丙烯腈对大鼠外周血内皮祖细胞功能的影响

发布时间：2019-01-13 08:49

【摘要】：第一部分大鼠丙烯腈染毒实验 [目的]选择合适剂量建立慢性丙烯腈染毒模型 [方法]在大鼠身上,丙烯腈LD50为100mg/kg,依此为依据,我们取25mg/kg、50mg/kg、75mg/kg、100mg/kg的丙烯腈剂量各染毒4只SD雄性大鼠。 [结果]给予一次100mg/kg丙烯腈后,SD大鼠2周内半数死亡；发现75mg/kg组雄鼠活动减少,萎靡,消瘦,然后陆续死亡,不适合慢性染毒；选择25mg/kg、50mg/kg组大鼠体重无明显消瘦或肥胖,无死亡。 [结论]选择25mg/kgi(A组)、50mg/kg(B组)丙烯腈进行灌胃染毒,染毒3个月,对照组(C组)以水灌胃。第二部分外周血内皮祖细胞的分离、培养、鉴定 [目的]分离并培养大鼠外周血EPCs,纯化扩增,并检测细胞表型。 [方法]抽取大鼠外周血,使用密度梯度离心法分离单个核细胞,并借助EPCs黏附于塑料瓶底这一特性进行纯化。相差显微镜观察形态变化,流式细胞仪检测CD133表达,鉴定EPCs。 [结果]将密度梯度离心法与贴壁法相结合,可获得较多的EPCs,通过传代后细胞进一步纯化,每个25cm2细胞培养瓶接种2×105个细胞,完全培养液胎牛血清浓度为10%,80%-90%细胞融合后按1：3或1：2比例传代,EPCs能稳定地传代扩增而无明显分化迹象。原代细胞培养2周左右可首次传代,以后7d左右传代一次。 [结论]应用密度梯度离心法与贴壁法相结合,可建立EPCs体外稳定的纯化扩增方法。第三部分丙烯腈对大鼠外周血内皮祖细胞增殖、迁移、体外血管形成能力及eNOS表达情况的实验研究 [目的]探讨大鼠慢性丙烯腈染毒模型外周血内皮祖CD133表达、增殖、迁移及体外血管形成能力的变化；研究内皮祖细胞eNOS的表达情况。 [方法]流式细胞仪检测P1细胞CD133表达变化；体外扩增培养EPCs,绘制生长曲线,四甲基偶氮唑盐[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT]比色法测定细胞增殖情况,Transwell小室进行细胞迁移实验,Matrigel实验观察细胞在体外血管形成能力,通过Western blot方法检测细胞eNOS的表达情况。 [结果](1)C组、A组、B组外周血单个核细胞可阳性表达CD133,其表达阳性率无显著学差异。(2)细胞增殖情况：传代细胞潜伏期为48h左右,对数增殖期约为接种后5-7d,至接种后第9d进入平台期。丙烯腈可使EPCs的增殖能力减弱；随着丙烯腈浓度的增加,其生长速度减慢(p0.05)。(3)迁移能力：C组、A组、B组培养24小时后迁移细胞数目分别为：45±2.76、42.5±3.08、42±4.32。丙烯腈处理后细胞迁移数目无明显减少(p均0.05)(4)体外血管形成情况：C组、A组、B组小管生成数分别为34+4.22,24±3.74,19±3.56。丙烯腈处理后细胞体外血管形成数目减少(p均0.05)(5)丙烯腈可以抑制细胞eNOS的表达,随着浓度的增加,抑制作用有增加趋势。 [结论]本实验表明：与对照组比较,ACN接触组EPCs增殖及体外血管形成能力减弱,且随着ACN浓度的增加而愈明显,同时引起细胞eNOS合成减少。同时,丙烯腈对大鼠细胞表型CD133表达及迁移能力无影响。
[Abstract]:Experimental study on the exposure of acrylonitrile in the first part of rats[Objective] To select the appropriate dose to establish the chronic acrylonitrile exposure. The model[method] was based on the LD50 of 100 mg/ kg in rats. We take 25mg/ kg, 50mg/ kg, 75mg/ kg and 100mg/ kg for 4 SD males at the dosage of 100 mg/ kg. sex rats.[Results] After administration of 100 mg/ kg of acrylonitrile, half of the SD rats died within 2 weeks; it was found that the activity of male mice in the 75mg/ kg group was reduced, listless, and emaciated, and then died in succession, and it was not suitable for chronic exposure; the weight of rats in the group of 25mg/ kg and 50mg/ kg was not significantly thinner or fat[Conclusion] 25mg/ kgi (Group A) and 50mg/ kg (Group B) of acrylonitrile were given intragastric administration for 3 months and control group (group B). Group C) Gavage with water. The second part of the peripheral blood was fine. The isolation, culture, identification[purpose] of the cells were isolated and cultured in rat peripheral blood, EPCs, pure. The peripheral blood of the rat was isolated from the peripheral blood of the rat, and the individual nuclear cells were isolated by density gradient centrifugation, and the cells were adhered with the aid of the EPCs. the characteristics of the bottom of the plastic bottle are purified, the change of the observation form of the phase difference microscope, the detection of the C by the flow cytometry, D133 expression and identification of EPCs.[Results] The density gradient centrifugation was combined with the wall-to-wall method to obtain more EPCs, and the cells were further purified by passage. Each 25cm2 cell culture flask was inoculated with 2 to 105 cells, and the serum concentration of the whole culture medium was 10%, and 80% to 90% of the cells. after the fusion, the mixture is passaged according to the ratio of 1: 3 or 1: 2, and the EPCs can Stable passage of amplification without obvious signs of differentiation. The primary cell culture may be left and right around 2 weeks. For the first passage, it was passaged around 7days.[Conclusion] The application of density gradient centrifugation is combined with the wall-wall method. A purification and amplification method for the in vitro stability of EPCs was established. The third part of acrylonitrile was used to promote the proliferation, migration and in vitro blood vessels of peripheral blood endothelial progenitor cells in rats. An experimental study of the formation and eNOS expression in rats[Objective] To study the expression of CD133 in peripheral blood of rats with chronic acrylonitrile exposure. The ability of proliferation, migration, and in vitro angiogenesis The expression of eNOS in the endothelial progenitor cells was studied. The ability of cells to form in vitro was observed by the rigel experiment, by means of Weste. The expression of eNOS was detected by rn-blot.[Results] (1) The peripheral blood mononuclear cells in group A, group A and B There was no significant difference in the expression of CD133 in the positive expression of CD133. (2) The proliferation of the cells: the incubation period of the passage cells was about 48h, and the log multiplication The period is about 5-7days after the inoculation, and the 9th day after the inoculation enters the platform period. The ability of the acrylonitrile to increase the proliferation ability of the EPCs can be reduced; The growth rate of acrylonitrile was slowed by the increase of acrylonitrile concentration (p0.05). (3) The migration ability: group C, group A and group B were cultured for 24 hours, and the number of migrated cells was 45. There was no significant reduction in the number of cell migration after treatment (p <0.05) (4) in the treatment of 2.76, 42.5, 3.08, 42-4.32. The number of small tubes in group C, group A and B were the same as those in group C, group A and group B, respectively. 34 + 4.22, 24-3.74, 19-3.56. The decrease in the number of in vitro vascular formation after treatment with acrylonitrile (p-0.05) (5) acrylonitrile inhibited the cell eN. The expression of the OS increased with the increase of the concentration.[Conclusion] This experiment shows that the proliferation of EPCs in the ACN contact group and the in vitro blood vessel forming ability decrease with the increase of the concentration. The higher the N concentration, the more obvious the synthesis of the eNOS of the cell,
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114

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