论文标题:重组蛋白Adinbitor对人肝癌细胞株SMMC-7721黏附与增殖影响的初步研究
Effects of Adinbitor on Adhesion and Proliferation in Human Hepatocellular Carcinoma Cell Strain SMMC-7721
论文作者 赵春玲
论文导师 赵宝昌,论文学位 硕士,论文专业 生物化学与分子生物学
论文单位 大连医科大学,点击次数 93,论文页数 45页File Size1813k
2004-05-01论文网 http://www.lw23.com/lunwen_383271397/ Adinbitor;黏附;增殖;细胞凋亡
Adinbitor; Adhesion; Proliferation; Apoptosis
目的:恶性肿瘤是严重威胁人类健康、造成人类死亡的主要原因之一。抗癌药物由于其不同程度的毒副作用而成为肿瘤治疗的一大主要障碍。利用新的靶点来寻找高效低毒的新型抗癌药物仍是当务之急。信号转导阻滞剂和细胞凋亡诱导剂都是新的研究领域。肿瘤细胞的黏附性在恶性肿瘤转移中起着极为重要的作用,而细胞黏附于细胞外基质是细胞生存与增殖的基础,有赖于细胞表面的黏附分子尤其是整合蛋白介导的黏附信号的转导。因此,阻断肿瘤细胞的黏附,防止肿瘤细胞定植,是发展抗癌药物的一个重要策略。含RGD模体的多肽,包括蛇毒去整合素的阻断瘤细胞与基质的黏附,从而发挥抗肿瘤转移的作用,引起了人们的关注。可溶性的RGD肽(soluble RGD peptide, sRGD)还能促使许多类型的细胞凋亡。为研究本实验室基因重组的RGD 肽—Adinbitor对肿瘤细胞的作用,本实验应用体外细胞培养技术,模拟体内环境,用纤连蛋白(fibronectin, FN)预刺激人肝癌细胞株SMMC-7721,旨在观察Adinbitor对SMMC-7721细胞黏附的影响。另外还初步研究了Adinbitor对SMMC-7721细胞生长增殖及诱导凋亡的作用,为Adinbitor抗肿瘤的作用机制提供实验依据。方法:实验分为两部分:(1)检测Adinbitor对SMMC-7721细胞黏附的影响。以细胞黏附实验观察纤连蛋白FN基质对SMMC-7721细胞的黏附影响;分别采用结晶紫染色法和MTT比色法测定重组蛋白Adinbitor对SMMC-7721向FN黏附的影响,及对已黏附于FN上的SMMC-7721的影响。(2)检测Adinbitor对SMMC-7721细胞增殖活性的影响。以MTT比色法检测不同浓度的Adinbitor对SMMC-7721细胞增殖的影响;HE染色观察Adinbitor诱导SMMC-7721细胞凋亡的形态变化;采用流式细胞仪检测Adinbitor对细胞凋亡的诱导作用。 结果:1.细胞黏附实验中,FN处理组OD值(黏附细胞数)均显著高于对照组(P<0.05),SMMC-7721细胞在20μg/ml的FN浓度时黏附即达到饱和,与40μg/ml 、60μg/ml无显著的组间差异,但此三种浓度下黏附的细胞数均高于5μg/ml 、10μg/ml 组(P(0.05)。2.Adinbitor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,Adinbitor的黏附抑制作用随其浓度的增高而增强,差异有显著性(P(0.05)。3.Adinbitor 对已黏附的细胞的影响:24h时, 200μg/ml Adinbitor组SMMC-7721细胞即有少量变成圆形, 体积缩小,细胞间隙增大,48h后有较多细胞脱落,悬浮于培养液中,细胞膜完整。MTT比色结果说明随着Adinbitor浓度的增加,脱落的细胞明显增多,细胞的增殖受到明显抑制(P<0.05)。4.Adinbitor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)。Adinbitor对SMMC-7721细胞作用48h的IC50值为177.83μg/ml。另外,Adinbitor作用48h的结果与包被FN后,Adinbitor促脱落、抑制增殖作用的结果一致。5. Adinbitor可诱导SMMC-7721细胞凋亡, 200μg/ml作用SMMC-7721细胞36h凋亡发生率达20.68%,明显高于未经处理的对照组的2.38%(P<0.05)。结论: 1.纤连蛋白FN对人肝癌细胞株SMMC-7721具有促黏附作用,且呈剂量依赖性。2.重组蛋白含RGD序列的Adinbitor能剂量依赖性地抑制SMMC-7721细胞在FN上的黏附。3.Adinbitor可促使已黏附的SMMC-7721细胞脱落、增殖受抑制。4.Adinbitor能抑制SMMC-7721细胞的增殖,且呈明显的剂量依赖关系,其作用48h的IC50值为177.83μg/ml,与Adinbitor促脱落作用、抑制作用一致。5.Adinbitor可以诱导SMMC-7721细胞凋亡发生,这可能是Adinbitor抑制SMMC-7721细胞增殖的机制之一。总之,本实验室基因重组蛋白含RGD序列的Adinbitor能够与FN竞争结合人肝癌细胞株SMMC-7721细胞表面的整合素受体,抑制该细胞在FN上的粘附;Adinbitor还能够抑制SMMC-7721细胞增殖并促进其凋亡。
Objective: Malignant tumor is one of the main reasons that threaten heavily health and cause death in humans. Toxic side effects of anti-cancer drugs have become one big and main obstacle to different extent. It is very urgent to look for new anti-cancer drugs with high efficiency and low toxicity. The agents inhibiting signal transduction and inducing apoptosis are both new research fields. The adhesion of tumor cells plays an important role in the metastasis of malignant tumor, while the adhesive interaction between cells and extracellular matrix (ECM) is the basis of cell survival and proliferation, which depends on adhesive molecules on cell surface, especially integrin–mediated signal transduction. So the block of tumor cell adhesion is one important strategy to develop anti-cancer drugs. The RGD-containing peptides including disintegrin catch the eyes of reseachers because they can block cell adhesion to ECM and prevent the metastasis of malignant tumors. Soluble RGD peptides can also induce apoptosis of many kinds of cells. To investigate the effects of Adinbitor, a novel RGD peptide obtained by genetic recombination, on the tumor cells , we first employed cell culture technique in vitro, simulated the environment in vivo and stimulated the human hepatocellular carcinoma cell strain SMMC-7721 with fibronectin to explore the effects of Adinbitor on SMMC-7721 cell adhesion. In addition, we studied its effects on the proliferation and apoptosis of SMMC-7721 cells. Our goal was to provide experimental and theoretical basis for Adinbitor in anti-cancer mechanisms.Methods: Our work was composed of two parts: (1) Detecting the effects of Adinbitor on the adhesion of SMMC-7721 cells. Cell adhesion assay was used to observe the adhesion of SMMC-7721 cells to fibronectin. Crystal violet staining was performed to detect the influence of Adinbitor on the adhesion of SMMC-7721 cells. MTT assay was employed to determine the effect of Adinbitor on SMMC-7721 cells that had been adhered. (2) Detecting the effects of Adinbitor on the proliferation of SMMC-7721 cells. SMMC-7721 cells were cultured in media with different concentrations of Adinbitor. The inhibition of proliferation was measured by colorimetric MTT assay. The morphologic changes were observed under light microscope. The apoptosis of SMMC-7721 cells was determined by flow cytometry.Results: 1. The numbers of the adhered cells ware higher in the treated group by fibronectin than the control group (P<0.05). The number of the adhered SMMC-7721 cells had reached to the highest at 20μg/ml, which had no significant difference compared with 40μg/ml and 60μg/ml group. But the numbers of the adhered cells at these three different concentrations are higher than 5μg/ml and 10μg/ml groups(P<0.05). 2. Adinbitor could inhibit the adhesion of SMMC-7721 cells to fibronectin dose-dependently. The higher the concentration was, the stronger the inhibition was. There was significant difference among the groups (P<0.05). 3. The influence of Adinbitor on the adhered SMMC-7721 cells. A few cells became round and smaller and the intercellular space extended at the dosage of 200μg/ml Adinbitor for 24h. After 48h, more detached cells were observed and suspended in the media. But the membranes were in integrity. The result from MTT assay showed that the number of detached cells became increased and the proliferation was strikingly inhibited at higher concentration of Adinbitor (P<0.05). 4. Adinbitor had a strong inhibition on the proliferation of SMMC-7721 cells dosage-dependently (P<0.05). After a 48h exposure, the IC50 value of Adinbitor was 177.83μg/ml. In addition, this result was consistent with the inhibition of Adinbitor on the proliferation of the adhered SMMC-7721 cells when the plates were coated with fibronectin. 5. After exposure of SMMC-7721 cells to Adinbitor at the dosage of 200μg/ml Adinbitor for 36h, the apoptosis rate was 20.68%, significantly higher than that of the control group (2.38%, P<0.05).Conclusions: 1. Fibronectin promotes the ad
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