家兔类胚胎干细胞分离培养及羊栖菜多糖对其生长增殖和分化的影响

Influence of rabbit embryonic stem cells and polysaccharides on the proliferation and differentiation of

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本实验的旨在分别造就取得家兔类胚胎干细胞及研讨羊栖菜多糖对家兔类ES细胞发展增殖和分化的影响,从细胞程度商量SFPS的养分感化和药理功效。经由过程研讨SFPS对家兔类ES细胞豢养层发展增殖和细胞因子排泄的影响及其对家兔类ES细胞物资分解、抗氧化和分化的感化,提醒SFPS调理家兔类胚胎干细胞发展增殖的感化机理,说明SFPS对家兔类ES细胞引诱分化为肝细胞的感化。1。从13一15dpc家兔胎儿生殖嵴分别出原始生殖细胞造就于小鼠胎儿成纤维细胞(MEF)豢养层和家兔胎儿成纤维细胞(REF)豢养层,以胰卵白酶消化法和机械分别一胰卵白酶消化法停止传代,并对造就获得的家兔类ES细胞停止判定。成果注解:以MEF作为豢养层,用机械分别。胰卵白酶消化法传代,更有益于家兔类ES细胞的体外造就,最高传至6代,造就获得的家兔类胚胎干细胞AKP染色和PAS染色阳性,坚持完全的染色体组(2n=44),具有多分化潜能,可构成类胚体,可分化为上皮样细胞、脂肪细胞、心肌细胞、神经样细胞。2。用含5、10、20、40、80mg/LSFPS的造就液造就家兔类ES细胞豢养层(即MEF),研讨SFPS对MEF发展增殖、养分物资接收应用和细胞因子排泄的影响。成果注解:5、10、20mg/LSFPS对MEF形状和发展增殖无明显影响(P》0。05),40、80mg/LSFPS对MEF表示出显著的克制感化(P0。05);低浓度SFPS有增进MEF碱性成纤维细胞因子(bFGF)、干细胞发展因子(SCF)、白血病克制因子(LIF)排泄的趋向,但高浓度SFPS有削减MEF排泄bFGF、SCF和LIF的的趋向。3。用分歧浓度SFPS造就家兔类ES细胞,不雅察家兔类ES细胞的形状和发展行动,测定其集落数和传代数,和家兔类ES细胞卵白质、DNA、RNA的含量。成果注解:5、10、20mg/L SFPS对家兔类ES细胞的发展增殖有增进感化,集落数目多,体积年夜,最高传至7代,40、80mg/LSFPS对家兔类ES细胞产生毒性感化,集落数目削减,易分化,传3一4代后丧失。SFPS对家兔类ES细胞卵白质的含量无明显影响(P》0。05),5、10、20mg/L SFPS可明显进步家兔类ES细胞DNA、RNA含量(P0。05),但明显下降RNA的含量(P《0。01)。4。以H2O2为家兔类ES细胞外源性氧化剂,制备氧化毁伤模子,并用分歧浓度SFPS对家兔类ES细胞停止掩护和修复,商量SFPS对家兔类ES细胞的抗氧化感化。成果显示:5、10、20mg/LSFPS对家兔类ES细胞具有掩护和修复感化,可明显进步氧化毁伤家兔类ES细胞的增殖活气和SOD和GSH一Px酶活性,削减MDA的发生,克制H2O2招致的DNA断链。40、80 mg/LSFPS对氧化毁伤家兔类ES细胞的掩护和修复感化不显著,有时反而加重对家兔类ES细胞的毁伤,对DNA断链的克制感化较弱。5。以家兔胎肝提取液为肝细胞引诱分化剂,同时与SFPS结合应用引诱家兔类胚胎干细胞向肝细胞分化,商量SFPS对家兔类胚胎干细胞引诱分化为肝细胞的影响。成果显示:胎肝提取液可引诱家兔类胚胎干细胞分化为肝细胞,引诱分化细胞呈圆形、星形、不规矩多边形,具有糖原储存、ICG摄取和渗出功效,可以或许分解白卵白和尿素。而且家兔类胚胎干细胞在体外也能自觉分化为肝细胞,但分化率较低。低浓度SFPS对胎肝提取液引诱家兔类胚胎干细胞分化为肝细胞有必定增进感化,可进步分化细胞PAS染色和ICG摄取阳性率,增进引诱分化构成的肝细胞白卵白和尿素的分解。综上所述,从胎儿生殖嵴分别造就的家兔类胚胎干细胞具有胚胎干细胞的特点。而且,低浓度SFPS增进家兔类ES细胞的发展增殖,高浓度SFPS起克制感化,SFPS增进豢养层细胞排泄细胞因子,同时施展抗氧化感化能够是其增进家兔类ES细胞发展增殖的感化机理。低浓度SFPS对胎肝提取液引诱家兔类胚胎干细胞分化为肝细胞有必定增进感化,可增进引诱分化构成的肝细胞的成熟。

Abstract:

The experiment of in order to create rabbit embryonic stem cells and research of Sargassum fusiforme polysaccharide of rabbit ES cell proliferation and differentiation effects from the cell level to discuss SFPs nutrient effect and pharmacological efficacy. Through the study of SFPs of rabbit es like cells were layer development proliferation and cytokine secretion and its influence on rabbit ES cell material decomposition and oxidation and differentiation effect remind SFPs conditioning rabbit embryonic stem cells proliferation mechanism of action of, of SFPs on rabbit es inducing cell differentiation for the action of liver cells. 1. From 13 15dpc rabbit fetal genital ridge respectively primordial germ cells created in fetal mice into fibroblast (MEF) collector layer and rabbit fetal fibroblasts (Ref) collector layer, to pancreatic albumen enzyme digestion and mechanical respectively a pancreatic albumen enzyme digestion method to stop passage, rabbits and to get the ES cells can judge. Note: results using MEF as collector layer, mechanical respectively. Pancreatic albumen enzyme digestion method in vitro and more beneficial to rabbit es like cells in vitro to create, the highest spread to six generations, creating the rabit embryonic stem cell AKP staining and PAS positive staining, insist on complete sets of chromosomes (2n = 44), with multiple differentiation potential, embryoid body composition, can differentiate into epithelial like cells, fat cells, myocardial cells, neuron like cells. 2. With 5, 10, 20, 40, 80mg/LSFPS solution to create rabbit ES cell collector layer (MEF), research of SFPs on proliferation of MEF development, nutrient material receiving application and cytokines excretion. Results: 5, 10, note 20mg/LSFPS had no obvious influence on the shape of MEF and the development of proliferation (P "0. 05, 40, 80mg/LSFPS) of MEF showed significant inhibition (P0. 05); low concentration of SFPs have enhance MEF basic fibroblast growth factor (bFGF), stem cell factor (SCF), leukemia restraint factor (LIF) excretion trend, but high concentration of SFPs have cut the MEF excretion of bFGF, SCF, LIF trend. 3. Cultivate rabbit es like cells with different concentration of SFPs, not Yacha rabbit class ES cell shape and development action, determination of the colony number and algebra, and rabbit ES cell albumen matter, DNA, RNA content. Notes: 5, 10, 20mg / L of SFPs on rabbit ES cell development proliferation have stimulative effect, colony number and volume is large, the highest spread to seven generations, 40, 80mg/LSFPS of rabbit ES cells produced toxicity effect, colony cuts the number, easy differentiation, 3 to 4 generations after the loss of. No significant effect of content on rabbit ES cell protein SFPS (P "0. 05, 5, 10), 20mg/L SFPS can significantly improve the rabbit ES cells DNA and RNA content (P0. 05), but significantly decreased the content of RNA (0 "P. 01). 4. H2O2 rabbit ES cells with exogenous oxidants, oxidative damage and mold preparation, with different concentrations of SFPS on rabbit ES cells stop protection and repair, discuss the antioxidant action of rabbit SFPS ES cells. Results: 5, 10, 20mg/LSFPS of rabbit ES cell has cover and repair effect, obvious progress of oxidative damage of rabbit es like cells proliferation vitality, and the activities of SOD and GSH PX enzyme activity, reduce MDA, H2O2 restraint causes DNA scission. 40, 80 mg/LSFPS on oxidative damage of rabbit ES cell cover and repair effect is not significant, sometimes aggravate the damage of rabbit ES cells, the DNA chain breaking restraining effect is weak. 5. Rabbit fetal liver extract as liver inducing cell differentiation agent, also with SFPs with attractive application of rabbit embryonic stem cells to differentiate into hepatocytes and talk of SFPs on rabbit embryonic stem inducing cell differentiation effects of liver cells. Results show that the fetal liver extract can lure rabbit embryonic stem cells to differentiate into liver cells lure cell differentiation is rounded, star shaped, irregular polygon, with glycogen storage, ICG uptake and exudation effect, can perhaps decomposition of albumin and urea. But the rabbit embryonic stem cells in vitro can consciously differentiate into liver cells, but the differentiation rate is low. Low concentration of SFPs on fetal liver extract liquid to lure rabbit embryonic stem cells to differentiate into liver cells have certain stimulative effect, progress differentiation cells, PAS staining and ICG uptake positive rate, enhance inducement differentiation constitutes the liver cell white albumen and urea decomposition. In summary, rabbit embryonic genital ridge were created from the fetal stem cells has the characteristics of embryonic stem cells. And low concentration of SFPs promotes the development and proliferation of rabbit es like cells, high concentrations of SFPs on the restraining effect, SFPs enhance captive layer cells of the excretory cell factor, and display their antioxidant action to improve the role of rabbit es like cells proliferation mechanism. Low concentration of SFPS on fetal liver extract induce rabit embryonic stem cells to differentiate into liver cells have certain stimulative effect, can enhance the differentiation of liver cells constitute the lure of mature.

目录:

封面1-2
文摘2-4
英文文摘4-7
论文说明:缩略词表7-8
声明8-9
第一部分文献综述9-29
    第一章羊栖菜多糖研究概况9-15
        1羊栖菜的生物学特性9-10
        2羊栖菜的营养成分10-10
        3羊栖菜多糖组分10-11
        4羊栖菜多糖的生物学功能11-14
        5羊栖菜多糖研究存在问题及其发展趋势14-15
    第二章兔胚胎干细胞研究进展15-24
        1兔胚胎干细胞的来源15-16
        2影响兔胚胎干细胞体外培养的因素16-18
        3兔胚胎干细胞的特征与鉴定18-22
        4兔胚胎干细胞应用前景22-24
    第三章中药活性物质对体外培养细胞影响作用研究概况24-29
        1中药活性物质对体外培养细胞生长增殖的影响24-25
        2中药活性物质对体外培养细胞蛋白质、核酸等合成的影响25-25
        3中药活性物质对体外培养细胞营养物质吸收利用的影响25-26
        4中药活性物质对体外培养细胞抗氧化作用的影响26-27
        5中药活性物质对体外培养细胞分化的影响27-27
        6本研究的目的和意义27-29
第二部分试验研究29-86
    第四章家兔类胚胎干细胞的分离、培养和鉴定29-40
        1材料与方法29-34
        2结果与分析34-37
        3讨论37-38
        4 小结38-40
    第五章羊栖菜多糖对家兔类胚胎干细胞饲养层生长增殖、营养物质利用及细胞因子分泌的影响40-52
        1材料与方法41-44
        2结果与分析44-48
        3讨论48-50
        4 小结50-52
    第六章羊栖菜多糖对家兔类胚胎干细胞生长增殖和物质合成的影响52-62
        1材料与方法53-56
        2结果与分析56-59
        3讨论59-61
        4小结61-62
    第七章羊栖菜多糖对家兔类胚胎干细胞抗氧化作用的研究62-75
        1材料与方法62-67
        2结果与分析67-71
        3讨论71-73
        4 小结73-75
    第八章羊栖菜多糖在家兔类胚胎干细胞诱导分化为肝细胞中的作用75-85
        1材料与方法76-79
        2结果与分析79-81
        3.讨论81-84
        4 小结84-85
    第九章 结论85-86
参考文献86-99
致谢99-100
附录 细胞图片100-105
作者简介105-106
导师简介106