【作者】 穆成龙；

【导师】 秦贵信；

【作者基本信息】 吉林农业大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 本研究分三个试验：1.以FITC-大豆凝集素为探针对SD大鼠肠上皮细胞系IEC-6表面的大豆凝集素受体进行标记,2.对本实验室纯化的大豆凝集素进行鉴定确定大豆凝集素的有效性,同时研究了培养基对大豆凝集素活性的影响。3.探讨了大豆凝集素对体外培养的大鼠小肠上皮细胞系(IEC-6)生长和迁移的影响。IEC-6的同步化处理及其大豆凝集素受体的荧光标记。细胞的同步化处理：试验通过单克隆方法对细胞进行同步化处理,进行形态学观察并用MTT比色法测定其增殖能力。荧光标记实验：用10μg·ml-1的FITC-大豆凝集素冷丙酮固定的IEC-6细胞爬片孵育30min。测定本实验室纯化大豆凝集素分子量、纯度、抗原特异性和凝集活性,并检测培养基对大豆凝集素凝血活性的影响。试验采用凝胶电泳方法对其分子量、纯度进行测定,用Western-blot方法对其鉴定,用红细胞凝集试验对其凝血活性进行检测,并将其与Sigma大豆凝集素标准品各指标进行比较。通过红细胞凝集实验检测并比较了大豆凝集素浓度均为1mg·ml-1的生长培养基和阳性对照的凝血活性。大豆凝集素对细胞增殖和细胞迁移的影响。细胞增殖试验：分别以大豆凝集素浓度为0,0.5,1.0,2.0,3.0,4.0,5.0,6.0mg·ml-1的培养基对IEC-6进行处理,其中0 mg·ml-1组为对照组,并在0,12,24,48,60,72,84h用MTT比色试验测定细胞活性；细胞迁移试验：分别用大豆凝集素浓度为0,0.5,1.0,1.5mg·ml-1的培养基对铺满细胞的盖玻片进行处理其中0mg·ml-1组为对照组,并在0d、1d、2d、3d观察上皮细胞的迁移数量。同步化处理结果：经过同步化处理细胞所有细胞生长形态单一,有1-2个核仁,细胞排列整齐,呈多边形铺路石状；生长曲线测定结果显示,细胞生命力旺盛,接种后12h后可全部贴壁生长,24h后进入对数期,48h进入平台期直到84h进入衰亡期。荧光标记结果为强阳性,说明细胞含有大量的大豆凝集素受体。大豆凝集素鉴定结果：试验测得本实验室纯化大豆凝集素分子量为30kDa,纯度在99%以上,Western-blot结果为阳性,凝集效价为1/210,各指标与Sigma标准品之间均未见差异且与理论值相同。红细胞凝集实验显示大豆凝集素浓度为1mg·ml-1的生长培养基和阳性对照的血凝效价均为1/210。说明试验所用大豆凝集素与标准品没有显著差异,所用培养基对大豆凝集素的活性没有显著影响。细胞增殖试验结果：各剂量组细胞活性随时间呈现先升高后下降的趋势,在72h出现最高值,84h后开始下降。同一测量时间,随着凝集素含量的升高,细胞活性呈现先升高后降低的趋势,并在0.5,1.0mg·ml-1显著高于对照组(P<0.05),2.0mg·ml-1虽然高于对照组但差异不显著(P>0.05),3.0mg·ml-1组显著低于对照组(P<0.05),4.0,5.0,6.0mg·ml-1组低于对照组且差异极显著(p<0.01)。说明低剂量大豆凝集素对细胞的增殖有一定促进作用,而当凝集素浓度高于一定范围后对细胞增殖有促进作用减弱,甚至对细胞增殖有抑制作用。细胞迁移试验结果,各剂量组与对照组细胞从盖玻片向周围迁移的数量随时间延长而增加；同一测量时间,随着大豆凝集素浓度增,细胞迁出数量呈现升高趋势,且各时间点当浓度为1.5 mg·ml-1时显著高于对照组和其他剂量组(P<0.05)。说明在试验浓度范围内,大豆凝集素可能促进细胞迁移。说明低浓度大豆凝集素可能促进细胞增殖,高浓度可能抑制细胞增殖,试验大豆凝集素浓度范围内对细胞迁移有促进作用。综上所述,IEC-6细胞存在大量的大豆凝集素受体低浓度(<2mg·ml-1)的大豆凝集素能够促进IEC-6的增殖,但较高浓度(>2mg·ml-1)大豆凝集素对其增殖的促进作用减弱或抑制细胞增殖。在一定范围内(<1.5mg·ml-1),大豆凝集素对细胞的迁移有促进作用,且随浓度的增加而加强。更多还原

【Abstract】 Three experiments were conducted. Firstly to label the soybean agglutinin receptors on the cells of intestinal epithelial cells line of SD-rat IEC-6, secondly to identify the purified soybean agglutinin, and evaluate the influence of the growth medium of the cell lines on the agglutinative activity of soybean agglutinin, and thirdly to investigate the effects of soybean agglutinin on the proliferation and immigration of IEC-6.IEC-6 cells were synchronized using a monoclonal method with the morphology and the growth curve recorded. The labeling of soybean agglutinin was done after fixing the cells with cold acetone and incubating them with FITC-SBA for about half an hour at 37℃.The molecular weight, purity and agglutinative activity of the soybean agglutinin purified in our laboratory were detected as well as the effect of growth medium on the agglutinative activity. SDS-PAGE gel electrophoresis was used to detect the molecular weight and purity of the soybean agglutinin purified, Western-blot for the antigenic specificity, and agglutination test for the agglutinative activity. The findings were then compared with the results of the Sigma standard Soybean agglutinin. The effect of growth medium on the agglutinative activity was also evaluated by comparing values of agglutination between the cell growth medium containing 1mg·ml-1 soybean agglutinin and the control group.The effects of soybean agglutinin on the cell proliferation and cell immigration were assessed after treating IEC-6 cells in logarithmic phase with growth medium containing SBA with the concentration of 0,0.5,1.0,2.0,3.0,4.0,5.0,6.0 mg·ml-1, and set 0 mg·ml-1 group as control. Proliferation curve of the cells was detected by the means of MTT method. For the cell immigration test, cells were treated with the medium with different concentrations of soybean agglutinin, i.e.0,0.5,1.0,1.5 mg·ml-1 and the number of immigration cells in different groups recorded.After synchronizing the growth of the cells, the growth curve showed that cells grew very well. At about 12h after seeding, the cells got into a logarithmic phase, from 48h to 84h the cells were in a plateau phase and after that they got into a decline phase. The results of labeling showed that there were a lot of soybean agglutinin receptors on the cells of IEC-6.The molecular weight of soya bean agglutinin was 30.78kDa, the level of purity was 98% and the value of agglutination was 1/210. All of these were not significantly different with those of Sigma standard soybean agglutinin (P>0.05). The values of agglutination of growth medium group and the control group were both 1/210. It indicated that the components of the cell growth medium had no influence on the agglutinative activity.With time, results of the proliferation test revealed that the number of activity in all cell groups rose at first and then declined later. After adding the growth medium at 72h, cell activity approached the summit, but begun to decline thereafter. In 0.5 and 1.0 mg·ml-1 groups the activity of cells was significantly higher than the control group (P<0.05), while significantly lower in the 3.0 mg·ml-1 (P<0.05), and the 4.0,5.0, 6.0mg·ml-1 groups (P<0.01), respectively. This indicated that low concentration of soybean agglutinin could enhance the proliferation of the cells and that high concentration of SBA does not or even inhibit it. Cells immigrated from the cover-glasses in all groups with the number of immigration becoming higher with the increase of the soybean agglutinin concentration. At the highest concentration group, the numbers of cells numbers were significantly higher than all the other groups (P<0.05). Using this concentration of soybean agglutinin could enhance the immigration of the cell.更多还原