【作者】 赵华山；

【导师】 余四九；

【作者基本信息】 甘肃农业大学 ， 动物医学工程， 2009， 硕士

【摘要】 本研究以小鼠和牦牛为研究对象,首先在小鼠上建立了单核细胞吞噬诱导凋亡卵泡颗粒细胞的模型,然后在此基础上,对牦牛单核细胞吞噬诱导凋亡颗粒细胞过程的形态变化做了深入的研究。在小鼠上,本实验采用体外培养和诱导凋亡的方式,模拟体内单核细胞吞噬由大蒜诱导凋亡的卵泡颗粒细胞的过程,在不同时点(1h,2h,3h,4h,5h)对共培养物进行瑞氏染色后,用显微镜观察并照相,对吞噬程度随时间变化的规律进行回归分析。结果显示,在颗粒细胞凋亡形变的初期,即可被单核细胞识别,形成吞噬泡,与此同时,颗粒细胞的凋亡程度也越来越大,在吞噬泡内可见到凋亡细胞崩解的碎片。对照片进行分类计数发现,单核细胞与颗粒细胞共同培养约3 h的时候,外接凋亡细胞的单核细胞个数与内吞凋亡细胞的单核细胞个数的比值接近1,说明此时单核细胞中半数呈现吞入状态,半数呈现外接(识别阶段)状态,笔者建议将此状态的时间点称为“半吞期”。SPSS13.0回归分析,可得线性回归方程y=-0.247x+1.644,R~2=0.912,回归方程显著性检验F=31.095,P=0.011(＜0.05),回归系数显著性检验T=-5.576,P=0.011(＜0.05)。在牦牛上,以先期的小鼠实验为基础,同样模拟了牦牛体内单核细胞吞噬由大蒜诱导凋亡的卵泡颗粒细胞的过程,在不同时点(1h,2h,3h,4h,5h)对共培养物进行瑞氏染色后,用显微镜观察并照相,对吞噬程度随时间变化的规律进行回归分析。同时,对牦牛单核细胞吞噬诱导凋亡的卵泡颗粒细胞的过程,进行了扫描电镜和透射电镜的观察。结果显示,牦牛单核细胞吞噬诱导凋亡卵泡颗粒细胞的光镜下的过程基本与在小鼠上观察到的相同,半吞期为4h。SPSS15回归分析,得线性回归方程y=-0.540x+2.958,R~2=0.937,回归方程显著性检验F=44.766,P=0.007(＜0.01),回归系数显著性检验T=-6.691,P=0.007(＜0.01)。在扫描电镜和透射电镜下,清晰可见颗粒细胞的凋亡和其被吞噬的过程。综上所述,这种对早期凋亡细胞吞噬过程的体外模拟可以作为一种定量分析的方法,判断吞噬能力的变化,从而可用于对一些影响因素,如促进或抑制吞噬的药物,进行定量分析。以小鼠模型为基础,可在许多的动物上研究其他吞噬细胞的吞噬能力。实验表明小鼠模型在牦牛上是适用的,且得出的半吞期及相关的形态学研究结果,一方面扩大了对牦牛生殖系统凋亡以及凋亡清除的认识,另一方面为深入的研究提供了依据。另外,实验过程中发现,反相处理瑞氏染色的照片,可获得单一荧光染色的效果。更多还原

【Abstract】 The present study firstly established a method for quantitative measurement of phagocytosis in mouse. Then the further research has been carried out about the morphologic change of phagocytosis in yak.On the mouse, the phagocytic process of apoptotic granulosa cells by monocytes was imitated in vitro. Monocytes and granulosa cells were isolated from Kunming mice and cultured. Granulosa cells were induced to apoptosis by garlic, and then co-cultured with monocytes. At different time points (1 h, 2 h, 3 h, 4 h, 5 h), co-cultured cells were assessed by Wright’s staining. The results showed that at the beginning of morphological changes in apoptotic granulosa cells, monocytes captured the apoptotic cells. Meanwhile, the apoptosis of granulosa cells were continuing. Debris was found in phagocytic vacuole. At the point of 3 h after co-culture, the ratio between the number of monocytes which attached apoptotic granulosa cells and that which engulfed the apoptotic cells was close to one. Namely, half of monocytes were in the state of recognition and half were in a state of engulfment. The author suggested to name it as ’half phagocytic period’. Regression analysis showed that the equation of linear regression was y = -0.247x + 1.644, R~2=0.912, F=31.095, P=0.011 (<0.05), T= -5.576, P=0.011 (<0.05).On the yak, according to the mode of mouse above, the phagocytic process of apoptotic granulosa cells by monocytes was also imitated in vitro. At different time points (1 h, 2 h, 3 h, 4 h, 5 h), co-cultured cells were assessed by Wright’s staining. In addition, the process was observed through scanning electron microscope (SEM) and transmission electron microscope (TEM). The results showed that the morphological changes under microscope were same with that of mouse in the main. At the point of 4 h after co-culture, the ratio between the number of monocytes which attached apoptotic granulosa cells and that which engulfed the apoptotic cells was close to one. Regression analysis showed that the equation of linear regression was y = -0.540x + 2.958, R~2 = 0.937, F=44.766, P=0.007 (<0.01), T= -6.691, P=0.007 (<0.01). Under the SEM and TEM, the apoptosis and phagocytosis were apparent.In conclusion, the present mode of phagocytosis can be used as a method of quantitative assay, which could analyze some effective factors such as medicines that could enhance or restrain phagocytosis. The mode of mouse fit on the study of yak. The result of half phagocytic period and morphological changes enlarged the understanding of apoptosis and phagocytosis, providing base for further research. Based on the mode, the ability of phagocytosis could be studied on other animals. In addition, it was notable that effect of fluorochrome stain could be got through picture processing of ’Invert’ on the pictures.更多还原