The construction of rat intestinal smooth muscle collagen band and evaluation of periodic stretching culture in vitro

doi:10.12307/2024.806

Abstract

Abstract: BACKGROUND: The in vitro construction of intestinal smooth muscle layer, as an important component of the intestinal wall, has attracted much attention in the bionic construction of tissue-engineered intestinal canal.
OBJECTIVE: To explore the effects of cyclic mechanical stretching on the growth activity of intestinal smooth muscle cells and the expression of functional genes within collagen strips.
METHODS: The collagen band culture system of intestinal smooth muscle cells was constructed using a self-designed collagen strip stretching culture device with self-made rat tail collagen as a scaffold and primary rat intestinal smooth muscle cells as seed cells. EthD-1/Calcein-AM cell activity staining, magenta staining, cytoskeleton-Ki67 immunofluorescence staining were used to observe the growth activity and proliferation of the cells, and quantitative RT-PCR was used to detect the expression of desmin, α-sma, and vimentin functional genes.
RESULTS AND CONCLUSION: The collagen band culture system of intestinal smooth muscle cells was successfully constructed, and intestinal smooth muscle cells in the band had good cell activity. The number of Ki67 positive cells increased and desmin, α-sma and vimentin were significantly overexpressed under cyclic stretching and dynamic culture conditions (P < 0.001). To conclude, mechanical stimulation is beneficial to maintain the growth phenotype of smooth muscle cells and promote their functional differentiation during three-dimensional culture in vitro.

Key words: tissue engineering, intestinal smooth muscle, collagen band, dynamic stretching, collagen scaffold, three-dimensional culture

CLC Number:

Yu Pengxin, Han Yuqiu, Guo Lina, Wang Xiuli. The construction of rat intestinal smooth muscle collagen band and evaluation of periodic stretching culture in vitro[J]. Chinese Journal of Tissue Engineering Research, 2024, 28(35): 5630-5635.

Figures/Tables 3

2.2 肠道平滑肌胶原条带的成功制备与表征倒置相差显微镜下可观察到：大鼠肠道平滑肌细胞均呈现圆形，均匀分布于胶原凝胶内。胶原在模具内凝固后，大体可见胶原条带紧紧收缩环绕在琼脂糖凝胶柱的周边，见图1A。由于收缩后的条带变得更为致密，导致透光性下降，因而在镜下难以进行清晰成像拍照。但镜下仍能够辨认出肠道平滑肌细胞形态变得更加伸展。随着培养时间的延长，胶原条带内的细胞数量有所增加，这提示胶原不但能够为肠道平滑肌细胞体外维持培养提供三维培养空间，而且还有利于细胞增殖与活性的体外维持。对胶原条带内肠道平滑肌细胞进行EthD-1/Calcein-AM活性染色发现：大多数细胞均显示绿色荧光，少有细胞被EthD-1染色而显示红色荧光，见图4A。该结果表明条带内肠道平滑肌细胞具有良好的细胞活性。 2.3 周期性拉伸维持培养对胶原条带平滑肌细胞生长表型与基因表达的影响为了优化胶原条带的体外维持培养条件，实验室自行设计搭建了可用于胶原条带拉伸的机械装置。当把胶原条带搭载到拉伸臂后，因为受到机械张力的作用，游离的条带不会发生“shrink”，从而利于胶原条带和条带内细胞保持其初始形态。 Whole-mount洋红组织学染色结果显示：不同培养条件下(动态拉伸培养或静态培养)，胶原条带内细胞的生长形态差异显著，见图4B。经拉伸培养后，肠道平滑肌细胞形态显示为典型的长梭形，细胞长轴多沿胶原条带的长轴反向进行纵向排列，显示出明显的排列方向性—该特征与其体内形态更为接近。与该结果相一致，细胞活性染色和细胞骨架荧光染色结果又进一步明确了这种定向排列的分布特征：细胞方向性排列，具有良好的细胞活性。而与之相对比，静态培养胶原条带内的细胞形态并未呈现出明显的延展拉伸形态特征，且在胶原条带内的分布排列随机，并不具备明显的方向性。此外研究结果还发现：对比静态培养条件下的平滑肌胶原条带，经体外周期性拉伸培养的胶原条带内Ki67阳性表达的细胞数量增多，见图4A。这提示周期性拉伸动态培养条件可能更利于肠道平滑肌细胞在体外三维培养过程中维持增殖活性。定量RT-PCR检测结果显示：与静态培养组相比，拉伸培养组内肠道平滑肌细胞可显著高表达标志性基因desmin、α-sma、vimentin，见图4C。该结果证明：周期性机械力学刺激能够促进肠道平滑肌细胞的功能性分化。 "

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