Batista Lobo S, Denyer M, Britland S, Javid F A
School of Pharmacy, University of Bradford and Institute of Pharmaceutical Innovation, Bradford, UK.
J Anat. 2007 Dec;211(6):819-29. doi: 10.1111/j.1469-7580.2007.00820.x. Epub 2007 Nov 1.
This paper reports on the development of an entirely new intestinal smooth muscle cell (ISMC) culture model using rat neonates for use in pharmacological research applications. Segments of the duodenum, jejunum and ileum were obtained from Sprague-Dawley rat neonates. The cell extraction technique consisted of ligating both ends of the intestine and incubating (37 degrees C) in 0.25% trypsin for periods of 30-90 min. Isolated cells were suspended in DMEM-HEPES, plated and allowed to proliferate for 7 days. Cell culture quality was assessed via a series of viability tests using the dye exclusion assay. In separate experiments, tissues were exposed to trypsin for varying durations and subsequently histological procedures were applied. Cell purification techniques included differential adhesion technique for minimizing fibroblasts. Selective treatments with neurotoxin scorpion venom (30 microg mL(-1)) and anti-mitotic cytosine arabinoside (6 microm) were also applied to purify respectively ISMC and myenteric neurones selectively. The different cell populations were identified in regard to morphology and growth characteristics via immunocytochemistry using antibodies to smooth muscle alpha-actin, alpha-actinin and serotonin-5HT3 receptors. Based on both viability and cell confluence experiments, results demonstrated that intestinal cells were best obtained from segments of the ileum dissociated in trypsin for 30 min. This provided the optimum parameters to yield highly viable cells and confluent cultures. The finding was further supported by histological studies demonstrating that an optimum incubation time of 30 min is required to isolate viable cells from the muscularis externae layer. When cell cultures were treated with cytosine arabinoside, the non-neuronal cells were abolished, resulting in the proliferation of cell bodies and extended neurites. Conversely, cultures treated with scorpion venom resulted in complete abolition of neurones and proliferation of increasing numbers of ISMC, which were spindle-shaped and uniform throughout the culture. When characterized by immunocytochemistry, neurones were stained with antibody to 5HT3 receptors but not with antibodies to alpha-smooth muscle actin and alpha-actinin. Conversely, ISMC were stained with antibodies to alpha-smooth muscle actin and alpha-actinin but not with antibody to 5HT3 receptors. The present study provides evidence that our method of dissociation and selectively purifying different cell populations will allow for pharmacological investigation of each cell type on different or defined mixtures of different cell types.
本文报道了一种全新的肠道平滑肌细胞（ISMC）培养模型的开发，该模型使用新生大鼠用于药理学研究应用。十二指肠、空肠和回肠段取自新生的Sprague-Dawley大鼠。细胞提取技术包括结扎肠道两端，并在0.25%胰蛋白酶中于37℃孵育30 - 90分钟。分离出的细胞悬浮于DMEM-HEPES中，接种并使其增殖7天。通过使用染料排除法的一系列活力测试来评估细胞培养质量。在单独的实验中，将组织暴露于胰蛋白酶不同时长，随后进行组织学处理。细胞纯化技术包括用于使成纤维细胞最少化的差异黏附技术。还分别应用神经毒素蝎毒（30μg mL⁻¹）和抗有丝分裂的阿糖胞苷（6μM）进行选择性处理，以分别选择性纯化ISMC和肌间神经细胞。通过使用针对平滑肌α-肌动蛋白、α-辅肌动蛋白和5-羟色胺5HT3受体的抗体进行免疫细胞化学，根据形态和生长特征鉴定不同的细胞群体。基于活力和细胞汇合实验，结果表明，肠道细胞最好从在胰蛋白酶中解离30分钟的回肠段获得。这提供了产生高活力细胞和汇合培养物的最佳参数。组织学研究进一步支持了这一发现，表明从外肌层分离活细胞需要30分钟的最佳孵育时间。当用阿糖胞苷处理细胞培养物时，非神经细胞被消除，导致细胞体和延长的神经突增殖。相反，用蝎毒处理的培养物导致神经细胞完全消除，并且纺锤形且在整个培养物中均匀分布的ISMC数量增加并增殖。当通过免疫细胞化学进行表征时，神经细胞用5HT3受体抗体染色，但不用α-平滑肌肌动蛋白和α-辅肌动蛋白抗体染色。相反，ISMC用α-平滑肌肌动蛋白和α-辅肌动蛋白抗体染色，但不用5HT3受体抗体染色。本研究提供了证据，表明我们分离和选择性纯化不同细胞群体的方法将允许对不同细胞类型或不同细胞类型的特定混合物中的每种细胞类型进行药理学研究。
