第一部分 成人心肌组织块培养法获取心肌干细胞 目的 自成人心肌组织采用组织块培养法获得具有心肌干细胞形态特征的小、圆、亮细胞。 方法 手术中取下的非心肌病患者心脏组织标本，剪碎后以0.2%胰蛋白酶及0.1% Ⅳ型胶原酶消化。将剩余组织块在CEM (Complete explant medium)培养基中进行培养，以倾斜去液及培养液递增法促进组织块贴壁，之后2～3天换液一次，培养约3～4周获得成纤维细胞层之上的小、圆、亮细胞。结果 心肌组织块消化后培养，约1周左右贴壁并开始有成纤维细胞爬出，3～4周可在组织块周围形成成纤维细胞层，并有小、圆、亮细胞在成纤维细胞层上由组织块爬出。 结论 通过组织块培养法可以由成人心肌组织培养出具有心肌干细胞形态特征的小、圆、亮细胞。 【关键词】心肌干细胞；组织块培养法；小、圆、亮细胞 第二部分 胎鼠成纤维细胞(MEF)的培养及条件培养基的制备 目的 采用单细胞悬液法及组织块培养法获得胎鼠成纤维细胞，通过观察细胞形态及绘制生长曲线比较两种方法，并通过该细胞制备条件培养基。 方法 取12.5～14.5天的孕小鼠解剖获得胚胎，分离后切除头、内脏(所有深色的东西)及四肢。单细胞悬液法：将组织剪碎，以0.05%胰蛋白酶/0.53mmol/L EDTA消化30分钟，直到有很少的组织物残留。以含血清的培养基终止消化后离心获得细胞，加入培养皿中以DMEM/F12培养，2～3天换液一次。组织块培养法：将组织剪成0.5～1㎜³的小块，以0.05%胰蛋白酶/0.53mmol/L EDTA溶液吹打消化约3～5min，以等体积含血清培养基终止消化。弃去上清液。将组织块接种于培养皿中培养。当培养皿中的细胞长到80%～90%融合时以0.025%胰蛋白酶/0.53mmol/L EDTA消化传代。当细胞传至第3代并生长达到80%左右融合时吸去原培养基，加入心球生长培养基CGM (Cardiosphere-growing medium)用于培养胎鼠成纤维细胞48小时以溶解来自成纤维细胞的旁分泌因子，培养后的培养基与CGM以3:1混合，通过滤网过滤除菌后再添加bFGF (5ng/ml)，即获得 CCGM (Conditioned cardiosphere-growing medium)。 结果 1.胎鼠成纤维细胞生长形态观察：单细胞悬液法12小时可见成纤维细胞贴壁并伸展成为梭形，细胞核为圆形，胞质透明，透光性较好。3～4天后可传代。组织块培养法48小时可见少许细胞自组织块周围游离出来，散在的细胞簇也已贴壁，细胞呈梭形为主，细胞生长缓慢，需要7～8天才能传代。2.细胞生长曲线检测：单细胞悬液法培养的细胞第3～5天进入对数生长期，第7天达到顶峰，经过平台期后，缓慢下降。组织块培养法培养的细胞开始生长缓慢，在第4～8天进入对数生长期，以后随着细胞的增多，形成接触抑制，细胞增殖缓慢下降。3.通过CGM培养48小时胎鼠成纤维细胞，可获得条件培养基，并可用于后续干细胞的培养。 结论 通过单细胞悬液法及组织块培养法均成功获得胎鼠成纤维细胞并可以培养传代。单细胞悬液法获得细胞周期短且不易老化，优于组织块培养法。以其制备的CCGM可以用来培养分离获得的心肌干细胞。 【关键词】胎鼠成纤维细胞；单细胞悬液法；组织块培养法；生长曲线；条件培养基 第三部分 成人心肌干细胞体外分离纯化方法及培养条件的研究 目的 1.分别比较直接培养法和磁珠分选法对于酶消化法及缓冲液冲洗法所获得细胞培养的差异。 2.研究对于心肌干细胞，CGM、CCGM何者更适合作为其培养基。 方法 1.酶消化法：分别以0.53mmol/L EDTA处理细胞1～2分钟，0.05%胰蛋白酶/0.53mmol/L EDTA溶液处理细胞2～3分钟，加入等量含血清培养基终止消化后收集液以40μm滤网过滤后，离心获取细胞；缓冲液冲洗法：通过D-Hanks液在灭菌后的相差倒置显微镜观察下冲洗组织块培养皿以获取小、圆、亮细胞，收集液以40μm滤网过滤后离心获取细胞；直接培养法：将获取的细胞直接接种于PDL包被的培养板中培养；磁珠分选法：将酶消化法或缓冲液冲洗法获取的细胞以anti human c-kit磁珠结合后通过autoMACS进行细胞分选纯化。 2.分别采用酶消化法+直接培养，缓冲液冲洗法+直接培养，酶消化法+磁珠分选，缓冲液冲洗法+磁珠分选四种不同的方法获得并处理小、圆、亮细胞，并分别在CGM、CCGM中培养，选取不同的时间点观察细胞的生长形态及形成心球样细胞团的能力。 结果 1.采用酶消化法+直接培养，所获得的细胞中杂细胞较多，开始在CGM和CCGM中培养细胞状态无明显差异，小、圆、亮细胞3天左右贴壁，观察2周可见成纤维细胞增殖活跃，CCGM培养中少量小、圆、亮细胞增殖，观察3周未见形成心球样细胞团结构；2.缓冲液冲洗法+直接培养，获得细胞数量少于酶消化法+直接培养，但细胞纯度略高，状态略好。小、圆、亮细胞3天左右贴壁，观察2周可见CCGM培养中少量小、圆、亮细胞出现分裂增殖，观察3周未见形成心球样细胞团结构。3.采用酶消化法+磁珠分选细胞纯度高，在CGM中培养，小、圆、亮细胞3天左右贴壁，2周少量增殖，未见形成心球样细胞团结构，在CCGM中培养，细胞2.5天左右贴壁，2周少量增殖，3周左右可见有多细胞簇形成，但继续培养不能形成心球样细胞团结构。4.缓冲液冲洗法+磁珠分选，细胞2天左右贴壁，在CGM中培养，2周左右开始增殖，但增殖缓慢，未见形成心球样细胞团结构。在CCGM中培养，1.5周左右开始增殖并逐渐形成心球样细胞团结构。 结论 四种方法均可获得小、圆、亮细胞，酶消化法+直接培养和缓冲液冲洗法+直接培养因杂细胞较多，且易贴壁增殖的成纤维细胞影响了目的细胞的生长。但缓冲液冲洗法较酶消化法获得的细胞更易贴壁。酶消化法+磁珠分选和缓冲液冲洗法+磁珠分选细胞纯度高，缓冲液冲洗法较酶消化法获得的细胞分选后更易贴壁和增殖且CCGM较CGM更适合心肌干细胞的生长增殖。因此缓冲液冲洗法+磁珠分选是分离及纯化心肌干细胞的较好方法，所获得的心肌干细胞更适合用CCGM培养。 【关键词】酶消化法；缓冲液冲洗法；磁珠分选；心球样细胞团 第四部分 成人心肌干细胞的鉴定及分化潜能的体外研究 目的 1.通过流式细胞仪鉴定免疫磁珠分选获得的成人心肌干细胞体外培养后的表面标记特征。2.通过细胞免疫荧光法检测c-kit⁺心肌干细胞的分化潜能。 方法 1.将磁珠分选获得的成人心肌干细胞在CCGM中培养，待其增殖至一定数量(约10⁵～10⁶)后，通过温和胰蛋白酶(0.025%胰蛋白酶/0.53mmol/LEDTA)消化收集细胞。将不同荧光抗体标记的细胞通过流式细胞仪鉴定其表面标记： c-kit、CD45、CD34、CD133、CD31、ABCG2、SSEA-4。2.利用多聚赖氨酸包被的盖玻片制作细胞爬片，通过固定、漂洗、透膜、封闭、一抗孵育、荧光二抗反应、制片、封片等步骤后，在荧光显微镜下观察GATA 4、Nkx 2.5、以及Flk-1/KDR的细胞阳性率。 结果 流式细胞检测结果表明磁珠分选后培养获得的细胞表面特征为c-kit⁺、 CD45⁻、CD34⁻、CD133⁻、CD31(4.46%±0.51%)、ABCG2 (25.86%±0.31%)、 SSEA-4 (11.56%+1.25%)部分阳性。细胞免疫荧光检测可见体外培养的c-kit⁺心肌干细胞中有部分细胞表达GATA 4 (52.63%±5.84%)、Nkx 2.5 (49.11%± 4.99%)、或 Flk-1/KDR (41.10%±4.33%)。 结论 c-kit磁珠分选获得的成人心肌干细胞体外培养后仍能保持干细胞c-kit的表面标记，且不具有造血干细胞、内皮祖细胞等的表面标记，证实了其心脏起源；其他干细胞标记(ABCG2、SSEA-4)的部分阳性说明其可能是具有多向分化潜能的。细胞免疫荧光结果证实该干细胞具有心脏早期的特征性的转录因子(GATA 4、Nkx 2.5)及早期内皮细胞标记(Flk-1/KDR)表明其可能进入了向心肌细胞及内皮细胞分化的途径。通过本实验的方法成功获得的细胞为一种成人心脏来源的多向分化潜能的干细胞，即心肌干细胞。 【关键词】流式细胞鉴定；细胞免疫荧光；分化潜能；转录因子

 Part 1 Using Explant Culture Technique to Obtain Cardiac Stem Cells from Adult Human Heart Objective: Using explant culture technique to obtain cardiac stem cells (CSCs) with the morphological characters of small, round and phase-bright from adult human heart. Methods: Adult human heart tissues were obtained in the operation from patients without cardiomyopathy. Tissues were minced into small pieces, and subjected to enzymatic dissociation with a mixture of 0.2% trypsin and 0.1% collagenase Ⅳ. After treatment, the remaining tissue fragments were cultured as explants in CEM. Also, culture dish inclining and a small amount medium were used to promote explants' adhesion. The medium was replaced every 3 days after the explants adhere to the surface of culture dish. Results: The tissue explants treated with enzymatic dissociation were adhered to the surface of culture dish after 4-5 days and the fibroblasts began to growth nearby the explants in 1 week's culture. At 3-4 weeks after explanting of minced human heart tissue, a layer of fibroblasts covered the culture dish, and small, round, phase-bright cells with different size migrated from the adherent explants. Conclusion: Our study has shown that the cardiac stem cells with morphological characters of small, round and phase-bright could be obtained from adult human heart by explant culture technique. Key words: Cardiac stem cells; Explant culture technique; Small, round and phase-bright cell Part 2 The Culture of Mouse Embryonic Fibroblasts and Preparation of Conditioned Medium Objective: Single cell suspension and explant cultural method could be used to isolate and culture mouse embryonic fibroblasts (MEF). This study aimed to compare the different effects of the two methods on cellular morphology, growth curve and prepare conditioned medium by exposure CGM to MEF culture. Methods: The pregnant mice for 12.5-14.5 days were killed for uterus but the head, visceral organs and four legs were then discarded. The remained trunk was cut into 1㎜³ tissue masses. The detailed method for isolating and culturing was as follows : 1.Single cell suspension method: The explants were subjected to enzymatic dissociation with 0.05% trypsin/0.53mM EDTA for 30 minutes and blown slightly until nearly no explants left, use medium with FBS to terminate digest reaction. Cells were centrifuged for 5 minutes at 1000 rpm. Supernatant was removed, and then 5 ml DMEM/F12 medium was added. After blowing uniformly dense, the cells were incubated in culture flask. 2. Explant cultural method: The explants were removed into the pipette. The explants were trypsinized with 1 ml digestive of 0.05% trypsin/0.53mM EDTA for 3-5 minutes. The explants were blown slightly. 1 ml of medium (with 10% FBS) were drawn into the pipette. The supernatant was removed and precipitate was left. The explants were drawn into culture flasks each 0.5 ㎝. Use culture dish inclining to promote explants' adhesion, sufficiently complete medium was added after explants' adhesion and medium was refreshed after 3 days. The different effects of two methods were assessed by cellular morphology and growth curve. 3. The DMEM/F12 medium was discarded when MEF passed to the 3rd generation and achieve 80% density. Cardiosphere-growing medium-CGM was conditioned by exposing to fibroblast culture for 48 hours, in order to dissolve paracrine factors from fibroblasts. The conditioned medium (CCGM) was mixed with fresh CGM medium at the ratio 3:1, sterilized by filtration, and supplemented with additional bFGF at 5 ng/ml. Results: 1. MEF cellular morphology: MEF cells isolated by cell suspension adhered to the surface of the culture dish and unfolded to fusiform. They had round nucleus and clearing cytoplasm and should be passed after 3-4 days. MEF cells isolated by explant cultural method emerged at day 2 and grew slowly. The cells shape are fusiform and could be passed after 7-8 days . 2. Curve of growth: The logarithmic phases of MEF cells isolated by single cell suspension method reached at logarithmic phase after 3-5 days, and cells by explant cultural method were 4-8 day. 3. We harvested CCGM by exposing CGM to MEF culture. Conclusion: We have successfully cultured MEF by both single cell suspension method and explant cultural method, but single cell suspension method could culture MEF for preparing CCGM in a shorter period and showed not prone to aging. By exposing CGM to MEF culture, we could harvest both CCGM and culture CSCs. Key words: Mouse embryonic fibroblasts; Single cell suspension method; Explant cultural method; Growth curve; Conditioned medium Part 3 The Study of Different Isolation, Purity Methods and Culture Condition of Cardiac Stem Cells from Adult Human Heart Objective: 1.Comparison of enzymatic dissociation + direct culture, buffer solution irrg+direct culture, enzymatic dissociation +magnetic bead sorting and buffer solution irrg+magnetic bead sorting , to observe the growth and the ability of forming clone by different methods. 2. Which medium is better for cardiac stem cells culture: CGM or CCGM ? Methods: 1. Enzymatic dissociation method: small, round, phase-bright cells were collected by pooling two washes with Ca²⁺ Mg²⁺ free PBS, one wash with0.53 mmol/L EDTA (1 to 2 minutes), and one wash with 0.5g/L trypsin and 0.53 mmol/L EDTA (2 to 3 minutes) at room temperature under visual control. The suspension was filtered through a 40-μm cell strainer .The cells were collected by centrifugalization ; Buffer solution irrg method: Harvest small, round, phase-bright cells by washing explants culture dish with D-Hanks under visual control. The washing liquid was filtered through a 40-μm cell strainer .The cells were collected by centrifugalization ; Direct culture: The cells collected by enzymatic dissociation and buffer solution irrg were seeded directly at multiwell plates precoated Poly-D-Lysine without purity. Magnetic bead sorting method: To sorted c-kit⁺ cells from collected cells by positive selection with anti-c-kit microbeads using a magnetic cell sorter device from Miltenyi Biotec. 2. Isolation and purity small, round, phase-bright cells by four different methods: Enzymatic dissociation + direct culture, buffer solution irrg + direct culture, enzymatic dissociation + magnetic bead sorting and buffer solution irrg + magnetic bead sorting. The cells were cultured in CGM or CCGM. The cellular morphology and ability to form clone were observed at different time point. Result: 1. A great number of fibroblasts were observed after enzymatic dissociation + direct culture. At the early stage, the growth state of small, round, phase-bright cells in CGM and CCGM was showed similar. The small, round, phase-bright cells adhered to the surface of the culture dish after around 3 days. After 2 weeks' culture, the fibroblasts occupied most of the culture surface and affected the growth of phase-bright cells due to active proliferation of fibroblasts. Only small amounts of phase-bright cells proliferated in CCGM. No cardiosphere was found after 3 weeks' culture in CGM and CCGM. 2. The quantity of collected cells by buffer solution irrg was less than by enzymatic dissociation and the intervention of fibroblasts also decreased. The small, round, phase-bright cells adhered to the surface of the culture dish at about 3 days both in CGM and CCGM. Some phase-bright cells could proliferate in CCGM but no cardiosphere formed after 3 weeks' culture in CGM and CCGM. 3. By the means of enzymatic dissociation + magnetic bead sorting, we could enrich very purity cardiac stem cells from collected cells. The small, round, phase-bright cells adhered to the surface of the culture dish at about 3 days in CGM and showed proliferation of a small quantity after 2 weeks' culture. No cardiosphere formed after 3 weeks in CGM. In CCGM phase-bright cells adhered to the surface of the culture dish at about 2.5 days and proliferated after 2 weeks' culture. Some cells were found to proliferate into small cluster at 3 weeks, but the cluster could not form cardiospheres in three-dimension. 4. The cells enriched by buffer solution irrg + magnetic sorting could adhere to the surface of the culture dish at about 2 days. In CGM the cell growth was similar to the cells enriched by enzymatic dissociation+ magnetic bead sorting. But in CCGM the sorted c-kit⁺ cells could proliferate at 1.5 weeks and form cardiospheres in three-dimension slowly. Conclusion: We could successfully harvest small, round, phase-bright cells through four methods. But fibroblasts affected the growth of small, round, phase-bright cells in direct culture methods. After magnetic sorting, we could enrich very purity cardiac stem cells from collected cells. The ability of cells' adherence and proliferation was better after buffer solution irrg than after enzymatic dissociation. Compared to CGM medium, the presence of CCGM medium improved the growth of c-kit⁺ cells. Therefore, buffer solution irrg + magnetic bead sorting and CCGM medium were a suitable protocol for the culture of c-kit⁺ cardiac stem cells. Key word: Enzymatic dissociation method; Buffer solution irrg method; Magnetic bead sorting method; Cardiospheres Part 4 Study on The Phenotype Characteristics and Differentiation Potential of c-kit⁺ Cardiac Stem Cells in Vitro Objective: 1. Identification the phenotype characteristics of c-kit⁺ cardiac stem cells cultured in vitro by flow cytometry. 2. Detecting differentiation potential of c-kit⁺ cardiac stem cells by cell immunofluorescence analyses. Methods: 1. The c-kit⁺ cells enriched by magnetic bead sorting were cultured in CCGM. When the quantity of cells reached 10⁵-10⁶, mild trypsin/EDTA (0.025%trypsin/0.53mmol/L EDTA) was used to digest and collect cells. After immunostain with a panel of antibodies including c-kit, CD45, CD34, CD133, CD31, ABCG2 and SSEA-4 or isotype controls respectively, the phenotype characteristics of cells were identified by flow cytometry. 2. Seeding single cell suspension on glass slides was precoated by Poly-D-Lysine. Primary antibodies specific for GATA 4, Nkx 2.5, and Flk-1/KDR for cultured slides were used in immunocytochemistry staining. Isotype-matched antibodies were used as control. Nuclei were counterstained with DAPI. Preparing samples as follow protocol: fixing, rinsing, penetrating cell membrane, blockage, immunostaining, counterstaining and mounting. Staining was observed by fluorescence microscope. Results: 1. The phenotype characteristics of sorted c-kit⁺ cells were identified by flow cytometry. They were included c-kit⁺, CD45⁻, CD34⁻, CD133⁻. Marker expression analysis indicated that c-kit⁺ cells also expressed another stem cell marker including ABCG2 (25.86% ±0.31%) 、 SSEA-4 (11.56% ± 1.25%) . The profile further suggested that a minority of cells expressed CD31 (4.46% ±0.51%) markers. 2. By immunofluorescence microscopy, phenotypic analysis of cultured c-kit⁺ cells revealed expression of GATA 4, Nkx 2.5 and Flk-1/KDR. The percentage of GATA 4, Nkx 2.5 and Flk-l/KDR positive cells were approximately 52.63%±5.84%, 49.11%±4.99% and 41.10% ±4.33%, respectively. Conclusion: The results demonstrated that the cells express stem cell marker (c-kit) but lack of CD45, CD34 and CD133, the markers for hematopoietic stem cells (HSCs) and endothelial progenitor cells (EPCs). These suggested c-kit⁺ cells that we cultured in vitro were cardiac origining. Aditional, the expressing of cardiac specific transcription marker (GATA 4, Nkx 2.5) and early endothelial cell marker (EC) indicated that c-kit⁺ cells origining from cardiospheres had entered a differentiation pathway toward a cardiomyocyte and endothelial cell phenotype. Therefore, we have successfully isolated and cultured cardiac stem cells (CSCs) with multi-directional differentiated potential from adult human heart. Key word: Flow cytometry identification; Cell immunofluorescence; Differentiation potential; Transcription marker