脂肪組織來源干細(xì)胞成瘤風(fēng)險性的相關(guān)實驗研究
發(fā)布時間：2018-07-23 16:16
【摘要】： 研究背景： 干細(xì)胞是一類具有自我更新和多向分化能力的原始未分化細(xì)胞,根據(jù)來源可分為胚胎干細(xì)胞(embryonic stem cells)與成體干細(xì)胞(adult stem cells)。間充質(zhì)干細(xì)胞(mesenchymal stem cells, MSCs)做為成體干細(xì)胞的主要組成部分,是現(xiàn)今干細(xì)胞研究的主要方向,其存在于人體的多種組織中,包括骨髓、脂肪、肝臟、臍血等,其中骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells, BMSCs)是研究相對集中和成熟的領(lǐng)域。BMSCs能夠在體外迅速擴(kuò)增并具有多項分化能力,在組織工程、細(xì)胞療法及再生醫(yī)學(xué)中都存在廣泛的臨床應(yīng)用前景。 脂肪組織來源干細(xì)胞(adipose-derived stem cells, ASCs)在體外培養(yǎng)條件下表現(xiàn)出與BMSCs極類似的多向分化潛能和迅速擴(kuò)增能力。且鑒于ASCs的組織來源更加廣泛、更易獲取等特點,使其迅速成為干細(xì)胞研究的新熱點。但有研究顯示,從脂肪瘤中提取的間充質(zhì)干細(xì)胞(Lipoma-derived mesenchymal stem cells, LMSCs)與正常ASCs在某些生物學(xué)特點上存在相似性。這就使我們對于涉及ASCs的細(xì)胞療法和組織工程的臨床應(yīng)用產(chǎn)生了疑慮。ASCs是否具有潛在成瘤性?其體內(nèi)的增殖和分化是否可控?形成的脂肪組織遠(yuǎn)期是否會出現(xiàn)類似脂肪瘤一樣的病理性生長?這些疑問已成為與ASCs相關(guān)的臨床應(yīng)用必須解答的關(guān)鍵問題。然而,對于ASCs的致瘤性問題,尚未見有國內(nèi)外相關(guān)實驗報告對此方面作出研究。所以本研究通過體外培養(yǎng)擴(kuò)增ASCs和LMSCs,對兩者的形態(tài)學(xué)、分化潛力、組織切片、生長動力學(xué)、表面標(biāo)記物、細(xì)胞周期分布、脂肪瘤特異性基因和端粒酶表達(dá)八個方面生物學(xué)特性進(jìn)行比較,對ASCs是否具有成瘤性進(jìn)行初步評估,旨在為ASCs的臨床應(yīng)用的安全性提供依據(jù)和支持。 目的： 評估體外培養(yǎng)的ASCs的致瘤性。通過對ASCs與LMSCs的形態(tài)學(xué)、分化潛力以及正常脂肪組織和脂肪瘤組織的切片觀察、生長動力學(xué)、表面標(biāo)記物、細(xì)胞周期分布、脂肪瘤特異性基因和端粒酶表達(dá)八個方面的對比來進(jìn)行鑒定。 方法： 分離培養(yǎng)ASCs和LMSCs,觀察細(xì)胞形態(tài)并使用油紅O染色、茜素紅染色、阿爾辛藍(lán)染色分別鑒定ASCs與LMSCs的成脂分化、成骨分化、成軟骨分化能力；對正常脂肪組織和脂肪瘤組織進(jìn)行切片染色；MTS比色法檢測細(xì)胞活性并繪制細(xì)胞生長曲線；流式細(xì)胞儀測定細(xì)胞周期及表面分子表達(dá)；定量RT-PCR檢測高遷移率族蛋白2(the high-mobility group AT-hook 2, HMGA2)及細(xì)胞殺傷誘導(dǎo)def45樣效應(yīng)因子(cell death-inducing def45-like effectors, CIDEA)表達(dá)水平；免疫組織化學(xué)染色法鑒定端粒酶逆轉(zhuǎn)錄酶(hTERT)的表達(dá)。 結(jié)果： 分離培養(yǎng)的ASCs與LMSCs細(xì)胞形態(tài)相似,多數(shù)細(xì)胞為長梭形,形態(tài)類似成纖維細(xì)胞,而LMSCs后期生長速度明顯快于ASCs; ASCs和LMSCs均具有向脂肪細(xì)胞、骨細(xì)胞、軟骨細(xì)胞誘導(dǎo)分化的能力；正常脂肪組織和脂肪瘤組織切片差異明顯,脂肪瘤組織存在完整包膜,可見豐富的纖維間隔和成束的梭形細(xì)胞,這些細(xì)胞大小較一致,核呈卵圓形或圓形,無核分裂象。而正常脂肪組織由大量成熟脂肪細(xì)胞細(xì)胞構(gòu)成；MTS活性測定ASCs增殖活性要遠(yuǎn)低于LMSCs細(xì)胞(P0.01)；流式細(xì)胞儀檢測結(jié)果顯示ASCs與LMSCs在干細(xì)胞標(biāo)志CD29、CD44、CD105上表達(dá)類似,而在腫瘤干細(xì)胞標(biāo)志CD133表達(dá)上,ASCs (5.35%)要低于LMSCs(26.87%);細(xì)胞周期顯示ASCs的增殖能力低于LMSCs;QRT-PCR顯示ASCs中HMGA2平均RQ值為1,遠(yuǎn)低于在LMSCs中的表達(dá)(1.79±0.279)(t=-6.329、P0.01); ASCs中CIDEA平均RQ值為1,高于在LMSCs中的表達(dá)(0.64±0.060)(t=13.324、P0.01),兩者差異均具有統(tǒng)計學(xué)意義；免疫細(xì)胞化學(xué)結(jié)果：hTERT在ASCs和LMSCs中的累計吸光度(IOD)分別為1379.597±498.617和3328.108±902.856(t=-7.317、P0.01),面積(area)分別為132390.27±35568.945和238000.53±49264.289(t=-6.732、P0.01),平均吸光度(density)分別為0.009±0.003和0.014±0.003(t=-4.683、P0.01), ASCs中hTERT表達(dá)遠(yuǎn)低于LMSCs,各指標(biāo)差異均具有顯著統(tǒng)計學(xué)意義。 結(jié)論： 體外實驗中沒有發(fā)現(xiàn)ASCs成瘤性證據(jù),其生物學(xué)特性與LMSCs存在顯著差別,臨床應(yīng)用成瘤風(fēng)險性極低,然而遠(yuǎn)期還需以體內(nèi)實驗來進(jìn)一步確證。
[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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R329
本文編號：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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R329
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