白曉東, 柳曉杰, 劉賢華, 王佳哲, 劉維維
實(shí)驗(yàn)研究
吸脂組織中脂肪干細(xì)胞分離和定向分化的研究
白曉東, 柳曉杰, 劉賢華, 王佳哲, 劉維維
目的探討吸脂組織中的干細(xì)胞分離和體外誘導(dǎo)分化為表皮樣細(xì)胞、成骨細(xì)胞及脂肪細(xì)胞的可能性。方法通過電動(dòng)負(fù)壓吸引獲取1例行吸脂手術(shù)的30歲女性腹部脂肪組織,酶消化法獲取脂肪來源干細(xì)胞,體外培養(yǎng)擴(kuò)增,通過流式細(xì)胞儀檢測(cè)表面抗原的表達(dá)。取生長(zhǎng)良好的第3代人脂肪來源干細(xì)胞,分別應(yīng)用成表皮誘導(dǎo)培養(yǎng)液(70%培養(yǎng)液A+30%成纖維細(xì)胞培養(yǎng)基上清液+10 ng/L表皮生長(zhǎng)因子),成骨誘導(dǎo)培養(yǎng)基(DMEM/10%FBS,0.1 μmol/L地塞米松,50 μmol/L維生素C,10 mmol/Lβ-甘油磷酸鈉, 100 U/ml 青霉素, 100 U/ml鏈霉素)和成脂肪細(xì)胞誘導(dǎo)培養(yǎng)基(DMEM+10%FBS+500 μmol/L 1-甲基-3-異丁基黃嘌呤+1 μmol/L吲哚美辛)誘導(dǎo)20 d后,分別對(duì)成表皮誘導(dǎo)組進(jìn)行免疫組化檢測(cè)CK19表達(dá),成骨誘導(dǎo)組進(jìn)行堿性磷酸酶檢測(cè),成脂誘導(dǎo)組進(jìn)行油紅O檢測(cè)。結(jié)果流式細(xì)胞儀鑒定結(jié)果示,人脂肪來源干細(xì)胞CD44和CD49d為陽性,CD34為陰性。誘導(dǎo)20 d后,成表皮誘導(dǎo)組示免疫組織化學(xué)鑒定結(jié)構(gòu)顯示有CK19的表達(dá);成骨誘導(dǎo)組示細(xì)胞堿性磷酸酶染色陽性;成脂肪細(xì)胞誘導(dǎo)組示油紅O染色,胞質(zhì)內(nèi)脂滴均被染成紅色,證實(shí)為脂性液體。結(jié)論從吸出的脂肪組織中分離出脂肪來源干細(xì)胞,在體外進(jìn)行了脂肪干細(xì)胞的擴(kuò)增和傳代,所分離的脂肪來源干細(xì)胞具備多向分化能力。
脂肪來源干細(xì)胞; 分化; 吸脂
吸脂是常見的整形手術(shù),脂肪來源干細(xì)胞(adipose derived stem cells, ADSCs)是成體間質(zhì)干細(xì)胞之一[1],存在于脂肪組織中,具備多項(xiàng)分化能力,可以分化為脂肪細(xì)胞以及其他類型細(xì)胞[2-4]。自2012年12月至2013年12月,我們對(duì)吸脂術(shù)中吸出的脂肪進(jìn)行分離,制備出具備擴(kuò)增和多向分化的脂肪干細(xì)胞,為解釋吸脂術(shù)后局部脂肪增生和利用脂肪干細(xì)胞修復(fù)組織提出依據(jù)。
1.1 ADSCs的體外分離培養(yǎng)
脂肪組織來自1例在武警總醫(yī)院燒傷整形科就診的30歲健康女性,已簽署知情同意書。電動(dòng)負(fù)壓吸引獲取腹部脂肪組織20 ml,用D-Hank′s液反復(fù)沖洗脂肪組織3次以上,盡量去除脂肪組織中肉眼可見的纖維及血管成分,剪碎脂肪組織,加入3倍體積的0.1%Ⅰ型膠原酶溶液,37 ℃振蕩消化60 min后液面分為3層,吸出下層液體,過濾,1000 r/min離心5 min,制成細(xì)胞懸液,加入紅細(xì)胞裂解液,靜置后離心5 min,以充分去除膠原酶及殘余紅細(xì)胞裂解液。所得細(xì)胞按2×107/L濃度接種于25 cm×25 cm培養(yǎng)瓶中,置于37 ℃、5%CO2培養(yǎng)箱中,生長(zhǎng)至70%~80%融合時(shí)進(jìn)行傳代,按照1∶3比例重新接種至培養(yǎng)瓶中[5]。
1.2 分離ADSCs的鑒定
加入CD34、CD49d及CD44的一抗,懸浮細(xì)胞,37 ℃孵育30 min,離心,棄上清。 PBS 1 ml,重新懸浮細(xì)胞,離心,棄上清,洗去多余一抗。按1∶100比例加入結(jié)合FITC的二抗,37 ℃孵育30 min,離心,棄上清。將細(xì)胞懸液移入流式細(xì)胞儀專用試管,上機(jī)檢測(cè)。CellQuest軟件分析結(jié)果。
1.3 干細(xì)胞的誘導(dǎo)分化
取生長(zhǎng)良好,達(dá)到90%融合的第3代人ADSCs,分別應(yīng)用成表皮誘導(dǎo)培養(yǎng)液(70%培養(yǎng)液A+30%成纖維細(xì)胞培養(yǎng)基上清液+10 ng/L 表皮生長(zhǎng)因子),成骨誘導(dǎo)培養(yǎng)基(DMEM/10%FBS,0.1 μmol/L地塞米松, 50 μmol/L維生素C, 10 mmol/L β-甘油磷酸鈉, 100 U/ml 青霉素, 100 U/ml鏈霉素),成脂肪細(xì)胞誘導(dǎo)培養(yǎng)基(DMEM+10% FBS+500 μmol/L 1-甲基-3-異丁基黃嘌呤+1 μmol/L吲哚美辛),各自設(shè)置對(duì)照組,對(duì)照組用基礎(chǔ)培養(yǎng)基。置培養(yǎng)箱中培養(yǎng),傳代,誘導(dǎo)20 d后分別對(duì)成表皮誘導(dǎo)組進(jìn)行免疫組化檢測(cè)CK19表達(dá),成骨誘導(dǎo)組進(jìn)行堿性磷酸酶檢測(cè),成脂誘導(dǎo)組進(jìn)行油紅O檢測(cè)。各自對(duì)照組也進(jìn)行相應(yīng)檢測(cè)。
1.4 誘導(dǎo)后細(xì)胞的測(cè)定
1.4.1 成表皮誘導(dǎo)組免疫細(xì)胞化學(xué)染色 取誘導(dǎo)20 d后的細(xì)胞,加入鼠抗人CK19一抗(陰性對(duì)照組加入PBS),4 ℃孵育過夜。次日取出,加入辣根過氧化物酶標(biāo)記羊抗鼠二抗室溫孵育30 min,加入DAB顯色。對(duì)照組進(jìn)行相同處理。
1.4.2 成骨誘導(dǎo)組堿性磷酸酶染色(鈣鈷法) 取誘導(dǎo)20 d后的細(xì)胞,加入95%乙醇固定30 min,干燥,加入37 ℃堿性磷酸酶孵育液2 h(pH 9.2~9.4,2%巴比妥鈉5 ml,3%β-甘油磷酸鈉5 ml,2%硝酸鈣10 ml,2%硫酸鎂5 ml,蒸餾水25 ml)。對(duì)照組進(jìn)行相同處理。
1.4.3 成脂誘導(dǎo)組油紅O染色 取誘導(dǎo)20 d后的細(xì)胞,1×104/ml接種于6孔培養(yǎng)板,培養(yǎng)2 d后去除孔內(nèi)培養(yǎng)基, 用預(yù)冷的10%甲醛固定10 min, 油紅O染液室溫染色30 min, 蘇木素復(fù)染5 min。倒置顯微鏡下觀察并及時(shí)拍照。對(duì)照組進(jìn)行相同處理。
2.1 吸脂組織中ADSCs的分離和培養(yǎng)
接種24 h后,多數(shù)已貼壁,呈短梭形或小多角形。接種48 h后,細(xì)胞體積增大,開始分裂增殖,細(xì)胞漸呈長(zhǎng)梭形,高倍鏡下可見單個(gè)細(xì)胞的胞漿較豐富,核較大且居中,核仁、核膜明顯。傳代24 h后,人ADSCs開始貼壁、伸展,恢復(fù)梭形形態(tài),大小較一致,增殖迅速,一般7、8 d即可形成單層匯合(圖1)。 所分離ADSCs的表面抗原特性, CD44、CD49d陽性,CD34陰性。
圖1 第3代脂肪干細(xì)胞 (×100)
Fig1 ADSCs at 3rd passage (×100).
2.2 ADSCs定向分化
2.2.1 ADSCs向表皮樣細(xì)胞誘導(dǎo)分化結(jié)果 經(jīng)誘導(dǎo)后,誘導(dǎo)組細(xì)胞由原來的長(zhǎng)梭形成纖維細(xì)胞狀結(jié)構(gòu)逐漸變短,細(xì)胞緊密排列成片,而對(duì)照組細(xì)胞未見變化,仍為原來的長(zhǎng)梭形成纖維細(xì)胞狀。誘導(dǎo)培養(yǎng)20 d后的細(xì)胞化學(xué)鑒定結(jié)構(gòu)顯示,有CK19陽性結(jié)果(圖2)。對(duì)照組未見CK19陽性細(xì)胞。
2.2.2 人ADSCs成骨誘導(dǎo)分化結(jié)果 誘導(dǎo)組誘導(dǎo)20 d后,部分細(xì)胞形態(tài)發(fā)生明顯變化,由梭形轉(zhuǎn)化為多角形,胞核變大變圓,細(xì)胞體積增大,突起增多,胞漿中可見細(xì)小黑色顆粒,細(xì)胞常圍繞集落中央呈島狀分布。對(duì)照組細(xì)胞未出現(xiàn)相應(yīng)變化,仍為長(zhǎng)梭形成纖維細(xì)胞狀。堿性磷酸酶染色顯示,誘導(dǎo)組存在陽性結(jié)果,細(xì)胞呈灰黑色,提示ADSCs有向成骨細(xì)胞分化的趨勢(shì)(圖3)。對(duì)照組示陰性結(jié)果。
2.2.3 人ADSCs成脂誘導(dǎo)分化結(jié)果 成脂誘導(dǎo)20 d后,倒置顯微鏡下可見部分細(xì)胞的胞漿內(nèi)出現(xiàn)多個(gè)高折光性的圓形小脂滴,細(xì)胞形狀也由原來的梭形逐漸變圓。隨誘導(dǎo)時(shí)間的延長(zhǎng),出現(xiàn)脂滴的細(xì)胞逐漸增多。油紅O染色示細(xì)胞內(nèi)出現(xiàn)亮紅色的顆粒,證明胞漿內(nèi)容物確為脂肪滴(圖4)。對(duì)照組無明顯變化,仍為梭形細(xì)胞,未見胞漿內(nèi)脂滴。
有些患者吸脂術(shù)后出現(xiàn)明顯的復(fù)發(fā),普遍認(rèn)為與脂肪內(nèi)存在干細(xì)胞有很大關(guān)系。目前分離常用酶消化法來提取ADSCs,本研究通過電動(dòng)負(fù)壓吸引獲取脂肪組織20 ml,用眼科剪盡量去除脂肪組織中肉眼可見的纖維及血管成分,剪碎脂肪組織,加入Ⅰ型膠原酶溶液,消化60 min后,吸出最下面含細(xì)胞層液體,裂解紅細(xì)胞后貼壁培養(yǎng),生長(zhǎng)至70%~80%融合時(shí)進(jìn)行傳代,我們所看到的細(xì)胞形態(tài)一致,很少有雜質(zhì)細(xì)胞。ADSCs不表達(dá)造血細(xì)胞表面抗原如造血前體細(xì)胞標(biāo)志抗原CD34、白細(xì)胞標(biāo)志抗原CD45、淋巴細(xì)胞表面抗原CD11a 等,只表達(dá)CD29、CD44、CD105、CD166和CD49d。
圖2 免疫組化示CK19 陽性 (×100)圖3 堿性磷酸酶陽性 (×100)圖4 油紅染色陽性(×200)
Fig2 Positive expression of CK19 (IHC×100).Fig3 Positive expression of ALP (IHC×100).Fig4 Positive expression of human ADSCs by oil red staining (×200).
為證明所分離細(xì)胞具有多向分化潛能,選取第3代的細(xì)胞,分別采用表皮細(xì)胞誘導(dǎo)液、骨細(xì)胞誘導(dǎo)液和脂肪細(xì)胞誘導(dǎo)液進(jìn)行定向誘導(dǎo),證實(shí)可以出現(xiàn)相應(yīng)的標(biāo)志,表皮表型CK19陽性,骨樣細(xì)胞的堿性磷酸酶陽性和脂肪油紅O染色陽性。
術(shù)中,我們?yōu)樘岢鯝DSCs,采用的是單純腫脹負(fù)壓吸脂,未采用超聲吸脂;在所吸出的20 ml脂肪液,經(jīng)過分離貼壁培養(yǎng)24 h,即可分離出ADSCs,連續(xù)傳代細(xì)胞活性沒有改變,說明在人體脂肪中大量存在干細(xì)胞,在體內(nèi)環(huán)境下可以分化為脂肪細(xì)胞,也可說明為什么吸脂術(shù)后有些患者效果不明顯,即所謂的“反彈”。
干細(xì)胞研究的快速發(fā)展,為組織工程技術(shù)構(gòu)建人體組織、器官提供了充足可靠的種子細(xì)胞[6-7]。根據(jù)干細(xì)胞的來源不同可分為胚胎干細(xì)胞(embryonic stem cell, ESC)和來源于出生后器官或成年個(gè)體組織的干細(xì)胞或成體干細(xì)胞(adult stem cell, ASC)。近年研究表明,ADSCs的多向分化潛能和方便獲取使其受到廣泛關(guān)注[8-9],ADSCs可通過負(fù)壓抽吸獲取,取材較易又避免了倫理問題,脂肪干細(xì)胞具有高度自我更新能力、免疫源性低等優(yōu)點(diǎn),是理想的組織工程學(xué)種子細(xì)胞[10-13],并分泌多種細(xì)胞因子[14-15]。若將此干細(xì)胞誘導(dǎo)分化為表皮細(xì)胞,作為創(chuàng)面修復(fù)的理想種子細(xì)胞將成為治療創(chuàng)面缺損的新思路。
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Researchofisolationanddirectionaldifferentiationofadiposederivedstemcellsaftersuctionlipectomy
BAIXiao-dong,LIUXiao-jie,LIUXian-hua,etal.
(DepartmentofBurnandPlasticSurgery,theGeneralHospitalofChinesePeopleArmedPoliceForce,Beijing100039,China)
ObjectiveTo observe the formation possibility of epidermic cells, skeletogenous cells and adipose cells by isolation and directional differentiation of adipose derived stem cells (ADSCs) in vitro.MethodsHuman abdominal adipose tissues were obtained by vacuum aspiration technique of suction lipectomy surgery from a 30-year-old woman. ADSCs were gotten by digestion and amplification. CD antigen was detected by flow cytometry. The ADSCs at 3rd passage were induced in epidermic cells′ culture solution (70% culture solution A+30% supernatant fluid from fibroblast medium+10 ng/LEGF) , skeletogenous cells′ culture solution (DMEM/10%FBS,0.1μmol/L desacort, 50μmol/L vitamin C, 10 mmol/L sodium β-glycerophosphate, 100 U/ml penicilin, 100 U/mlphytomycin)and adipose cells culture solution (DMEM+10% FBS+500 μmol/L IBMX+1 μmol/L antinfan) respectively. the expression CK19 in the group which was induced into epidermic cells was detected by IHC, ALP in the group which was induced into skeletogenous cells was detected and the rathonum red in the third group was checked.ResultsThe expression of CD44 and CD49 was positive and CD34 was negative by flow cytometry. The expression of CK19 in the group which was induced into epidermic cells was positive at 20 days after induction, ALP in the group which was induced into skeletogenous cells was positive and the rathonum red in the third group was positive.ConclusionHuman adipose derived stem cells can be obtained from human adipose tissue after suction lipectomy. The ADSCs can be differentiated into epidermic cells, skeletogenous cells and adipose cells in vitro.
Adipose derived stem cells; Differentiation; Suction lipectomy
武警部隊(duì)資助項(xiàng)目(WZ2006006)
100039 北京, 武警總醫(yī)院 (燒傷整形科:白曉東,柳曉杰,王佳哲,劉維維;醫(yī)療科:劉賢華)
白曉東(1969-),男,山西太原人,主任醫(yī)師,博士.
10.3969/j.issn.1673-7040.2014.07.022
R622.9
A
1673-7040(2014)07-0443-04
2014-03-25)