An acellular protocol of whole organ perfusion in rat heart and evaluation of its acellular effect
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摘要:
目的 用一种新的灌注脱细胞方案对离体SD大鼠心脏进行脱细胞处理，对其脱细胞效果进行检测与评估。 方法 取12只2月龄同窝雄性SD大鼠，按随机数字表法分为脱细胞组和对照组，每组6只。将各组大鼠麻醉并行全身肝素化后取出心脏。取脱细胞组心脏称取重量，然后将灌注针逆行插入升主动脉，连接好灌注装置进行脱细胞，观察脱细胞过程中心脏外观的变化，结束后称取脱细胞后的质量。在各心脏左心耳、右心耳、心尖部及右室流出道的同一部位取少量组织测定dsDNA含量，余组织制作石蜡切片并行HE染色及Masson染色后采集图像分析。 结果 脱细胞过程中，心脏颜色逐渐变得淡红，再变得相对透明，最后变成乳白色，肉眼可见冠脉血管结构保存完好。脱细胞组脱细胞前后心脏质量分别为（1.27±0.03）g、（0.49±0.02）g，差异有统计学意义（t=111.164, P < 0.001）。2组心脏各处测得的dsDNA含量差异均有统计学意义（均P < 0.01），且脱细胞组各处测得的dsDNA含量均显著低于50 ng/mg。对照组心脏HE染色切片及Masson染色切片均可见典型的心肌组织切片图像。而脱细胞组HE染色切片及Masson染色切片图像呈网状，均无明显细胞及核结构，其内可见冠状动脉轮廓。 结论 该脱细胞方案操作简单，效果可靠，是一种较为理想的心脏全器官脱细胞生物支架制备方案。 Abstract:Objective A new perfusion decellularization protocol was used to decellularize isolated SD rat hearts, and the decellularization effect was tested and evaluated. Methods Twelve 2-month-old male SD rats from the same litter were randomly divided into decellularized and control groups according to the random number table method, with 6 rats in each group. The hearts of each rat were removed after anesthesia and systemic heparinization. The hearts of the decellularized group were weighed, and then the perfusion needle was inserted retrograde into the ascending aorta and the perfusion device was connected for decellularization, and the changes of heart appearance were observed during the decellularization process. A small amount of tissue was taken from the same part of the left auricle, right auricle, apical region and right ventricular outflow tract of each heart to determine the dsDNA content, and the remaining tissue was prepared in paraffin sections and stained with HE and Masson stain, and then images were collected for analysis. Results During decellularization, the heart color gradually became light red, then became relatively transparent, and finally became milky white, and the coronary vascular structure was well preserved as seen by the naked eye. The heart mass before and after decellularization in the decellularized group were (1.27±0.03) g and (0.49±0.02) g, respectively, with statistically significant differences (t=111.164, P < 0.01). The differences in dsDNA content measured at each site in the hearts of both groups were statistically significant (all P < 0.01), and the dsDNA content measured at each site in the decellularized group was significantly lower than 50 ng/mg. Typical myocardial tissue sections were seen in HE-stained sections and Masson stained sections of the control group. In contrast, HE-stained sections and Masson stained sections of the decellularized group were reticulated, with no obvious cellular or nuclear structures, and the coronary artery contours were visible within them. Conclusion This decellularization protocol is simple and reliable, and is a more ideal solution for the preparation of cardiac whole-organ decellularized biological scaffolds. -
Key words:
- Acellular /
- Biological scaffold /
- Extracellular matrix /
- Tissue engineering /
- Artificial heart
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图 1 脱细胞过程心脏变化情况
注：图中箭头表示脱细胞步骤，数字1~9表示具体试剂，其前后图片分别表示经该步骤处理前后心脏照片。1~9具体表示为：1为去离子水30 mL；2为PBS 60 mL；3为0.05% EDTA和0.02%胰蛋白酶混合液60 mL；4为1% SDS 30 mL；5为0.05% EDTA和3% Triton X-100混合液30 mL；6为2.4%去氧胆酸30 mL；7为4%乙醇和0.1%过氧乙酸混合液30 mL；8为PBS 60 mL；9为去离子水60 mL。
Figure 1. Cardiac changes during decellularization
图 2 2组心脏心肌切片HE染色图像比较(×400)
注：A为对照组心肌切片HE染色图，可见心肌细胞纤维及细胞基质被染成红色(红色箭头所指)，细胞核被染成蓝色(蓝色箭头所指)；B为脱细胞组心肌切片HE染色图，图中未见明显心肌细胞及细胞核结构，仅可见被染成淡红色网格状结构。
Figure 2. Comparison of HE staining images of cardiac myocardial slices between the two groups (×400)
图 3 2组心脏心肌切片Masson染色图像比较(×400)
注：A为对照心肌切片Masson染色图，心肌细胞被染成红色(红色箭头所指)，细胞核被染成蓝黑色(黑色箭头所指)，胶原纤维被染成蓝色(蓝色箭头所指)，其中可见冠状动脉结构(白色箭头所指)。B为脱细胞后心肌切片Masson染色图，未见明显的心肌细胞及核结构，可见被染成蓝色的胶原纤维及保存较完好的冠状动脉结构。
Figure 3. Comparison of Masson stained images of heart myocardial sections between the two groups(×400)
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