【摘要】：第一部分大鼠丙烯腈染毒實(shí)驗(yàn) [目的]選擇合適劑量建立慢性丙烯腈染毒模型 [方法]在大鼠身上,丙烯腈LD50為100mg/kg,依此為依據(jù),我們?nèi)?5mg/kg、50mg/kg、75mg/kg、100mg/kg的丙烯腈劑量各染毒4只SD雄性大鼠。 [結(jié)果]給予一次100mg/kg丙烯腈后,SD大鼠2周內(nèi)半數(shù)死亡；發(fā)現(xiàn)75mg/kg組雄鼠活動(dòng)減少,萎靡,消瘦,然后陸續(xù)死亡,不適合慢性染毒；選擇25mg/kg、50mg/kg組大鼠體重?zé)o明顯消瘦或肥胖,無(wú)死亡。 [結(jié)論]選擇25mg/kgi(A組)、50mg/kg(B組)丙烯腈進(jìn)行灌胃染毒,染毒3個(gè)月,對(duì)照組(C組)以水灌胃。 第二部分外周血內(nèi)皮祖細(xì)胞的分離、培養(yǎng)、鑒定 [目的]分離并培養(yǎng)大鼠外周血EPCs,純化擴(kuò)增,并檢測(cè)細(xì)胞表型。 [方法]抽取大鼠外周血,使用密度梯度離心法分離單個(gè)核細(xì)胞,并借助EPCs黏附于塑料瓶底這一特性進(jìn)行純化。相差顯微鏡觀察形態(tài)變化,流式細(xì)胞儀檢測(cè)CD133表達(dá),鑒定EPCs。 [結(jié)果]將密度梯度離心法與貼壁法相結(jié)合,可獲得較多的EPCs,通過傳代后細(xì)胞進(jìn)一步純化,每個(gè)25cm2細(xì)胞培養(yǎng)瓶接種2×105個(gè)細(xì)胞,完全培養(yǎng)液胎牛血清濃度為10%,80%-90%細(xì)胞融合后按1：3或1：2比例傳代,EPCs能穩(wěn)定地傳代擴(kuò)增而無(wú)明顯分化跡象。原代細(xì)胞培養(yǎng)2周左右可首次傳代,以后7d左右傳代一次。 [結(jié)論]應(yīng)用密度梯度離心法與貼壁法相結(jié)合,可建立EPCs體外穩(wěn)定的純化擴(kuò)增方法。第三部分丙烯腈對(duì)大鼠外周血內(nèi)皮祖細(xì)胞增殖、遷移、體外血管形成能力及eNOS表達(dá)情況的實(shí)驗(yàn)研究 [目的]探討大鼠慢性丙烯腈染毒模型外周血內(nèi)皮祖CD133表達(dá)、增殖、遷移及體外血管形成能力的變化；研究?jī)?nèi)皮祖細(xì)胞eNOS的表達(dá)情況。 [方法]流式細(xì)胞儀檢測(cè)P1細(xì)胞CD133表達(dá)變化；體外擴(kuò)增培養(yǎng)EPCs,繪制生長(zhǎng)曲線,四甲基偶氮唑鹽[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT]比色法測(cè)定細(xì)胞增殖情況,Transwell小室進(jìn)行細(xì)胞遷移實(shí)驗(yàn),Matrigel實(shí)驗(yàn)觀察細(xì)胞在體外血管形成能力,通過Western blot方法檢測(cè)細(xì)胞eNOS的表達(dá)情況。 [結(jié)果](1)C組、A組、B組外周血單個(gè)核細(xì)胞可陽(yáng)性表達(dá)CD133,其表達(dá)陽(yáng)性率無(wú)顯著學(xué)差異。(2)細(xì)胞增殖情況：傳代細(xì)胞潛伏期為48h左右,對(duì)數(shù)增殖期約為接種后5-7d,至接種后第9d進(jìn)入平臺(tái)期。丙烯腈可使EPCs的增殖能力減弱；隨著丙烯腈濃度的增加,其生長(zhǎng)速度減慢(p0.05)。(3)遷移能力：C組、A組、B組培養(yǎng)24小時(shí)后遷移細(xì)胞數(shù)目分別為：45±2.76、42.5±3.08、42±4.32。丙烯腈處理后細(xì)胞遷移數(shù)目無(wú)明顯減少(p均0.05)(4)體外血管形成情況：C組、A組、B組小管生成數(shù)分別為34+4.22,24±3.74,19±3.56。丙烯腈處理后細(xì)胞體外血管形成數(shù)目減少(p均0.05)(5)丙烯腈可以抑制細(xì)胞eNOS的表達(dá),隨著濃度的增加,抑制作用有增加趨勢(shì)。 [結(jié)論]本實(shí)驗(yàn)表明：與對(duì)照組比較,ACN接觸組EPCs增殖及體外血管形成能力減弱,且隨著ACN濃度的增加而愈明顯,同時(shí)引起細(xì)胞eNOS合成減少。同時(shí),丙烯腈對(duì)大鼠細(xì)胞表型CD133表達(dá)及遷移能力無(wú)影響。
[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,
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R114

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