CAJ | 학술논문

目的：原代心肌细胞培养是一项重要的体外实验技术，为建造心血管疾病相关模型提供了重要的基础保障。文中旨在通过探讨不同消化时间对原代心肌细胞培养的影响，得到一套简便易行并且能获得较高成活率、纯度及活性的原代心肌细胞的培养方法。方法采用单消化酶多次消化心肌组织，根据消化时间分为8 min组和10 min组，差速贴壁法和5-溴-2-脱氧尿苷抑制法获得纯度较高的心肌细胞。光学显微镜下观察心肌细胞形态，记录镜下不同区域心肌细胞搏动频率测定细胞活力。用台盼蓝染色法检测培养细胞的存活率，横纹肌肌动蛋白鉴定心肌细胞。最后建立心肌细胞肥大模型，比较实验组（血管紧张素Ⅱ）和对照组[10％FBS DMEM （ H＋）培养基]的细胞截面平均面积。结果与8 min组比较，10 min组原代心肌细胞大部分伸出伪足相互接触交织成网，逐渐形成细胞簇，立体感明显，细胞搏动及收缩更为明显而有力。分离纯化后原代心肌细胞的平均存活率均＞90％，10 min组平均存活率显著高于8 min组[（95．4±0．8）％sv （93．0±0．8）％，P＜0．05]。α-actin免疫细胞化学染色鉴定，8 min组和10 min组原代心肌细胞的阳性率均＞95％。血管紧张素Ⅱ作用48 h后，10 min组原代心肌细胞实验组心房钠尿肽（atrial natriuretic peptide， ANP）、细胞截面平均面积较对照组表达均显著升高[（20．8±0．7） vs （15．8±0．5），（34．77±8．43）μm2 vs （14．11±4．29）μm2，P＜0．05]，而消化时间为8 min的原心肌细胞截面平均面积和ANP值未见显著变化。结论采用消化时间为8 min的原代心肌细胞培养方法具有简便、省时、易操作等特点，最重要的是能够获得较高质量的心肌细胞。
목적：원대심기세포배양시일항중요적체외실험기술，위건조심혈관질병상관모형제공료중요적기출보장。문중지재통과탐토불동소화시간대원대심기세포배양적영향，득도일투간편역행병차능획득교고성활솔、순도급활성적원대심기세포적배양방법。방법채용단소화매다차소화심기조직，근거소화시간분위8 min조화10 min조，차속첩벽법화5-추-2-탈양뇨감억제법획득순도교고적심기세포。광학현미경하관찰심기세포형태，기록경하불동구역심기세포박동빈솔측정세포활력。용태반람염색법검측배양세포적존활솔，횡문기기동단백감정심기세포。최후건립심기세포비대모형，비교실험조（혈관긴장소Ⅱ）화대조조[10％FBS DMEM （ H＋）배양기]적세포절면평균면적。결과여8 min조비교，10 min조원대심기세포대부분신출위족상호접촉교직성망，축점형성세포족，입체감명현，세포박동급수축경위명현이유력。분리순화후원대심기세포적평균존활솔균＞90％，10 min조평균존활솔현저고우8 min조[（95．4±0．8）％sv （93．0±0．8）％，P＜0．05]。α-actin면역세포화학염색감정，8 min조화10 min조원대심기세포적양성솔균＞95％。혈관긴장소Ⅱ작용48 h후，10 min조원대심기세포실험조심방납뇨태（atrial natriuretic peptide， ANP）、세포절면평균면적교대조조표체균현저승고[（20．8±0．7） vs （15．8±0．5），（34．77±8．43）μm2 vs （14．11±4．29）μm2，P＜0．05]，이소화시간위8 min적원심기세포절면평균면적화ANP치미견현저변화。결론채용소화시간위8 min적원대심기세포배양방법구유간편、성시、역조작등특점，최중요적시능구획득교고질량적심기세포。
Objective Primary myocardial cell culture , as an important technique for in vitro experiment , provides an essen-tial basis for the establishment of models of cardiovascular diseases .This study aimed to investigate the influence of digestion time on primary myocardial cell culture and to find some simple primary culture methods for obtaining myocardial cells with high survival rate , purity, and activity. Methods We used only one collagenase for repeated digestion of myocardial tissue for 8 and 10 minutes, re-spectively , and obtained high-purity cardiomyocytes by differential adhesion and Brdu inhibition .We observed the morphological chan-ges of the cardiomyocytes under the optical microscope , measured their vitality according to the beating frequency in different regions , and counted the living cells after typan blue staining. Finally, we established a model of myocyte hypertrophy for verification of cell activity and compared the cell surface area between the experimental ( angiotensinⅡ) and control ( 10%FBS DMEM [H +] medium ) groups. Results After cultured for 48 hours, the primary cardimyocytes in the 10 min group mostly extended pseudopodia , in-terwoven into a network and gradually forming cell clusters , with obviously powerful beating and contraction .After isolation and purifi-cation, the average survival rate of the cells was >90%, significantly higher in the 10 min group than in the 8 min ([95.4 ±0.8]%vs [93.0 ±0.8]%, P<0.05).The positive expression rate of cardiac α-actin was >95%in both of the groups.After cultured with angiotensinⅡfor 48 hours, the 10 min group showed a significantly increased level of atrial natriuretic peptide (ANP) (20.8 ±0.67 vs 15.8 ±0.48, P<0.05) and cell surface area ([34.77 ±8.43] μm2 vs [14.11 ±4.29] μm2, P<0.05) as compared with the 8 min group. Conclusion Primary culture of cardimyocytes with 8 min digestion is simple and time-saving, and what is most important , can obtain high-quality cardiomyocytes .