三种方法培养慢性阻塞性肺疾病大鼠气道平滑肌细胞的比较

doi:10.3969/j.issn.2095-4344.2012.50.025

摘要/Abstract

摘要：

背景：原代培养方法被广泛应用于正常和哮喘大鼠气道平滑肌细胞的培养，但少见用于慢性阻塞性肺疾病大鼠气道平滑肌细胞培养的报道。
目的：建立大鼠慢性阻塞性肺疾病模型，比较组织块法、酶消化法和改良组织块消化法原代培养模型大鼠气道平滑肌细胞生长情况的差异。
方法：将16只清洁级雄性健康SD大鼠随机分成2组，对照组正常饲养，慢性阻塞性肺疾病组用熏烟法建立大鼠慢性阻塞性肺疾病模型，显微镜下观察大鼠肺组织病理学特点。分别应用上述3种方法原代培养慢性阻塞性肺疾病大鼠气道平滑肌细胞，相差显微镜下观察细胞形态，用α-平滑肌肌动蛋白免疫组织化学染色鉴定细胞类型。
结果与结论：经病理学证实成功构建慢性阻塞性肺疾病大鼠模型。在倒置相差显微镜下见培养的细胞表现为典型的 “谷-峰状”生长。经免疫细胞化学染色后，可见胞质呈棕色阳性反应，所培养的细胞有94%以上为气道平滑肌细胞。3种方法在耗时和细胞质量方面均无明显差别，但组织块法更经济、稳定可靠和简单。

Abstract:

BACKGROUND: Methods of primary culture have been extensively used to culture airway smooth muscle cells in normal or asthma rats, but the same way to culture airway smooth muscle cells in chronic obstructive pulmonary disease rats has rarely been reported.
OBJECTIVE: To establish the rat chronic obstructive pulmonary disease model, and to compare the different growth conditions of airway smooth muscle cells in rat chronic obstructive pulmonary disease models by three methods: attachment-block culture and enzymatic dispersion as well as improved tissue-piece digestion inoculation.
METHODS: Sixteen healthy male clean-grade Sprague-Dawley rats were randomly divided into chronic obstructive pulmonary disease group and control group with eight rats in each group. Rats in the control group were fed normally. The rat chronic obstructive pulmonary disease models were established in the chronic obstructive pulmonary disease group by cigarette smoke exposure. The pathologic characteristics of lung tissues of the rat models were observed under microscope. The airway smooth muscle cells of rat chronic obstructive pulmonary disease model were cultured from primary generation using the three ways mentioned above, respectively. The morphology of the cultured cells was observed under phase contrast microscope and α-smooth muscle actin immunostaining was used to identify the cell types.
RESULTS AND CONCLUSION: The rat chronic obstructive pulmonary disease models were established successfully, which was proved by pathology. The cultured cells demonstrated the typical “hill and valley” appearance under phase contrast microscope. After α-smooth muscle actin immunostaining, brown positive reaction was observed in the cytoplasm. There were over 94% cultured cells identified to be airway smooth muscle cells. There was no significant difference of consuming time and quality of cells in these three ways. Compared with the other two methods, attachment-block culture was a more economical and stable and less complicated way to culture airway smooth musclecells in rat chronic obstructive pulmonary disease models.

杨诚，梁瑞韵，黄林洁，江山平. 三种方法培养慢性阻塞性肺疾病大鼠气道平滑肌细胞的比较[J]. 中国组织工程研究, 2012, 16(50): 9448-9452.

Yang Cheng, Liang Rui-yun, Huang Lin-jie, Jiang Shan-ping. Comparison of three methods for culturing the air smooth muscle cells of rats with chronic obstructive pulmonary disease[J]. Chinese Journal of Tissue Engineering Research, 2012, 16(50): 9448-9452.

参考文献

引言

材料方法

讨论

文章快阅

延伸阅读

原代培养的要点是要获得高纯度的气道平滑肌细胞，且不损伤细胞活力，几点技巧总结如下。

培养方法比较:酶消化法可快速获得稳定数量的气道平滑肌细胞，不过平滑肌细胞较脆弱，消化时间较难掌控。胰酶的主要作用是使细胞间的蛋白质水解，使细胞相互离散。胰酶消化时间最好为10～25 min，消化耐受时间一般不超过0.5 h，并且使用酶的批次不同、存放时间长短不同、甚至实验室的环境不同均会对消化时间产生影响，可根据具体情况适当增减时间，常用浓度为0.25%。胶原酶消化作用较温和，它能在生理pH和温度条件下特异性地水解天然胶原蛋白的三维螺旋结构，却不损伤其他蛋白质和组织。消化15～60 min效果均较好，文献报道称胶原酶消化时间可长达1 d，常用浓度为1～2 mg/mL。胶原酶-胰酶混合消化法效果最好。贴块法细胞萌出时间漫长，且原代培养时细胞密度不均匀，故需大量细胞培养时一般不采用此法。

组织取材:气道越向下，平滑肌含量相对越多。如何取细小的支气管平滑肌标本，并除去其内膜和外膜而取得较纯的气道原代平滑肌细胞是气道平滑肌细胞培养的难点。因此在处理组织过程中注意要尽量去除气管周围脂肪和结缔组织，内外膜尽可能刮除干净，否则容易长出成纤维细胞、上皮细胞等杂细胞，人工纯化效果并不十分理想。使用眼科虹膜剪处理组织块，能更快速地将组织块剪得均匀细小，增加与酶的接触表面积，提高消化效率。

原代培养:培养液只需满过组织即可，一般1～2 mL，不可多加，否则静置后细胞及组织块易悬浮，不易贴壁。若液体不够，可在细胞贴壁后再临时添加。一般头3 d需要绝对静置，等待细胞贴壁牢固后方可换液。原代培养时对细胞密度的要求其实并不严格，只要有少量细胞贴壁就能生长至贴满瓶底，浓度高则传代速度快些，照本方法，1瓶原代细胞可传4～5瓶，3 d内就能长满。

传代培养:在传代过程中胰酶消化5 min即可，否则对细胞损伤太大。镜下观察细胞悬浮于液体中，胞质回缩，如还有细胞贴附于瓶底，可轻轻晃动瓶身，细胞便会掉落。传代过程中胰酶消化后可直接加培养基终止消化，直接培养。

无菌操作：本试验的关键点在于坚持做好每一步骤的无菌操作，否则一旦发生污染很难查找污染源，且有可能殃及培养箱内其余细胞。