人脂肪組織與脂肪瘤組織來源間充質(zhì)干細胞生物學(xué)特性比較
發(fā)布時間：2018-09-02 09:39
【摘要】：目的:1.通過膠原酶消化法提取、分離人脂肪與脂肪瘤來源間充質(zhì)干細胞并培養(yǎng),對提取培養(yǎng)的細胞進行初步鑒定;2.對分離培養(yǎng)的人脂肪與脂肪瘤來源間充質(zhì)干細胞的生物學(xué)特性進行對比,研究兩種細胞的不同,有助于脂肪瘤間充質(zhì)干細胞的進一步研究,并對探討脂肪瘤發(fā)生的相關(guān)機制提供重要基礎(chǔ)。方法:將同等體積的脂肪及脂肪瘤組織經(jīng)膠原酶充分消化,對兩種間充質(zhì)干細胞進行分離、培養(yǎng)及擴增,鏡下觀察人脂肪間充質(zhì)干細胞(human adipose derived mesenchymal stem cells,hASCs)及人脂肪瘤間充質(zhì)干細胞(human lipoma derived mesenchymal stem cells,hLMSCs)的細胞形態(tài)及生長情況。經(jīng)成脂成骨誘導(dǎo)后,對培養(yǎng)至14天后的hASCs及hLMSCs油紅O染色及茜素紅染色,觀察hASCs及hLMSCs成脂成骨分化程度。流式細胞儀檢測細胞免疫表型C34、CD45、CD90及CD133的表達情況,觀察兩種干細胞表面標志物表達情況。CCK8檢測hASCs及hLMSCs的增殖情況。實時定量PCR檢測hASCs及hLMSCs中HMGA2的表達量的不同。結(jié)果:1.通過膠原酶消化法分離提取了人脂肪及脂肪瘤來源的間充質(zhì)干細胞。在MSCs培養(yǎng)基中培養(yǎng)時發(fā)現(xiàn):(1)原代細胞開始貼壁時間:hASCs及hLMSCs均于24小時內(nèi)完成貼壁;(2)提取數(shù)量及首次融合時間:原代細胞提取后發(fā)現(xiàn)每毫升人脂肪瘤組織中提取的干細胞數(shù)多于人脂肪組織中提取的干細胞數(shù),但hASCs及hLMSCs首次融合的時間基本一致,即6d~8d細胞即可達到80%融合,并向一定方向生長;(3)原代培養(yǎng)的hASCs及hLMSCs兩種細胞均成長梭形,并出現(xiàn)旋渦狀排列生長;(4)兩種細胞在成骨成脂誘導(dǎo)后均可分化為脂肪細胞及成骨細胞。(5)流式細胞儀檢測hASCs及hLMSCs表面標志物CD45及CD90的表達量,表達類似。表明:hASCs及hLMSCs具有相似的特性;2.CCK8法檢測、對比hASCs及hLMSCs的增殖能力,實驗結(jié)果表明于96h~144h hLMSCs增殖能力明顯高于hASCs。流式細胞儀檢測hASCs及hLMSCs細胞表面標志物CD34與CD133表達情況。hASCs中CD34與CD133表達分別為2.14%及3.17%,hLMSCs中CD34與CD133表達分別為20.62%及16.04%。實時定量PCR檢測hASCs及hLMSCs組的HMGA2表達量變化,可見hLMSCs組HMGA2表達量顯著高于hASCs組表達量。結(jié)論:1.運用膠原酶消化法成功將hASCs及hLMSCs分離、培養(yǎng)及擴增,hASCs及hLMSCs均為貼壁生長,誘導(dǎo)后均具有成脂成骨能力,具有多向分化潛能,表達相似的細胞表面標志物,該兩種細胞具有相似的生物學(xué)特性;2.表面標志物表達量及HMGA2表達量不同,對探討脂肪瘤發(fā)生發(fā)展及良、惡性腫瘤發(fā)生發(fā)展提供重要基礎(chǔ)。
[Abstract]:Purpose 1. Mesenchymal stem cells derived from human adipose and lipoma were isolated and cultured by collagenase digestion. The biological characteristics of mesenchymal stem cells derived from lipoma and human adipose cells were compared, and the differences between the two types of cells were studied, which was helpful to the further study of mesenchymal stem cells of lipoma. It provides an important basis for exploring the mechanism of lipoma. Methods: two kinds of mesenchymal stem cells were isolated, cultured and expanded by collagenase digestion in the same volume of fat and lipoma tissue. The morphology and growth of human adipose mesenchymal stem cells (human adipose derived mesenchymal stem cells,hASCs) and human lipoma mesenchymal stem cells (human lipoma derived mesenchymal stem cells,hLMSCs) were observed under microscope. After induced by adipogenic osteogenesis, the degree of hASCs and hLMSCs adipogenic osteogenesis was observed by hASCs and hLMSCs oil red O staining and alizarin red staining after cultured for 14 days. Flow cytometry was used to detect the expression of CD45, CD90 and CD133, and the proliferation of hASCs and hLMSCs were detected by CCK8. The expression of HMGA2 in hASCs and hLMSCs was detected by real-time quantitative PCR. The result is 1: 1. Mesenchymal stem cells derived from human adipose and lipoma were isolated by collagenase digestion. When cultured in MSCs medium, we found that: (1) the initial cell adhesion time was 1: hASCs and hLMSCs were completed within 24 hours; (2) the number of extracted cells and the first fusion time: after the primary cells were extracted, it was found that the cells were extracted from every milliliter of human lipoma tissue. The number of stem cells is more than the number of stem cells extracted from human adipose tissue. However, the time of first fusion of hASCs and hLMSCs was basically the same, that is, 6d~8d cells could reach 80% fusion and grow in a certain direction. (3) hASCs and hLMSCs cells in primary culture both grew fusiform. (4) the two kinds of cells could differentiate into adipocytes and osteoblasts after adipogenic induction. (5) the expressions of CD45 and CD90 on hASCs and hLMSCs surface markers were detected by flow cytometry, and the expression of CD45 and CD90 were similar. The results show that hLMSCs and hASCs have similar characteristics. 2. The proliferative ability of 96h~144h hLMSCs is higher than that of hASCs. 2. The proliferation ability of hASCs and hLMSCs is higher than that of 96h~144h hLMSCs. 2. The results of CCK8 method show that the proliferative ability of 96h~144h hLMSCs is higher than that of hASCs.. The expression of CD34 and CD133 in hASCs and hLMSCs cells were 2.14% and 3.17%, respectively. The expression of CD34 and CD133 were 20.62% and 16.04%, respectively. Real-time quantitative PCR was used to detect the changes of HMGA2 expression in hASCs and hLMSCs groups. It was found that the expression of HMGA2 in hLMSCs group was significantly higher than that in hASCs group. Conclusion 1. HASCs and hLMSCs were isolated successfully by collagenase digestion and cultured and amplified for adherent growth. Both of them had the ability of adipogenic osteogenesis, the potential of multiple differentiation and the expression of similar cell surface markers after induction. The two kinds of cells have similar biological characteristics. The difference of surface marker expression and HMGA2 expression provides an important basis for the development of lipoma, benign and malignant tumor.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R329.2
本文編號：2218967
[Abstract]:Purpose 1. Mesenchymal stem cells derived from human adipose and lipoma were isolated and cultured by collagenase digestion. The biological characteristics of mesenchymal stem cells derived from lipoma and human adipose cells were compared, and the differences between the two types of cells were studied, which was helpful to the further study of mesenchymal stem cells of lipoma. It provides an important basis for exploring the mechanism of lipoma. Methods: two kinds of mesenchymal stem cells were isolated, cultured and expanded by collagenase digestion in the same volume of fat and lipoma tissue. The morphology and growth of human adipose mesenchymal stem cells (human adipose derived mesenchymal stem cells,hASCs) and human lipoma mesenchymal stem cells (human lipoma derived mesenchymal stem cells,hLMSCs) were observed under microscope. After induced by adipogenic osteogenesis, the degree of hASCs and hLMSCs adipogenic osteogenesis was observed by hASCs and hLMSCs oil red O staining and alizarin red staining after cultured for 14 days. Flow cytometry was used to detect the expression of CD45, CD90 and CD133, and the proliferation of hASCs and hLMSCs were detected by CCK8. The expression of HMGA2 in hASCs and hLMSCs was detected by real-time quantitative PCR. The result is 1: 1. Mesenchymal stem cells derived from human adipose and lipoma were isolated by collagenase digestion. When cultured in MSCs medium, we found that: (1) the initial cell adhesion time was 1: hASCs and hLMSCs were completed within 24 hours; (2) the number of extracted cells and the first fusion time: after the primary cells were extracted, it was found that the cells were extracted from every milliliter of human lipoma tissue. The number of stem cells is more than the number of stem cells extracted from human adipose tissue. However, the time of first fusion of hASCs and hLMSCs was basically the same, that is, 6d~8d cells could reach 80% fusion and grow in a certain direction. (3) hASCs and hLMSCs cells in primary culture both grew fusiform. (4) the two kinds of cells could differentiate into adipocytes and osteoblasts after adipogenic induction. (5) the expressions of CD45 and CD90 on hASCs and hLMSCs surface markers were detected by flow cytometry, and the expression of CD45 and CD90 were similar. The results show that hLMSCs and hASCs have similar characteristics. 2. The proliferative ability of 96h~144h hLMSCs is higher than that of hASCs. 2. The proliferation ability of hASCs and hLMSCs is higher than that of 96h~144h hLMSCs. 2. The results of CCK8 method show that the proliferative ability of 96h~144h hLMSCs is higher than that of hASCs.. The expression of CD34 and CD133 in hASCs and hLMSCs cells were 2.14% and 3.17%, respectively. The expression of CD34 and CD133 were 20.62% and 16.04%, respectively. Real-time quantitative PCR was used to detect the changes of HMGA2 expression in hASCs and hLMSCs groups. It was found that the expression of HMGA2 in hLMSCs group was significantly higher than that in hASCs group. Conclusion 1. HASCs and hLMSCs were isolated successfully by collagenase digestion and cultured and amplified for adherent growth. Both of them had the ability of adipogenic osteogenesis, the potential of multiple differentiation and the expression of similar cell surface markers after induction. The two kinds of cells have similar biological characteristics. The difference of surface marker expression and HMGA2 expression provides an important basis for the development of lipoma, benign and malignant tumor.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R329.2
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