本文選題：脂肪源性間充質(zhì)干細(xì)胞　切入點：分化　出處：《中國人民解放軍軍醫(yī)進修學(xué)院》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： 背景:干細(xì)胞治療急性心肌梗死是近年來研究的熱點。脂肪源性間充質(zhì)干細(xì)胞(adipose tissue-derived mesenchymal stem cells,ADMSCs)作為一種新的細(xì)胞工程的種子細(xì)胞因為取材方便、來源充足、更易被人們所接受等優(yōu)點逐漸被大家接受。國內(nèi)、外對于ADMSCs的研究還外于起步階段,其治療機制的基礎(chǔ)研究還很有限,在臨床應(yīng)用前尚有許多問題有待解決。目前干細(xì)胞移植治療心梗的可能機制主要有兩種:1、MSCs可分化為心肌細(xì)胞。2、通過旁分泌生長因子(如HGF、VEGF)等來發(fā)揮作用。最近科學(xué)家們發(fā)現(xiàn)干細(xì)胞的分化能力存在性別差異,從雌性小鼠肌肉中提取的干細(xì)胞比雄性肌肉中提取的更易分化為身體組織。那么人干細(xì)胞是否也存在分化的性別差異呢?性別或其它個體因素(如年齡、是否有高血壓、糖尿病、冠心病等)對于干細(xì)胞增殖、分化、旁分泌、抗凋亡能力等生物學(xué)特性是否有影響呢?這些問題的解決對于了解ADMSCs本身的生物學(xué)特性,指導(dǎo)ADMSCs基礎(chǔ)研究和臨床應(yīng)用具有十分重要的意義。 目的:一是要建立從人腹部皮下脂肪組織中分離、培養(yǎng)ADMSCs方法,并從表面標(biāo)志物及多向分化潛能方面對其進行鑒定,為后續(xù)研究做準(zhǔn)備。二是觀察比較不同人ADMSCs增殖、分化、旁分泌、抗凋亡能力的差別,從細(xì)胞水平探索ADMSCs是否存在個體差異,為今后干細(xì)胞的基礎(chǔ)研究及臨床應(yīng)用提供一種新的視角。 內(nèi)容和方法:根據(jù)以上目的,本研究分成為兩個部分: 第一部分研究人ADMSCs的分離及體外培養(yǎng);并通過對獲取的細(xì)胞進行CD13、CD44、CD45、HLA-DR和VWF免疫組化細(xì)胞表面間充質(zhì)干細(xì)胞相關(guān)標(biāo)志的鑒定及成脂、成骨、成心肌多向分化潛能的檢驗來驗證其是否為間充質(zhì)干細(xì)胞。 第二部分對第一部分獲取的人ADMSCs分別采用MTT比色實驗、ELIsA雙抗體夾心法、流式細(xì)胞計數(shù)技術(shù)來比較不同人ADMSCs在增殖、旁分泌、分化及抗凋亡能力方面的差別。 結(jié)果: 從人腹部皮下脂肪組織分離培養(yǎng)出長梭形細(xì)胞。這些細(xì)胞的細(xì)胞表面分子CD13、CD44皆陽性,CD45、HLA-DR、VWF均陰性;具有成脂、成骨、成心肌的多向分化能力。 MTT比色實驗示:人ADMSCs由OD值描繪的生長曲線大致形狀相似,呈S型。在第5d、7d、9d女性h-ADMSCs的OD值明顯高于相應(yīng)的男性h-ADMSCs(P＜0.01),同時年輕個體ADMSCs的OD值更高。經(jīng)10umol/L 5-aza誘導(dǎo)24小時后,流式細(xì)胞技術(shù)示女性h-ADMScs的TNT的表達率普遍高于男性h-ADMSCs(27.3%±2.4%VS 18.9%±2.5%,P＜0.01),而且年輕個體的ADMSCs更容易被誘導(dǎo)并表達TNT。Elisa結(jié)果示:女性h-ADMSCs分泌VEGF和HGF的能力要強于男性h-ADMSCs,前者為1582±132 pg/10~6個細(xì)胞VS 1265±133 pg/10~6個細(xì)胞P＜0.01,后者為13316±1404 pg/10~6個細(xì)胞VS 11790±1541 pg/10~6個細(xì)胞P＜0.01。用含500umol/L H_2O_2的培養(yǎng)基誘導(dǎo)30分鐘后,流式細(xì)胞技術(shù)示女性h-ADMSCs的凋亡率低于男性h-ADMSCs(18.68%±1.35%VS 25.21%±1.57%,P＜0.01),同時年輕個體的ADMSCs更容易被誘導(dǎo)凋亡。 結(jié)論: 1.通過酶消化的方法成功地從人皮下脂肪組織分離培養(yǎng)出具有多向分化潛能的ADMSCs。 2.h-ADMSCs在增殖、分化、旁分泌、抗凋亡能力上存在性別差異。年齡也是影響h-ADMSCs增殖、分化和凋亡的一個重要因素。個體本身的冠心病、高血壓、糖尿病狀況并不會影響其ADMSCs的生物學(xué)特性。
[Abstract]:Background: stem cell therapy for acute myocardial infarction is a research hotspot in recent years. Adipose derived mesenchymal stem cells (adipose tissue-derived mesenchymal stem cells, ADMSCs) as a new seed cell for cell engineering because of convenient, abundant and easier to be accepted and other advantages is gradually accepted in China. Outside, for the research of ADMSCs is in the initial stage, the treatment mechanism of basic research is still very limited, which need to be solved before clinical application. At present, there are many problems stem cells transplantation mechanism mainly has two kinds: 1, MSCs can differentiate into myocardial cells.2 by paracrine growth factors (such as HGF VEGF, etc.) to play a role. Recently, scientists found that stem cell differentiation due to gender differences, extracted from female mice in muscle cells than male muscle extracted more easily divided into body tissues. So whether there is a gender difference in stem cell differentiation? Gender or other individual factors (such as age, whether hypertension, diabetes, coronary heart disease etc.) for stem cell proliferation, differentiation, paracrine, whether it will affect the biological characteristics of anti apoptosis ability? To solve these problems for understanding the biological characteristics of ADMSCs itself. Has very important significance in basic research and clinical application of ADMSCs guidance.
Objective: to establish a is isolated from human adipose tissue culture, ADMSCs method, and the surface markers and multilineage differentiation potential of their identification, for the further research. The two is to observe and compare the different ADMSCs proliferation, differentiation, paracrine, anti apoptosis ability difference, to explore whether there was ADMSCs individual differences in the cell level, provide a new perspective for the basic research and clinical application of stem cells in the future.
Content and method: according to the above purpose, this study is divided into two parts:
The first part of the isolation and in vitro culture of human ADMSCs; and through CD13, the cell CD44, CD45, HLA-DR and VWF and adipogenicdifferentiation, immunohistochemistry of cell surface mesenchymal stem cell associated markers identification of osteogenic differentiation potential, inspection to verify whether the myocardial mesenchymal stem cells.
The second part of the first part of the human ADMSCs is MTT colorimetric test, ELIsA double antibody sandwich method, flow cytometry technology to compare ADMSCs proliferation, paracrine, differentiation and anti apoptotic ability of different people.
Result:
The spindle cells from human abdominal subcutaneous adipose tissue were isolated and cultured. All the cell surface molecules CD13 and CD44 were positive, but CD45, HLA-DR and VWF were negative. They had the ability to differentiate into adipocytes, osteoblasts, and into cardiomyocytes.
MTT assay showed the growth curve of ADMSCs by depicting the general shape of OD is similar to that of a S type. In 5D, 7d, 9D was significantly higher than those of the female h-ADMSCs male h-ADMSCs (P < 0.01), while the higher ADMSCs od young individuals. By 10umol/L 5-aza after 24 hours of induction, the expression of flow cytometry shows female h-ADMScs TNT was generally higher than that of male h-ADMSCs (27.3% + 18.9% 2.4%VS + 2.5%, P < 0.01), and younger individuals are more likely to be induced by ADMSCs and expression of TNT.Elisa showed: female h-ADMSCs and HGF secretion of VEGF was better than that of male h-ADMSCs, the former is 1582 + 132 pg/10~6 cells VS 1265 + 133 pg/10~6 cell P < 0.01, the medium was 13316 + 1404 pg/10~6 11790 + 1541 pg/10~6 cell VS cell P < 0.01. with 500umol/L H_2O_2 induced apoptosis after 30 minutes, flow cytometry shows the rate is lower than the female h-ADMSCs Male h-ADMSCs (18.68% + 1.35%VS 25.21% + 1.57%, P < 0.01), while ADMSCs in young individuals was more likely to be induced to apoptosis.
Conclusion:
1. ADMSCs. was successfully isolated and cultured from human subcutaneous adipose tissue by enzymatic digestion.
There are gender differences in proliferation, differentiation, paracrine and anti apoptotic ability of 2.h-ADMSCs. Age is also an important factor affecting h-ADMSCs proliferation, differentiation and apoptosis. The individual's coronary heart disease, hypertension and diabetes do not affect the biological characteristics of ADMSCs.

【學(xué)位授予單位】：中國人民解放軍軍醫(yī)進修學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R329

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本文編號：1609789