诱导人乳腺脂肪来源干细胞向乳腺上皮样细胞分化的实验研究
发布时间：2018-10-12 12:55
【摘要】：第一部分 人肥大乳房乳腺组织中脂肪来源干细胞的分离、培养及鉴定 目的建立人乳腺脂肪来源干细胞(hbASCs)原代培养的方法,并对其进行体外培养及鉴定,观察其形态特点、生物学特性及间充质来源干细胞表面相关标志的表达。 方法本实验于2011.03-2012.04在华中科技大学附属协和医院外科实验中心整形外科实验室完成。①对象：实验期间在我科行乳房缩小术患者7例,年龄23-53岁,BMI指数20.01-23.26kg/m2,术后病理检查无恶性肿瘤发生,患者知情同意。②方法：于无菌条件下获取人乳腺脂肪组织块约10-15毫升,采用Ⅰ型胶原酶消化+贴壁法分离原代脂肪来源干细胞,生长至85%融合时传代,倒置相差显微镜观察形态特点、生物学特性,采用CCK-8法绘制细胞生长曲线,计算细胞倍增时间；选择生长状态良好的第3代hbASCs分别行荧光免疫法和流式细胞仪检测间充质来源干细胞特异性表面标志物的表达,并向成脂、成骨定向诱导。 结果hbASCs呈长梭形贴壁生长,可阳性表达间充质干细胞生长特性相对特异性标志物,体外可成脂、成骨定向分化。经冻存复苏后仍可维持细胞活性、生长和增值能力。 结论可从人乳腺脂肪组织中成功分离获取干细胞,为组织工程化乳房提供了新的取材方便、不增加供区的种子细胞来源。 第二部分 共培养诱导hbASCs向乳腺上皮样细胞定向分化的实验研究 目的探讨通过Transwell建立hbASCs和HBL-100细胞系共培养体系,诱导hbASCs向乳腺上皮样细胞转化的可能性。 方法分对照组和实验组,每组3复孔。实验组：将第3代hbASCs和HBL-100细胞系分别接种于Transwell共培养系统的下室和上室中,调整细胞比例为1：1,使用DMEM/F12培养基；对照组：分别在Transwell上、下室接种第3代hbASCs,使用DMEM/F12培养基；以上两组均隔日换液。共培养15天后,观察实验组和对照组Transwell下室中hbASCs的光镜特征、电镜结构和荧光免疫细胞化学染色,对下室被诱导细胞进行鉴定。 结果Transwell体系中各细胞均可良好贴壁生长。共培养15天后,实验组下室hbASCs细胞数量较对照组明显增多,细胞外基质合成丰富,部分细胞形态上表现出乳腺上皮样细胞特征；电镜下可见微绒毛、桥粒和张力丝等典型上皮细胞结构特征；免疫荧光结果显示,实验组共培养环境下分化细胞可阳性表达乳腺上皮细胞特异性标记物CK-18CK-19和间充质细胞标记物Vimentin;对照组下室hbASCs上述检测结果均为阴性； 结论本实验在体外成功建立了hbASCs和HBL-100细胞系的Transwell共培养体系,并证实在该体系中可诱导hbASCs向乳腺上皮样细胞定向分化。 第三部分 EGF促进hbASCs向乳腺上皮样细胞定向分化的实验研究 目的探讨在体外间接共培养体系中添加表皮生长因子(EGF)对促进hbASCs向乳腺上皮样细胞定向分化的影响。 方法研究分两组。实验组：取生长良好第3代hbASCs,加入条件培养基(HBL-100细胞系上清液)+10ug/L EGF+1％双抗；对照组：取生长良好第3代hbASCs,仅加入条件培养基(HBL-100细胞系上清液)+1%双抗；两组细胞均隔日换液。共培养15天后,观察各组中hbASCs的光镜特征、荧光免疫细胞化学染色、流式细胞仪检测CK-18阳性细胞比例以及实时定量PCR (real-time quantitative reverse-transcription polymerase chain reaction, QRT-PCR)检测CK-18mRNA水平,对结果进行配对t检验。结果共培养体系中各细胞均可良好贴壁生长。共培养15天后,两组中均可发现部分hbASCS细胞形态、排列方式发生改变,实验组中细胞各项改变较对照组明显；实验组诱导细胞荧光免疫细胞化学染色乳腺上皮细胞特异性角蛋白-18、角蛋白-19阳性细胞数和QRT-PCR基因表达量(△△CT法)分别为683.000±58.014个/视野和3.5350+0.3737倍,明显高于对照组的298.167±27.272个/视野和1倍,有统计学意义(P＜0.05)。结论表皮生长因子(EGF)可促进体外间接共培养体系中hbASCs向乳腺上皮样细胞的定向分化。
[Abstract]:The first part Isolation and culture of adipose-derived stem cells from breast tissue of human hypertrophic breast Objective To establish primary culture method of human breast fat-derived stem cells (hbASCs) and to culture and identify them in vitro. Its morphological characteristics, biological characteristics and the surface-related surface of mesenchymal stem cells Expression of markers. Methods This experiment was conducted in 2011. 03-2012. 04 at Central Huazhong University of Science and Technology Co., Ltd. and Hospital Surgical Experiment Center. Results: 7 patients with breast reduction, 23-53 years of age and 20. 01-23.26kg/ m2 of BMI were performed during the experiment. There was no malignant tumor in postoperative pathological examination. Method: Obtain human mammary adipose tissue mass of about 10-15 ml under aseptic conditions, isolate primary adipose-derived stem cells with type I collagenase digestion + apposition method, grow to 85% fusion, and observe the morphology by inverted phase contrast microscope. Characteristics, biological characteristics, using CCK-8 method to plot the cell growth curve, calculating the cell doubling time, selecting the third generation hbASCs with good growth state, respectively carrying out fluorescence immunoassay and flow cytometry to detect the expression of the specific surface marker of the mesenchymal stem cells, The results showed that hbASCs had a long shuttle-shaped adherent growth, and the growth characteristics of positive expression mesenchymal stem cells were relatively specific markers. It can be made into fat or bone-oriented differentiation. After frozen storage, it can still be maintained fine Conclusion The stem cells can be isolated from human breast adipose tissue successfully, and new materials are provided for tissue engineered breast.-No, no, no, no, no. The source of seed cells in the donor region. The second portion was co-cultured to induce hb. An experimental study of ASCs directed differentiation to mammary epithelial-like cells was conducted to investigate the co-culture system of hbASCs and HBL-100 cell lines by Transwell inducing hbASCs to epithelial-like cells in breast The possibility of transformation was divided into two groups: control group and experimental group, 3 complex pores in each group. The third generation hbASCs and HBL-100 cell lines were respectively inoculated into the lower and upper chambers of the Transwell co-culture system, the cell ratio was 1: 1, DMEM/ F12 medium was used, and the control group: the 3rd generation hbAS was inoculated on Transwell and the lower chamber respectively. Cs, DMEM/ F12 medium were used; two groups of the above two groups were used to exchange liquid. After 15 days of co-culture, the light microscope characteristics of hbASCs in the experimental group and control group were observed, and the electron microscope Structure and fluorescence immunocytochemistry staining to identify cells to be induced in the lower chamber Results After 15 days of co-culture, the number of hbASCs in the experimental group was higher than that in the control group, the extracellular matrix was rich, and the morphology of some cells showed the mammary epithelium-like cells. Characteristics: The structural characteristics of typical epithelial cells, such as microvilli, bridges and tension wires, were observed under electron microscope. The results showed that the differentiated cells in the experimental group could express the specific markers CK-18CK-19 and the marker V of the mesenchymal epithelial cells in the co-culture environment of the experimental group. The results of hbASCs and hbASCs were negative in the control group. Conclusion Transwell co-culture of hbASCs and HBL-100 cell lines was successfully established in vitro. the system, and It was confirmed that hbASCs were induced to differentiate into breast epithelial-like cells in the system. The third part of EGF promotes the directional differentiation of hbASCs to mammary epithelial-like cells, and discusses the indirect co-culture in vitro. Add epidermis in the system The effects of growth factor (EGF) on the orientation and differentiation of hbASCs to breast epithelial-like cells were studied. The experimental groups were divided into two groups: the third generation hbASCs, the addition condition medium (supernatant of HBL-100 cell line) + 10ug/ L EGF + 1% double resistance, the control group: the third generation of growth was good. BASCs were added only with conditioned medium (supernatant of HBL-100 cell line) + 1% double resistance; two groups of cells were incubated on day-to-day exchange. After 15 days of co-culture, the light microscope characteristics of hbASCs in each group were observed, the chemical staining of fluorescence immunocytochemistry, the ratio of CK-18 positive cells detected by flow cytometry and real-time quantitative PCR (real-time quantitative reverse-trans-transcription policy) were observed. ain reaction, QRT-PCR) CK-18mRNA level was detected and paired t-test was performed on the results. Results All cells in co-culture system could grow well. After 15 days of co-culture, the morphology and arrangement of partial hbASCS cells could be found in both groups. In the experimental group, the specific cytokeratin-18, keratin-19 positive cells and QRT-PCR gene expression were 683,000 and 584.014/ visual field and 3,5350 + 0.3737 times, respectively. It was significantly higher than that of the control group (298. 167, 27. 272)/ field of view (P <0.05).
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R329
本文编号：2266191
[Abstract]:The first part Isolation and culture of adipose-derived stem cells from breast tissue of human hypertrophic breast Objective To establish primary culture method of human breast fat-derived stem cells (hbASCs) and to culture and identify them in vitro. Its morphological characteristics, biological characteristics and the surface-related surface of mesenchymal stem cells Expression of markers. Methods This experiment was conducted in 2011. 03-2012. 04 at Central Huazhong University of Science and Technology Co., Ltd. and Hospital Surgical Experiment Center. Results: 7 patients with breast reduction, 23-53 years of age and 20. 01-23.26kg/ m2 of BMI were performed during the experiment. There was no malignant tumor in postoperative pathological examination. Method: Obtain human mammary adipose tissue mass of about 10-15 ml under aseptic conditions, isolate primary adipose-derived stem cells with type I collagenase digestion + apposition method, grow to 85% fusion, and observe the morphology by inverted phase contrast microscope. Characteristics, biological characteristics, using CCK-8 method to plot the cell growth curve, calculating the cell doubling time, selecting the third generation hbASCs with good growth state, respectively carrying out fluorescence immunoassay and flow cytometry to detect the expression of the specific surface marker of the mesenchymal stem cells, The results showed that hbASCs had a long shuttle-shaped adherent growth, and the growth characteristics of positive expression mesenchymal stem cells were relatively specific markers. It can be made into fat or bone-oriented differentiation. After frozen storage, it can still be maintained fine Conclusion The stem cells can be isolated from human breast adipose tissue successfully, and new materials are provided for tissue engineered breast.-No, no, no, no, no. The source of seed cells in the donor region. The second portion was co-cultured to induce hb. An experimental study of ASCs directed differentiation to mammary epithelial-like cells was conducted to investigate the co-culture system of hbASCs and HBL-100 cell lines by Transwell inducing hbASCs to epithelial-like cells in breast The possibility of transformation was divided into two groups: control group and experimental group, 3 complex pores in each group. The third generation hbASCs and HBL-100 cell lines were respectively inoculated into the lower and upper chambers of the Transwell co-culture system, the cell ratio was 1: 1, DMEM/ F12 medium was used, and the control group: the 3rd generation hbAS was inoculated on Transwell and the lower chamber respectively. Cs, DMEM/ F12 medium were used; two groups of the above two groups were used to exchange liquid. After 15 days of co-culture, the light microscope characteristics of hbASCs in the experimental group and control group were observed, and the electron microscope Structure and fluorescence immunocytochemistry staining to identify cells to be induced in the lower chamber Results After 15 days of co-culture, the number of hbASCs in the experimental group was higher than that in the control group, the extracellular matrix was rich, and the morphology of some cells showed the mammary epithelium-like cells. Characteristics: The structural characteristics of typical epithelial cells, such as microvilli, bridges and tension wires, were observed under electron microscope. The results showed that the differentiated cells in the experimental group could express the specific markers CK-18CK-19 and the marker V of the mesenchymal epithelial cells in the co-culture environment of the experimental group. The results of hbASCs and hbASCs were negative in the control group. Conclusion Transwell co-culture of hbASCs and HBL-100 cell lines was successfully established in vitro. the system, and It was confirmed that hbASCs were induced to differentiate into breast epithelial-like cells in the system. The third part of EGF promotes the directional differentiation of hbASCs to mammary epithelial-like cells, and discusses the indirect co-culture in vitro. Add epidermis in the system The effects of growth factor (EGF) on the orientation and differentiation of hbASCs to breast epithelial-like cells were studied. The experimental groups were divided into two groups: the third generation hbASCs, the addition condition medium (supernatant of HBL-100 cell line) + 10ug/ L EGF + 1% double resistance, the control group: the third generation of growth was good. BASCs were added only with conditioned medium (supernatant of HBL-100 cell line) + 1% double resistance; two groups of cells were incubated on day-to-day exchange. After 15 days of co-culture, the light microscope characteristics of hbASCs in each group were observed, the chemical staining of fluorescence immunocytochemistry, the ratio of CK-18 positive cells detected by flow cytometry and real-time quantitative PCR (real-time quantitative reverse-trans-transcription policy) were observed. ain reaction, QRT-PCR) CK-18mRNA level was detected and paired t-test was performed on the results. Results All cells in co-culture system could grow well. After 15 days of co-culture, the morphology and arrangement of partial hbASCS cells could be found in both groups. In the experimental group, the specific cytokeratin-18, keratin-19 positive cells and QRT-PCR gene expression were 683,000 and 584.014/ visual field and 3,5350 + 0.3737 times, respectively. It was significantly higher than that of the control group (298. 167, 27. 272)/ field of view (P <0.05).
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R329
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相关期刊论文 前3条
1 郭子宽,杨靖清,刘晓丹,李秀森,侯春梅,唐佩弦,毛宁;骨髓间叶干细胞的生物学特性(英文)[J];Chinese Medical Journal;2001年09期
2 孙家明,乔群,戚可名;肥大乳房和小乳房乳腺组织中雌激素受体的表达[J];中华整形外科杂志;2004年06期
3 郝伟;胡蕴玉;魏义勇;庞龙;白建萍;吕荣;王军;张大伟;;兔脂肪干细胞的分离培养鉴定及成骨诱导分化研究[J];中国矫形外科杂志;2006年21期
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