Wan Juyi, Zhong Xiaolin, Liao Bin, Wang Mingyong, Deng Mingbin
Department of Cardiac Surgery, Affiliated Hospital of Luzhou Medical College, Luzhou Sichuan 646000, PR China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2009 Aug;23(8):980-4.
To locate sinoatrial node (SAN) in suckling pigs, to develop a reliable method for isolation, purification and cultivation of SAN cells and to observe the compatibility of SAN cells and Col I fiber scaffold.
Five newborn purebred ChangBaiShan suckling pigs (male and female), aged less than 1-day-old and weighing 0.45-0.55 kg, were used. Multi-channels electrophysiological recorder was applied to detect the original site of atrial waves. Primary SAN cells harvested from that area were cultured by the conventional culture method and the purification culture method including differential velocity adherent technique and 5-BrdU treatment, respectively. Atrial myocytes isolated from the left atrium underwent purified culture. Cell morphology, time of cell attachment, time of unicellular pulsation, and pulsation frequency were observed using inverted microscope. The purified cultured SAN cells (5 x 10(5) cells/mL) were co-cultured with pre-wetted Col I fiber scaffold for 5 days, and then the cells were observed by HE staining and scanning electron microscope (SEM).
The atrial waves occurred firstly at the area of SAN. The purified cultured SAN cells were spindle, triangular, and irregular in morphology, and the spindle cells comprised the greatest proportion. Atrial myocytes were not spindle-shaped, but primarily triangular and irregular. The proportion of spindle cells in the conventional cultured SAN cells was decreased from 73.0% +/- 2.9% in the purified cultured SAN cells, to 44.7% +/- 2.3% (P < 0.01), and the proportion of irregular cells increased from 7.0% +/- 1.7% in the purified cultured SAN cells to 36.1% +/- 2.6% (P < 0.01) . The proportion of the triangular cells in the purified and the conventional cultured SAN cells was 20.0% +/- 2.1% and 19.2% +/- 2.5%, respectively (P > 0.05). At 5 days after co-culture, HE staining displayed lots of SAN cells in Col I fiber scaffold, and SEM demonstrated conglobata adherence of the cells to the surface and lateral pore wall of scaffold, mutual connections of the cell processes, or attachment of cells to lateral pore wall of scaffold through pseudopodia.
With accurate SAN location, the purification culture method containing differential velocity adherent technique and 5-BrdU treatment can increase the proportion of spindle cells and is a reliable method for the purification and cultivation of SAN cells. The SAN cells and Col I fiber scaffold have a good cellular compatibility.
定位乳猪的窦房结（SAN），建立一种可靠的窦房结细胞分离、纯化及培养方法，并观察窦房结细胞与I型胶原纤维支架的相容性。
选用5只出生1天以内、体重0.45 - 0.55 kg的纯种长白山新生乳猪（雌雄不限）。应用多通道电生理记录仪检测心房波的起源部位。从该区域获取的原代窦房结细胞分别采用常规培养方法以及包括差速贴壁技术和5-溴脱氧尿嘧啶核苷（5-BrdU）处理的纯化培养方法进行培养。从左心房分离的心房肌细胞进行纯化培养。使用倒置显微镜观察细胞形态、细胞贴壁时间、单细胞搏动时间及搏动频率。将纯化培养的窦房结细胞（5×10⁵个细胞/mL）与预先湿润的I型胶原纤维支架共培养5天，然后通过苏木精-伊红（HE）染色和扫描电子显微镜（SEM）观察细胞。
心房波首先出现在窦房结区域。纯化培养的窦房结细胞形态呈纺锤形、三角形和不规则形，其中纺锤形细胞所占比例最大。心房肌细胞不是纺锤形，主要呈三角形和不规则形。常规培养的窦房结细胞中纺锤形细胞比例从纯化培养的窦房结细胞中的73.0%±2.9%降至44.7%±2.3%（P<0.01），不规则形细胞比例从纯化培养的窦房结细胞中的7.0%±1.7%增至36.1%±2.6%（P<0.01）。纯化培养和常规培养的窦房结细胞中三角形细胞比例分别为20.0%±2.1%和19.2%±2.5%（P>0.05）。共培养5天后，HE染色显示I型胶原纤维支架中有大量窦房结细胞，SEM显示细胞紧密粘附于支架表面和侧孔壁，细胞突起相互连接，或细胞通过伪足附着于支架侧孔壁。
通过准确的窦房结定位，包含差速贴壁技术和5-BrdU处理的纯化培养方法可提高纺锤形细胞比例，是一种可靠的窦房结细胞纯化及培养方法。窦房结细胞与I型胶原纤维支架具有良好的细胞相容性。
