基于原子力顯微術(shù)對(duì)干細(xì)胞分化與黑色素瘤細(xì)胞凋亡的研究
發(fā)布時(shí)間：2018-10-15 07:24
【摘要】：細(xì)胞的分化是個(gè)體發(fā)育的一個(gè)重要階段,胚層細(xì)胞的分化導(dǎo)致組織形成、器官發(fā)生和系統(tǒng)建成,細(xì)胞的異常分化會(huì)導(dǎo)致癌變。細(xì)胞的衰老和凋亡也與人體健康有著極為密切的關(guān)系。為了深入了解正常細(xì)胞的分化和腫瘤細(xì)胞的凋亡,探索細(xì)胞分化與凋亡的作用過程和機(jī)制,本研究以豬脂肪前體細(xì)胞、豬精原干細(xì)胞(SSCs)、小鼠黑色素瘤細(xì)胞B16為模型,采用原子力顯微術(shù)(AFM)、激光共聚焦顯微術(shù)(LSCM)、流式細(xì)胞術(shù)(FCS)、透射電子顯微術(shù)(TEM)、CCK-8法及Western-blot等方法,對(duì)分化及凋亡過程中細(xì)胞的表面形貌、內(nèi)部結(jié)構(gòu)、力學(xué)性能和相關(guān)蛋白進(jìn)行檢測(cè),獲得了以下具有創(chuàng)新性的成果： 1.深入探討脂肪前體細(xì)胞向成熟脂肪細(xì)胞分化前后細(xì)胞在納米水平的變化,應(yīng)用AFM掃描發(fā)現(xiàn)細(xì)胞核周圍出現(xiàn)孔洞,膜表面變得光滑,粗糙度減小顆粒大小降低,膜表面粘附力由(146.33+11.23)PN降低到(119.95+8.67)PN。硬度與楊氏模量方面也降低了約20%,分化后的成熟脂肪細(xì)胞膜流動(dòng)性更強(qiáng),更易變形。本研究首次獲得脂肪前體細(xì)胞分化前后細(xì)胞的超微結(jié)構(gòu)和力學(xué)性能,為理解體內(nèi)能量平衡和肥胖疾病的發(fā)生及診斷提供新的實(shí)驗(yàn)依據(jù)。 2.首次運(yùn)用AFM研究SSCs的多能性,分析SSCs向脂肪細(xì)胞分化后細(xì)胞形貌及內(nèi)部結(jié)構(gòu)、分子的變化。成脂誘導(dǎo)后,SSCs由立體球形變?yōu)楸馄讲灰?guī)則的圓盤狀,貼壁性能提高,內(nèi)部出現(xiàn)大量脂滴,膜表面粗糙度及顆粒大小增加。粘附力由(47.31士19.75)PN增加至(64.64±1.44)PN,硬度、楊氏模量均升高。TEM掃描得到細(xì)胞內(nèi)部線粒體、內(nèi)質(zhì)網(wǎng)及空泡大量增多。完全分化后,成熟脂肪細(xì)胞內(nèi)部特異性分子PPARy與C/EBPa出現(xiàn)�？偨Y(jié)來說,我們從定性及定量?jī)蓚�(gè)角度表征了SSCs成脂分化所產(chǎn)生的一系列變化。 3.以B16細(xì)胞為研究對(duì)象分析替莫唑胺(TMZ)對(duì)其致凋亡作用,發(fā)現(xiàn)TMZ對(duì)能有效抑制B16細(xì)胞的生長(zhǎng),50μMTMZ作用細(xì)胞時(shí),凋亡率達(dá)到最大為34.6%。隨著藥物濃度的增加,細(xì)胞膜及細(xì)胞核都產(chǎn)生不可逆損傷,細(xì)胞膜電位、內(nèi)部Ca2+濃度和Caspase-3蛋白含量也發(fā)生相應(yīng)的變化。此工作首次推斷TMZ對(duì)B16細(xì)胞膜和核的毒性,可能是藥物造成細(xì)胞死亡的一個(gè)機(jī)制。 綜上所述,本課題采用原子力顯微術(shù)為基礎(chǔ)對(duì)細(xì)胞形貌、超微結(jié)構(gòu)及力學(xué)性能變化進(jìn)行探測(cè),結(jié)合生物技術(shù)手段對(duì)分化及凋亡過程表征,可以直觀的看到在這兩個(gè)重要階段中細(xì)胞的變化,為生命活動(dòng)的深入研究提供方法和依據(jù)。
[Abstract]:Differentiation of cells is an important stage of ontogeny. The differentiation of embryonic cells leads to tissue formation, organogenesis and system development, and abnormal differentiation of cells leads to carcinogenesis. Cell aging and apoptosis are also closely related to human health. In order to understand the differentiation of normal cells and apoptosis of tumor cells, and to explore the process and mechanism of differentiation and apoptosis, the model of melanoma cell B16 of porcine adipose precursor cells and porcine spermatogonial stem cells (SSCs) of (SSCs), mice was used in this study. Atomic force microscopy (AFM), (AFM), laser confocal microscopy (AFM), (LSCM), flow cytometry (LSCM), transmission electron microscopy (FCS), transmission electron microscopy (TEM), CCK-8) and Western-blot were used to detect the surface morphology, internal structure, mechanical properties and related proteins of cells during differentiation and apoptosis. The following innovative results have been obtained: 1. The changes of adipose progenitor cells before and after differentiation into mature adipocytes were studied. AFM scanning showed that there were holes around the nucleus, the membrane surface became smooth, and the roughness decreased. Surface adhesion force of membrane decreased from (146.33 11.23) PN to (119.95 8.67) PN. The hardness and Young's modulus also decreased by about 20%, and the mature adipose cell membrane was more fluidity and deformation after differentiation. In this study, the ultrastructure and mechanical properties of adipose precursor cells before and after differentiation were obtained for the first time, which provided a new experimental basis for understanding the energy balance in vivo and the occurrence and diagnosis of obesity diseases. 2. The pluripotency of SSCs was studied by AFM for the first time, and the morphology, internal structure and molecular changes of SSCs after differentiation into adipocytes were analyzed. After fat-forming, SSCs changed from a three-dimensional sphere to a flat and irregular disk, and the adhesion property was improved, a large number of lipid droplets appeared inside the membrane, and the surface roughness and particle size of the membrane were increased. The adhesion force increased from (47.31 鹵19.75) PN to (64.64 鹵1.44) PN, hardness, and Young's modulus increased. Mitochondria, endoplasmic reticulum and vacuoles were obtained by TEM scanning. After full differentiation, specific molecules PPARy and C/EBPa appeared in mature adipocytes. In conclusion, we characterize a series of changes in adipogenic differentiation of SSCs from both qualitative and quantitative perspectives. The apoptotic effect of temozolidomide (TMZ) on B16 cells was analyzed. It was found that TMZ could effectively inhibit the growth of B16 cells. When 50 渭 MTMZ treated the cells, the apoptotic rate reached the maximum of 34.6%. With the increase of drug concentration, the cell membrane and nucleus were irreversibly damaged. The cell membrane potential, the concentration of Ca2 and the content of Caspase-3 protein also changed correspondingly. This work for the first time inferred that the toxicity of TMZ to the cell membrane and nucleus of B16 may be a mechanism of cell death caused by drugs. In conclusion, the changes of cell morphology, ultrastructure and mechanical properties were detected by atomic force microscopy (AFM), and the differentiation and apoptosis process were characterized by biotechnology. The changes of cells in these two important stages can be seen intuitively, which provides the method and basis for the further study of life activities.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R739.5
本文編號(hào)：2271796
[Abstract]:Differentiation of cells is an important stage of ontogeny. The differentiation of embryonic cells leads to tissue formation, organogenesis and system development, and abnormal differentiation of cells leads to carcinogenesis. Cell aging and apoptosis are also closely related to human health. In order to understand the differentiation of normal cells and apoptosis of tumor cells, and to explore the process and mechanism of differentiation and apoptosis, the model of melanoma cell B16 of porcine adipose precursor cells and porcine spermatogonial stem cells (SSCs) of (SSCs), mice was used in this study. Atomic force microscopy (AFM), (AFM), laser confocal microscopy (AFM), (LSCM), flow cytometry (LSCM), transmission electron microscopy (FCS), transmission electron microscopy (TEM), CCK-8) and Western-blot were used to detect the surface morphology, internal structure, mechanical properties and related proteins of cells during differentiation and apoptosis. The following innovative results have been obtained: 1. The changes of adipose progenitor cells before and after differentiation into mature adipocytes were studied. AFM scanning showed that there were holes around the nucleus, the membrane surface became smooth, and the roughness decreased. Surface adhesion force of membrane decreased from (146.33 11.23) PN to (119.95 8.67) PN. The hardness and Young's modulus also decreased by about 20%, and the mature adipose cell membrane was more fluidity and deformation after differentiation. In this study, the ultrastructure and mechanical properties of adipose precursor cells before and after differentiation were obtained for the first time, which provided a new experimental basis for understanding the energy balance in vivo and the occurrence and diagnosis of obesity diseases. 2. The pluripotency of SSCs was studied by AFM for the first time, and the morphology, internal structure and molecular changes of SSCs after differentiation into adipocytes were analyzed. After fat-forming, SSCs changed from a three-dimensional sphere to a flat and irregular disk, and the adhesion property was improved, a large number of lipid droplets appeared inside the membrane, and the surface roughness and particle size of the membrane were increased. The adhesion force increased from (47.31 鹵19.75) PN to (64.64 鹵1.44) PN, hardness, and Young's modulus increased. Mitochondria, endoplasmic reticulum and vacuoles were obtained by TEM scanning. After full differentiation, specific molecules PPARy and C/EBPa appeared in mature adipocytes. In conclusion, we characterize a series of changes in adipogenic differentiation of SSCs from both qualitative and quantitative perspectives. The apoptotic effect of temozolidomide (TMZ) on B16 cells was analyzed. It was found that TMZ could effectively inhibit the growth of B16 cells. When 50 渭 MTMZ treated the cells, the apoptotic rate reached the maximum of 34.6%. With the increase of drug concentration, the cell membrane and nucleus were irreversibly damaged. The cell membrane potential, the concentration of Ca2 and the content of Caspase-3 protein also changed correspondingly. This work for the first time inferred that the toxicity of TMZ to the cell membrane and nucleus of B16 may be a mechanism of cell death caused by drugs. In conclusion, the changes of cell morphology, ultrastructure and mechanical properties were detected by atomic force microscopy (AFM), and the differentiation and apoptosis process were characterized by biotechnology. The changes of cells in these two important stages can be seen intuitively, which provides the method and basis for the further study of life activities.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R739.5
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