基于原子力显微术对干细胞分化与黑色素瘤细胞凋亡的研究
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摘要
细胞的分化是个体发育的一个重要阶段,胚层细胞的分化导致组织形成、器官发生和系统建成,细胞的异常分化会导致癌变。细胞的衰老和凋亡也与人体健康有着极为密切的关系。为了深入了解正常细胞的分化和肿瘤细胞的凋亡,探索细胞分化与凋亡的作用过程和机制,本研究以猪脂肪前体细胞、猪精原干细胞(SSCs)、小鼠黑色素瘤细胞B16为模型,采用原子力显微术(AFM)、激光共聚焦显微术(LSCM)、流式细胞术(FCS)、透射电子显微术(TEM)、CCK-8法及Western-blot等方法,对分化及凋亡过程中细胞的表面形貌、内部结构、力学性能和相关蛋白进行检测,获得了以下具有创新性的成果：
1.深入探讨脂肪前体细胞向成熟脂肪细胞分化前后细胞在纳米水平的变化,应用AFM扫描发现细胞核周围出现孔洞,膜表面变得光滑,粗糙度减小颗粒大小降低,膜表面粘附力由(146.33+11.23)PN降低到(119.95+8.67)PN。硬度与杨氏模量方面也降低了约20%,分化后的成熟脂肪细胞膜流动性更强,更易变形。本研究首次获得脂肪前体细胞分化前后细胞的超微结构和力学性能,为理解体内能量平衡和肥胖疾病的发生及诊断提供新的实验依据。
2.首次运用AFM研究SSCs的多能性,分析SSCs向脂肪细胞分化后细胞形貌及内部结构、分子的变化。成脂诱导后,SSCs由立体球形变为扁平不规则的圆盘状,贴壁性能提高,内部出现大量脂滴,膜表面粗糙度及颗粒大小增加。粘附力由(47.31士19.75)PN增加至(64.64±1.44)PN,硬度、杨氏模量均升高。TEM扫描得到细胞内部线粒体、内质网及空泡大量增多。完全分化后,成熟脂肪细胞内部特异性分子PPARy与C/EBPa出现。总结来说,我们从定性及定量两个角度表征了SSCs成脂分化所产生的一系列变化。
3.以B16细胞为研究对象分析替莫唑胺(TMZ)对其致凋亡作用,发现TMZ对能有效抑制B16细胞的生长,50μMTMZ作用细胞时,凋亡率达到最大为34.6%。随着药物浓度的增加,细胞膜及细胞核都产生不可逆损伤,细胞膜电位、内部Ca2+浓度和Caspase-3蛋白含量也发生相应的变化。此工作首次推断TMZ对B16细胞膜和核的毒性,可能是药物造成细胞死亡的一个机制。
综上所述,本课题采用原子力显微术为基础对细胞形貌、超微结构及力学性能变化进行探测,结合生物技术手段对分化及凋亡过程表征,可以直观的看到在这两个重要阶段中细胞的变化,为生命活动的深入研究提供方法和依据。
The cellular differentiation is an important stage in individual development and growth. The differentiation of endoderm cell leads to tissue and system formation and organogenesis. The abnormal differentiation of cell can cause cancer. Senescence and apoptosis of cell also has a very close relationship with human health. In order to better understand and explore the process and mechanism of normal cell differentiation and tumor cell apoptosis, we use atomic force microscopy (AFM), laser scanning confocal microscopy (LSCM), flow cytometry (FCS), transmission electron microscopy (TEM), CCK-8, western-blot and other methods to study the cells. The surface morphology, internal structure, mechanical properties and the related protein of adipocyte precursor cells, porcine spermatogonial stem cells and mouse melanoma cells were detected during the process of cell differentiation or apoptosis. The following results were obtained:
1. The changes of surface structure of adipocyte precursor cells were examined during differentiation. There were some holes around the nucleus. The cellular surface became smooth and the roughness of the membrane and the particle size were all decreased. The adhesion force was reduced from (146.33±11.23) PN to (119.95±8.67) PN. The stiffness and Young's modulus of cellular membrane were also reduced about 20%. The results indicate that mature adipocytes are more fluid and stronger deformation. This is the first time to obtain the changes of cellular ultrastructure and mechanical properties after differentiation of adipocyte precursor cells and the results provide new experimental evidence to understand the energy balance and obesity occurrence and diagnosis.
2. The pluripotent of SSCs was firstly studied by AFM. After the SSCs differentiated to adipocytes, the roughness, particle size, adhesion force, stiffness and Young's modulus of the membrane were all increased. The cellular morphology was changed from round spheroid to long spindle. The number of intracellular mitochondria, endoplasmic and vacuoles were also increased to provide energy, which was required with the formation of adipocytes. After the complete differentiation, the mature adipocytes'specific molecule PPARy and C/EBPa appeared. These qualitative and quantitative results can improve understanding of stem cells pluripotent.
3. After analysis of temozolomide (TMZ) on the apoptosis of B16 cells, the results demonstrated that TMZ could significantly suppress the proliferation of B16 cells in a dose-dependent manner. The TMZ concentration of 0-50μM induced apoptosis and 100-200μM induced cell death. With the concentration of TMZ increase, changes in morphology and nucleus were irreversibly damaged. The membrane potential, intercellular Ca2+ concentration and caspase-3 protein content were accordingly changed. Our study firstly deduced that the cellular membrane and nucleus toxicity may be the mechanism of B16 cell apoptosis.
In summary, we use AFM to detect cell morphology, ultrastructure and mechanical properties. Then combined with biotechnology, we characterized the process of differentiation and apoptosis. These visual data provide basis to in-depth study of life activities.
1.深入探讨脂肪前体细胞向成熟脂肪细胞分化前后细胞在纳米水平的变化,应用AFM扫描发现细胞核周围出现孔洞,膜表面变得光滑,粗糙度减小颗粒大小降低,膜表面粘附力由(146.33+11.23)PN降低到(119.95+8.67)PN。硬度与杨氏模量方面也降低了约20%,分化后的成熟脂肪细胞膜流动性更强,更易变形。本研究首次获得脂肪前体细胞分化前后细胞的超微结构和力学性能,为理解体内能量平衡和肥胖疾病的发生及诊断提供新的实验依据。
2.首次运用AFM研究SSCs的多能性,分析SSCs向脂肪细胞分化后细胞形貌及内部结构、分子的变化。成脂诱导后,SSCs由立体球形变为扁平不规则的圆盘状,贴壁性能提高,内部出现大量脂滴,膜表面粗糙度及颗粒大小增加。粘附力由(47.31士19.75)PN增加至(64.64±1.44)PN,硬度、杨氏模量均升高。TEM扫描得到细胞内部线粒体、内质网及空泡大量增多。完全分化后,成熟脂肪细胞内部特异性分子PPARy与C/EBPa出现。总结来说,我们从定性及定量两个角度表征了SSCs成脂分化所产生的一系列变化。
3.以B16细胞为研究对象分析替莫唑胺(TMZ)对其致凋亡作用,发现TMZ对能有效抑制B16细胞的生长,50μMTMZ作用细胞时,凋亡率达到最大为34.6%。随着药物浓度的增加,细胞膜及细胞核都产生不可逆损伤,细胞膜电位、内部Ca2+浓度和Caspase-3蛋白含量也发生相应的变化。此工作首次推断TMZ对B16细胞膜和核的毒性,可能是药物造成细胞死亡的一个机制。
综上所述,本课题采用原子力显微术为基础对细胞形貌、超微结构及力学性能变化进行探测,结合生物技术手段对分化及凋亡过程表征,可以直观的看到在这两个重要阶段中细胞的变化,为生命活动的深入研究提供方法和依据。
The cellular differentiation is an important stage in individual development and growth. The differentiation of endoderm cell leads to tissue and system formation and organogenesis. The abnormal differentiation of cell can cause cancer. Senescence and apoptosis of cell also has a very close relationship with human health. In order to better understand and explore the process and mechanism of normal cell differentiation and tumor cell apoptosis, we use atomic force microscopy (AFM), laser scanning confocal microscopy (LSCM), flow cytometry (FCS), transmission electron microscopy (TEM), CCK-8, western-blot and other methods to study the cells. The surface morphology, internal structure, mechanical properties and the related protein of adipocyte precursor cells, porcine spermatogonial stem cells and mouse melanoma cells were detected during the process of cell differentiation or apoptosis. The following results were obtained:
1. The changes of surface structure of adipocyte precursor cells were examined during differentiation. There were some holes around the nucleus. The cellular surface became smooth and the roughness of the membrane and the particle size were all decreased. The adhesion force was reduced from (146.33±11.23) PN to (119.95±8.67) PN. The stiffness and Young's modulus of cellular membrane were also reduced about 20%. The results indicate that mature adipocytes are more fluid and stronger deformation. This is the first time to obtain the changes of cellular ultrastructure and mechanical properties after differentiation of adipocyte precursor cells and the results provide new experimental evidence to understand the energy balance and obesity occurrence and diagnosis.
2. The pluripotent of SSCs was firstly studied by AFM. After the SSCs differentiated to adipocytes, the roughness, particle size, adhesion force, stiffness and Young's modulus of the membrane were all increased. The cellular morphology was changed from round spheroid to long spindle. The number of intracellular mitochondria, endoplasmic and vacuoles were also increased to provide energy, which was required with the formation of adipocytes. After the complete differentiation, the mature adipocytes'specific molecule PPARy and C/EBPa appeared. These qualitative and quantitative results can improve understanding of stem cells pluripotent.
3. After analysis of temozolomide (TMZ) on the apoptosis of B16 cells, the results demonstrated that TMZ could significantly suppress the proliferation of B16 cells in a dose-dependent manner. The TMZ concentration of 0-50μM induced apoptosis and 100-200μM induced cell death. With the concentration of TMZ increase, changes in morphology and nucleus were irreversibly damaged. The membrane potential, intercellular Ca2+ concentration and caspase-3 protein content were accordingly changed. Our study firstly deduced that the cellular membrane and nucleus toxicity may be the mechanism of B16 cell apoptosis.
In summary, we use AFM to detect cell morphology, ultrastructure and mechanical properties. Then combined with biotechnology, we characterized the process of differentiation and apoptosis. These visual data provide basis to in-depth study of life activities.
引文
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