【摘要】：研究背景：間充質(zhì)干細(xì)胞（Mesenchymalstemcells,MSCs）也稱為多潛能間充質(zhì)基質(zhì)細(xì)胞，為一種非造血成體干細(xì)胞，在組織中分布廣泛，具有自我更新和多向分化能力。由于具有免疫調(diào)節(jié)、多向分化潛能、易于獲取、體外增殖快、凍存后活性損失小、免疫原性低并且無倫理問題及毒副作用等特點(diǎn)已經(jīng)在臨床上被廣泛用于多種疾病的治療性研究。成體骨髓來源的MSCs（Bonemarrow-derivedMSCs,BM-MSCs）是研究最早且較為深入的MSCs，但由于抽取骨髓對供者損傷較大、易受病毒感染、細(xì)胞數(shù)量和增殖/分化能力隨年齡顯著下降等原因限制了BM-MSCs的臨床應(yīng)用。大量研究已表明，多種組織均含有MSCs，如臍帶（Umbilicalcord,UC）、脂肪等。而臍帶作為胎兒娩出后的醫(yī)療廢棄物，具有易于獲取、倫理限制小、體外易于擴(kuò)增及培養(yǎng)等優(yōu)勢。因此，本實驗擬對臍帶來源的MSCs（UC-derivedMSCs,UC-MSCs）與成人BM-MSCs的生物學(xué)特性進(jìn)行比較，以期望獲得更佳的MSCs種子細(xì)胞來源。同時，許多研究均表明，MSCs分泌的細(xì)胞因子在MSCs治療效應(yīng)中發(fā)揮至關(guān)重要的作用，但目前尚無UC-MSCs細(xì)胞因子表達(dá)譜的研究報道。因此，本實驗擬采用細(xì)胞因子抗體芯片對不同代次UC-MSCs分泌的細(xì)胞因子進(jìn)行高通量篩選，為UC-MSCs傳代次數(shù)的確定并為后續(xù)治療機(jī)制的研究奠定理論基礎(chǔ)。 目的：在MSCs臨床應(yīng)用中，為種子細(xì)胞來源提供實驗依據(jù)；我們比較了體外分離培養(yǎng)UC-和BM-MSCs在形態(tài)特征、增殖能力、表面標(biāo)志和誘導(dǎo)分化能力等方面的差異；為給體外長期培養(yǎng)UC-MSCs的穩(wěn)定性提供實驗證據(jù)，我們進(jìn)一步研究了體外長期培養(yǎng)UC-MSCs的生物特性及活性變化等特點(diǎn)；為了確定體外培養(yǎng)UC-MSCs的較佳傳代次數(shù)并為治療機(jī)制提供一定的理論依據(jù)，我們對不同代次的UC-MSCs體外分泌的細(xì)胞因子進(jìn)行細(xì)胞因子抗體芯片篩查和表達(dá)譜的差異比較。 方法：我們分別應(yīng)用酶消化法和密度梯度離心法分離UC-MSCs和BM-MSCs，并用貼壁培養(yǎng)方法進(jìn)一步純化兩種來源MSCs。采用顯微鏡技術(shù)觀察比較了兩者的形態(tài)特點(diǎn)；應(yīng)用細(xì)胞計數(shù)法檢測了兩者的增殖能力；通過體外連續(xù)傳代試驗研究兩者的傳代能力；采用流式細(xì)胞術(shù)分析了兩者免疫表型；通過染色法鑒定比較體外誘導(dǎo)UC-和BM-MSCs向脂肪、軟骨和成骨細(xì)胞分化能力。我們進(jìn)一步對UC-MSCs進(jìn)行體外連續(xù)培養(yǎng)，檢測了細(xì)胞生物特性和功能，并采用細(xì)胞因子抗體芯片對不同代次UC-MSCs的上清液進(jìn)行了高通量篩查（目標(biāo)蛋白為174種細(xì)胞因子，RayBio），根據(jù)熒光強(qiáng)度信號情況，篩選出MSCs上清液中陽性表達(dá)的細(xì)胞因子，并采用統(tǒng)計學(xué)方法分析不同代次UC-MSCs間的分泌陽性細(xì)胞因子的信號差異。 結(jié)果：細(xì)胞形態(tài)結(jié)果顯示，UC-和BM-MSCs細(xì)胞形態(tài)相似，均表現(xiàn)為長梭型、貼壁生長；體外長期傳代試驗表明，BM-MSCs體外增殖能力較差，傳代至9~10代基本失去增殖能力，而UC-MSCs增殖及更新能力明顯強(qiáng)于BM-MSCs，傳至15代時仍保持原有的特性；細(xì)胞表型結(jié)果表明，，UC-和BM-MSCs具有均一的免疫表型：不表達(dá)造血和內(nèi)皮標(biāo)志CD34、CD45、CD11b、CD31以及免疫共刺激分子CD80、CD86和MHCⅡ類分子（HLA-DR），表達(dá)間質(zhì)細(xì)胞標(biāo)記CD44、CD105和CD166和CD73。但BM-MSCs的CD80顯著高于UC-MSCs的（P=0.0137）；誘導(dǎo)分化結(jié)果顯示，UC-和BM-MSCs均可向脂肪、成骨和軟骨三系分化；細(xì)胞因子抗體芯片檢測結(jié)果表明，UC-MSCs上清液中陽性分泌的細(xì)胞因子有102種（強(qiáng)陽性34種，一般陽性68種），主要涉及促細(xì)胞遷移類（24.5%）、受體類（20.6%）、促細(xì)胞生長和分化類（20.6%）和免疫調(diào)節(jié)類（12.7%）；統(tǒng)計分析結(jié)果表明，第3、5和9代UC-MSCs分泌的細(xì)胞因子有顯著統(tǒng)計學(xué)差異的只有11種：TNF-beta（P=0.0147）、IL-12p70（P=0.0265）、MSP-alpha（P=0.0289）、HCC-4/CCL16（P=0.027）、IP-10/CXCL10（P=0.0202）、GITR（P=0.024）、PDGFRalpha（P=0.0277）、VEGFR2（P=0.038）、IL-9（P=0.0291）、MIP-1beta/CCL4（P=0.0497）和CNTF（P=0.0073）。 結(jié)論：UC-MSCs能替代BM-MSCs作為臨床上MSCs的種子細(xì)胞；不同代次UC-MSCs分泌的絕大多數(shù)細(xì)胞因子水平無差異；蛋白芯片能以極小的樣本量檢測高通量的細(xì)胞因子，是篩查MSCs細(xì)胞因子表達(dá)譜比較理想的技術(shù)方法。
[Abstract]:BACKGROUND: Mesenchymal stem cells (MSCs), also known as pluripotent mesenchymal stromal cells, are non-hematopoietic adult stem cells. They are widely distributed in tissues and have the ability of self-renewal and multi-differentiation. Adult bone marrow-derived MSCs (BM-MSCs) are the earliest and more in-depth MSCs. However, bone marrow extraction is more harmful to the donor, susceptible to viral infection, and increases the number of cells. The clinical application of BM-MSCs is limited by the decrease of reproductive/differentiation ability with age. Many studies have shown that many kinds of tissues contain MSCs, such as umbilical cord (UC), fat and so on. The biological characteristics of umbilical cord derived MSCs (UC-derived MSCs) and adult BM-MSCs were compared in order to obtain a better source of MSCs seed cells. In this study, cytokines secreted by different generations of UC-MSCs were screened by cytokine antibody chips in order to determine the number of passages of UC-MSCs and lay a theoretical foundation for the follow-up study of treatment mechanism.
OBJECTIVE: To provide experimental evidence for the origin of seed cells in the clinical application of MSCs; We compared the morphological characteristics, proliferation, surface markers and differentiation induction ability of UC-MSCs and BM-MSCs isolated and cultured in vitro; To provide experimental evidence for the stability of long-term cultured UC-MSCs in vitro, we further studied the in vitro growth of UC-MSCs. In order to determine the best passage times of UC-MSCs cultured in vitro and provide a theoretical basis for the treatment mechanism, we screened cytokines secreted by different generations of UC-MSCs in vitro and compared the differences of cytokine antibody chip and expression profiles.
METHODS: UC-MSCs and BM-MSCs were separated by enzyme digestion and density gradient centrifugation, and further purified by adherent culture. The morphological characteristics of UC-MSCs and BM-MSCs were observed and compared by microscopy, their proliferation ability was detected by cell counting, and the proliferation of UC-MSCs and BM-MSCs was studied by continuous passage test in vitro. The ability of UC-and BM-MSCs to differentiate into adipose, cartilage and osteoblasts in vitro was compared by staining. We further cultured UC-MSCs in vitro, detected their biological characteristics and functions, and used cytokine antibody chips for different generations. The supernatant of secondary UC-MSCs was screened by high-throughput screening (the target protein was 174 cytokines, RayBio). According to the fluorescence intensity signal, the positive cytokines expressed in the supernatant of secondary UC-MSCs were screened. The signal difference of secreting positive cytokines between different generations of UC-MSCs was analyzed by statistical method.
Results: Cell morphology showed that UC-and BM-MSCs were similar in morphology, both of which were spindle-shaped and adherent to the wall. Long-term passage test in vitro showed that BM-MSCs had poor proliferative ability and basically lost proliferative ability from passage 9 to 10, while UC-MSCs had stronger proliferative and renewable ability than BM-MSCs, and still maintained their original characteristics at passage 15. The results of cell phenotype showed that UC-and BM-MSCs had homogeneous immunophenotype: CD34, CD45, CD11b, CD31, CD80, CD86 and HLA-DR were not expressed in UC-and BM-MSCs, but CD80 of BM-MSCs was significantly higher than that of UC-MSCs (P=0.0137). The results showed that both UC-and BM-MSCs could differentiate into adipose, osteogenic and cartilage trilineage; cytokine antibody chip assay showed that there were 102 cytokines (34 strongly positive, 68 generally positive) secreted in the supernatant of UC-MSCs, mainly involved in promoting cell migration (24.5%), receptors (20.6%), promoting cell growth and differentiation (20.6%) and immunity. Statistical analysis showed that only 11 cytokines secreted by UC-MSCs in the 3rd, 5and 9generations were significantly different: TNF-beta (P = 0.0147), IL-12p70 (P = 0.0265), MSP-alpha (P = 0.0289), HCC-4/CCL16 (P = 0.027); IP-10/CXCL10 (P = 0.0202), GITR (P = 0.024), GITR (P = 0.0.024), PDGFRalpha (P = 0.77 (P = 0.77), IL-12p70 (P = 0.77), VEGF R2 (P = 0.0265), MSP = 0.0265, MSP-alpha (P = 0.02890.0289), HCC-4/CCL16 (P 0.0291), MI P-1beta/CCL4 (P=0.0497) and CNTF (P=0.0073).
CONCLUSION: UC-MSCs can replace BM-MSCs as the seed cells of MSCs in clinic; the levels of most cytokines secreted by different generations of UC-MSCs are not different; protein chip can detect high-throughput cytokines with very small sample size, which is an ideal technique for screening the cytokine expression profiles of MSCs.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 張衛(wèi)光;間充質(zhì)干細(xì)胞與生物人工肝的研究進(jìn)展[J];解剖科學(xué)進(jìn)展;2004年03期

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