發(fā)布時(shí)間：2018-04-22 22:14

  本文選題：hADSCs + 細(xì)胞培養(yǎng)��； 參考：《重慶醫(yī)科大學(xué)》2015年博士論文

【摘要】：第一部分人脂肪干細(xì)胞體外培養(yǎng)及細(xì)胞載體復(fù)合物誘導(dǎo)培養(yǎng)的研究目的：探討人脂肪干細(xì)胞體外培養(yǎng)方法以及對細(xì)胞載體復(fù)合物誘導(dǎo)成軟骨分化的研究。方法：通過選擇性吸脂或其他腹部手術(shù)獲取3個(gè)樣品的脂肪組織,分離、消化獲取入脂肪間充質(zhì)干細(xì)胞(hADSCs),并經(jīng)過原代和傳代培養(yǎng)MTT法檢測細(xì)胞生長情況并繪制hADSCs的生長曲線；收集第3代hADSCs,利用流式細(xì)胞術(shù)檢測hADSCs細(xì)胞表面分子標(biāo)志(CD29、 CD34、CD45、CD90和CD105); hADSCs成脂誘導(dǎo)分化并使用油紅O染色法鑒定,hADSCs成骨誘導(dǎo)分化并使用免疫熒光檢測骨唾液酸蛋白(BSP)、骨橋蛋白(OPN)、骨連接蛋白(ON)的表達(dá),利用阿利新藍(lán)染色和免疫組化檢測Ⅱ型膠原蛋白的表達(dá)正式ADSCs的成軟骨誘導(dǎo)分化作用；將第3代hADSCs與不同濃度膠原纖維蛋白溶液混合制成細(xì)胞載體復(fù)合物凝膠,14d后進(jìn)行阿利新藍(lán)染色以及免疫組化檢測Ⅱ型膠原蛋白表達(dá)。結(jié)果：體外分離、培養(yǎng)出高活性hADSCs,增值曲線呈S形。細(xì)胞CD29、CD90、CD105呈陽性表達(dá),CD34、CD45呈陰性表達(dá)。hADSCs經(jīng)成脂誘導(dǎo)后,細(xì)胞呈類圓形,細(xì)胞漿內(nèi)出現(xiàn)有透亮的脂滴,油紅O染色色脂滴呈鮮紅色。成骨誘導(dǎo)后細(xì)胞呈為不規(guī)則形,免疫熒光檢測細(xì)胞表達(dá)骨唾液酸蛋白、骨橋蛋白、骨連接蛋白。hADSCs成軟骨誘導(dǎo)后細(xì)胞形態(tài)變?yōu)殇伮肥瘶?阿利新藍(lán)染色細(xì)胞基質(zhì)呈藍(lán)色,免疫組化檢測Ⅱ型膠原蛋白染色呈陽性。阿利新藍(lán)染色細(xì)胞載體復(fù)合物成藍(lán)色,免疫組化檢測Ⅱ型膠原蛋白結(jié)果顯示,混合膠原蛋白組較單一膠原蛋白組更強(qiáng)陽性表達(dá)。結(jié)論：成功分離培養(yǎng)出高度同源性ADSCs,具有多分化潛能。膠原纖維蛋白復(fù)合的支架載體,通過混合培養(yǎng)誘導(dǎo)hADSCs成軟骨細(xì)胞分化,可以作為修復(fù)軟骨缺損的理想載體支架材料。第二部分人脂肪干細(xì)胞軟骨分化過程中MicroRNA的表達(dá)目的：檢測人脂肪干細(xì)胞向軟骨細(xì)胞分化過程中的MicroRNA (miRNA或者miR)表達(dá)譜,并確認(rèn)該過程中MicroRNA影響軟骨分化的潛在機(jī)制。方法：(1)通過選擇性吸脂或其他腹部手術(shù)獲取3個(gè)樣品的脂肪組織,分離和培養(yǎng)人脂肪干細(xì)胞(hADSCs);(2)利用流式細(xì)胞儀檢測hADSCs表面標(biāo)記物(CD29、CD44、CD49和CD45);(3) hADSCs經(jīng)過成軟骨誘導(dǎo)后分化,免疫組化檢測軟骨細(xì)胞相關(guān)蛋白的表達(dá)；(4)然后通過MicroRNA列陣獲得MicroRNA表達(dá)譜,篩選出20個(gè)miRNAs具有至少2倍的差異性表達(dá),這些miRNA包括12上調(diào)miRNAs和8下調(diào)miRNAs。 Northern印跡分析進(jìn)一步證實(shí)了miRNAs的表達(dá)水平,并通過Northern印跡分析加以證實(shí)；(5)利用TargetScan (www.targetscan.org)、MiRanda (www.microrna.org)和miRBase (microma.sanger.ac.uk)等算法預(yù)測miRNAs的假定靶基因,并利用Western印跡分析檢測所預(yù)知靶基因,熒光素酶報(bào)告基因分析加以證實(shí)；(6)利用功能分析來檢測在hADSCs向軟骨細(xì)胞誘導(dǎo)分化中miR-196a和miR-490-5p的作用。用miR-490-5p'慢病毒轉(zhuǎn)染這些細(xì)胞,并用ELISA量化測定軟骨細(xì)胞誘導(dǎo)分化標(biāo)志物(Col2A1, Col10A1 and aggrecan)的表達(dá)；(7)用BMPR2 siRNA慢病毒轉(zhuǎn)染hADSCs,并經(jīng)過向軟骨細(xì)胞誘導(dǎo)分化孵育后,Western印跡分析檢測BMPR2蛋白的表達(dá)。ELISA量化測定軟骨細(xì)胞誘導(dǎo)分化標(biāo)志物(Col2A1,Col10A1 and aggrecan)的表達(dá)。結(jié)果：(1)第3代hADSCs是包括小的,單個(gè)圓形或幾個(gè)成團(tuán)懸浮在培養(yǎng)基中,經(jīng)過流式細(xì)胞儀檢測細(xì)胞表面標(biāo)記物：細(xì)胞陽性表達(dá)CD29, CD44和CD49,陰性表達(dá)CD45；(2)hADSCs經(jīng)軟骨誘導(dǎo)培養(yǎng)后細(xì)胞形態(tài)呈多邊形(這是軟骨細(xì)胞典型的形態(tài)學(xué)特征),利用免疫組化染色發(fā)現(xiàn)Ⅱ型膠原蛋白表達(dá)明顯升高,證明hADSCs向軟骨細(xì)胞分化；(3)利用miRNA微陣列芯片進(jìn)行檢測3組樣品的hADSCs經(jīng)過軟骨誘導(dǎo)分化前、后的hADSCs的miRNA的表達(dá)水平,軟骨誘導(dǎo)分化前、后差異表達(dá)至少2倍的hADSCs的miRNA,包括12個(gè)上調(diào)miRNA (miR-196a, miR-143, miR-383, miR-193b, miR-7i, miR-26a, miR-539, miR-199a-3p, miR-337-5p, miR-146a-5p, miR-646和miR-381)和8個(gè)下調(diào)miRNA (miR-490-5p, miR-1307, miR-125b, miR-96-3p, miR-302-3p, miR-23a-3p, miR-590和miR-510)。(4)使用Northern印跡分析檢測8個(gè)已利用微列陣證實(shí)了,具有代表性差異性表達(dá)的miRNAs,包括4個(gè)上調(diào)的miRNAs (miR-196a, miR-193b, miR-383和miR-143)和4個(gè)下調(diào)的miRNAs (miR-490-5p, miR-1307, miR-125b和miR-590)。Northern印跡分析顯示miR-196a在3個(gè)樣品中均過表達(dá),miR-490-5p在3個(gè)樣品中均明顯下調(diào)。(5)我們利用功能分析來檢測在hADSCs向軟骨細(xì)胞誘導(dǎo)分化中這2個(gè)miRNAs (miR-490-5p和miR-196a)的作用。然而,我們僅發(fā)現(xiàn)miR-490-5p在軟骨誘導(dǎo)分化過程中具有明顯的作用,miR-196a卻沒有(數(shù)據(jù)未表明)。但是,在分化的第12天,用miR-490-5p慢病毒轉(zhuǎn)染這些細(xì)胞,與對照慢病毒組比較,到第18天時(shí)細(xì)胞形態(tài)學(xué)發(fā)生逆轉(zhuǎn)。我們使用ELISA量化測定軟骨細(xì)胞誘導(dǎo)分化標(biāo)志物(Col2A1, Col10A1 and aggrecan),在第12天,第15天和第18天,細(xì)胞表面標(biāo)記物的水平逐漸上調(diào)；但是,經(jīng)過miR-490-5p慢病毒轉(zhuǎn)染的細(xì)胞,這些標(biāo)志物表達(dá)下調(diào)。這些結(jié)果表明miR-490-5p抑制ADSCs向軟骨細(xì)胞分化。(6)生物信息學(xué)分析證實(shí)了SOX-2和BMPR2作為miR-490-5p的假定的靶基因,與軟骨細(xì)胞分化有關(guān)。利用Western印跡分析檢測所預(yù)知靶基因SOX-2和BMPR2的蛋白表達(dá),結(jié)果表明經(jīng)過軟骨細(xì)胞誘導(dǎo)分化的hADSCs,其SOX-2和BMPR2的表達(dá)水平上調(diào)；將這些誘導(dǎo)分化后的hADSCs用miR-490-5p慢病毒轉(zhuǎn)染后,其BMPR2的表達(dá)水平下調(diào),而SOX-2的表達(dá)并未受到明顯影響。熒光素酶報(bào)告分析證實(shí)了miR-490-5p抑制BMPR23'UTR螢光素酶40%的活性,而用突變的BMPR23'UTR轉(zhuǎn)染后,熒光素酶活性被完全逆轉(zhuǎn)。這些結(jié)果說明BMPR2是的miR-490-5p的直接靶標(biāo)。(7)用BMPR2 siRNA慢病毒轉(zhuǎn)染hADSCs,并經(jīng)過向軟骨細(xì)胞誘導(dǎo)分化孵育后,免疫組化檢測表明BMPR2蛋白表達(dá)以時(shí)間依賴的方式顯著下降,其Ⅱ型膠原蛋白表達(dá)下調(diào)；ELISA分析發(fā)現(xiàn)Col 2A1、Col 10A1和aggrecan的表達(dá)水平在第12天,第15天和第18天均下調(diào)。結(jié)論：我們證實(shí)了一組在調(diào)節(jié)hADSCs向軟骨細(xì)胞分化的過程中起關(guān)鍵作用的miRNA。我們的研究結(jié)果為進(jìn)一步研究在hADSC軟骨形成過程中發(fā)揮作用的miRNAs的分子機(jī)制奠定了基礎(chǔ)。
[Abstract]:The purpose of this study was to investigate the in vitro culture of human adipose - derived stem cells and to study the induction culture of human adipose - derived stem cells . Methods : The adipose tissue , separation and digestion of human adipose - derived stem cells were studied .
hADSCs were collected by flow cytometry to detect the molecular markers of hADSCs ( CD29 , CD34 , CD45 , CD90 and CD105 ) . hADSCs were induced and differentiated by oil - red O staining . hADSCs were differentiated into bone - induced differentiation and the expression of bone sialoprotein ( BSP ) , osteo - bridge protein and bone connexin ( ON ) were detected by immunofluorescence staining .
The expression of microRNAs ( CD29 , CD44 , CD49 and CD45 ) in human adipose - derived mesenchymal stem cells ( hADSCs ) was detected by immunohistochemistry .
( 4 ) MicroRNA expression profiles were then obtained through the MicroRNA array to screen out a differential expression of at least 2 - fold in 20 microRNAs , which included 12 up - regulation and 8 - down - downgrades . Northern blot analysis further confirmed the level of miRNA expression and confirmed by Northern blot analysis .
( 5 ) The putative target gene was predicted using an algorithm such as TargetScan ( www.targetscan . org ) , MiRanda ( www.microrna . org ) and miRBase ( microma . sanger . ac.uk ) , and confirmed by Western blot analysis using the predicted target gene , luciferase reporter gene analysis ;
( 6 ) Using the function analysis to detect the role of miR - 196a and miR - 490 - 5p in the differentiation of hADSCs into chondrocytes . These cells were transfected with miR - 490 - 5p ' lentivirus , and the expression of the differentiation markers ( Col2A1 , Col10A1 and aggrecan ) was determined by ELISA .
( 7 ) The expression of BMPR2 protein was detected by Western blot analysis after transfection of hADSCs with BMPR2 siRNA lentivirus , and the expression of BMPR2 protein was determined by Western blot analysis .
( 2 ) The cell morphology of hADSCs was polygonal ( which is a typical morphological feature of chondrocytes ) after cartilage - induced culture , and the expression of type 鈪，

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