蜗轴螺旋动脉平滑肌的培养及其鉴定

发布时间：2018-03-27 23:02

本文选题：贴壁培养法　切入点：酶消化法　出处：《第四军医大学》2010年硕士论文

【摘要】：1.目的本研究的目的是通过联合两种常用的细胞培养方法(酶消化培养法和组织贴壁培养法),建立了一个体外的蜗轴螺旋动脉平滑肌细胞原代培养的模型。 2.方法 2.1用于进行实验的的蜗轴螺旋动脉的平滑肌来自于豚鼠。将蜗轴螺旋动脉血管组织切碎并且放入37度二氧化碳培养箱中在溶液浓度为0.1%胰蛋白酶溶液中消化20分钟。接着,将这些消化后的血管组织块在35-mm的培养皿中贴壁,组织块中间间隔3-5mm。 2.2联合运用这两种细胞培养方法,我们发现其中混杂的细胞几乎都为成纤维细胞,所以如何去除成纤维细胞是本实验需要克服的问题。经过查阅文献和多次的实践,我们将蜗轴螺旋动脉血管平滑肌细胞原代培养过程中混杂的成纤维细胞通过其与血管平滑肌细胞不同的贴壁能力和成纤维细胞对无血清的培养液不耐受,因为可在在传代的时候被去除。当35mm培养皿中细胞长满80%-90%的时候,就可以进行传代。在传代过程中应用上述的去除成纤维细胞的方法。我们将细胞悬液加入到35mm的培养皿中,15分钟后,吸出液体。将吸出的液体加入另外一个35mm的培养皿中,静置15分钟。吸出液体并加入到第三个35mm的培养皿中。收集第二和第三个培养皿继续培养。反复应用纯化的程序,可以在第三代得到纯的并且活性好的蜗轴螺旋动脉平滑肌细胞。 3.结果 3.1通过倒置显微镜的观察,我们发现在7-10天后,可以见到有细胞从组织块中长出来。大约在3周左右,这些细胞可长满培养皿,大约在细胞长满培养皿80%-90%时,可以进行传代。通过方法中介绍的血管平滑肌细胞纯化的方法,在第三代培养的血管平滑肌细胞中,纯的并且活性好的细胞可以被获得。 3.2经过形态学,免疫荧光化学对于上述方法得到的蜗轴螺旋动脉血管血管平滑肌细胞进行了鉴别。这些细胞呈现梭形的细胞形态,每个细胞含有1-2个核。结果显示了典型的平滑肌的细胞的特点:形态学显示了平滑肌细胞在细胞密度大的时候出现的典型的“峰-谷”样的生长形态。免疫荧光化学显示这些原代培养的蜗轴螺旋动脉血管平滑肌细胞表达平滑肌细胞的特异性标志物(α-SM- actin和myosin)。 3.3透射电镜结果显示的培养的蜗轴螺旋动脉平滑肌细胞中具有密斑,密体,肌丝这些平滑肌细胞的标志性结构。 4.结论本文通过联合两种常见的细胞培养方法,建立了一种有效并且简单的细胞培养方法。应用这种原代培养的方法,大量纯的并且活性良好的蜗轴螺旋动脉血管平滑肌细胞可以在原代培养第三代的时候得到。这些血管平滑肌细胞是一个很好的研究血管平滑肌细胞在内耳循环紊乱过程生理功能的一个体外模型。除此之外,这些活性良好的平滑肌细胞还可以是一些作用于血管平滑肌药物评价的体外模型。
[Abstract]:1. Purpose. The aim of this study was to establish a primary culture model of cochlear spiral artery smooth muscle cells in vitro by combining two common cell culture methods (enzyme digestion culture and tissue adherence culture). 2. Methodology. 2.1 the smooth muscle of the cochlear axial spiral artery used for the experiment comes from the guinea pig. The vascular tissue of the cochlear axial spiral artery is chopped and digested in a 37 degree carbon dioxide incubator in a 0.1% trypsin solution for 20 minutes. Then, These digested vascular tissue blocks were adhered to the 35-mm dish with an interval of 3 to 5 mm. 2.2 in combination with these two methods of cell culture, we found that almost all of the mixed cells were fibroblasts, so how to remove fibroblasts is a problem to overcome in this experiment. In the primary culture of vascular smooth muscle cells of cochlear axis spiral artery, the mixed fibroblasts were treated by their different adherent ability from vascular smooth muscle cells and the intolerance of fibroblasts to serum-free culture medium. Because it can be removed during passage. When the cells in the 35mm dish are 80 to 90 percent longer, We put the cell suspensions in the culture dish of 35mm for 15 minutes, and then we suck out the liquid, and we add the liquid to another dish of 35mm. Sit still for 15 minutes. Suck out the liquid and add it to the third 35mm dish. Collect the second and third dishes and continue the culture. Repeat the purification process. Pure and active smooth muscle cells of the cochlear axial spiral artery can be obtained in the third generation. 3. Results. 3.1 from the observation of the inverted microscope, we found that after 7-10 days, cells could be seen growing out of the tissue mass. In about 3 weeks, these cells could be filled with petri dishes, about 80 to 90 percent of the time when the cells were full of petri dishes. In the third passage of vascular smooth muscle cells, pure and active cells can be obtained. 3.2 morphologically, immunofluorescence was used to identify vascular smooth muscle cells of the cochlear axial spiral artery obtained by the above method. Each cell contains 1-2 nuclei. The results show the characteristics of typical smooth muscle cells: morphology shows typical "peak-valley" growth patterns of smooth muscle cells at high cell density. Immunofluorescence. Chemical analysis showed that these primary cultured vascular smooth muscle cells of the cochlear spiral artery expressed specific markers of smooth muscle cells (伪 -SM- actin and myosinine). 3.3 the results of transmission electron microscope showed that the cultured smooth muscle cells of the cochlear axial spiral artery had the iconic structures of dense spots, dense bodies and myofilms. 4. Conclusions. In this paper, an effective and simple cell culture method was established by combining two common cell culture methods. A large number of pure and active vascular smooth muscle cells of spiral cochlear artery can be obtained in the third generation of primary culture. These vascular smooth muscle cells are a good study of vascular smooth muscle cells in the inner ear circulation. An in vitro model of the physiological function of chaotic processes. These active smooth muscle cells may also be in vitro models for the evaluation of vascular smooth muscle drugs.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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