-124-3p 的表达水平升高（P<0.05）,克隆形成数与侵袭细胞数减少（均P<0.05）,划痕愈合率与N-cadherin蛋白水平降低（P<0.05）,circASH2L的表达水平降低（P<0.05）,且不同浓度组间比较差异有统 计学意义（P<0.05）。circASH2L可负向调控 miR-124-3p。与 si-NC 组比较,si-circASH2L组细胞增殖抑制率升高（P<0.05）,凋亡 率与 cleaved-caspase3、cleaved-caspase9、E-cadherin 蛋白水平升高（均 P<0.05）,划痕愈合率与 N-cadherin 蛋白水平降低（均 P<0.05）,克隆形成数与侵袭细胞数减少（均 P<0.05）。与 LH+pcDNA 组比较,LH+pcDNA-circASH2L 组 miR-124-3p的表达水平 降低（P<0.05）,细胞增殖抑制率降低（P<0.05）,凋亡率与 cleaved-caspase3、cleaved-caspase9、E-cadherin 蛋白水平降低（均 P<0.05）,克隆形成数与侵袭细胞数增多（均 P<0.05）,划痕愈合率与N-cadherin蛋白水平升高（均 P<0.05）。结论：LH可通过调控 circASH2L/miR-124-3p轴来抑制肝癌细胞HCCLM3的增殖、迁移、侵袭并诱导其凋亡。     

转化生长因子-β3对增生性瘢痕成纤维细胞的生物学作用

背景哺乳类动物有3种TGF-β异构体即TGF-β1,β2,β3,这3种异构体各自具有独特而不同的生物学作用,TGF-β1是明显的促瘢痕形成因子,而TGF-β3可减少TGF-β1,β2的产生,从而减少细胞外基质(ECM)合成,因此,TGF-β3有可能是人体内天然的抗瘢痕形成因子.目的通过外源性TGF-β3对增生性瘢痕成纤维细胞(HSFB)生长增殖及Ⅰ,Ⅲ型胶原及TGF-β1蛋白表达的影响,了解其生物学行为,为临床治疗提供理论依据.设计随机对照实验研究.地点和对象汕头大学医学院第二附属医院整形烧伤外科完成,对象为新鲜无菌增生性瘢痕组织,来源于本科收治及门诊手术的增生性瘢痕患者6例,男3例,女3例;年龄5～45岁.干预6例增生性瘢痕成纤维细胞体外培养,分为4组,实验组又分为5,10,50 mg/L组分别加TGF-β3 5,10,及50 mg/L,对照组加入FBM1.5 mL,采用MTT比色法测定TGF-β3对HSFB增殖活性的影响,3H-脯氨酸掺人法测定其胶原合成,免疫组化检测Ⅰ,Ⅲ型胶原及TGF-β1蛋白表达情况.主要观察指标TGF-β3对HSFB体外增殖,HSFB Ⅰ,Ⅲ型胶原及TGF-β1蛋白表达的影响,TGF-β3作用后HSFB胶原合成情况.结果转化生长因子β3可抑制HSFB细胞增殖,作用120 h后实验组细胞密度分别为(6.34±0 51),(6.01±0.38),(5.24±0.65)×105/瓶,明显低于对照组(10.69±0.76)x105/瓶(F=102.432～163 024,P＜0.01);转化生长因子β3可抑制胶原蛋白合成,实验组(10,50mg/L组)脯氨酸含量分别为(164.01±70.27),(151 02±49 85)min-1明显低于对照组9231.56±67.83)min-1(q=32.124,31.021,P＜0.01);下调TGF-β1蛋白及Ⅰ型胶原蛋白表达;而对Ⅲ型胶原影响较小.结论TGF-β3可抑制HSFB细胞增殖,下调TGF-β1蛋白表达,减少胶原合成,显示其具有抗组织纤维化的作用,具有临床应用价值.     

基质金属蛋白酶及其抑制物在子宫内膜异位症中的作用

近来 ,一些研究表明基质金属蛋白酶 (MMPs)及其抑制物在子宫内膜异位症的发生中起着重要的作用。现就MMPs及其抑制物的分类、分布、生物学功能及参与子宫内膜异位症形成的机理做一综述。     

The activity of a designer tissue inhibitor of metalloproteinases (TIMP)-1 against native membrane type 1 matrix metalloproteinase (MT1-MMP) in a cell-based environment

The surface-anchored membrane type 1 matrix metalloproteinase (MT1-MMP) degrades a wide range of extracellular matrix components that includes collagens, laminins, fibronectin and the structural proteoglycan aggrecan. The enzyme modulates cell motility and plays an important role in tumour invasion and proliferation. We have previously designed a variant of tissue inhibitor of metalloproteinase (TIMP)-1 bearing a triple mutation (V4A + P6V + T98L, or N-TIMP-1^mt1) that forms tight binary complex with the soluble catalytic domain of MT1-MMP [M.H. Lee, M. Rapti, G. Murphy, J. Biol. Chem. 278 (2003) 40224–40230]. Here, we report our latest findings on the cellular potency of this mutant against native MT1-MMP in cell-based environment. We show that N-TIMP-1^mt1 is a highly potent inhibitor against the ectodomain form of MT1-MMP (K_i 9.53 nM) with potential for further development as a therapeutic agent. The mutant is devoid of pro-MMP-2-activating capability but is highly effective in blocking MT1-MMP-mediated FITC-labelled collagen and gelatin film degradation in HTC75 fibrosarcoma and MCF7 breast cancer models. Most encouragingly, N-TIMP-1^mt1 is also effective against CD44 shedding in HTC75 cells and able to prevent tubule formation in human umbilical vascular endothelial cells (HUVEC) in a 3D fibrin gel model. We are interested in the development of the TIMPs as therapeutic agents against MT1-MMP related disorders such as cancers. Our findings here indicate the potential for the design of selective TIMPs with refined specificity and possibility for future therapeutic application.     

Liu-He-Tang inhibited plaque formation by human respiratory syncytial virus infection in cell lines of the human respiratory tract

Ethnopharmacological relevance

Liu-He-Tang (LHT) has been used to treat adult respiratory tract infection with productive cough and fever for a thousand years in ancient China. Adults with respiratory tract infection of human respiratory syncytial virus (HRSV) can have symptoms similar to those managed by LHT. Therefore, LHT is supposed to be beneficial for adult HRSV infection. However, LHT does not have any antiviral activity to support its use against HRSV infection.

Aim of the study

HRSV is the most important virus causing serious pediatric respiratory tract infections worldwide. HRSV also contributes considerably to respiratory tract illness in adults. There is no effective therapeutic modality against HRSV infection. In order to find readily available agents to manage adult HRSV infection, this study tested the hypothesis that LHT has antiviral activity against HRSV-induced cytopathy.

Materials and methods

Effect of the hot water extract of LHT on HRSV was tested by plaque reduction assay in both human upper (HEp-2) and low (A549) respiratory tract cell lines and also a human normal fibroblast cell line (WI-38). Ability of LHT to stimulate anti-viral cytokines was evaluated by enzyme-linked immunosorbent assay (ELISA).

Results

LHT could dose-dependently inhibit HRSV-induced plaque formation (p < 0.0001), especially in A549 cell. 300 μg/ml LHT nearly abolished plaque formation in A549 cells. LHT was more effective when given before viral inoculation (p < 0.0001). LHT dose-dependently inhibited viral attachment (p < 0.0001). Besides, LHT could inhibit HRSV internalization both time-dependently and dose-dependently (p < 0.0001). Furthermore, LHT stimulated epithelial cells to secrete IFN-β and TNF-α to counteract HRSV infection before infection becomes established.

Conclusions

LHT has anti-HRSV activity that provides a basic support of its possible use in managing adult HRSV infection.     

Ge-Gen-Tang has anti-viral activity against human respiratory syncytial virus in human respiratory tract cell lines

Ethnopharmacological relevance

Ge-Gen-Tang (GGT) has been used against adult respiratory tract infection for thousand years in ancient China. However, GGT is unable to inhibit influenza virus. The effect of GGT to manage respiratory tract viral infection has been questioned. Several ingredients of GGT and their constituents are able to inhibit various viruses. Therefore, GGT might have antiviral activity against other viruses causing respiratory tract illness. Human respiratory syncytial virus (HRSV) is one of the most important airway viruses. However, it is unknown whether GGT is effective against HRSV.

Aim of the study

HRSV contributes considerably to respiratory tract illness of the elderly and immunocompromised adults. There is no effective therapeutic modality for HRSV infection. In order to find a readily available agent to manage HRSV infection, the authors tested the hypothesis that GGT can effectively minimize airway pathology by preventing HRSV-induced plaque formation in respiratory mucosal cell lines.

Materials and methods

Effect of the hot water extract of GGT on HRSV was tested by plaque reduction assay in both human upper (HEp-2) and low (A549) respiratory tract cell lines. Ability of GGT to stimulate anti-viral cytokines was evaluated by enzyme-linked immunosorbent assay (ELISA).

Results

GGT dose-dependently inhibited HRSV-induced plaque formation in both cell lines (p < 0.0001), especially in A549 cells. GGT was more effective when given before viral infection (p < 0.0001). GGT could dose-dependently inhibit viral attachment (p < 0.0001) with or without heparin. GGT could further inhibit HRSV internalization time-dependently and dose-dependently (p < 0.0001). GGT could stimulate mucosal cells to secrete IFN-β to counteract viral infection before and after viral inoculation.

Conclusions

GGT is effective against HRSV-induced plaque formation in airway epithelium.     

c-Myc反义寡核苷酸对人肝癌Bel-7402细胞增殖的双向调节作用

目的观察c-Myc反义寡核苷酸(c-Myc ASODN)对人肝癌Bel-7402细胞增殖的双重调节作用,探讨c-Myc基因的生物学功能.方法台盼兰拒染细胞计数,CCK-8细胞检测试剂盒检测各组细胞增殖抑制率,计算单纯黄连素组及合用c-Myc ASODN后IC50.结果单纯黄连素及c-Myc ASODN均能明显抑制细胞的增殖(P＜0.05),将黄连素合用c-Myc ASODN后,细胞增殖抑制率低于单用黄连素组,但仍高于c-Myc ASODN组.黄连素作用Bel-7402细胞的IC50值在合用c-Myc ASODN后增加1.42倍.结论c-Myc基因在Bel-7402细胞细胞生长环境良好时可促进细胞的生长,在细胞生长环境不利的情况下则诱导细胞的的凋亡,抑制细胞的生长.依据环境不同,对Bel-7402细胞增殖具有不同的调节作用,c-Myc ASODN通过下调c-Myc基因的表达,在不同的环境下亦表现出不同的生物学效应.c-Myc ASODN与黄连素合用能明显降低Bel-7402细胞对黄连素的敏感性.