脂肪干细胞作为生物起搏器种子细胞的筛选研究
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摘要
背景：随着分子生物学和细胞生物学的发展,尤其是近年来对干细胞研究的不断深化,生物起搏心脏的梦想已不再遥远。目前已有众多研究通过转染不同的起搏基因到种子细胞中,然后再将其移植到心脏模型中,从而使其表现出异位起搏活动,这些研究为成功构建生物起搏器迈出了第一步。构建生物起搏器有两个重要因素,一个是要找到好的种子细胞,另一个是选择理想的起搏基因。由于骨髓间充质干细胞(bone marrow stromal cells, BMSCs)是最早发现的成体干细胞,对其研究也最多,因此以往大多数学者将其作为构建生物起搏器的种子细胞,而对种子细胞本身的研究却至今无人涉及。目前对于生物起搏器研究的重点则被更多的放在了起搏基因的选择搭配上。现阶段,对心脏的起搏的主要认识均集中在内向超极化电流(起搏电流,If),而HCN(超极化激活的环状核苷酸调控通道的基因家族)是编码主要起搏电流的基因,其在膜超极化时被激活。HCN有4种亚型,其中3种(HCN1,HCN2,HCN4)在心脏表达,这其中最受关注的基因当属HCN2。但随着近两年来对窦房结区域的研究逐步深入,结果发现HCN4是窦房结和房室结中表达最丰富的亚型,HCN4不仅对产生正常起搏电位和基础心率至关重要,而且也参与交感神经对心率的调节作用,因此,HCN4越来越受到生物起搏研究者的重视。蓦然回首,生物起搏研究从开始出现已有10余年,但至今为止一直未有较大突破,体内外研究均证实转染HCN基因作为生物起搏在动物模型上是一种可行的实验方法,但目前的体内实验都没有超过6周,这可能与研究者所选用的基因载体有关,因此选择合适的载体有待进一步研究。近年来,脂肪干细胞(Adipose tissue derived mesenchymal stem cells, ADSCs)越来越受到人们的关注,其已经广泛应用到脂肪、骨、软骨、心脏、神经的组织工程研究中,人们推测它将成为一种更好的种子细胞。但是,目前对其自身的属性还不十分清楚,是否每一种属的ADSCs都适合作为种子细胞呢?它们彼此之间有何优势?对此我们并无统一认识。因此,进一步研究不同种属ADSCs的生物学特征便显得越发重要,藉此能够筛选出适合心脏组织工程的种子细胞。进而,再将该ADSCs与同种属BMSCs相比较,尤其是对比其在构建生物起搏器中各自的优劣,从而找到理想的生物起搏器种子细胞。
目的：本研究比较来源于大鼠、犬、人ADSCs在形态学、表面标志物和多向分化潜能,明确三种ADSCs的基本生理学差异。然后,通过比较体外扩增能力、多向分化能力、起搏基因质粒pcDNA3.0-EGFP-hHCN4转染效率和基因转染后凋亡水平的差异,找到适合心脏组织工程和生物起搏工程ADSCs。然后,将其与同种属的BMSCs进行对比研究,再次比较两种干细胞的成心肌细胞分化能力、起搏基因HCN2和HCN4的表达水平,明确其作为心脏组织工程和生物起搏工程种子细胞的基本属性。最后再次比较质粒pcDNA3.0-EGFP-hHCN4的转染效率、凋亡水平以及基因转染后hHCN4 mRNA表达水平和随时间延长衰减的程度,从而找到理想的生物起搏器种子细胞。
方法：通过对来源于大鼠、犬、人的脂肪组织进行分离培养,获取各种ADSCs。光镜下观察每种ADSCs的形态。经过流式细胞仪检测每种ADSCs的表面抗原,明确差异,然后,将其分别向成脂和成骨诱导分化,分别进行红油O和碱性磷酸酶染色,证实其干细胞的属性。通过CCK-8检测绘制生长曲线,比较不同ADSCs的体外倍增时间。对不同种属ADSCs进行成脂、成骨和成心肌诱导4周,分别通过红油O和碱性磷酸酶染色以及cTnl免疫荧光染色比较各种属ADSCs成脂、成骨和成心肌能力,找到适合心脏组织工程的ADSCs。构建pcDNA3.0-EGFP-hHCN4质粒,并进行双酶切来鉴定克隆的正确性。将重组质粒用电穿孔法转染三个种属的ADSCs,分别比较转染效率及转染后ADSCs凋亡水平,从而进一步找到适合生物起搏器研究的ADSCs。然后,将初步筛选出的ADSCs与同种属的BMSCs进行对比,光学显微镜下比较不同代数ADSCs和BMSCs的形态学,CCK-8检测绘制生长曲线从而比较增殖速度,通过流式细胞仪技术检测表面标志物,同时给予成脂及成骨诱导,从而确定其为BMSCs。然后进行细胞周期检测,进一步精确了解两种干细胞经多次传代后的增殖特性,通过免疫荧光染色比较两种干细胞在不同代数的成心肌分化率,然后,使用Real-time PCR技术了解不同代数的大鼠ADSCs和BMSCs在HCN2和HCN4基因上的表达水平,再用western blot法检测向心肌细胞诱导分化后HCN2和HCN4蛋白含量,从而验证Real-time PCR的结果。将pcDNA3.0-EGFP-hHCN4质粒分别转染不同代数的大鼠ADSCs和BMSCs,比较转染效率以及转染后细胞凋亡水平,此后,再通过Real-time PCR来检测HCN4基因导入后的mRNA表达水平及2周后的衰减程度。
结果：1.大鼠、犬、人ADSCs在形态学上无明显差异,均似成纤维细胞。流式细胞仪检测CD29,CD44,CD73均呈阳性表达,而CD34呈阴性表达。同时,CD29,CD44在三个种属的ADSCs表达水平无显著差异,而CD73表达量却存在较大的变异性。人ADSCs表面抗原CD73的表达水平较高,而在动物的ADSCs中表达水平较低。三种来源的ADSCs均能够向成脂和成骨分化,从而进一步证实其为ADSCs。
2.人、犬、大鼠三种ADSCs经体外培养均能迅速扩增,并且其速度无明显差异。大鼠ADSCs与其他两种ADSCs相比有着更强的成脂和成心肌分化能力,犬ADSCs则具有更强的成骨分化能力。构建了重组质粒pcDNA3.0-EGFP-hHCN4,通过EcoRI(?)口XBal双酶切后电泳分析,质粒pcDNA3.0-EGFP-hHCN4可见3700bp条带,与预期的目的基因大小一致。经电穿孔转染后,荧光显微镜下可见转染后的干细胞呈绿色荧光,三种ADSCs转染效率在30-40%之间,无明显的差异。对成功转染质粒的ADSCs进行凋亡检测,人ADSCs的凋亡率明显高于其它两种ADSCs (p<0.05)。
3.大鼠ADSCs和BMSCs在形态和表面抗原的表达上无明显差异,通过CCK-8绘制生长曲线,提示第5代和第10代大鼠ADSCs在体外均有较高的增殖能力。流式细胞仪检测细胞周期,大鼠ADSCs更多的处于S期,证实其自我复制的能力较BMSCs强,而且其高速增殖能力一直持续至第10代。对大鼠ADSCs和BMSCs向成心肌方向诱导,ADSCs向心肌细胞分化率比BMSCs高。质粒pcDNA3.0-EGFP-hHCN4分别转染大鼠ADSCs和BMSCs,第5代大鼠ADSCs和BMSCs均有较高的转染效率,而第10代大鼠BMSCs质粒的转染效率明显降低,同时,基因转染后大鼠BMSCs凋亡水平均高于同代数的ADSCs。Real-time PCR提示同代数大鼠ADSCs和BMSCs的HCN2 mRNA表达水平基本相同,第5代大鼠ADSCs的HCN4 mRNA表达水平与同代数BMSCs相同,但第10代BMSCs的HCN4 mRNA表达却较ADSCs明显降低,蛋白定量检测证实了上述结果。质粒pcDNA3.0-EGFP-hHCN4转染后,两种干细胞均检测到hHCN4 mRNA表达,第10代ADSCs和BMSCs表达水平较第5代低,并且所有第5代、第10代大鼠ADSCs和BMSCs在hHCN4基因转染2周后,其自身表达量出现明显的衰减,但以BMSCs衰减的程度更重。
结论：人、犬、大鼠三种ADSCs均能通过标准方法获得,其均具有较强的多向分化能力。大鼠ADSCs向心肌细胞分化率较高,HCN4基因转染后细胞凋亡的水平低,是较为优秀的种子细胞。进一步与大鼠BMSCs相比,其更具有体外扩增能力强,HCN4基因转染效率高,转染后mRNA表达幅度高、衰减慢以及转染后细胞凋亡水平低等诸多优势,最终确定大鼠ADSCs较BMSCs更适合作为构建生物起搏器的种子细胞。
Backgrouds:Recent advances in molecular and cell biology and in tissue engineering technologies have paved the way to develope a new and exciting field in biological pacemaker. Many researches used the method by carrying different genes to seed cells to build biological pacemaker. They transplanted seed cells transferred pacemaker channel into heart model and made it showing ectopic pacing activities. These studies took the first step toward successful constructing biological pacemaker. Thus, choose good seed cells and an ideal pacing gene are particularly important. Because bone marrow stem cells are the first adult stem cells to be found in the world, many scholars choose it as seed cells to build biological pacemakers. More focused on the choice of pacing related genes. The most popular genes are HCN2. In recent decades, with the in-depth study on sinus node, HCN4 become more and more attractive. So far, there was no large breakthrough about building biological pacemaker. The reason is probably that there are not ideal seed cells and genetic combinations. In other words, seed cells may be the most important factor in building biological pacemakers. In recent years, Adipose tissue derived mesenchymal stem cells (ADSCs) are more and more concerned. People speculate that it will become a kind of better seed cells. But, whether each species ADSCs are suitable to serve as the seed cells? Therefore, further study about biological characteristics of ADSCs in different genus and became important. In addition, we compared the biological features between ADSCs and BMSCs, especially in constructing biological pacemaker. At last we could find the most ideal biological pacemakers seeding cells.
Objective:The aim of this study was to analyze the differences in proliferation, phenotype and differentiate capacity of ADSCs from three different species respectively. A comparative study of cultured rat, canine and human ADSCs were carried out, and the main morphological parameters, proliferative activity, expression of surface markers, multi-differentiate capacity, gene transfection efficiency and apoptosis rate were characterized. Then, we compared BMSCs and ADSCs cultured under identical conditions with respect to culture characteristics, proliferation capacity, cell cycle, multi-differentiation potential, cardiomyogenic differentiation capacity, gene transfection efficiency, apoptosis rate, gene and protein expression of HCN2 and HCN4 in BMSCs and ADSCs in vitro at different passages. We also studied the relationship between HCN2 and HCN4 mRNA level and time index after gene transfection. So we could find out which are suitable seed cells in biological pacemakers study.
Methods:Adipose-derived stem cells (ADSCs) were isolated with standard methods. To further study the proliferation and multi-differentiation potentials of ADSCs, their growth curves were achieved with cck-8. Surface protein expression was analyzed by flow cytometry and the multi-lineage potential of ADSCs were testified by differentiating cells with adipogenic and osteogenic inducers. Alkaline phosphatase (ALP) staining and Oil-red O staining were carried out 28 days later. After transfecting the three kinds of ADSCs with plasmid of pacemaker gene pcDNA3.0-EGFP-hHCN4, we compared the gene transfection efficiency and apoptosis rate. Then we extensively evaluated the differences between rat BMSCs and ADSCs in culture characteristics, proliferation capacity and cardiomyogenic differentiation capacity. The expression of cardiac pacing-related genes, including hyperpolarization-activated cyclic nucleotide-gated 2 (HCN2) and HCN4 were detected by realtime-PCR. Western blot was used to detect the corresponding protein expression. The cell apoptosis rate and gene transfection efficiency were detected by flow cytometry after transfecting plasmid. At last, we detected the expression of HCN4 mRNA 2 weeks after transfecting plasmid.
Results: 1. The results showed that no significant difference was observed for the morphology of ADSCs under light microscope. We examined the expression of CD29, CD44, CD73 and CD34 of three species ADSCs. CD29, CD44 and CD73 were positive in expression, while CD34 was negative. The expression level of CD29 and CD44 had no significant differences among three ADSCs, but the percentage of CD73 was detected with great variability. CD73 was expressed highly in human ADSCs, but the expressions in canine's ADSCs were much lower. In this study, the cells were able to differentiate into adipocytes and osteoblasts in vitro, strongly suggesting that the cells we obtained were ADSCs.
2. The proliferation capacity was similar in all animals. Rat ADSCs showed higher adipogenic and cardiomyogenic ability and dog showed higher osteogenic ability. There were a 3700bp balteum and a 5400bp balteum in pcDNA3.0 hHCN4 after cutting by EcoRI and XBaI, which was identical with the size of the objective gene. Many cells gave out green fluorescent after treated by pcDNA3.0-EGFP-hHCN4, the carrying efficiency between 30-40%. The apoptosis rate of human ADSCs was much higher than the other two species ADSCs.
3. CCK-8 test found group showed that ADSCs possess high self-propagating potential. Cell circle stage was measured by flow cytometry assays. The ratio of S phase of rat ADSCs was higher than BMSCs. The capacity of ADSCs differentiated into cardiomyocytes was significantly higher than that of BMSC; meanwhile, ADSCs retained the high cardiac differentiation capacity up to passage 10. However, the cardiomyogenic differentiation capacity of BMSCs decreased significantly at passage 10. We found that the gene transfection efficiency declined in BMSCs at passage 10 and the apoptosis rate of BMSCs was higher than ADSCs at the same passage. We also found the protein expression levels of HCN2 and HCN4 were higher in the ADSCs than BMSCs at the same passage. Meanwhile, the expression levels of these proteins decreased in BMSCs at passage 10. In all groups, we detected the expression of HCN4 mRNA after treating by pcDNA3.0-EGFP-hHCN4. The transfer efficiency of ADSCs and BMSCs at passage 5 was significantly higher than ADSCs and BMSCs at passage 10. Two weeks after transferring with pcDNA3.0-EGFP-hHCN4, the hHCN4 mRNA expression decreased in ADSCs and BMSCs at passage 10, but the decreasing level was higher in BMSCs.
Conclusion:We could obtain ADSCs from human, canine and rat by standard method. ADSCs have strong multiplex differentiation capacity. Because rat ADSCs have higher cardiomyogenic differentiation ability, but the apoptosis rate was lower. Therefore, ADSCs maybe good kind of seeding cells. After further compared with BMSCs, ADSCs have higher self-propagating potential, gene transfection efficiency, HCN4 mRNA expression level and lower apoptosis rate. We concluded that rat ADSCs were more suitable for biological pacemakers study as seeding cells.
目的：本研究比较来源于大鼠、犬、人ADSCs在形态学、表面标志物和多向分化潜能,明确三种ADSCs的基本生理学差异。然后,通过比较体外扩增能力、多向分化能力、起搏基因质粒pcDNA3.0-EGFP-hHCN4转染效率和基因转染后凋亡水平的差异,找到适合心脏组织工程和生物起搏工程ADSCs。然后,将其与同种属的BMSCs进行对比研究,再次比较两种干细胞的成心肌细胞分化能力、起搏基因HCN2和HCN4的表达水平,明确其作为心脏组织工程和生物起搏工程种子细胞的基本属性。最后再次比较质粒pcDNA3.0-EGFP-hHCN4的转染效率、凋亡水平以及基因转染后hHCN4 mRNA表达水平和随时间延长衰减的程度,从而找到理想的生物起搏器种子细胞。
方法：通过对来源于大鼠、犬、人的脂肪组织进行分离培养,获取各种ADSCs。光镜下观察每种ADSCs的形态。经过流式细胞仪检测每种ADSCs的表面抗原,明确差异,然后,将其分别向成脂和成骨诱导分化,分别进行红油O和碱性磷酸酶染色,证实其干细胞的属性。通过CCK-8检测绘制生长曲线,比较不同ADSCs的体外倍增时间。对不同种属ADSCs进行成脂、成骨和成心肌诱导4周,分别通过红油O和碱性磷酸酶染色以及cTnl免疫荧光染色比较各种属ADSCs成脂、成骨和成心肌能力,找到适合心脏组织工程的ADSCs。构建pcDNA3.0-EGFP-hHCN4质粒,并进行双酶切来鉴定克隆的正确性。将重组质粒用电穿孔法转染三个种属的ADSCs,分别比较转染效率及转染后ADSCs凋亡水平,从而进一步找到适合生物起搏器研究的ADSCs。然后,将初步筛选出的ADSCs与同种属的BMSCs进行对比,光学显微镜下比较不同代数ADSCs和BMSCs的形态学,CCK-8检测绘制生长曲线从而比较增殖速度,通过流式细胞仪技术检测表面标志物,同时给予成脂及成骨诱导,从而确定其为BMSCs。然后进行细胞周期检测,进一步精确了解两种干细胞经多次传代后的增殖特性,通过免疫荧光染色比较两种干细胞在不同代数的成心肌分化率,然后,使用Real-time PCR技术了解不同代数的大鼠ADSCs和BMSCs在HCN2和HCN4基因上的表达水平,再用western blot法检测向心肌细胞诱导分化后HCN2和HCN4蛋白含量,从而验证Real-time PCR的结果。将pcDNA3.0-EGFP-hHCN4质粒分别转染不同代数的大鼠ADSCs和BMSCs,比较转染效率以及转染后细胞凋亡水平,此后,再通过Real-time PCR来检测HCN4基因导入后的mRNA表达水平及2周后的衰减程度。
结果：1.大鼠、犬、人ADSCs在形态学上无明显差异,均似成纤维细胞。流式细胞仪检测CD29,CD44,CD73均呈阳性表达,而CD34呈阴性表达。同时,CD29,CD44在三个种属的ADSCs表达水平无显著差异,而CD73表达量却存在较大的变异性。人ADSCs表面抗原CD73的表达水平较高,而在动物的ADSCs中表达水平较低。三种来源的ADSCs均能够向成脂和成骨分化,从而进一步证实其为ADSCs。
2.人、犬、大鼠三种ADSCs经体外培养均能迅速扩增,并且其速度无明显差异。大鼠ADSCs与其他两种ADSCs相比有着更强的成脂和成心肌分化能力,犬ADSCs则具有更强的成骨分化能力。构建了重组质粒pcDNA3.0-EGFP-hHCN4,通过EcoRI(?)口XBal双酶切后电泳分析,质粒pcDNA3.0-EGFP-hHCN4可见3700bp条带,与预期的目的基因大小一致。经电穿孔转染后,荧光显微镜下可见转染后的干细胞呈绿色荧光,三种ADSCs转染效率在30-40%之间,无明显的差异。对成功转染质粒的ADSCs进行凋亡检测,人ADSCs的凋亡率明显高于其它两种ADSCs (p<0.05)。
3.大鼠ADSCs和BMSCs在形态和表面抗原的表达上无明显差异,通过CCK-8绘制生长曲线,提示第5代和第10代大鼠ADSCs在体外均有较高的增殖能力。流式细胞仪检测细胞周期,大鼠ADSCs更多的处于S期,证实其自我复制的能力较BMSCs强,而且其高速增殖能力一直持续至第10代。对大鼠ADSCs和BMSCs向成心肌方向诱导,ADSCs向心肌细胞分化率比BMSCs高。质粒pcDNA3.0-EGFP-hHCN4分别转染大鼠ADSCs和BMSCs,第5代大鼠ADSCs和BMSCs均有较高的转染效率,而第10代大鼠BMSCs质粒的转染效率明显降低,同时,基因转染后大鼠BMSCs凋亡水平均高于同代数的ADSCs。Real-time PCR提示同代数大鼠ADSCs和BMSCs的HCN2 mRNA表达水平基本相同,第5代大鼠ADSCs的HCN4 mRNA表达水平与同代数BMSCs相同,但第10代BMSCs的HCN4 mRNA表达却较ADSCs明显降低,蛋白定量检测证实了上述结果。质粒pcDNA3.0-EGFP-hHCN4转染后,两种干细胞均检测到hHCN4 mRNA表达,第10代ADSCs和BMSCs表达水平较第5代低,并且所有第5代、第10代大鼠ADSCs和BMSCs在hHCN4基因转染2周后,其自身表达量出现明显的衰减,但以BMSCs衰减的程度更重。
结论：人、犬、大鼠三种ADSCs均能通过标准方法获得,其均具有较强的多向分化能力。大鼠ADSCs向心肌细胞分化率较高,HCN4基因转染后细胞凋亡的水平低,是较为优秀的种子细胞。进一步与大鼠BMSCs相比,其更具有体外扩增能力强,HCN4基因转染效率高,转染后mRNA表达幅度高、衰减慢以及转染后细胞凋亡水平低等诸多优势,最终确定大鼠ADSCs较BMSCs更适合作为构建生物起搏器的种子细胞。
Backgrouds:Recent advances in molecular and cell biology and in tissue engineering technologies have paved the way to develope a new and exciting field in biological pacemaker. Many researches used the method by carrying different genes to seed cells to build biological pacemaker. They transplanted seed cells transferred pacemaker channel into heart model and made it showing ectopic pacing activities. These studies took the first step toward successful constructing biological pacemaker. Thus, choose good seed cells and an ideal pacing gene are particularly important. Because bone marrow stem cells are the first adult stem cells to be found in the world, many scholars choose it as seed cells to build biological pacemakers. More focused on the choice of pacing related genes. The most popular genes are HCN2. In recent decades, with the in-depth study on sinus node, HCN4 become more and more attractive. So far, there was no large breakthrough about building biological pacemaker. The reason is probably that there are not ideal seed cells and genetic combinations. In other words, seed cells may be the most important factor in building biological pacemakers. In recent years, Adipose tissue derived mesenchymal stem cells (ADSCs) are more and more concerned. People speculate that it will become a kind of better seed cells. But, whether each species ADSCs are suitable to serve as the seed cells? Therefore, further study about biological characteristics of ADSCs in different genus and became important. In addition, we compared the biological features between ADSCs and BMSCs, especially in constructing biological pacemaker. At last we could find the most ideal biological pacemakers seeding cells.
Objective:The aim of this study was to analyze the differences in proliferation, phenotype and differentiate capacity of ADSCs from three different species respectively. A comparative study of cultured rat, canine and human ADSCs were carried out, and the main morphological parameters, proliferative activity, expression of surface markers, multi-differentiate capacity, gene transfection efficiency and apoptosis rate were characterized. Then, we compared BMSCs and ADSCs cultured under identical conditions with respect to culture characteristics, proliferation capacity, cell cycle, multi-differentiation potential, cardiomyogenic differentiation capacity, gene transfection efficiency, apoptosis rate, gene and protein expression of HCN2 and HCN4 in BMSCs and ADSCs in vitro at different passages. We also studied the relationship between HCN2 and HCN4 mRNA level and time index after gene transfection. So we could find out which are suitable seed cells in biological pacemakers study.
Methods:Adipose-derived stem cells (ADSCs) were isolated with standard methods. To further study the proliferation and multi-differentiation potentials of ADSCs, their growth curves were achieved with cck-8. Surface protein expression was analyzed by flow cytometry and the multi-lineage potential of ADSCs were testified by differentiating cells with adipogenic and osteogenic inducers. Alkaline phosphatase (ALP) staining and Oil-red O staining were carried out 28 days later. After transfecting the three kinds of ADSCs with plasmid of pacemaker gene pcDNA3.0-EGFP-hHCN4, we compared the gene transfection efficiency and apoptosis rate. Then we extensively evaluated the differences between rat BMSCs and ADSCs in culture characteristics, proliferation capacity and cardiomyogenic differentiation capacity. The expression of cardiac pacing-related genes, including hyperpolarization-activated cyclic nucleotide-gated 2 (HCN2) and HCN4 were detected by realtime-PCR. Western blot was used to detect the corresponding protein expression. The cell apoptosis rate and gene transfection efficiency were detected by flow cytometry after transfecting plasmid. At last, we detected the expression of HCN4 mRNA 2 weeks after transfecting plasmid.
Results: 1. The results showed that no significant difference was observed for the morphology of ADSCs under light microscope. We examined the expression of CD29, CD44, CD73 and CD34 of three species ADSCs. CD29, CD44 and CD73 were positive in expression, while CD34 was negative. The expression level of CD29 and CD44 had no significant differences among three ADSCs, but the percentage of CD73 was detected with great variability. CD73 was expressed highly in human ADSCs, but the expressions in canine's ADSCs were much lower. In this study, the cells were able to differentiate into adipocytes and osteoblasts in vitro, strongly suggesting that the cells we obtained were ADSCs.
2. The proliferation capacity was similar in all animals. Rat ADSCs showed higher adipogenic and cardiomyogenic ability and dog showed higher osteogenic ability. There were a 3700bp balteum and a 5400bp balteum in pcDNA3.0 hHCN4 after cutting by EcoRI and XBaI, which was identical with the size of the objective gene. Many cells gave out green fluorescent after treated by pcDNA3.0-EGFP-hHCN4, the carrying efficiency between 30-40%. The apoptosis rate of human ADSCs was much higher than the other two species ADSCs.
3. CCK-8 test found group showed that ADSCs possess high self-propagating potential. Cell circle stage was measured by flow cytometry assays. The ratio of S phase of rat ADSCs was higher than BMSCs. The capacity of ADSCs differentiated into cardiomyocytes was significantly higher than that of BMSC; meanwhile, ADSCs retained the high cardiac differentiation capacity up to passage 10. However, the cardiomyogenic differentiation capacity of BMSCs decreased significantly at passage 10. We found that the gene transfection efficiency declined in BMSCs at passage 10 and the apoptosis rate of BMSCs was higher than ADSCs at the same passage. We also found the protein expression levels of HCN2 and HCN4 were higher in the ADSCs than BMSCs at the same passage. Meanwhile, the expression levels of these proteins decreased in BMSCs at passage 10. In all groups, we detected the expression of HCN4 mRNA after treating by pcDNA3.0-EGFP-hHCN4. The transfer efficiency of ADSCs and BMSCs at passage 5 was significantly higher than ADSCs and BMSCs at passage 10. Two weeks after transferring with pcDNA3.0-EGFP-hHCN4, the hHCN4 mRNA expression decreased in ADSCs and BMSCs at passage 10, but the decreasing level was higher in BMSCs.
Conclusion:We could obtain ADSCs from human, canine and rat by standard method. ADSCs have strong multiplex differentiation capacity. Because rat ADSCs have higher cardiomyogenic differentiation ability, but the apoptosis rate was lower. Therefore, ADSCs maybe good kind of seeding cells. After further compared with BMSCs, ADSCs have higher self-propagating potential, gene transfection efficiency, HCN4 mRNA expression level and lower apoptosis rate. We concluded that rat ADSCs were more suitable for biological pacemakers study as seeding cells.
引文
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