新生SD大鼠心肌细胞原代培养方法的比较

Comparison of primary culture methods of neonatal SD rat cardiomyocytes

目的建立并比较新生SD大鼠心肌细胞的原代培养方法。方法采用胰酶法和胰酶+Ⅱ型胶原酶法分别消化心肌组织,两次90 min差速贴壁纯化心肌细胞,倒置显微镜下观察细胞形态变化,分别采用台盼蓝和免疫荧光染色评估心肌细胞的存活率和纯度。结果与胰酶法相比,胰酶+Ⅱ型胶原酶法可以获得形态良好且结构完整的心肌细胞;胰酶+Ⅱ型胶原酶法分离心肌细胞的获得率[(1.21±0.03)×10^(6)vs(0.65±0.02)×10^(6),P<0.01]和心肌细胞存活率[(93.37±0.40)%vs(86.40±0.36)%,P<0.01]明显高于胰酶法;两种方法获得的心肌细胞纯度均达到95%以上,但是胰酶+Ⅱ型胶原酶法获得心肌细胞所耗时间明显多于胰酶法[(14.00±0.40)h vs(4.59±0.10)h,P<0.01]。结论胰酶+Ⅱ型胶原酶法比胰酶法分离心肌细胞耗时长,但胰酶+Ⅱ型胶原酶法分离的心肌细胞获得率高、存活率高、细胞结构完整,是一种简单易行且高效的培养方法。

Objective To establish and compare the primary culture methods of neonatal SD rat cardiomyocytes.Methods Myocardial tissue was digested by trypsin method and trypsin+type Ⅱ collagenase method,respectively.Cardiomyocytes were purified with differential adhesion method with 90 minutes by twice.The morphological changes of cardiomyocytes were observed under inverted microscope.The survival rate and purity of cardiomyocytes were evaluated by trypan blue and immunofluorescence staining,respectively.Results Compared with trypsin method,trypsin+type Ⅱ collagenase method could obtain cardiomyocytes with good morphology and complete structure.The rate of isolation of cardiomyocytes by trypsin+type Ⅱ collagenase method is[(1.21±0.03)×10^(6) vs(0.65±0.02)×10^(6),P<0.01]and the survival rate of cardiomyocytes[(93.37±0.40)%vs(86.40±0.36)%,P<0.01]were significantly higher than trypsin method.The purity of cardiomyocytes obtained by the two methods was more than 95%,but the time of obtaining cardiomyocytes by trypsin+type Ⅱ collagenase method was significantly longer than that by trypsin method[(14.00±0.40)h vs(4.59±0.10)h,P<0.01].Conclusion The separation of cardiomyocytes by trypsin+type Ⅱ collagenase method takes longer than that by trypsin method,but the cardiomyocytes separated by trypsin+type Ⅱ collagenase method has high acquisition rate,high survival rate and complete cell structure.It is a simple,easy and efficient culture method.