中国组织工程研究
中國組織工程研究
중국조직공정연구
Journal of Clinical Rehabilitative Tissue Engineering Research
2014年
20期
3127-3132
,共6页
组织构建%组织工程%成年%腺病毒%多段式酶消化%心肌细胞%国家自然科学基金
組織構建%組織工程%成年%腺病毒%多段式酶消化%心肌細胞%國傢自然科學基金
조직구건%조직공정%성년%선병독%다단식매소화%심기세포%국가자연과학기금
myocytes,cardiac%adenoviruses,human%hypoxia-inducible factor 1,alpha subunit%anoxia%tissue engineering
背景：采用成年心肌细胞构建组织工程细胞已成为心脏疾病领域新的科研热点。<br> 目的：探讨简单、快捷的成年大鼠心肌细胞分离方法，并初步探索组织工程化成年心肌细胞的构建方式。<br> 方法：采用分段式酶消化方法消化成年大鼠心室肌心肌细胞。分别转染腺病毒和脂质体介导的红色荧光蛋白基因，倒置荧光显微镜、流式细胞仪检测组织工程细胞的构建效率。转染腺病毒介导的缺氧诱导因子1a，采用Western blot检测目标蛋白的表达。<br> 结果与结论：采用分段式酶消化方法可获得大量心肌细胞。流式细胞分析表明所获得的成年心肌细胞的存活率为(87.03±0.70)%。与脂质体转染相比(转染效率为0)，腺病毒感染效率高，为(70.31±1.39)%，荧光显微镜下细胞发出红色荧光。在腺病毒转染第4天后，可有效表达目标蛋白缺氧诱导因子1a。以上结果表明分段式酶消化法是成熟心肌细胞的快速分离方式，并明确重组腺病毒载体是转染心肌细胞的最佳基因载体。
揹景：採用成年心肌細胞構建組織工程細胞已成為心髒疾病領域新的科研熱點。<br> 目的：探討簡單、快捷的成年大鼠心肌細胞分離方法，併初步探索組織工程化成年心肌細胞的構建方式。<br> 方法：採用分段式酶消化方法消化成年大鼠心室肌心肌細胞。分彆轉染腺病毒和脂質體介導的紅色熒光蛋白基因，倒置熒光顯微鏡、流式細胞儀檢測組織工程細胞的構建效率。轉染腺病毒介導的缺氧誘導因子1a，採用Western blot檢測目標蛋白的錶達。<br> 結果與結論：採用分段式酶消化方法可穫得大量心肌細胞。流式細胞分析錶明所穫得的成年心肌細胞的存活率為(87.03±0.70)%。與脂質體轉染相比(轉染效率為0)，腺病毒感染效率高，為(70.31±1.39)%，熒光顯微鏡下細胞髮齣紅色熒光。在腺病毒轉染第4天後，可有效錶達目標蛋白缺氧誘導因子1a。以上結果錶明分段式酶消化法是成熟心肌細胞的快速分離方式，併明確重組腺病毒載體是轉染心肌細胞的最佳基因載體。
배경：채용성년심기세포구건조직공정세포이성위심장질병영역신적과연열점。<br> 목적：탐토간단、쾌첩적성년대서심기세포분리방법，병초보탐색조직공정화성년심기세포적구건방식。<br> 방법：채용분단식매소화방법소화성년대서심실기심기세포。분별전염선병독화지질체개도적홍색형광단백기인，도치형광현미경、류식세포의검측조직공정세포적구건효솔。전염선병독개도적결양유도인자1a，채용Western blot검측목표단백적표체。<br> 결과여결론：채용분단식매소화방법가획득대량심기세포。류식세포분석표명소획득적성년심기세포적존활솔위(87.03±0.70)%。여지질체전염상비(전염효솔위0)，선병독감염효솔고，위(70.31±1.39)%，형광현미경하세포발출홍색형광。재선병독전염제4천후，가유효표체목표단백결양유도인자1a。이상결과표명분단식매소화법시성숙심기세포적쾌속분리방식，병명학중조선병독재체시전염심기세포적최가기인재체。
BACKGROUND:Construction of tissue engineering adult cardiac myocytes has been a new research hotspot in cardiovascular fields. <br> OBJECTIVE:To explore a simple, fast method for the separation of adult rat cardiomyocytes, and preliminarily explore the construction methods of tissue engineering adult cardiac myocytes. <br> METHODS:Segmented enzymatic digestion method was used to isolate cardiac myocytes from adult rats. Subsequently, cardiac myocytes were transfected with adenovirus and liposome-mediated red fluorescent protein gene. Construction efficiency of tissue engineering cells was qualified using inverted fluorescence microscopy and flow cytometry. Final y, cardiac myocytes were transfected with adenovirus-mediated hypoxia-inducible factor-1a, and the expression of hypoxia-inducible factor-1a was examined by western blot analysis. <br> RESULTS AND CONCLUSION:A lot of cardiac myocytes were col ected using the segmented enzyme digestion method. Flow cytometric analysis showed that the survival of adult cardiomyocytes was (87.03±0.70)%. Compared with liposomal transfection (transfection efficiency was 0), adenovirus infection efficiency was (70.31± 1.39)%, and the cells expressed red fluorescence under fluorescence microscope. After 4 days of adenovirus infection, transfected cells expressed hypoxia-inducible factor-1a protein. These results showed that segmented enzyme digestion is a fast way to isolate adult cardiac myocytes, and recombinant adenovirus vector is a good vector to transfect cardiac myocytes.
