周艷華, 許婷婷, 李 軍, 何冬梅, 譚廣銷(xiāo), 劉革修
(暨南大學(xué)醫(yī)學(xué)院血液病研究所,廣東 廣州 510632)
人臍帶間充質(zhì)干細(xì)胞低氧條件培養(yǎng)液對(duì)小鼠造血系統(tǒng)自然衰老的影響*
周艷華, 許婷婷, 李 軍, 何冬梅, 譚廣銷(xiāo), 劉革修△
(暨南大學(xué)醫(yī)學(xué)院血液病研究所,廣東 廣州 510632)
目的通過(guò)探討人臍帶間充質(zhì)干細(xì)胞(hUC-MSCs)低氧條件培養(yǎng)液對(duì)小鼠造血系統(tǒng)自然衰老的延緩作用,進(jìn)一步了解hUC-MSCs對(duì)機(jī)體造血的支持作用機(jī)制。方法將6月齡的BALB/c小鼠隨機(jī)分為實(shí)驗(yàn)組和對(duì)照組,前者腹腔注射hUC-MSCs低氧條件培養(yǎng)液,每4 d 1次,持續(xù)6個(gè)月;后者注射培養(yǎng)基。干預(yù)后觀察小鼠一般情況,并于干預(yù)后第3個(gè)月和第6個(gè)月分別比較兩組的外周血象(白細(xì)胞與血紅蛋白)、單側(cè)股骨骨髓有核細(xì)胞(BMCN)計(jì)數(shù)、造血祖細(xì)胞集落(CFU-GM、 CFU-E、 CFU-MK)培養(yǎng)、成纖維細(xì)胞集落(CFU-F)培養(yǎng)和外源性脾集落形成單位(CFU-S)計(jì)數(shù)。于第6個(gè)月時(shí)觀察骨髓病理變化、測(cè)定比較骨髓細(xì)胞重建造血能力及P16蛋白表達(dá)情況。結(jié)果實(shí)驗(yàn)組小鼠的一般情況優(yōu)于對(duì)照組;干預(yù)后第3個(gè)月,實(shí)驗(yàn)組的BMNC、CFU-GM和CFU-MK多于對(duì)照組,而外周血象、CFU-F和CFU-E無(wú)明顯差別;干預(yù)后第6個(gè)月,外周血象仍無(wú)顯著差別,其余的上述指標(biāo)實(shí)驗(yàn)組均明顯高于對(duì)照組,且各組的這些指標(biāo)隨時(shí)間變化呈不斷下降的趨勢(shì),但實(shí)驗(yàn)組的下降速度明顯慢于對(duì)照組,差異顯著(P<0.05)。組織學(xué)觀察發(fā)現(xiàn),實(shí)驗(yàn)組造血組織較對(duì)照組豐富,P16蛋白的表達(dá)也明顯低于對(duì)照組,而對(duì)照組骨髓脂肪化顯著增加;實(shí)驗(yàn)組骨髓細(xì)胞的造血重建能力明顯優(yōu)于對(duì)照組。結(jié)論hUC-MSCs分泌的細(xì)胞因子能夠延緩小鼠造血系統(tǒng)的衰退,有望應(yīng)用于延緩衰老的研究。
間充質(zhì)干細(xì)胞; 臍帶; 衰老; 細(xì)胞因子類(lèi)
間充質(zhì)干細(xì)胞(mesenchymal stem cells,MSCs)因具有獨(dú)特的、多樣的生物學(xué)活性而成為近年醫(yī)學(xué)研究熱點(diǎn),因?yàn)榫哂忻庖哒{(diào)節(jié)作用而被應(yīng)用于免疫性疾病治療,因?yàn)榫哂卸嘞蚍只瘽撃芏粦?yīng)用于再生醫(yī)學(xué)研究與應(yīng)用,因?yàn)榭煞置诙喾N細(xì)胞因子和生長(zhǎng)因子而被應(yīng)用于調(diào)節(jié)組織特異細(xì)胞生物學(xué)活性研究[1,2]。然而在培養(yǎng)MSCs過(guò)程中換液常常將這些細(xì)胞因子、生長(zhǎng)因子等丟棄了。這些培養(yǎng)液是否存在潛在價(jià)值呢?本研究采用人臍帶間充質(zhì)干細(xì)胞(human umbilical cord mesenchymal stem cells,hUC-MSCs)低氧條件培養(yǎng)液對(duì)小鼠自然衰老進(jìn)行干預(yù)研究,觀察是否對(duì)造血系統(tǒng)有積極作用,以期為延緩造血干細(xì)胞衰老的研究開(kāi)辟一條新思路。
1材料
足月剖宮產(chǎn)健康新生兒的臍帶取自暨南大學(xué)華僑醫(yī)院;6月齡雌性BALB/c純系小鼠40只、8周齡雌性BALB/c小鼠80只,SPF級(jí),體重(20±2) g,購(gòu)自廣東省醫(yī)學(xué)實(shí)驗(yàn)動(dòng)物中心,動(dòng)物合格證號(hào):SCXK(粵)2008-0002,所有動(dòng)物實(shí)驗(yàn)均按照中國(guó)科學(xué)技術(shù)委員會(huì)頒發(fā)的實(shí)驗(yàn)動(dòng)物管理?xiàng)l例執(zhí)行,在無(wú)菌條件下飼養(yǎng);DMEM/F12培養(yǎng)基、IMDM培養(yǎng)基、膠原酶Ⅳ和胰蛋白酶均購(gòu)自Gibco;胎牛血清(fetal bovine serum,F(xiàn)BS)購(gòu)自PAA;甲基纖維素、地塞米松、胰島素、抗壞血酸、β-磷酸甘油與油紅O購(gòu)自Sigma;促紅細(xì)胞生成素(erythropoietin,EPO)、粒細(xì)胞集落刺激因子(granulocyte colony-stimulating factor,G-CSF)、干細(xì)胞因子(stem cell factor,SCF)和促血小板生成素(thrombopoietin,TPO)均購(gòu)自Peprotech;P16免疫組織化學(xué)試劑盒購(gòu)自福州邁新生物技術(shù)公司。
2方法
2.1臍帶間充質(zhì)干細(xì)胞的分離培養(yǎng)及低氧條件培養(yǎng)液的制備 臍帶間充質(zhì)干細(xì)胞的分離培養(yǎng)及鑒定按文獻(xiàn)報(bào)道[3]的方法進(jìn)行。經(jīng)過(guò)鑒定的第5代細(xì)胞生長(zhǎng)至80%融合時(shí)換不含血清的營(yíng)養(yǎng)豐富培養(yǎng)液并置于37 ℃、5% O2、5% CO2、飽和濕度培養(yǎng)箱中培養(yǎng)24 h,收集培養(yǎng)液保存于-80°備用。
2.2hUC-MSCs低氧條件培養(yǎng)液干預(yù)小鼠造血系統(tǒng)自然衰老的觀察 隨機(jī)取8只6月齡雌性BALB/c小鼠,于干預(yù)前測(cè)定其單側(cè)股骨骨髓有核細(xì)胞計(jì)數(shù)、成纖維細(xì)胞集落、造血祖細(xì)胞集落培養(yǎng)、外源性脾集落形成單位計(jì)數(shù),剩余的隨機(jī)分為實(shí)驗(yàn)組和對(duì)照組,每組16 只。實(shí)驗(yàn)組為hUC-MSCs低氧培養(yǎng)液干預(yù)組:經(jīng)腹腔輸注制備好的hUC-MSCs 低氧條件培養(yǎng)液0.5 mL,每4 d 1次,持續(xù)輸注6個(gè)月;對(duì)照組為未干預(yù)組: 經(jīng)腹腔相應(yīng)地輸注0.5 mL培養(yǎng)基。2組動(dòng)物分別于干預(yù)開(kāi)始后第3個(gè)月、第6個(gè)月測(cè)定外周血象、骨髓有核細(xì)胞計(jì)數(shù)、成纖維細(xì)胞集落培養(yǎng)和造血祖細(xì)胞集落培養(yǎng)計(jì)數(shù),并比較各組小鼠生存情況;第6月時(shí),觀察骨髓病理變化及P16蛋白表達(dá)情況、對(duì)骨髓細(xì)胞造血重建能力進(jìn)行測(cè)定比較。
2.3外周血象測(cè)定及骨髓有核細(xì)胞(bone marrow nucleated cell,BMNC)計(jì)數(shù) 實(shí)驗(yàn)組和對(duì)照組分別于干預(yù)前、干預(yù)后第3個(gè)月和第6個(gè)月尾靜脈取血,常規(guī)測(cè)定其外周血白細(xì)胞(white blood cell,WBC)和血紅蛋白(hemoglobin,Hb)。于第3個(gè)月時(shí)每組各處死8只小鼠,無(wú)菌條件下取出左側(cè)股骨,按照常規(guī)方法,計(jì)數(shù)BMNC,調(diào)整細(xì)胞濃度用于祖細(xì)胞集落培養(yǎng)、成纖維細(xì)胞集落培養(yǎng)。于第6個(gè)月時(shí)處死各組剩余的8只小鼠,方法同前,計(jì)數(shù)BMNC,并進(jìn)行祖細(xì)胞集落培養(yǎng)、成纖維細(xì)胞集落培養(yǎng)及骨髓細(xì)胞造血重建能力的測(cè)定,且另一側(cè)股骨用于做骨髓病理切片。
2.4造血祖細(xì)胞集落(colony-forming units-granulocyte and macrophage, CFU-GM;colony-forming units-erythroid,CFU-E;colony-forming units-megakaryocyte,CFU-MK)培養(yǎng) 將上述方法得到的骨髓有核細(xì)胞,接種于24孔板,細(xì)胞濃度為2×108cells/L,0.5 mL cells/well,1個(gè)復(fù)孔,分別在以下3種體系中進(jìn)行培養(yǎng):(1)CFU-GM培養(yǎng)體系:含20%的FBS、G-CSF 60 μg/L、SCF 20 μg/L和0.8%甲基纖維素的IMDM培養(yǎng)基;(2)CFU-E培養(yǎng)體系:含20%的FBS、EPO 4 kU/L、SCF 20 μg/L和0.8%甲基纖維素的IMDM培養(yǎng)基;(3)CFU-MK培養(yǎng)體系:含20%的FBS、TPO 60 μg/L、SCF 20 μg/L和0.8%甲基纖維素的IMDM培養(yǎng)基。置于37 ℃、5% CO2、飽和濕度培養(yǎng)箱中孵育,3 d后計(jì)數(shù)CFU-E集落數(shù),大于8個(gè)細(xì)胞計(jì)數(shù)為1個(gè)集落;7 d后計(jì)數(shù)CFU-GM集落數(shù),大于50個(gè)細(xì)胞計(jì)數(shù)為1個(gè)集落;14 d 后計(jì)數(shù)CFU-MK集落數(shù),大于3個(gè)細(xì)胞計(jì)數(shù)為1個(gè)集落。
2.5成纖維細(xì)胞集落形成單位( fibroblast colony-forming units,CFU-F)培養(yǎng) 建立CFU-F培養(yǎng)體系[4],再加入已制備好的骨髓細(xì)胞懸液,以5×104cells/cm2的密度接種于6孔板,置于37 ℃、7% CO2、飽和濕度培養(yǎng)箱中培養(yǎng),每3 d換液1次,第14 d在倒置顯微境下計(jì)數(shù)大于30個(gè)細(xì)胞的集落。
2.6外源性脾集落形成單位(exogenous spleen colony-forming units,CFU-S)計(jì)數(shù) CFU-S計(jì)數(shù)反映供體小鼠骨髓細(xì)胞居留在受體小鼠脾臟造血組織中的干細(xì)胞數(shù)量。受體為8周齡雌性BALB/c小鼠,在接受致死量[60Coγ](8 GY)照射后4 h左右,由尾靜脈注射上述過(guò)程中得到的骨髓有核細(xì)胞各0.2 mL(含2×106cells)。2周后,斷頸處死小鼠取出脾臟,浸在Bouin液中24 h,80%乙醇中脫色3 d(每天換液1次),然后進(jìn)行CFU-S 計(jì)數(shù)。
2.7骨髓病理變化及P16蛋白表達(dá)測(cè)定 在上述過(guò)程中得到的股骨快速放入體積分?jǐn)?shù)為4%鹽酸甲醛脫鈣固定液中,12 h后進(jìn)行常規(guī)脫水、浸蠟、石蠟包埋、切片,HE染色后在顯微鏡下觀察骨髓造血組織情況并攝像。骨髓組織的衰老程度觀察通過(guò)檢測(cè)其P16蛋白的表達(dá)情況,采用免疫組織化學(xué)染色檢測(cè),操作步驟按試劑盒說(shuō)明進(jìn)行,DAB顯色,封片后并攝像。
2.8骨髓細(xì)胞的造血重建能力測(cè)定 干預(yù)后第6個(gè)月,進(jìn)行骨髓移植實(shí)驗(yàn),受體為8周齡雌性BALB/c小鼠,40只,分為5 組(8只/組);供體為上述得到的骨髓有核細(xì)胞以及另外制備的年輕小鼠的骨髓細(xì)胞。受體小鼠在接受致死量[60Coγ](8 GY)照射后4 h內(nèi)進(jìn)行移植。(1)Ⅰ組:供體小鼠為經(jīng)hUC-MSCs培養(yǎng)液干預(yù)后的小鼠,受體接受照射后,輸注細(xì)胞0.1 mL(含5×106cells);(2)Ⅱ組:供體小鼠為未經(jīng)hUC-MSCs培養(yǎng)液干預(yù)的小鼠,受體接受照射后,輸注細(xì)胞0.1 mL(含5×106cells);(3)Ⅲ組:供體為年輕的8周齡小鼠,受體接受照射后,輸注細(xì)胞0.1 mL(含5 ×106cells);(4)Ⅳ組:?jiǎn)渭冋丈浣M,受體接受照射后,輸注IMDM 0.1 mL;(5)Ⅴ組:空白對(duì)照組,不接受照射,僅輸注IMDM 0.1 mL。移植后,小鼠每天飲慶大霉素酸化水(4×105U/L)。觀察各組小鼠一般情況更及存活狀態(tài);各組分別在照射前(+0 d)及照射后+7 d、+14 d、+21 d、+28 d測(cè)定小鼠外周血WBC及Hb變化,并進(jìn)行各組生存率比較。
3統(tǒng)計(jì)學(xué)處理
1小鼠MSCs輸注實(shí)驗(yàn)中一般情況觀察
對(duì)照組小鼠在觀察期內(nèi)(6-12月齡)出現(xiàn)毛發(fā)粗糙無(wú)光澤,脫毛較多,體重減輕,運(yùn)動(dòng)逐漸遲緩,精神狀態(tài)較差;而實(shí)驗(yàn)組小鼠在觀察期內(nèi)毛發(fā)保持光亮,較少脫毛,體重保持穩(wěn)定,運(yùn)動(dòng)能力及精神狀態(tài)改變不明顯。
2hUC-MSCs低氧條件培養(yǎng)液輸注對(duì)小鼠造血系統(tǒng)的影響
經(jīng)測(cè)定比較兩組小鼠外周血象,無(wú)顯著差異,隨著時(shí)間的變化也未出現(xiàn)顯著波動(dòng),見(jiàn)表1。但是其余指標(biāo)變化明顯,干預(yù)后第3個(gè)月測(cè)定,實(shí)驗(yàn)組的BMNC、CFU-GM、CFU-MK計(jì)數(shù)均高于對(duì)照組(P<0.05),而CFU-F、CFU-E、CFU-S計(jì)數(shù)無(wú)顯著差異;第6個(gè)月測(cè)定實(shí)驗(yàn)組的指標(biāo)均明顯高于對(duì)照組(P<0.05)。隨時(shí)間變化各組指標(biāo)變化呈不同的趨勢(shì),實(shí)驗(yàn)組下降速度明顯慢于對(duì)照組,差異顯著(P<0.05),見(jiàn)表2。
3hUC-MSCs低氧條件培養(yǎng)液輸注對(duì)小鼠骨髓病理變化及P16蛋白的影響
骨髓切片顯示,實(shí)驗(yàn)組有核細(xì)胞較多,造血組織明顯多于對(duì)照組;而對(duì)照組脂肪組織明顯多于實(shí)驗(yàn)組,見(jiàn)圖1A-C。P16蛋白與造血組織衰老程度密切相關(guān),大都表達(dá)在細(xì)胞漿中,經(jīng)免疫組化染色后,陽(yáng)性細(xì)胞呈棕黃色,實(shí)驗(yàn)組P16蛋白表達(dá)量明顯低于對(duì)照組,但高于年輕的8周齡小鼠,見(jiàn)圖2A-C。
表1hUC-MSCs條件培養(yǎng)液干預(yù)對(duì)小鼠外周血象的影響
ExperimentalgroupControlgroupWBCHbWBCHbBeforeintervention9.73±3.08103.90±15.959.26±1.73100.46±2.073rdmonth9.50±1.13109.34±13.658.99±1.91104.72±14.076thmonth9.74±2.67114.85±18.989.59±2.49105.48±3.37
表2hUC-MSCs條件培養(yǎng)液對(duì)小鼠BMNC計(jì)數(shù)、造血祖細(xì)胞集落生長(zhǎng)、CFU-F和CFU-S的影響
ExperimentalgroupControlgroupBeforeintervention3rdmonth6thmonthBeforeintervention3rdmonth6thmonthBMNC9.9±2.210.0±1.2▲9.5±0.8◆10.5±1.98.9±1.37.9±0.8CFU-GM37.0±8.835.2±6.6##31.7±8.6??36.5±7.827.5±8.421.4±9.6CFU-E29.9±6.125.9±7.925.7±6.9??31.9±5.825.4±7.019.1±6.1CFU-MK54.5±11.557.8±12.4▲52.5±11.6??53.4±10.549.8±12.241.2±9.1CFU-S44.3±9.342.5±10.842.5±4.9??45.1±10.340.6±11.133.7±8.4CFU-F63.4±12.756.8±12.050.1±11.0??61.9±11.952.1±11.740.6±8.9
▲P<0.05vscontrol group at 3rd month;##P<0.01vscontrol group at 3rd month;◆P<0.05vscontrol group at 6th months;**P<0.01vscontrol group at 6th months.The change trends were different with time,P<0.05.
Figure 1. Histological observation of mouse bone marrow (HE staining,×100). A:the bone marrow biopsy of young mice,with very rich hematopoietic tissue and almost invisible adipose tissue;B:the bone marrow biopsy of control group, less hematopoietic tissue was observed and much more adipose tissue was filled in almost the whole marrow cavity;C:the bone marrow biopsy of experimental group, more hematopoietic tissue and less adipose tissue were found compared with that of control group.
圖1小鼠骨髓組織學(xué)觀察
Figure 2. The expression of P16 protein in mouse bone marrow(×200). A:young mice;B:conrtrol group;C:experimental group;D:negative control.
圖2骨髓P16蛋白的表達(dá)量
4hUC-MSCs低氧條件培養(yǎng)液輸注后的供體對(duì)小鼠骨髓細(xì)胞造血重建能力的影響
不同時(shí)點(diǎn)WBC、Hb量及其兩者動(dòng)態(tài)變化的比較可以反映不同供體骨髓重建造血的能力。移植后1-2周為小鼠骨髓抑制的嚴(yán)重期,Ⅳ組小鼠死亡時(shí)間為第+6 d-第+14 d,主要死于造血衰竭。Ⅰ、Ⅱ、Ⅲ各組的指標(biāo)隨時(shí)間恢復(fù)的趨勢(shì)不同(P<0.01),Ⅲ組WBC與Hb恢復(fù)最快,Ⅱ組恢復(fù)最慢,Ⅰ組介于兩者之間,見(jiàn)表3、4。Ⅳ組生存率為0,Ⅲ組與Ⅴ組的小鼠全部存活,Ⅰ、Ⅱ組均有小鼠死亡,但Ⅰ組生存率(88.5%)高于Ⅱ組(63.5%)。
表3 各組小鼠不同時(shí)點(diǎn)外周血WBC的變化
▲▲P<0.01vsⅡ group;##P<0.01vsⅢ group;○P<0.05vsⅣ group;●●P<0.01vsⅣ group;**P<0.01vsⅤ group.
表4 各組小鼠不同時(shí)點(diǎn)外周血Hb的變化
▲▲P<0.01vsⅡ group;○○P<0.01vsⅢ group;#P<0.05vsⅢ group;●●P<0.01vsⅣ group;**P<0.01vsⅤ group.
衰老(aging or senescence)是機(jī)體的細(xì)胞、組織與器官在結(jié)構(gòu)和功能上逐漸出現(xiàn)不可逆轉(zhuǎn)地、全面地退化,其關(guān)鍵在于組織特異干細(xì)胞衰老、再生能力下降。造血系統(tǒng)作為人體重要的系統(tǒng)之一,隨著年齡的增長(zhǎng)會(huì)出現(xiàn)功能不斷衰退。造血系統(tǒng)的核心是造血干細(xì)胞(hematopoietic stem cells,HSCs),通過(guò)增殖與發(fā)育分化以持續(xù)補(bǔ)充機(jī)體各種血細(xì)胞需要;而骨髓MSCs則與基質(zhì)等組成造血微環(huán)境,支持調(diào)節(jié)HSCs生物學(xué)活性。造血系統(tǒng)漸進(jìn)的衰老表現(xiàn)為造血細(xì)胞的產(chǎn)生及造血微環(huán)境穩(wěn)態(tài)調(diào)控能力的下降[4,5],其最終結(jié)果是造血功能衰退、不能滿足機(jī)體需要。HSCs衰老表現(xiàn)為自我復(fù)制與增殖能力下降,導(dǎo)致骨髓中造血干/祖細(xì)胞數(shù)量減少;MSCs衰老則表現(xiàn)為分泌重要細(xì)胞因子和生長(zhǎng)因子的能力下降、對(duì)HSCs調(diào)節(jié)支持作用下降,而且向脂肪細(xì)胞分化、抑制造血[6]。那么,有什么措施延緩造血系統(tǒng)衰老、調(diào)整機(jī)體健康狀態(tài)?
幾十年國(guó)內(nèi)外骨髓移植結(jié)果顯示,HSCs具有極強(qiáng)的免疫原性和免疫力、可產(chǎn)生移植排斥反應(yīng);而且進(jìn)行骨髓移植難度極大。所以,直接運(yùn)用年輕HSCs取代機(jī)體內(nèi)衰老HSCs具有極大難度,很難被應(yīng)用于抗衰老。既然衰老MSCs分泌重要細(xì)胞因子和生長(zhǎng)因子的能力下降、對(duì)HSCs調(diào)節(jié)支持作用下降,那么,是否可以通過(guò)補(bǔ)充相應(yīng)重要細(xì)胞因子和生長(zhǎng)因子以增強(qiáng)對(duì)HSCs支持作用呢?本研究給中老年老鼠定期輸注間充質(zhì)干細(xì)胞低氧條件培養(yǎng)液,結(jié)果發(fā)現(xiàn)這不僅可改善其一般情況,還可以調(diào)節(jié)骨髓造血干/祖細(xì)胞數(shù)量與質(zhì)量以及骨髓造血微環(huán)境:實(shí)驗(yàn)組小鼠骨髓造血祖細(xì)胞集落數(shù)、外源性脾集落形成單位數(shù)、造血重建能力和CFU-F數(shù)均顯著高于同期對(duì)照組小鼠;組織學(xué)觀察顯示實(shí)驗(yàn)組造血組織比同期對(duì)照組豐富、脂肪化輕,而且未經(jīng)MSCs低氧條件培養(yǎng)液干預(yù)的小鼠骨髓P16蛋白表達(dá)量明顯高于干預(yù)小鼠。P16蛋白是抑癌基因P16編碼,是細(xì)胞周期蛋白依賴激酶抑制因子,是細(xì)胞衰老的重要指標(biāo),通過(guò)阻止pRb蛋白磷酸化,進(jìn)而抑制從G1期進(jìn)入S期所需的下游基因的表達(dá),將細(xì)胞周期阻斷在G1期、細(xì)胞則逐漸衰老;抑制P16,細(xì)胞周期蛋白依賴激酶活性增加,pRb磷酸化失活,轉(zhuǎn)錄因子EZF活性增高,多種生命必需基因得以表達(dá),DNA修復(fù)能力增強(qiáng),端粒縮短減慢,從而延緩細(xì)胞衰老[7,8]。所以,本研究結(jié)果說(shuō)明hUC-MSCs低氧條件培養(yǎng)液干預(yù)可延緩小鼠造血系統(tǒng)自然衰老。由于hUC-MSCs可被干擾素-γ激活而提高M(jìn)HCⅠ類(lèi)分子的表達(dá)水平并表達(dá)MHCⅡ類(lèi)分子,如將其多次注射于炎癥區(qū)域或注射前應(yīng)用干擾素-γ則能誘發(fā)免疫反應(yīng)[9],因此,與直接應(yīng)用hUC-MSCs本身相比,其條件培養(yǎng)液在臨床應(yīng)用方面可能具有其優(yōu)點(diǎn),如在不能直接應(yīng)用hUC-MSCs時(shí)選擇其條件培養(yǎng)液。
hUC-MSCs低氧條件培養(yǎng)液為什么具有這些作用呢?研究顯示,hUC-MSCs 不僅具有極強(qiáng)的增殖和分化能力[10,11],表達(dá)原始干細(xì)胞標(biāo)志,如白血病抑制因子受體通路、胚胎干細(xì)胞特異基因Ⅰ、Nanog、Oct-4等[12,13],具有干細(xì)胞特性;而且能分泌多種細(xì)胞因子,如白細(xì)胞介素-6、單核細(xì)胞趨化蛋白-1、生長(zhǎng)相關(guān)癌基因、肝細(xì)胞生長(zhǎng)因子、胰島素樣生長(zhǎng)因子結(jié)合蛋白4和白細(xì)胞介素-8等[14],支持CD34+臍帶血HSCs擴(kuò)增[15];支持ESCs發(fā)育分化[16]。這些資料說(shuō)明hUC-MSCs具有極強(qiáng)的生物學(xué)活性,可產(chǎn)生重要因子發(fā)揮支持調(diào)節(jié)作用。最近研究發(fā)現(xiàn)干細(xì)胞在低氧環(huán)境中具有更好的生物學(xué)活性。MSCs培養(yǎng)在低氧環(huán)境中不僅增殖更快,而且干細(xì)胞標(biāo)志性基因表達(dá)水平更高,端粒逆轉(zhuǎn)錄酶的活性增加[17,18]。利用人體皮膚細(xì)胞培養(yǎng)iPS細(xì)胞時(shí)把培養(yǎng)環(huán)境的氧濃度從通常的21%降到5%,iPS細(xì)胞的生成效率可提高到原來(lái)的2.5-4.2倍[19]。劉革修等[20-23]研究發(fā)現(xiàn)MSCs在低氧環(huán)境中多種生長(zhǎng)因子表達(dá)增高。所以,推測(cè)hUC-MSCs低氧條件培養(yǎng)液延緩小鼠造血系統(tǒng)自然衰老作用可能與hUC-MSCs在低氧條件下分泌重要細(xì)胞因子和生長(zhǎng)因子有關(guān)。其詳細(xì)情況有待進(jìn)一步研究。
[1] Noort WA, Feye D, Van Den Akker F, et al. Mesenchymal stromal cells to treat cardiovascular disease:strategies to improve survival and therapeutic results[J]. Panminerva Med,2010,52(1):27-40.
[2] Kode JA, Mukherjee S, Joglekar MV, et al. Mesenchymal stem cells:immunobiology and role in immunomodulation and tissue regeneration[J]. Cytotherapy,2009,11(4):377-391.
[3] 許婷婷,周艷華,劉革修,等. 人臍帶間充質(zhì)干細(xì)胞對(duì)小鼠自然衰老過(guò)程中骨髓脂肪化的影響[J].中國(guó)病理生理雜志,2010,26(4):816-818.
[4] Naveiras O, Nardi V, Wenzel PL, et al. Bone-marrow adipocytes as negative regulators of the haematopoietic microenvironment[J]. Nature,2009,460(7252):259-263.
[5] Ksiazek K. A comprehensive review on mesenchymal stem cell growth and senescence[J]. Rejuvenation Res,2009,12(2):105-116.
[6] Fehrer C, Lepperdinger G. Mesenchymal stem cell aging[J]. Exp Gerontol,2005,40(12):926-930.
[7] Krishnamurthy J, Torrice C, Ramsey MR, et al. Ink4a/Arf expression is a biomarker of aging[J]. J Clin Invest,2004, 114(9):1299-1307.
[8] Ohtani N, Yamakoshi K, Takahashi A, et al. The p16INK4a-RB pathway: molecular link between cellular senescence and tumor suppression[J]. J Med Invest,2004,51(3-4):146-153.
[9] Cho PS, Messina DJ, Hirsh EL, et al. Immunogenicity of umbilical cord tissue derived cells[J]. Blood,2008,111(1):430-438.
[10]Lu LL, Liu YJ, Yang SG, et al. Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesis-supportive function and other potentials[J]. Haematologica,2006,91(8):1017-1026.
[11]Kestendjieva S, Kyurkchiev D, Tsvetkova G, et al. Characterization of mesenchymal stem cells isolated from the human umbilical cord[J]. Cell Biol Int,2008,32(7):724-732.
[12]Weiss ML, Medicetty S, Bledsoe AR, et al. Human umbilical cord matrix stem cells:preliminary characterization and effect of transplantation in a rodent model of Parkinson’s disease[J]. Stem Cells,2006,24(3):781-792.
[13]Weiss ML, Anderson C, Medicetty S, et al. Immune properties of human umbilical cord Wharton’s jelly-derived cells[J]. Stem Cells,2008,26(11):2865-2874.
[14]Chao KC, Chao KF, Chen CF, et al. A novel human stem cell coculture system that maintains the survival and function of culture islet-like cell clusters[J]. Cell Transplant,2008,17(6):657-664.
[15]Bakhshi T, Zabriskie RC, Bodie S, et al. Mesenchymal stem cells from the Wharton’s jelly of umbilical cord segments provide stromal support for the maintenance of cord blood hematopoietic stem cells during long-termexvivoculture[J]. Transfusion,2008,48(12):2638-2644.
[16]Hiroyama T, Sudo K, Aoki N, et al. Human umbilical cord-derived cells can often serve as feeder cells to maintain primate embryonic stem cells in a state capable of producing hematopoietic cells[J]. Cell Biol Int,2008,32(1):1-7.
[17]D’Ippolito G, Diabira S, Howard GA, et al. Low oxygen tension inhibits osteogenic differentiation and enhances stemness of human MIAMI cells[J]. Bone,2006,39(3):513-522.
[18]Grayson WL, Zhao F, Izadpanah R, et al. Effects of hypoxia on human mesenchymal stem cell expansion and plasticity in 3D constructs[J]. J Cell Physiol,2006,207(2):331-339.
[19]Yoshida Y, Takahashi K, Okita K, et al. Hypoxia enhances the generation of induced pluripotent stem cells[J]. Cell Stem Cell,2009,5(3):237-241.
[20]劉革修, 張 洹, 何冬梅, 等. 低氧誘導(dǎo)胎肝間質(zhì)細(xì)胞表達(dá)堿性成纖維生長(zhǎng)因子[J]. 第二軍醫(yī)大學(xué)學(xué)報(bào),2005,26(9):1012-1014.
[21]劉革修, 張 洹, 何冬梅, 等. 低氧誘導(dǎo)胎肝基質(zhì)細(xì)胞表達(dá)轉(zhuǎn)化生長(zhǎng)因子β1表達(dá)升高[J]. 第二軍醫(yī)大學(xué)學(xué)報(bào),2005,26(10):1148-1151.
[22]劉革修,張 洹, 何冬梅. 人骨髓間充質(zhì)細(xì)胞在低氧培養(yǎng)條件下腦源性神經(jīng)營(yíng)養(yǎng)因子的表達(dá)[J]. 中國(guó)臨床康復(fù),2006,10(29):1-3.
[23]劉革修, 張 洹, 何冬梅. 低氧誘導(dǎo)胎肝間質(zhì)細(xì)胞表達(dá)胰島素樣生長(zhǎng)因子[J]. 中國(guó)老年學(xué)雜志,2007,27(2):129-131.
Effectsofhypoxic-conditionedmediumofmesenchymalstemcellsfromhumanumbilicalcordonnaturalsenescenceofmousehematopoieticsystem
ZHOU Yan-hua, XU Ting-ting, LI Jun, HE Dong-mei, TAN Guang-xiao, LIU Ge-xiu
(InstituteofHematology,SchoolofMedicine,JinanUniversity,Guangzhou510632,China.E-mail:tliugx@jnu.edu.cn)
AIM: To explore the anti-aging roles of hypoxic-conditioned medium of mesenchymal stem cells from human umbilical cord (hUC-MSCs) on the natural senescence of hematopoietic system in aging mice.METHODSThe 6-month-old BALB/c mice were randomly divided into experimental group and control group. The mice in experimental group were intraperitoneally injected with hypoxic-conditioned medium of hUC-MSCs every 4 days for 6 months, and the mice in control group were injected with medium. After treated with hypoxic-conditioned medium of hUC-MSCs, the general situations of the mice were observed. Three months and 6 months after treatment, peripheral hemogram(white blood cell and hemoglobin), bone marrow nucleated cell (BMNC)count, hematopoietic progenitor cell colony (CFU-GM, CFU-E, CFU-MK)culture, fibroblast colony (CFU-F)culture, and exogenous spleen colony-forming unit (CFU-S)count were compared between the 2 groups. Bone marrow histology, P16 expression, and the capability of hematopoietic reconstruction of the bone marrow cells were also compared at the sixth month.RESULTSThe general status of the mice in experimental group was better than that in control group. At the third month, BMNC, CFU-GM and CFU-MK of the experimental group were higher than those in control group. No significant difference of the peripheral hemogram, CFU-F, CFU-E and CFU-S was observed. At the sixth month, all the indexes mentioned above in experimental group were higher than those in control group except peripheral hemogram. All the indexes had a similar descending-tendency during 6 months, but the tendency of experimental group was much slower than that of control group with significant difference (P<0.05). In experimental group, more hematopoietic tissue and less adipose tissue were observed, and the expression level of P16 was lower than those in control group. The hematopoietic reconstruction capability of bone marrow cells in experimental group was better than that in control group.CONCLUSIONThe cytokines from hUC-MSCs slow down the natural recession of hematopoietic system in aging mice, which may be considered to be a potential strategy of anti-aging research in the future.
Mesenchymal stem cells; Umbilical cord; Aging; Cytokines
R322
A
1000-4718(2011)03-0469-06
2010-10-08
2011-01-13
國(guó)家自然科學(xué)基金資助項(xiàng)目(No. 30670902)
△通訊作者 Tel:020-85220262;E-mail: tliugx@jnu.edu.cn
10.3969/j.issn.1000-4718.2011.03.010