Effect of transforming growth factor beta and bone morphogenetic proteins on rat hepatic stellate cell proliferation and transdifferentiation

AIM: To explore different roles of TGF-β (transforming growth factor beta) and bone morphogenetic proteins (BMPs)in hepatic stellate cell proliferation and trans-differentiation.METHODS: Hepatic stellate cells were isolated from male Sprague-Dawley rats. Sub-cultured hepatic stellate cells were employed for cell proliferation assay with WST-1 reagent and Western blot analysis with antibody against smooth muscle alpha actin (SMA).RESULTS: The results indicated that TGF-β1 significantly inhibited cell proliferation at concentration as low as 0.1 ng/ml, but both BMP-2 and BMP-4 did not affect cell proliferation at concentration as high as 10 ng/ml. The effect on hepatic stellate cell trans-differentiation was similar between TGFβ1 and BMPs. However, BMPs was more potent at transdifferentiation of hepatic stellate cells than TGF-β1. In addition, we observed that TGF-β1 transient reduced the abundance of SMA in hepatic stellate cells.CONCLUSION: TGF-β may be more important in regulation of hepatic stellate cell proliferation while BMPs may be the major cytokines regulating hepatic stellate cell transdifferentiation.

Hepatitis C virus infection of human hepatoma cell line 7721 in vitro

AIM: To establish a cell culture system with long-term replication of hepatitis C virus in vitro. METHODS: Human hepatoma cell line 7721 was tested for its susceptibility to HCV by incubating with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various phases during the culturing periods. The presence of HCV RNA, the expression of HCV antigens in cells and/or supernatant were examined by RTPCR, in situ hybridization and immunohistochemistry respectively. RESULTS: The intracellular HCV RNA was first detected on d2 after infection and then could be intermittently detected in both cells and supernatant over a period of at least three months. The expression of HCV NS3,CP10 antigens could be observed in cells. The fresh cells could be infected by supernatant from cultured infected cells and the transmission of viral genome from HCV-infected 7721 cells to PBMCs was also observed. CONCLUSION: The hepatoma line 7721 is not only susceptible to HCV but also supports its long-term replication in vitro.

Transcription factor EGR-1 inhibits growth of hepatocellular carcinoma and esophageal carcinoma cell lines

AIM: The transcription factor EGR-1 (early growth response gene-1) plays an important role in cell growth, differentiation and development. It has identified that EGR-1 has significant transformation suppression activity in some neoplasms, such as fibrosarcoma, breast carcinoma. This experiment was designed to investigate the role of egr-1 in the cancerous process of hepatocellular carcinoma (HCC) and esophageal carcinoma (EC), and then to appraise the effects of EGR-1 on the growth of these tumor cells. METHODS: Firstly, the transcription and expression of egr-1 in HCC and EC, paracancerous tissues and their normal counterpart parts were detected by in situ hybridization and immunohistochemistry, with normal human breast and mouse brain tissues as positive controls. Egr-1 gene was then transfected into HCC (HHCC, SMMC7721) and EC (ECa109) cell lines in which no egr-1 transcription and expression were present. The cell growth speed, FCM cell cycle, plate clone formation and tumorigenicity in nude mice were observed and the controls were the cell lines transfected with vector only. RESULTS: Little or no egr-1 transcription and expression were detected in HCC, EC and normal liver tissues. The expression of egr-1 were found higher in hepatocellular paracancerous tissue (transcription level P=0.000; expression level P=0.143, probably because fewer in number of cases) and dysplastic tissue of esophageal cancer (transcription level P=0.000; expression level P=0.001). The growth rate of egr-1-transfected HHCC (HCC cell line) cells and ECa109 (EC cell line) cells was much slower than that of the controls. The proportion of S phase cell, clone formation and tumorigenicity were significantly lower than these of the controls' (decreased 45.5% in HHCC cells and 34.1% in ECa109 cells; 46.6% and 41.8%; 80.4% and 72.6% respectively). There were no obvious differences between SMMC7721 (HCC) egr-1-transfected cells and the controls with regard to the above items. CONCLUSION: The decreased expression of egr-1 might play a role in the dysregulation of normal growth in the cancerous process of HCC and EC. Egr-1 gene of transfected HHCC and ECa109 cells showed obvious suppression of the cell growth and malignant phenotypes, but no suppression in SMMC7721 (HCC cell line) cells.

Reduction of tumorigenicity of SMMC-7721 hepatoma cells by vascular endothelial growth factor antisense gene therapy

AIM: To test the hypothesis to block VEGF expression of SMMC-7721 hepatoma cells may inhibit tumor growth using the rat hepatoma model. METHODS: Amplify the 200 VEGF cDNA fragment and insert it into human U6 gene cassette in the reverse orientation transcribing small antisense RNA which could specifically interact with VEGF165, and VEGF121 mRNA. Construct the retroviral vector containing this antisense VEGF U6 cassette and package the replication-deficient recombinant retrovirus. SMMC-7721 cells were transduced with these virus and positive clones were selected with G418. PCR and Southern blot analysis were performed to determine if U6 cassette integrated into the genomic DNA of positive clone. Transfected tumor cells were evaluated for RNA expression by ribonuclease protection assays. The VEGF protein in the supernatant of parental tumor cells and genetically modified tumor cells was determined with ELISA. In vitro and in vivo growth properties of antisense VEGF cell clone in nude mice were analyzed. RESULTS: Restriction enzyme digestion and PCR sequencing verified that the antisense VEGF RNA retroviral vector was successfully constructed.After G418 selection, resistant SMMC-7721 cell clone was picked up. PCR and Southern blot analysis suggested that U6 cassette was integrated into the cell genomic DNA. Stable SMMC-7721 cell clone transduced with U6 antisense RNA cassette could express 200 bp small antisense VEGF RNA and secrete reduced levels of VEGF in culture condition. Production of VEGF by antisense transgene-expressing cells was 65+/-10 ng/L per 10(6) cells, 42045 ng/L per 10(6) cells in sense group and 485+/-30 ng/L per 10(6) cells in the negative control group, (P【 0.05). The antisense-VEGF cell clone appeared phenotypically indistinguishable from SMMC-7721 cells and SMMC-7721 cells transfected sense VEGF. The growth rate of the antisense-VEGF cell clone was the same as the control cells. When S.C. was implanted into nude mice, growth of antisense-VEGF cell lines was greatly inhibited compared with control cells. CONCLUSION: Expression of antisense VEGF RNA in SMMC-7721 cells could decrease the tumorigenicity, and antisense-VEGF gene therapy may be an adjuvant treatment for hepatoma.

Effects of retinoic acid on proliferation,phenotype and expression of cyclin-dependent kinase inhibitors in TGF-β1-stimulated rat hepatic stellate cells

AIM To study the molecular mechanisms ofretinoic acid(RA)on proliferation andexpression of cyclin-dependent kinase inhibitors(CKI),i.e.p16,p21 and p27 in cultured rathepatic stellate cells(HSC)stimulated withtransforming growth factor beta 1(TGF-β1).METHODS HSC were isolated from healthy ratlivers and cultured.After stimulated with1 mg/L TGF-β1,subcultured HSC were treatedwith or without 1 nmol/L RA.MTT assay,immunocytochemistry(ICC)for p16,p21,p27and α-smooth muscle actin(α-SMA)protein,insitu hybridization(ISH)for retinoic acidreceptor beta 2(RAR-β2)and p16,p21 and p27mRNA and quantitative image analysis(partially)were performed.RESULTS RA inhibited HSC proliferation(41.50%,P【0.05),decreased the protein levelof α-SMA(55.09%,P【0.05),and induced HSCto express RAR-β2 mRNA.In addition,RAincreased the protein level of p16(218.75%,P【0.05)and induced p21 protein expression;meanwhile,p27 was undetectable by ICC in bothcontrol and RA-treated HSC.However,RA hadno influence on the mRNA levels of p16,p21 orp27 as determined by ISH.CONCLISION Up-regulation of p16 and p21 on post-transcriptional level may contribule, in part to RA inhibition of TGF-β1-initiated rat HSC activation in vitro.

Alterations in metastatic properties of hepatocellular carcinoma cell following H-ras oncogene transfection

AIM: To demonstrate the relationship between H-ras oncogene and hepatocellular carcinoma (HCC) metastasis. METHODS: Activated H-ras oncogene was transfected into SMMC 7721, a cell line derived from human HCC, by calcium phosphate transfection method. Some metastasis-related parameters were detected in vitro, including adhesion assay, migration assay, expression of collagenase IV(c IV ase) and epidermal growth factor receptor (EGFR). RESULTS: The abilities of H-ras-transfected cell clones in adhesion to laminin (LN) or fibronectin (FN), migration, c IV ase secretion increased markedly, and the expression of EGFR elevated moderately. More importantly, these alterations were consistent positively with the expression of p21, the protein product of H-ras oncogene. CONCLUSION: H-ras oncogene could induce the metastatic phenotype of HCC cell in vitro to raise its metastatic potential.

SF/HGF-c-Met autocrine and paracrine promote metastasis of hepatocellular carcinoma

AIM: To explore the role of SF/HGF-Met autocrine and paracrine in metastasis of hepatocellular carcinoma (HCC). METHODS: SF/HGF and c-met transcription and protein expression in HCC were examined by RT-PCR and Western Blot in 4 HCC cell lines, including HepG2, Hep3B, SMMC7721 and MHCC-1, the last cell line had a higher potential of metastasis. sf/hgf cDNA was transfected by the method of Lipofectin into SMMC7721. SF/HGF and c-met antibody were used to stimulate and block SF/HGF-c-met signal transduction. Cell morphology, mobility, and proliferation were respectively compared by microscopic observation, wound healing assay and cell growth curve. RESULTS: HCC malignancy appeared to be relative to its met-SF/HGF expression. In MHCC-1, c-met expression was much stronger than that in other cell lines with lower potential of metastasis and only SF/HGF autocrine existed in MHCC-1. After sf/hgf cDNA transfection or conditioned medium of MHCC-1 stimulation, SMMC7721 changed into elongated morphology, and the abilities of proliferation (P 【 0.05) and mobility increased. Such bio-activity could be blocked by c-met antibody (P 【 0.05). CONCLUSION: The system of SF/HGF-c-met autocrine and paracrine played an important role in development and metastasis potential of HCC. Inhibition of SF/HGF-c-met signal transduction system may reduce the growth and metastasis of HCC.

Antihepatoma effect of alpha-fetoprotein antisense phosphorothioate oligodeoxyribonucleotides in vitro and in mice

AIM: To evaluate antihepatoma effect of antisense phosphorothioate oligodeoxyribonucleotides (S-ODNs) targeted to alpha-fetoprotein (AFP) genes in vitro and in nude mice. METHODS: AFP gene expression was examined by immunocytochemical method or enzyme-linked immunosorbent assay. Effect of S-ODNs on SMMC-7721 human hepatoma cell growth in vitro was determined using microculture tetrazolium assay. In vitro antitumor activities of S-ODNs were monitored by measuring tumor weight differences in treated and control mice bearing SMMC-7721 xenografts. Induction of cell apoptosis was evaluated by fluorescence-activated cell sorter (FACS) analysis. RESULTS: Antisense S-ODN treatment led to reduced AFP gene expression. Specific antisense S-ODNs, but not control S-ODNs, inhibited the growth of hepatoma cells in vitro. In vitro, only antisense S-ODNs exhibited obvious antitumor activities. FACS analysis revealed that the growth inhibition by antisense S-ODNs was associated with their cell apoptosis induction. CONCLUSION: Antisense S-ODNs targeted to AFP genes inhibit the growth of human hepatoma cells and solid hepatoma, which is related to their cell apoptosis induction.

Serum deprivation enhances DNA synthesis of human hepatoma SMMC-7721 cells

AIM To determine the relationship between serum deprivation or serum levels and cell proliferation of human hepatoma SMMC 7721 cells. METHODS Human hepatoma SMMC 7721 cells were grown in RPMI 1640 supplemented with 10% fetal calf (FCS) in 5% CO 2 incubator at 37℃ for 24h , and culture media were replaced to serum free or different serum FCS levels (2 5%, 5%, 10%, 20% and 25%). Six h, 12h , 18h and 24h after the culture, the cells were incorporated TdR for 4h . At last TdR incorporation was detected with liquid scintillation counting. RESULTS DNA synthesis of SMMC 7721 cells could be sharply stimulated by short time (6h) serum deprivation (the cpm value of 3H TdR incorporation of cells in serum free was 39 32 fold higher than cells in 25% serum), and the incorporation of 3H TdR was negatively related to the serum levels. Longer time serum starvation ( 12h , 18h and 24h ) also greatly stimulated DNA synthesis, although the cpm value of 3H TdR incroporation was less than that in 6h serum deprivation. Morphology of cells cultured in different serum levels also showed significant difference. CONCLUSIONS Compared with other cell lines such as BEL7404 and Swiss 3T3, human hepatoma SMMC 7721 cells had different response to the serum deprivation. Short time serum deprivation could greatly stimulate DNA synthesis of human hepatoma SMMC 7721 cells. Precautions must be given to the changes of serum levels for the detection of growth factors and drugs using SMMC 7721 cells as a model.

Cellular Forms in Cultivation in Suspension of Bougainvillea glabra Choisy Variety Surprise

The objective of this work was to describe the predominant cell forms in the phases of growth kinetics in a suspension culture of the Bougainvillea glabra Choisy variety Surprise.Treatments in suspension with Murashige and Skoog(MS)basal culture medium were supplemented with six different concentrations and combinations of vegetable growth regulator(VGR)of type auxin(2,4-D and NAA)and cytokinin(BAP)(0.2-5.3 mg/L)and a control.Friable callus was obtained from leaf explants ex vitro to in vitro culture in solid MS medium using PhytagelTM as gelling agent to 2.2 g/L.A portion of callus(1.0 g)was used as cellular inoculum and grown under controlled conditions(50 mL,120 rpm,25°C and luminous intensity of 48μmol m2/s).The best treatment with significant differences(p≤0.05)were with a hormonal relation of 2:1 of BAP and NAA,respectively,with a fresh weight yield ranging from 1.7905 g to 5.8340 g,which represents around 70%.An adaptation phase was observed from day 0 to day 14 on the curve of fresh weight;an exponential phase at day 14 to day 19 and a declination phase at day 21.Cellular forms in the adaptation phase were elongated cells,a few globular and a few with kidney shape.In the exponential phase,these cells formed small aggregates of globular cells.In the death phase,brown,elongated,damaged and fragmented cells were found.Whit this data obtained it is possible to establish the subculture time in fresh medium.

Identification and characterization of a novel isoform of hepatopoietin

AIM: To isolate a novel isoform of human HPO (HPO-205) from human fetal liver Marathon-ready cDNA and characterize its primary biological function. METHODS: 5'-RACE (rapid amplification of cDNA 5' ends) was used to isolate a novel isoform of hHPO in this paper. The constructed pcDNA(HPO-205), pcDNA(HPO) and pcDNA eukaryotic expression vectors were respectively transfected by lipofectamine method and the stimulation of DNA synthesis was observed by (3)H-TdR incorporation assay. Proteins extracted from different cells were analyzed by Western blot. RESULTS: A novel isoform of hHPO (HPO-205) encoding a 205 amino acid ORF corresponding to a translated production of 23 kDa was isolated and distinguished from the previous HPO that lacked the N-terminal 80 amino acids. The dose-dependent stimulation of DNA synthesis of HepG2 hepatoma cells by HPO-205 demonstrated its similar biological activity with HPO in vitro. The level of MAPK (Mitogen-activated protein kinase) phosphorylation by Western blot analysis revealed that HPO-205 might have the stronger activity of stimulating hepatic cell proliferation than that of HPO. CONCLUSION: A novel isoform of hHPO (HPO-205) was isolated from hepatic-derived cells. The comparison of HPO-205 and HPO will lead to a new insight into the structure and function of hHPO, and provide the new way of thinking to deeply elucidate the biological roles of HPO/ALR.

In vitro culture of isolated primary hepatocytes and stem cell-derived hepatocyte-like cells for liver regeneration

Various liver diseases result in terminal hepatic failure, and liver transplantation, cell transplantation and artificial liver support systems are emerging as effective therapies for severe hepatic disease. However, all of these treatments are limited by organ or cell resources, so developing a sufficient number of functional hepatocytes for liver regeneration is a priority. Liver regeneration is a complex process regulated by growth factors （GFs）, cytokines, transcription factors （TFs）, hormones, oxidative stress products, metabolic net- works, and microRNA. It is well-known that the function of isolated primary hepatocytes is hard to maintain; when cultured in vitro, these cells readily undergo dedifferentia- tion, causing them to lose hepatocyte function. For this mason, most studies focus on inducing stem cells, such as embryonic stem cells （ESCs）, induced pluripotent stem cells （iPSCs）, hepatic progenitor cells （HPCs）, and mesenchymal stem cells （MSCs）, to differentiate into hepatocyte-like cells （HLCs） in vitro. In this review, we mainly focus on the nature of the liver regeneration process and discuss how to main- tain and enhance in vitro hepatic function of isolated primary hepatocytes or stem cell-derived HLCs for liver regenera- tion. In this way, hepatocytes or HLCs may be applied for clinical use for the treatment of terminal liver diseases and may prolong the survival time of patients in the near future.

高糖对乳牙牙髓干细胞生长及成骨分化能力的影响

目的探讨高糖对乳牙牙髓干细胞(SHED)生长及成骨分化能力的影响。方法从河南大学赛思口腔医院口腔颌面外科收集拔除的6~8岁无牙体牙髓牙周疾病的健康儿童的滞留乳牙,体外培养获得SHED,实验分为5.5 mmol·L^(-1)低糖组及25 mmol·L^(-1)高糖组。用细胞计数CCK-8试剂盒(CCK-8)检测两组细胞的增殖速度。分别用含两种不同糖浓度的成骨矿化液诱导21 d后,行茜素红染色实验,实时荧光定量PCR(QPCR)技术检测骨向分化相关基因碱性磷酸酶(ALP)、Runt相关转录因子2(RUNX2)和骨钙素(OCN)、骨桥蛋白(OPN)表达的差异。结果CCK-8检测结果显示,培养2~6 d时高糖组的OD值小于低糖组(P<0.05);成骨诱导后,高糖组的钙化结节数量少于低糖组,且高糖组的RUNX-2、ALP、OCN和OPN的相对表达量低于低糖组(P<0.05)。结论高糖抑制SHED的生长及成骨分化能力。

一种小鼠原代肝细胞的分离与培养方法

目的:探索一种高效、稳定的小鼠原代肝细胞分离方法,并延长其体外培养的时间。方法:取成年雄性C57BL/6小鼠,以含双抗的D-Hanks灌流液经肝门静脉充分灌注肝脏,然后以0.2 mg/mL的Ⅳ型胶原酶灌注消化,接着过滤、离心、洗涤,获得小鼠原代肝细胞。通过台盼蓝染色检验小鼠原代肝细胞活率,利用倒置显微镜观察肝细胞的形态变化,糖原染色鉴定细胞纯度,添加ITS-X和HGF细胞因子延长小鼠原代肝细胞的体外培养时间。结果:平均每只成年小鼠肝脏可提取获得原代肝细胞约2×107个,台盼蓝染色显示细胞活率均超过95%,糖原染色结果表明此方法获得的原代肝细胞纯度高于95%,通过添加HGF和ITS-X两种细胞因子能够有效地延长肝细胞形态的体外维持时间。结论:本实验在原有小鼠原代肝细胞两步灌流分离法和培养的基础上,经过优化,成功建立了一种高效、稳定的小鼠原代肝细胞分离和培养方法,为肝脏的体外研究提供了技术保障。

Com bined effect of alphafetoprotein antisense oligodeoxynucleotidesand 5-fluorouracil on human hepato ma cell growth

Objective To investigate the effect of alpha fetoprotein (AFP) antisense phosphorothioate oligodeoxynucleotides in combination with 5 fluorouracil (5 FU) on the growth of BEL 7404 human hepatoma cells in vitro Methods Phosphorothioate oligodeoxynucleotides were synthesized by β cyanoethyl phosphoramidite chemistry AFP gene expression in human hepatoma cells was determined by avidin biotin peroxidase complex (ABC) immunocytochemical method Cell growth in the presence or absence of experimental agents was measured using 3 (4, 5 dimethylthiazol 2 yl) 2, 5 dipheny ltetrazolium (MTT) microculture tetrazolium assay Results AFP antisense oligomers markedly suppressed the growth of BEL 7404 human hepatoma cells in vitro by sequence specific blocking of the AFP gene expression in the cells (P<0 05) 5 FU also inhibited the hepatoma cell growth in a dose dependent manner when used alone (P<0 05) The combined treatment with AFP antisense oligomers and 5 FU showed significantly enhanced hepatoma cell growth inhibition than either AFP antisense or 5 FU treated cells alone (P<0 05) Conclusion Combined use of AFP antisense oligomers and 5 FU could more effectively inhibit the growth of BEL 7404 human hepatoma cells in vitro

健脾活血中药抗肝纤维化的体外实验研究

目的研究健脾活血中药抗肝纤维化的作用机制。方法体外培养LX-2细胞,采用CCK-8法检测健脾活血中药冻干粉促进LX-2细胞增殖的最佳浓度;实验分为对照组、模型组[5 ng/mL转化生长因子-β_(1)(transforming growth factor-beta 1,TGF-β_(1))诱导]和中药组(5 ng/mL TGF-β_(1)+健脾活血中药冻干粉),采用Western blot法和免疫荧光法检测各组Ⅰ型胶原蛋白(CollagenⅠ,Col-Ⅰ)、Ⅲ型胶原蛋白(Collagen Ⅲ,Col-Ⅲ)、α-平滑肌肌动蛋白(alpha smooth muscle actin,α-SMA)的表达水平。结果CCK-8法检测结果显示,1.5 mg/mL健脾活血中药冻干粉诱导的LX-2细胞增殖率最高(P<0.05)。Western blot法和免疫荧光法检测结果均显示,与对照组比较,模型组LX-2细胞中Col-Ⅰ、Col-Ⅲ、α-SMA蛋白表达水平明显升高(P<0.05);与模型组比较,中药组LX-2细胞中Col-Ⅰ、Col-Ⅲ、α-SMA的表达水平均显著降低(P<0.05)。结论健脾活血中药通过抑制Col-Ⅰ、Col-Ⅲ、α-SMA的表达改善LX-2细胞纤维化。

阻断类胰岛素生长因子-1-受体系统对肌腱细胞生长的影响

为了进一步探索调控肌腱细胞生长的方法，在肌腱细胞体外培养条件下，采用多种手段阻断类胰岛素生长因子１（ＩＧＦ１）受体系统的不同环节，观察其对肌腱细胞生长的负调节作用。结果发现，ＩＧＦ１受体的抗体（ＩＧＦ１Ｒα）和合成的ＩＧＦ１受体的ｍＲＮＡ的反义寡核苷酸链，对肌腱细胞的增殖有负调节作用。认为，在人工肌腱的构建或防止肌腱粘连的实践中，可以考虑使用上述两种物质。

干细胞样肝原始细胞的分离和鉴定

目的 :证实小鼠胎肝中肝干细胞的存在。方法 :小鼠胎肝细胞的分离培养和免疫细胞化学等。结果 :从小鼠胎肝组织中成功地分离得到了AFP、CD34及Albumin等特异性分子标记阳性、并呈集落样生长的细胞系。结论 :小鼠胎肝中存在具有干细胞特性的原始细胞 。

体外培养条件下SCF、LIF与bFGF对昆明白小鼠精原干细胞增殖的影响

The present study identified the favorable environment conditions for Spermatogomial stem cells in vitro according to their unique biological properties. Three growth factors, stem cell factor (SCF), leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF) were all found to independently contribute to the proliferation of mouse spermatogonial stem cell. The percentage of cell proliferation significantly enhanced by SCF at 30 ng/mL but decreased with heightening its combination after cultured 120 hours. The mice spermatogonial stem cells were significantly proliferated after 120 hours’ culture with 10 ng/mL and 20 ng/mL (P<0.01) of LIF, between 20 ng/mL and 50 ng/mL (P<0.01) for bFGF. SCF and bFGF were significantly enhanced mice spermatogonial stem cells proliferation after these three factors combination. For LIF, no obvious effect was abserved.

柴胡皂甙d对肝癌细胞VEGF和Ang-2表达的影响

目的观察柴胡皂甙d(saikosaponins-d,SSd)对人肝癌SMMC-7721细胞血管内皮生成因子(vascular endo-thelial growth factor,VEGF)和血管生成素-2(angiopoietin-2,Ang-2)表达的影响,探讨其抗癌作用机制。方法肝癌SMMC-7721细胞株经LPS诱导,加入不同终浓度的SSd(2.5、5、10、20 mg/L)作用48 h,并以300μmol/L终浓度的Nimesulide(COX-2选择性抑制剂)作为阳性对照,采用MTT法检测细胞增殖,免疫细胞化学法测定肝癌细胞内VEGF和Ang-2蛋白表达,RT-PCR法检测VEGF mRNA表达,ELISA法检测培养上清中VEGF含量。结果 SSd可显著抑制肝癌SMMC-7721细胞的生长、增殖。与LPS诱导组比较,10 mg/L SSd作用组肝癌细胞VEGF蛋白表达率、mRNA表达水平和培养上清中VEGF含量均显著降低[VEGF蛋白:80.0%vs 60.0%;mRNA表达:(0.83±0.07)vs(0.09±0.03);培养上清中VEGF含量:(655.00±5.56)vs(446.15±5.05)pg/ml,P<0.05,P<0.01],同时Ang-2蛋白表达亦显著降低(86.67%vs 53.33%,P<0.05)。在2.5～10 mg/L作用终浓度范围内,SSd对培养上清中VEGF含量降低的影响呈现一定的剂量依耐性。结论 SSd可能通过VEGF和Ang-2信号通路,发挥其抗癌作用。