脂肪组织来源干细胞成瘤风险性的相关实验研究
发布时间：2018-07-23 16:16
【摘要】： 研究背景： 干细胞是一类具有自我更新和多向分化能力的原始未分化细胞,根据来源可分为胚胎干细胞(embryonic stem cells)与成体干细胞(adult stem cells)。间充质干细胞(mesenchymal stem cells, MSCs)做为成体干细胞的主要组成部分,是现今干细胞研究的主要方向,其存在于人体的多种组织中,包括骨髓、脂肪、肝脏、脐血等,其中骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)是研究相对集中和成熟的领域。BMSCs能够在体外迅速扩增并具有多项分化能力,在组织工程、细胞疗法及再生医学中都存在广泛的临床应用前景。 脂肪组织来源干细胞(adipose-derived stem cells, ASCs)在体外培养条件下表现出与BMSCs极类似的多向分化潜能和迅速扩增能力。且鉴于ASCs的组织来源更加广泛、更易获取等特点,使其迅速成为干细胞研究的新热点。但有研究显示,从脂肪瘤中提取的间充质干细胞(Lipoma-derived mesenchymal stem cells, LMSCs)与正常ASCs在某些生物学特点上存在相似性。这就使我们对于涉及ASCs的细胞疗法和组织工程的临床应用产生了疑虑。ASCs是否具有潜在成瘤性?其体内的增殖和分化是否可控?形成的脂肪组织远期是否会出现类似脂肪瘤一样的病理性生长?这些疑问已成为与ASCs相关的临床应用必须解答的关键问题。然而,对于ASCs的致瘤性问题,尚未见有国内外相关实验报告对此方面作出研究。所以本研究通过体外培养扩增ASCs和LMSCs,对两者的形态学、分化潜力、组织切片、生长动力学、表面标记物、细胞周期分布、脂肪瘤特异性基因和端粒酶表达八个方面生物学特性进行比较,对ASCs是否具有成瘤性进行初步评估,旨在为ASCs的临床应用的安全性提供依据和支持。 目的： 评估体外培养的ASCs的致瘤性。通过对ASCs与LMSCs的形态学、分化潜力以及正常脂肪组织和脂肪瘤组织的切片观察、生长动力学、表面标记物、细胞周期分布、脂肪瘤特异性基因和端粒酶表达八个方面的对比来进行鉴定。 方法： 分离培养ASCs和LMSCs,观察细胞形态并使用油红O染色、茜素红染色、阿尔辛蓝染色分别鉴定ASCs与LMSCs的成脂分化、成骨分化、成软骨分化能力；对正常脂肪组织和脂肪瘤组织进行切片染色；MTS比色法检测细胞活性并绘制细胞生长曲线；流式细胞仪测定细胞周期及表面分子表达；定量RT-PCR检测高迁移率族蛋白2(the high-mobility group AT-hook 2, HMGA2)及细胞杀伤诱导def45样效应因子(cell death-inducing def45-like effectors, CIDEA)表达水平；免疫组织化学染色法鉴定端粒酶逆转录酶(hTERT)的表达。 结果： 分离培养的ASCs与LMSCs细胞形态相似,多数细胞为长梭形,形态类似成纤维细胞,而LMSCs后期生长速度明显快于ASCs; ASCs和LMSCs均具有向脂肪细胞、骨细胞、软骨细胞诱导分化的能力；正常脂肪组织和脂肪瘤组织切片差异明显,脂肪瘤组织存在完整包膜,可见丰富的纤维间隔和成束的梭形细胞,这些细胞大小较一致,核呈卵圆形或圆形,无核分裂象。而正常脂肪组织由大量成熟脂肪细胞细胞构成；MTS活性测定ASCs增殖活性要远低于LMSCs细胞(P0.01)；流式细胞仪检测结果显示ASCs与LMSCs在干细胞标志CD29、CD44、CD105上表达类似,而在肿瘤干细胞标志CD133表达上,ASCs (5.35%)要低于LMSCs(26.87%);细胞周期显示ASCs的增殖能力低于LMSCs;QRT-PCR显示ASCs中HMGA2平均RQ值为1,远低于在LMSCs中的表达(1.79±0.279)(t=-6.329、P0.01); ASCs中CIDEA平均RQ值为1,高于在LMSCs中的表达(0.64±0.060)(t=13.324、P0.01),两者差异均具有统计学意义；免疫细胞化学结果：hTERT在ASCs和LMSCs中的累计吸光度(IOD)分别为1379.597±498.617和3328.108±902.856(t=-7.317、P0.01),面积(area)分别为132390.27±35568.945和238000.53±49264.289(t=-6.732、P0.01),平均吸光度(density)分别为0.009±0.003和0.014±0.003(t=-4.683、P0.01), ASCs中hTERT表达远低于LMSCs,各指标差异均具有显著统计学意义。 结论： 体外实验中没有发现ASCs成瘤性证据,其生物学特性与LMSCs存在显著差别,临床应用成瘤风险性极低,然而远期还需以体内实验来进一步确证。
[Abstract]:Research background:
Stem cells are primitive undifferentiated cells with self-renewal and multidirectional differentiation, which can be divided into embryonic stem cells (embryonic stem cells) and adult stem cells (adult stem cells) according to their sources. Mesenchymal stem cells (mesenchymal stem cells, MSCs) are the main components of adult stem cells. In the direction, it exists in a variety of tissues of the human body, including bone marrow, fat, liver, cord blood and so on, in which bone marrow mesenchymal stem cells, BMSCs is a field of relatively concentrated and mature research,.BMSCs can expand rapidly in vitro and have a number of differentiation ability in tissue engineering, cell therapy and regenerative medicine. There is a wide range of clinical applications in the study.
Adipose-derived stem cells (ASCs), which is derived from adipose tissue derived stem cells (cells, ASCs), displays the multidirectional differentiation potential and rapid amplification ability similar to BMSCs in vitro. And in view of the wider and easier access to ASCs, it has become a new hot spot in the research of stem cells. Lipoma-derived mesenchymal stem cells (LMSCs) is similar to normal ASCs in some biological characteristics. This gives us a doubt about the potential tumorigenicity of.ASCs in the clinical application of cell therapy and tissue engineering involving ASCs? Is the proliferation and differentiation in the body controllable? Is the fat tissue similar to the pathological growth of lipoma in the long term? These questions have become a key problem to be answered in ASCs related clinical applications. However, there have been no domestic and foreign related experimental reports on the tumorigenicity of ASCs. Therefore, this study amplified ASCs by culture in vitro. LMSCs, the eight biological characteristics of both morphology, differentiation potential, tissue section, growth kinetics, surface markers, cell cycle distribution, lipoma specific genes and telomerase expression are compared, and the preliminary evaluation of whether ASCs has a tumorigenicity is carried out to provide the basis and support for the safety of clinical application of ASCs.
Objective:
To assess the tumorigenicity of ASCs in vitro, by comparing the morphology of ASCs with LMSCs, the potential of differentiation and the observation of normal fat tissue and lipoma tissue, growth kinetics, surface markers, cell cycle distribution, lipoma specific genes and telomerase expression of eight sides.
Method:
ASCs and LMSCs were isolated and cultured. The cells were stained with oil red O, alizarin red staining, alizarin blue staining. The differentiation of ASCs and LMSCs was identified, osteogenesis differentiation and chondrodifferentiation were identified, and normal adipose tissue and lipoma tissue were stained with sliced tissue; MTS colorimetric assay was used to detect cell activity and plot cell growth curve. The expression of cell cycle and surface molecules was measured by flow cytometry; quantitative RT-PCR was used to detect high mobility group protein 2 (the high-mobility group AT-hook 2, HMGA2) and cell killing induced def45 like effect factor (cell death-inducing def45-like effectors, CIDEA) expression level; immunohistochemical staining method was used to identify telomerase reverse transcriptase. HTERT) expression.
Result:
The morphology of the isolated ASCs and LMSCs cells was similar. Most of the cells were long spindle shaped, similar to fibroblasts, while the growth rate of LMSCs was faster than that of ASCs, and ASCs and LMSCs had the ability to induce differentiation to adipocytes, bone cells and chondrocytes, and the normal fat tissue and lipoma sections were distinct, lipoma tissues existed. In the complete envelope, there are abundant fibrous septum and beam forming spindle cells, the size of these cells is more consistent, the nucleus is oval or round, without nuclear mitosis, and the normal adipose tissue is made up of a large number of mature adipocyte cells; MTS activity determination of ASCs proliferation activity is far lower than that of LMSCs cells (P0.01); flow cytometry results show AS The expression of Cs and LMSCs was similar on the stem cell markers CD29, CD44, and CD105, while ASCs (5.35%) was lower than LMSCs (26.87%) in the expression of tumor stem cell marker CD133; cell cycle showed that the proliferation of ASCs was lower than LMSCs, and QRT-PCR showed that the average value of the ASCs was 1, which was far lower than that of the LMSCs (1.79 + 0.279). The average RQ value was 1, which was higher than that in LMSCs (0.64 + 0.060) (t=13.324, P0.01), and the differences were statistically significant. The cumulative absorbance (IOD) of hTERT in ASCs and LMSCs (IOD) was 1379.597 + 498.617 and 3328.108 + 902.856 (t= -7.317, P0.01) respectively, and the area (area) was 132390.27 + 35568.945 and 238000.5, respectively. The average absorbance (density) of 3 + 49264.289 (t=-6.732, P0.01) was 0.009 + 0.003 and 0.014 + 0.003 (t=-4.683, P0.01) respectively. The expression of hTERT in ASCs was much lower than that of LMSCs, and the difference of each index had significant statistical significance.
Conclusion:
No evidence of ASCs tumorigenicity was found in the experiment in vitro, and its biological characteristics were significantly different from that of LMSCs, and the risk of clinical application of tumor was very low. However, in the long term, it should be further confirmed by the experiment in vivo.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329
本文编号：2139949
[Abstract]:Research background:
Stem cells are primitive undifferentiated cells with self-renewal and multidirectional differentiation, which can be divided into embryonic stem cells (embryonic stem cells) and adult stem cells (adult stem cells) according to their sources. Mesenchymal stem cells (mesenchymal stem cells, MSCs) are the main components of adult stem cells. In the direction, it exists in a variety of tissues of the human body, including bone marrow, fat, liver, cord blood and so on, in which bone marrow mesenchymal stem cells, BMSCs is a field of relatively concentrated and mature research,.BMSCs can expand rapidly in vitro and have a number of differentiation ability in tissue engineering, cell therapy and regenerative medicine. There is a wide range of clinical applications in the study.
Adipose-derived stem cells (ASCs), which is derived from adipose tissue derived stem cells (cells, ASCs), displays the multidirectional differentiation potential and rapid amplification ability similar to BMSCs in vitro. And in view of the wider and easier access to ASCs, it has become a new hot spot in the research of stem cells. Lipoma-derived mesenchymal stem cells (LMSCs) is similar to normal ASCs in some biological characteristics. This gives us a doubt about the potential tumorigenicity of.ASCs in the clinical application of cell therapy and tissue engineering involving ASCs? Is the proliferation and differentiation in the body controllable? Is the fat tissue similar to the pathological growth of lipoma in the long term? These questions have become a key problem to be answered in ASCs related clinical applications. However, there have been no domestic and foreign related experimental reports on the tumorigenicity of ASCs. Therefore, this study amplified ASCs by culture in vitro. LMSCs, the eight biological characteristics of both morphology, differentiation potential, tissue section, growth kinetics, surface markers, cell cycle distribution, lipoma specific genes and telomerase expression are compared, and the preliminary evaluation of whether ASCs has a tumorigenicity is carried out to provide the basis and support for the safety of clinical application of ASCs.
Objective:
To assess the tumorigenicity of ASCs in vitro, by comparing the morphology of ASCs with LMSCs, the potential of differentiation and the observation of normal fat tissue and lipoma tissue, growth kinetics, surface markers, cell cycle distribution, lipoma specific genes and telomerase expression of eight sides.
Method:
ASCs and LMSCs were isolated and cultured. The cells were stained with oil red O, alizarin red staining, alizarin blue staining. The differentiation of ASCs and LMSCs was identified, osteogenesis differentiation and chondrodifferentiation were identified, and normal adipose tissue and lipoma tissue were stained with sliced tissue; MTS colorimetric assay was used to detect cell activity and plot cell growth curve. The expression of cell cycle and surface molecules was measured by flow cytometry; quantitative RT-PCR was used to detect high mobility group protein 2 (the high-mobility group AT-hook 2, HMGA2) and cell killing induced def45 like effect factor (cell death-inducing def45-like effectors, CIDEA) expression level; immunohistochemical staining method was used to identify telomerase reverse transcriptase. HTERT) expression.
Result:
The morphology of the isolated ASCs and LMSCs cells was similar. Most of the cells were long spindle shaped, similar to fibroblasts, while the growth rate of LMSCs was faster than that of ASCs, and ASCs and LMSCs had the ability to induce differentiation to adipocytes, bone cells and chondrocytes, and the normal fat tissue and lipoma sections were distinct, lipoma tissues existed. In the complete envelope, there are abundant fibrous septum and beam forming spindle cells, the size of these cells is more consistent, the nucleus is oval or round, without nuclear mitosis, and the normal adipose tissue is made up of a large number of mature adipocyte cells; MTS activity determination of ASCs proliferation activity is far lower than that of LMSCs cells (P0.01); flow cytometry results show AS The expression of Cs and LMSCs was similar on the stem cell markers CD29, CD44, and CD105, while ASCs (5.35%) was lower than LMSCs (26.87%) in the expression of tumor stem cell marker CD133; cell cycle showed that the proliferation of ASCs was lower than LMSCs, and QRT-PCR showed that the average value of the ASCs was 1, which was far lower than that of the LMSCs (1.79 + 0.279). The average RQ value was 1, which was higher than that in LMSCs (0.64 + 0.060) (t=13.324, P0.01), and the differences were statistically significant. The cumulative absorbance (IOD) of hTERT in ASCs and LMSCs (IOD) was 1379.597 + 498.617 and 3328.108 + 902.856 (t= -7.317, P0.01) respectively, and the area (area) was 132390.27 + 35568.945 and 238000.5, respectively. The average absorbance (density) of 3 + 49264.289 (t=-6.732, P0.01) was 0.009 + 0.003 and 0.014 + 0.003 (t=-4.683, P0.01) respectively. The expression of hTERT in ASCs was much lower than that of LMSCs, and the difference of each index had significant statistical significance.
Conclusion:
No evidence of ASCs tumorigenicity was found in the experiment in vitro, and its biological characteristics were significantly different from that of LMSCs, and the risk of clinical application of tumor was very low. However, in the long term, it should be further confirmed by the experiment in vivo.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329
【参考文献】
相关期刊论文 前3条
1 史春梦,麦跃,程天民,粟永萍,徐辉,郑怀恩,王军平,黄跃生,蒋建新;真皮来源成体多能干细胞体外自发恶性转化现象及机制研究[J];第三军医大学学报;2004年01期
2 朱茗;高建华;鲁峰;李华;;脂肪组织来源干细胞的细胞生物学研究[J];南方医科大学学报;2007年04期
3 呼莹,张丽艳,马冠杰,姜学英,赵春华;胎儿骨髓和肝脏间充质干细胞的表型和生物学性状研究[J];中国实验血液学杂志;2001年04期
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