Over-expression of Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger 1 and its clinicopathologic significance in hepatocellular carcinoma

The activation of Na⁺/H⁺ exchanger 1 (NHE1) is involved in the formation of tumor cell pseudopodia, possibly resulting in their malignant potential. The clinicopathological significance of NHE1, however, is not yet well known in the case of hepatocellular carcinoma (HCC). In this study, we first analyzed the NHE1 expression in 100 cases of hepatocellular carcinoma (HCC) and found that NHE1, at both messenger RNA and protein levels, was over-expressed in majority of HCC tissues compared with matched non-cancerous tissue. In addition, the increased expression of NHE1 correlated with tumor size, venous invasion and advanced pTNM stage (P < 0.05). Kaplan–Meier curves demonstrated that patients with increased NHE1 expression were at significantly increased risk for shortened survival time (P < 0.05). This is the first demonstration that the expression level of NHE1 is correlated with tumor progression and prognosis in HCC. We proposed that NHE1 may have diagnostic and therapeutic potential for patients with HCC.     

Role of Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger in resveratrol-induced growth inhibition of human breast cancer cells

Cancer cells maintain low intracellular pH [pHi]; therefore, it is likely that resveratrol [RSVL] inhibits cell growth through interference with regulation of pHi. Na–H exchanger [NHE] regulates pHi and NaCl uptake. In this study, we investigated a putative role of NHE-1 and NHE-3 isoforms in the RSVL-induced cell death using MDA-MB-231 estrogen receptor-negative [ER−] and MCF-7 [ER+] human breast cancer cell lines. ECL Western blot analysis and fluorescence morphometeric analysis were used. Cell viability and counting were performed using standard procedures. RSVL caused a dose- and time-dependent induction of NHE-1 and NHE-3 proteins in both cancer cell lines as shown by ECL Western blot analysis and fluorescence measurement. Interestingly, the level of actin, an internal control, remains unaltered. Thus, it is concluded that RSVL-inhibited cell growth and viability, increased cell size, and volume along with an increased apoptotic activity are due to the induction of NHE-1 and NHE-3 isoforms in the present breast cancer cell lines. Induction of NHE will increase the uptake of NaCl and decrease pHi leading to disturbance in Ca²⁺ homeostasis, which is responsible for cell death.     

黄芩素诱导人肝癌细胞株SMMC-7721凋亡作用的研究

目的:研究12-脂氧合酶(12-lipoxygenase,12-LOX)在人肝癌细胞株SMMC-7721中的表达,以及选择性12-LOX抑制剂黄芩素对人肝癌细胞SMMC-7721生长、凋亡的影响.方法:采用免疫细胞化学染色及RT-PCR检测12-LOX的蛋白质和mRNA在SMMC-7721中的表达.应用MTT、流式细胞术及TUNEL检测细胞生长及凋亡.蛋白印迹法检测分析Caspase-3,Bcl-2,Bax,phospho-ERK1/2,ERK1/2和β-actin蛋白质的表达.结果:免疫细胞化学技术显示12-LOX蛋白表达于SMMC-7721细胞质.RT-PCR检测到黄芩素呈浓度-时间依赖性地抑制12-LOX mRNA表达.采用黄芩素小同浓度或不同时间段干预细胞SMMC-7721,MTT法检测细胞增殖呈剂量、时问依赖性的下降.原位末端转移酶标记技术及流式细胞仪分析证实12-LOX抑制剂诱导细胞凋亡.蛋白印迹法检测显示,黄芩素干预后,Bcl-2蛋白表达明显下降和Bax蛋白轻微上升,细胞凋亡蛋白酶-3活性水平与黄芩素浓度正相关,黄芩素对细胞凋亡相关蛋白的影响被12 (S)-HETE逆转,12(5)-HETE可能激活ERK1/2磷酸化.结论:人肝癌细胞株SMMC-7721存在12-LOX的表达,12-LOX抑制剂黄芩素可抑制细胞生长和诱导细胞凋亡.     

沙利度胺对人肝癌细胞株SMMC-7721体外生长的抑制作用

目的 研究沙利度胺对人肝癌细胞株SMMC-7721体外生长的抑制作用及其可能的机制.方法 将不同浓度的沙利度胺作用于人肝癌细胞株SMMC-7721,采用四甲摹偶氮唑蓝(MTT)法检测沙利度胺对SMMC-7721细胞的增殖抑制作用.将SMMC-7721细胞培养至对数生长期,采用DNA琼脂糖凝胶电泳、荧光显微镜观察、流式细胞仪检测等方法 观察沙利度胺处理后SMMC-7721细胞的凋亡梯度、形态学变化和凋亡率,并对凋亡调控蛋白caspase-3的表达进行测定.采用酶联免疫吸附(ELISA)法测定不同浓度的沙利度胺处理后SMMC-7721细胞表达血管内皮生长因子(VEGF)的变化.结果 沙利度胺的浓度从3.125μg/ml增至200μg/ml时,其对SMMC-7721细胞的增殖抑制率从11.7%增至34.2%;当沙利度胺的浓度>25 μg/ml时,其对SMMC-7721细胞的增殖抑制作用明显强于空白对照组(P<0.05).200 μg/ml的沙利度胺处理SMMC-7721细胞24 h后,行琼脂糖凝胶电泳,可见到DNA梯形条带;48 h后梯形条带更明显,并且在荧光显微镜下可见SMMC-7721细胞出现核固缩和核裂解现象.200μg/ml的沙利度胺处理SMMC-7721细胞12、24、48和72 h时,碘化丙啶(PI)法检测SMMC-7721细胞的凋亡率分别为3.1%±0.5%、8.4%±1.3%、19.4%±3.5%和25.8%±2.1%,24 h起的凋亡率均明显高于空白对照组SMMC-7721细胞48 h的自然凋亡率(1.6%±0.6%,均P<0.05).50、100和200μg/ml的沙利度胺处理SMMC-7721细胞48 h时,Annexin V-FITC/PI双标法检测SMMC-7721细胞的凋亡率分别为8.7%±1.2%、16.8%±2.5%和25.4%±4.5%,均明显高于空白对照组SMMC-7721细胞48 h的自然凋亡率(2.1%±0.5%,均P<0.05).随着沙利度胺浓度的增加,表达caspase-3蛋白的SMMC-7721细胞数量不断增加,而SMMC-7721细胞中VEGF的含量却逐渐下降.结论 沙利度胺可能通过诱导肝癌细胞的凋亡、抑制肿瘤血管的生成而发挥双重抗肿瘤生长的作用.     

反义RNA技术对抑制人肝癌细胞SMMC-7721中POLD1基因表达的研究

目的:应用反义RNA技术抑制人肝癌细胞SMMC-7721中POLD1基因的表达,探索采用该技术干预肝癌的可行性。方法:制备人POLD1基因的反义RNA表达质粒,同时建立空白对照组与阴性对照组,由此将实验分为阴性对照组(转染空载体的SMMC-7721细胞)、空白对照组(肝癌细胞SMMC-7721)和实验组(转染人POLD1基因反义RNA表达质粒的肝癌SMMC-7721细胞)3组。MTT实验分析细胞增殖变化,并在转染SMMC-7721细胞48h后,实时荧光定量PCR和蛋白质印迹法检测POLD1基因的表达。结果:MTT实验显示,与空白对照和阴性对照组相比,转染了反义RNA的实验组细胞增殖受到明显抑制。在转染后24～72h,实验组吸光度24h为0.306 7±0.015 4,48h为0.459 2±0.033 2,72h为0.567 1±0.061 1,与空白和阴性对照组相比,P值均<0.05。实时荧光定量PCR实验显示,实验组POLD1的mRNA表达明显下调为0.142 5±0.020 5,阴性对照组为1.017±0.188,空白对照组为1.00±0.00(F=26.5,P<0.05),说明POLD1基因表达受抑制。蛋白质印迹实验显示,POLD1基因蛋白水平(p125)变化趋势与mRNA相同,均为下调,其中实验组为0.222 3±0.009 7,阴性对照组为0.237 9±0.005 9,空白对照组为0.235 4±0.003 4(F=1 365.754,P值均<0.05),说明反义RNA可抑制p125蛋白表达,结论与基因水平趋势一致。结论:针对POLD1基因设计的反义RNA体外抑制了肝癌细胞SMMC-7721的增殖,这与POLD1在基因和蛋白水平的表达下调有关。     

Immunohistochemical Analysis of Foxp3<Superscript>+</Superscript>, CD4<Superscript>+</Superscript>, CD8<Superscript>+</Superscript> Cell Infiltrates and PD-L1 in Oral Squamous Cell Carcinoma

The immunoexpression of the PD-L1 and the number of immune infiltrating cells have been shown to be a significant prognostic factors in various human cancers. Immunohistochemical method was used to examine the immunoexpression of PD-L1 and number of Foxp3+, CD4+, CD8+ cells in 78 cases of oral squamous cell carcinomas (OSCCs): with better prognosis - OSCCBP (n = 37), and with poorer prognosis - OSCCPP (n = 41), and 18 cases of normal mucosa as a control. The immunoexpression of PD-L1 and the mean number of Foxp3+ cells was significantly increased in OSCCPP group in comparison to OSCCBP and control groups. The mean number of CD4+ cells was significantly increased in OSCCPP group in comparison to OSCCBP and control groups. CD8+ cells were significantly more numerous in OSCCBP group in comparison to OSCCPP and control group. In both OSCCPP and OSCCBP groups there were positive significant correlations between number of Foxp3+ and CD4+ cells. We found positive correlations between the immunoexpression of PD-L1 and numbers of Foxp3+ cells, and negative correlation between the immunoexpression of PD-L1 and numbers of CD8+ cells in both OSCCPP and OSCCBP groups. We found also significant positive correlation between immunoexpression of PD-L1 and the number of CD4+ cells in OSCCPP group. In conclusion, our findings support the hypothesis of involvement of Tregs and PD-L1 in OSCC development and progression.     

Effects of silencing cyclooxygenase-2 expression via RNA interference on the tumorigenicity of the SMMC-7721 human hepatocarcinoma cell line

We constructed a vector carrying a shRNA sequence against cyclooxygenase-2 (COX-2) that was subsequently transfected into the human hepatocarcinoma cell line SMMC?7721. Furthermore, we established a COX-2-deficient stable cell line and a model of tumor-shRNA transplantation in nude mice. Negative shRNA was used as the control. The tumor volume in the experimental group was smaller compared to that in the control group. Hematoxylin and eosin staining indicated that the cells in the experimental group differentiated better than those in the control group. The COX-2 mRNA level in the tumor tissues injected with SMMC-7721/COX-2i was markedly downregulated compared to that in the tumor tissues injected with SMMC-7721/negative shRNA. The inhibition rate reached 68.6%. Immunohistological study showed a significantly strong COX-2 expression in the control group tumor cells, whereas the experimental group exhibited moderate expression, indicating the inhibition of COX-2 expression after transfection of cells with shRNA against COX-2. Western blot analysis further proved the inhibition of COX-2 expression. In conclusion, RNAi-mediated regulation of COX-2 expression could efficiently inhibit liver-transplanted tumor growth in BALB/c nude mice.     

Apoptosis in human hepatoma cell line SMMC-7721 induced by water-soluble macromolecular components of Artemisia capillaris Thunberg.

The aim of this study was to investigate the effect of water-soluble macromolecular components of Artemisia capillaris Thunberg (ACT) on human hepatoma cell line SMMC-7721 (SMMC-7721). The morphological changes of SMMC-7721 were observed under a light microscope and an electron microscope. Inhibition of proliferation was measured with a colorimetric MTT assay. It was discovered that ACT extract-treated cells exhibit morphological changes typical of apoptosis, including condensed chromatin and a reduction in volume. ACT extract at 25-200 microg/ml dose-dependently inhibited the proliferation of SMMC-7721. The 50% effective dose, evaluated on day 3 of exposure to the extract, was 64.52+/-3.53 microg/ml. Upon gel electrophoresis, the fragmented DNA showed a characteristic ladder pattern. Cell cycle analyses revealed that ACT induced cell cycle arrest at the G0/G1 phase.     

RNAi沉默MCM7基因对人肝癌细胞SMMC-7721裸鼠移植瘤影响研究

目的探讨微小染色体维持蛋白7(miniehromosome maintenanceprotein7,MCM7)基因RNAi(RNA interference)的重组慢病毒载体,对人肝癌细胞SMMC-7721 MCM7基因表达和裸鼠移植瘤生长的影响。方法构建重组逆转录慢病毒载体MCM7-shRNA,以MCM7基因沉默重组慢病毒颗粒(LV-shRNA-MCM7)感染SMMC-7721细胞,作为实验组;以对照慢病毒颗粒(LV-shRNA-NC)感染SMMC-7721细胞,作为阴性对照组;空白对照组常规培养,不做任何处理。通过嘌呤霉素筛选出稳定转染细胞株。3组细胞分别接种至裸鼠皮下,建立人肝癌裸鼠移植瘤模型。观察裸鼠成瘤情况、移植瘤生长情况并绘制肿瘤生长曲线;4周后测定肿瘤体积和质量,并用RT-PCR、实时荧光定量PCR、蛋白质印迹法及免疫组织化学法检测移植瘤中MCM7的表达情况。结果 MCM7-shRNA慢病毒载体构建成功。裸鼠接种癌细胞后第6天均有肿瘤形成,与空白对照组和阴性对照组相比,实验组的肿瘤生长速度明显减慢,实验组、阴性对照组和空白对照组的瘤体平均体积分别为(27.72±7.80)、(81.86±10.91)和(79.75±16.61)mm3,差异有统计学意义,F=61.949,P<0.05;实验组、阴性对照组和空白对照组的瘤体平均质量分别为(0.19±0.06)、(0.501±0.14)和(0.509±0.18)g,差异有统计学意义,F=18.41,P<0.05。实验组MCM7mRNA的相对表达量为0.253±0.198,阴性对照组1.213±0.548,空白对照组1.201±0.744,实验组相比阴性对照组和空白对照组明显下降,差异有统计学意义,F=4.091,P<0.05;实验组MCM7蛋白相对表达量为0.207±0.015,阴性对照组1.116±0.062,空白对照组1.088±0.040,实验组相比阴性对照组和空白对照组明显下降,差异有统计学意义,F=292.778,P<0.05。MCM7蛋白在阴性对照组和空白对照组中阳性表达率均为100%(10/10),在实验组中为30%(3/10),实验组明显低于阴性对照组和空白对照组,差异有统计学意义,χ2=18.261,P<0.001。结论慢病毒沉默SMMC-7721细胞MCM7基因表达能有效抑制人肝癌裸鼠移植瘤的生长,MCM7基因可能成为肝癌基因治疗的有效靶点。     

Expression of the Na<Superscript>+</Superscript>/l<Superscript>-</Superscript>symporter (NIS) is markedly decreased or absent in gastric cancer and intestinal metaplastic mucosa of Barrett esophagus

Background  

The sodium/iodide symporter (NIS) is a plasma membrane glycoprotein that mediates iodide (I^-) transport in the thyroid, lactating breast, salivary glands, and stomach. Whereas NIS expression and regulation have been extensively investigated in healthy and neoplastic thyroid and breast tissues, little is known about NIS expression and function along the healthy and diseased gastrointestinal tract.     

PTEN在三氧化二砷诱导肝癌细胞(SMMC-7721)凋亡中的作用

目的 研究PTEN在三氧化二砷(arsenic trioxide,As2O3)诱导肝癌细胞凋亡中的作用.方法 采用四甲基偶氮唑盐(MTT)方法检测As2O3对SMMC-7721细胞增殖的抑制作用;流式细胞仪定量检测各组细胞的凋亡率;Western blot方法检测各组细胞中PTEN蛋白的表达情况;用PTEN siRNA转染SMMC-7721细胞,观察其对As2O3诱导细胞凋亡作用的影响.结果 As2O3能显著抑制SMMC-7721细胞存活率,其抑制率呈剂量依赖关系;As2O3剂量依赖性诱导肝癌细胞凋亡;Western blot检测显示As2O3明显增加细胞中PTEN蛋白的表达;PTEN siRNA转染可减弱As2O3对SMMC-7721细胞凋亡的诱导作用.结论 As2O3诱导SMMC-7721细胞凋亡可能与其诱导PTEN表达有关.     

冬凌草甲素抑制人肝癌SMMC．7721细胞增殖及诱导细胞凋亡的机制研究

目的研究冬凌草甲素对人肝癌细胞株SMMC-7721细胞的抑制作用及诱导凋亡机制。方法采用不同浓度的冬凌草甲素作用于人肝癌SMMC-7721细胞,以四甲基偶氮唑蓝（MTT）法检测细胞抑制率,倒置显微镜观察不同浓度组的细胞形态,RT—PCR法检测细胞Survivin、Bcl-2、BaxmRNA的表达,WesternBlot法检测细胞Survivin、Bcl-2、Bax的蛋白表达。结果MTT比色法显示不同浓度的冬凌草甲素可抑制人肝癌SMMC．7721细胞的增殖,抑制作用呈时间和剂量依赖性。冬凌草甲素作用48h后细胞表现出特异性凋亡。RT—PCR法和WesternBlot法显示,人肝癌SMMC-7721细胞的BaxmRNA和Bax蛋白随冬凌草甲素浓度增加而表达增加,Bcl-2、SurvivinmRNA和Bcl-2、Survivin蛋白随药物浓度增加而表达下降。结论冬凌草甲素能抑制人肝癌SMMC-7721细胞的生长及诱导细胞发生凋亡,降低Sur—vivin、Bcl-2表达和上调Bax表达可能是诱导细胞发生凋亡的机制。     

INFLUENCE OF p53 SMALL DOUBLE STRANDED RNA INTERFERENCE ON HEPATOMA CELL LINE SK-HEP-1

Hepatocellular carcinoma is one of the most frequentmalignant diseases worldwide,especially with annualgradually increasing incidence in China.From the firstconcept of tumor suppressor being set up by Knudson[1],many studies have showed that there in very highmutation frequency in the evolutionarily conservedcodons of the p53tumor suppressor gene,which iscommon in diverse types of human cancers[2].The p53gene encodes a nuclear phosphoprotein of53-kDa masswhich belongs to a diverse family of n…     

Proteinase-activated receptor-1 mediates allogeneic CD8<Superscript>+</Superscript> T cell-induced apoptosis of vascular endothelial cells

Vascular endothelial-cells injury plays a pivotal role in the pathogenesis of graft-versus-host disease (GVHD) and transplant-associated endothelial injury syndrome. Vascular endothelial cells are an exposed target tissue for immune-mediated injury during GVHD. Early endothelial injury syndromes share common features with acute GVHD. Chronic GVHD leads to a rarefaction of microvessels caused by the infiltration of alloreactive cytotoxic T lymphocytes. In this context, allogeneic reactive cytotoxic T cell may contribute to apoptosis of vascular endothelial cells. The involvement of proteinase-activated receptor (PAR-1) in regulation of apoptosis has been recently recognized in many cell types. We hypothesized that apoptosis of vascular endothelial cells induced by allogeneic cytotoxic T cell are mediated via the PAR-1. Allogeneic CD8⁺ T cell, PAR-1 agonist peptide (SFLLRN) induced apoptosis of human umbilical vein endothelial cells (HUVECs) and human dermal microvascular endothelial cells (HDMECs) as assessed by AnnexinV-FITC labeling. To ascertain the mechanism of endothelial apoptosis, we determined that allogeneic CD8⁺ T cell, SFLLRN enhanced cleavage of caspase-3 and led to p38MAPK activation as assessed by Western blot. The effects of allogeneic CD8⁺ T cell and SFLLRN on apoptosis of vascular endothelial cells were largely prevented by a cleavage-blocking anti-human PAR-1-antibody (ATAP2) and a specific inhibitor of p38MAPK. In concert, these observations provide strong evidence that allogeneic CD8⁺ T cell induces apoptosis of human vascular endothelial cells through PAR-1-dependent modulation of intrinsic apoptotic pathway via alterations of p38MAPK and caspase-3.     

An evaluation of the clinical significance of FOXP3<Superscript>+</Superscript> infiltrating cells in human breast cancer

Studies in mice have shown that thymic-derived CD4+ CD25+ regulatory T cells (T reg; FOXP3⁺ lymphocytes) inhibit an antitumour immune response. Additional studies have also reported that the T reg population increases in peripheral blood and tumour tissues from patients with cancer. However, the relationship between the T reg population and the patient prognosis remains controversial. Our aim was to determine the prognostic value of T reg cell density in breast cancer using immunohistochemical assessment of FOXP3, which has been shown to be the optimal marker for T regs. Tissue microarrays were used, and the density of FOXP3⁺ cells was determined in a series of 1445 cases of well-characterised primary invasive breast carcinoma cases with long-term follow up. FOXP3⁺ cell numbers were counted in tumour nests, in tumour-adjacent stroma, and in distant stroma. The total number of FOXP3⁺ cells significantly correlated with higher tumour grade (r _s = 0.37, P < 0.001) and ER negativity (Mann–Whitney U test, P < 0.001). In addition, FOXP3 infiltration positively correlated with HER2 expression and basal phenotype subclass. On univariate analysis, FOXP3⁺ cells were associated with a worse prognosis (P = 0.012, log rank = 6.36). This association was found for intratumoural FOXP3⁺ and for tumour-adjacent stromal FOXP3⁺-cells (tumour-cell associated FOXP3, P = 0.001 and log rank 10.35). However, the number of FOXP3⁺ cells was not found to be an independent prognostic factor in multivariate analysis. We therefore conclude that FOXP3⁺ infiltrating cells do not have a dominant role in breast cancer prognosis and suggest that other inflammatory cell subsets may be more critical variables.     

Identification of immunodominant HLA-B7-restricted CD8<Superscript>+</Superscript> cytotoxic T cell epitopes derived from mammaglobin-A expressed on human breast cancers

Mammaglobin-A (MGBA), a 10-kD protein, is over expressed in 80% of primary and metastatic human breast cancers. Breast cancer patients demonstrate high frequencies of CD8⁺ cytotoxic T lymphocytes (CTL) specific to MGBA. Defining CD8⁺ CTL responses to HLA class I-restricted MGBA-derived epitopes assumes significance in the context of our ongoing efforts to clinically translate vaccine strategies targeting MGBA for prevention and/or treatment of human breast cancers. In this study, we define the CD8⁺ CTL response to MGBA-derived candidate epitopes presented in the context of HLA-B7, which has a frequency of 17.7% in Caucasian and 15.5% in African American populations. We identified seven MGBA-derived candidate epitopes with high predicted binding scores for HLA-B7 using a computer algorithm. Membrane stabilization studies with TAP-deficient T2 cells transfected with HLA-B7 indicated that MGBA B7.3 (VSKTEYKEL), B7.6 (KLLMVLMLA), B7.7 (NPQVSKTEY), and B7.1 (YAGSGCPLL) have the highest HLA-B7 binding affinities. Further, two CD8⁺ CTL cell lines generated in vitro against T2.B7 cells individually loaded with MGBA-derived candidate epitopes showed significant cytotoxic activity against MGBA B7.1, B7.3, B7.6, and B7.7. In addition, the same CD8⁺ CTL lines lysed the HLA-B7⁺/MGBA⁺ human breast cancer cell line DU-4475 but had no significant cytotoxicity against HLA-B7⁻ or MGBA⁻ breast cancer cell lines. Cold-target inhibition studies strongly suggest that MGBA B7.3 is an immunodominant epitope. In summary, our results define HLA-B7-restriced, MGBA-derived, CD8⁺ CTL epitopes with all of the necessary features for developing novel vaccine strategies against HLA-B7 expressing breast cancer patients.     

白藜芦醇通过下调PRMT5表达抑制肝胆管癌SMMC-7721细胞的增殖、侵袭和细胞周期

目的：探究白藜芦醇（Res）通过调控PRMT5表达对肝胆管癌SMMC-7721细胞增殖、侵袭、细胞周期的影响及其机制。方法：常规培养正常肝细胞LO2和SMMC-7721细胞,用0、20、40、80μmol/L的Res进行处理,用qPCR法、MTT法、Transwell实验、流式细胞术和WB法分别检测Res处理后PRMT5 mRNA在LO2和SMMC-7721细胞中的表达,Res对SMMC-7721细胞增殖能力、侵袭能力、细胞周期和凋亡,以及PRMT5、cyclin D1和cyclin E1蛋白表达的影响。结果：PRMT5在SMMC-7721细胞中呈高表达（P<0.01）;20、40、80μmol/L Res均能明显抑制PRMT5 mRNA和蛋白在SMMC-7721细胞中的表达（均P<0.01）,抑制SMMC-7721细胞的增殖能力（P<0.01）和侵袭能力（P<0.05）,阻滞SMMC-7721细胞周期于G0/G1期并促进其凋亡（P<0.01）,明显抑制SMMC-7721细胞中周期蛋白cyclin D1、cyclin E1蛋白的表达（P<0.01）。...     

白眉蝮蛇去整合素rAdinbitor对人肝癌细胞系SMMC-7721细胞黏附与增殖的影响

背景与目的：蛇毒去整合素是一类从多种蛇毒和水蛭中分离到的含RGD（Arg—Gly—Asp）或KGD（Lys—Gly—Asp）模体的小分子蛋白质，其由于能竞争性地结合细胞表面的整合蛋白受体，阻断肿瘤细胞与细胞外基质的黏附，从而发挥抗肿瘤转移的作用。rAdinbitor是我实验室采用基因克隆方法从大连产白眉蝮蛇（Gloydius blomhoffi brevicaudus）的毒腺中获得的一种含有RGD模体的去整合素。本实验的目的是探讨rAdinbitor对人肝癌细胞系SMMC-7721黏附、增殖的抑制作用，为rAdinbitor抗肿瘤转移的作用机制提供实验依据。方法：以细胞黏附实验观察纤连蛋白（FN）基质对SMMC-7721细胞黏附的影响。分别采用结晶紫染色法和MTT比色法测定重组蛋白rAdinbitor对SMMC-7721向FN黏附的影响，及对已黏附于FN上的SMMC-7721的影响。以MTT比色法检测不同浓度的rAdinbitor对SMMC-7721细胞增殖的影响；HE染色观察200μg／ml Adinbitor诱导SMMC-7721细胞36h凋亡的形态变化；采用流式细胞仪检测rAdinbitor对细胞凋亡的诱导作用。结果：①rAdinbitor对SMMC-7721细胞在FN基质上的黏附有一定的抑制作用，并呈浓度依赖关系，25、50、100、200μg／ml浓度组rAdinbitor的黏附抑制率分别为4．90％、11．73％、22．84％、37．28％，各组差异有显著性（P〈0．05）。②rAdinbitor作用后，已黏附的SMMC-7721细胞变圆、脱落，各浓度组黏附细胞的存活率的平均值分别为98．89％、90．01％、63．37％、35．13％，细胞的增殖受到明显抑制（P〈0．05）。③rAdinbitor对SMMC-7721细胞的增殖具有较强的抑制作用，且随着药物浓度的增加，其抑制作用增强（P〈0．05）。rAdinbitor对SMMC-7721细胞作用48h的IC50值为177．83μg／ml。④200μg／ml rAdinbitor作用SMMC-7721细胞36h呈现早期细胞凋亡的形态改变，凋亡率达20．68％，明显高于未经处理的对照组的2．38％（P〈0．05）。结论：重组白眉蝮蛇去整合素rAdinbitor能够剂量依赖性地抑制SMMC-7721细胞在FN上的黏附，并促使已黏附的SMMC-7721细胞脱落，剂量依赖性抑制SMMC-7721细胞增殖并诱导其凋亡。