【作者】 刘黎黎；

【导师】 阮秋蓉；

【作者基本信息】 华中科技大学 ， 病理学与病理生理学， 2009， 硕士

【摘要】 目的:通过观察脂多糖(1ipopolysaccharides,LPS)对离体小鼠骨髓源性平滑肌祖细胞(vascular smooth progenitor muscle cell,SPC)膜表面基质细胞衍生因子受体表达的影响,探讨SDF-1/CXCR4轴在动脉粥样硬化发生和发展过程中所起的作用,从而探索抗动脉粥样硬化的可能方法。方法:复苏本实验室已成功分选出的平滑肌祖细胞,并将其传代培养,按照实验设计分别分为六个组:①正常对照组:加入普通培养基;②低剂量组(L组):加入LPS终浓度为1ug/mL,孵育6h;③中剂量组(M组):加入LPS终浓度为10ug/mL,孵育6 h;④高剂量组(H组):加入LPS终浓度为30ug/mL,孵育6 h;⑤12 h组:加入LPS终浓度为10ug/mL,孵育12 h;⑥24 h组:加入LPS终浓度为10ug/mL,孵育24 h。用免疫荧光染色及共聚焦激光扫描显微镜分析和蛋白免疫印迹检测平滑肌祖细胞基质细胞衍生因子受体蛋白表达,逆转录聚合酶链反应检测平滑肌祖细胞基质细胞衍生因子受体mRNA的表达,观察脂多糖对平滑肌祖细胞表达基质细胞衍生因子受体的时效和浓度关系。结果:免疫荧光染色和共聚焦激光扫描显微镜分析测得的结果与RT-PCR和Western blot检测的结果相一致,未给予脂多糖刺激的平滑肌祖细胞有基础水平的基质细胞衍生因子受体表达,脂多糖是基质细胞衍生因子受体的强诱导物。当刺激时间恒定时(6h),随着刺激浓度的增加,L组和M组的基质细胞衍生因子受体表达逐渐增强,与正常对照组相比均具有显著性差异(P<0.01),H组开始下降。当刺激浓度恒定时(10ug/mL),随着刺激时间的延长,M组和12h组的表达量也逐级增强,与正常对照组相比均具有显著性差异(P<0.01)。在所有组中,12h组表达最强,其mRNA和蛋白水平分别为基础水平的6.10倍和4.76倍,24h组又下降,但仍高于基础水平。结论:平滑肌祖细胞能表达低水平的CXCR4,动脉粥样硬化的危险因子脂多糖能够使平滑肌祖细胞的CXCR4水平上调,其作用在一定范围内呈浓度时间依赖性,提示SDF-1/CXCR4轴可能参与了平滑肌祖细胞在血管损伤部位的归巢。更多还原

【Abstract】 Aims:Through observing the effects of 1ipopolysaccharides on exsomatize mice spinal marrow non-odontogenic smooth progenitor muscle cell membrane surface stroma cells derived factor receptor expression, to discuss the role that SDF-1/CXCR4 axis plays in the developing process of atherosclerosis formation, thus to seek the possible ways of anti atherosclerosis.Methods:Using enhancement type P green-fluorescing cells plasmid recombinant to select the smooth progenitor muscle cell out from the mice marrow mesenchyma stem cells , then subculture them and divide them into six groups as the experiment’s design:①Normal control group: add ordinary culture medium;②Low dosage group(group L): final concentration of the LPS is 1ug/mL, incubating for 6h;③Medium dosage group(group M):final concentration of the LPS is 10ug/mL, incubating for 6h;④High dosage group(group H): final concentration of the LPS is 30ug/mL, incubating for 6h;⑤group12h: final concentration of the LPS is 10ug/mL, incubating for 12h;⑥group24h: final concentration of the LPS is 10ug/mL, incubating for 24h. Use Western blotting and immunofluorescence staining and confocal laser scanning microscope to detect smooth progenitor muscle cell membrane surface stroma cells derived factor receptor protein expression and to use reverse transcription polymerase chain reaction to detect smooth progenitor muscle cell membrane surface stroma cells derived factor receptor mRNA’s expression, thus to observe the relations between lipopolysaccharide’s aging and concentration when the smooth progenitor muscle cell is expressing stroma cells derived factor receptor. Results:The immunofluorescence staining and confocal laser scanning microscope method have got the identical results with RT-PCR and Western blot detection, those smooth muscle progenitor cells that haven’t been given lipopolysaccharide stimulation have stroma cells derived factor receptor expression of foundation level, lipopolysaccharide is a strong inducer of stroma cells derived factor receptor. When the stimulus time is invariable(6h), as the increasing of the stimulus concentration, stroma cells derived factor receptor expression of group L and group M strengthened gradually, and has significant difference with the normal control group(P<0.01), but group H began to decrease. When the stimulus concentration is invariable(10ug/mL), as the extend of the time, the expression volume of group M and group 12h increased as well, and has significant difference with normal control group(P<0.01). In all the groups, group 12h has the strongest expression, and it’s mRNA and protein level are 6.10 times and 4.76 times as the foundation level respectively, the group 24h decreased, while still higher than the foundation level.Conclusion:Smooth muscle progenitor cells can express CXCR4 of low level, Atherosclerotic risk factors can lipopolysaccharide smooth muscle progenitor cells increases the level of CXCR4, the effect present concentration and time-dependent, SDF-1/CXCR4 axis involved in the smooth muscle progenitor cells vascular injury in the homing site.更多还原