姚舜，靳安民，張輝，閔少雄，黃帥，周治來

(南方醫(yī)科大學(xué)珠江醫(yī)院，廣州510282)

低氧預(yù)處理對(duì)臍帶間充質(zhì)干細(xì)胞表型、增殖及神經(jīng)營養(yǎng)因子分泌功能的影響

姚舜，靳安民，張輝，閔少雄，黃帥，周治來

(南方醫(yī)科大學(xué)珠江醫(yī)院，廣州510282)

目的探討低氧預(yù)處理對(duì)臍帶間充質(zhì)干細(xì)胞表型、增殖、神經(jīng)營養(yǎng)因子分泌功能的影響。方法收集分娩過程中丟棄的臍帶組織，分離培養(yǎng)臍帶間充質(zhì)干細(xì)胞，將原代培養(yǎng)的間充質(zhì)干細(xì)胞進(jìn)行傳代。取傳至第3代的臍帶間充質(zhì)干細(xì)胞，隨機(jī)分為低氧組和常氧組，分別置于氧濃度為5%、21%的恒溫培養(yǎng)箱中培養(yǎng)6天。取對(duì)數(shù)生長期的兩組細(xì)胞，采用流式細(xì)胞術(shù)檢測(cè)CD44、CD45、CD90、HLA-DR表達(dá)，細(xì)胞免疫熒光法檢測(cè)CD34及層粘連蛋白、波形蛋白表達(dá)，實(shí)時(shí)熒光定量PCR法檢測(cè)腦源性神經(jīng)營養(yǎng)因子(BDNF)、血管內(nèi)皮生長因子(VEGF)、肝細(xì)胞生長因子(HGF)、睫狀節(jié)神經(jīng)細(xì)胞營養(yǎng)因子(CNTF)mRNA表達(dá)。兩組培養(yǎng)1～6天進(jìn)行細(xì)胞計(jì)數(shù)，并采用CCK-8法檢測(cè)細(xì)胞增殖活性。結(jié)果兩組均高表達(dá)CD44、CD90、層粘連蛋白、波形蛋白，低表達(dá)CD45、HLA-DR、CD34，證實(shí)兩組細(xì)胞均具備間充質(zhì)干細(xì)胞表型特性；兩組上述細(xì)胞表型相關(guān)指標(biāo)表達(dá)比較P均>0.05。兩組培養(yǎng)1～6天細(xì)胞計(jì)數(shù)及細(xì)胞增殖活性均先升高后降低；低氧組培養(yǎng)1～6天細(xì)胞計(jì)數(shù)均高于常氧組，培養(yǎng)2～6天細(xì)胞增殖活性均高于常氧組(P均<0.05)。低氧組BDNF、VEGF、HGF、CNTF mRNA相對(duì)表達(dá)量均高于常氧組(P均<0.05)。結(jié)論低氧預(yù)處理對(duì)臍帶間充質(zhì)干細(xì)胞的表型特征無明顯影響，但可提高其增殖能力及神經(jīng)營養(yǎng)因子分泌功能。

間充質(zhì)干細(xì)胞；臍帶；低氧；細(xì)胞增殖；細(xì)胞表型；神經(jīng)營養(yǎng)因子

Abstract:ObjectiveTo investigate the influences of hypoxic preconditioning on cell phenotype, proliferation, and secretion of neurotrophic factors of human umbilical cord mesenchymal stem cells (UCMSCs).MethodsThe discarded umbilical cord tissues during delivery process were collected for UCMSCs culture, and the primary cultured mesenchymal stem cells were passaged. Cells at passage 3 were randomly divided into the normoxia group and hypoxia group, which were growing in 21% and 5% O2, respectively. Cells in logarithmic growth phase were used. The expression of CD44, CD45, CD90, and HLA-DR was detected by flow cytometry; the expression of CD34, laminin, and vimentin was determined by immunocytochemistry; the mRNA expression of brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and ciliary neurotrophic factor (CNTF) was determined by real-timer fluorescent quantitative PCR. After the cells were cultured for 1 to 6 days, we did the cell counting, and determined the cell proliferation by CCK-8.ResultsCD44, CD90, laminin, and vimentin were highly expressed, and CD45, HLA-DR, and CD34 were low expressed in both of the two groups, which showed that cells in the two groups had the characteristics of mesenchymal stem cells. There was no significant difference in expression of phenotype-related indexes between the two groups (allP>0.05). The CCK-8 results indicated that the cell counting and proliferation rate first increased and then decreased in 1-6 days′ culture in the two groups. When the cells were cultured for 1-6 days, the cell counting in the hypoxia group was higher than that in the normoxia group; when the cells were cultured for 2-6 days, the proliferation activity of the hypoxia group was higher than that of the normoxia group (allP<0.05). The mRNA expression of HGF, BDNF, VEGF, and CNTF in the hypoxia group was higher than that of the normoxia group (allP<0.05).ConclusionHypoxic preconditioning has little effect on the phenotypic features of UCMSCs, but can significantly enhance the proliferation rate and the secretion of neurotrophic factors.

Keywords: mesenchymal stem cells; umbilical cord; hypoxia; cell proliferation; cell phenotype; neurotrophic factors

間充質(zhì)干細(xì)胞最早是從人骨髓中提取，但之后研究發(fā)現(xiàn)人體許多組織均能分離、培養(yǎng)間充質(zhì)干細(xì)胞，包括脂肪、臍帶、牙髓、韌帶、皮膚、肌肉等[1]。不同組織來源的間充質(zhì)干細(xì)胞在細(xì)胞形態(tài)、表型、免疫源性等方面大體相同，但增殖速度及分泌功能等則差別較大。臍帶組織為分娩過程中丟棄的組織，取材方便；臍帶間充質(zhì)干細(xì)胞增殖速度較快[2,3]，但存活率較低，分泌能力、炎癥調(diào)節(jié)能力有限。體外分離培養(yǎng)間充質(zhì)干細(xì)胞的氧濃度一般為20%，而宿主體內(nèi)氧濃度為1.5%～8%，細(xì)胞移植到宿主體內(nèi)后氧濃度急劇下降，細(xì)胞適應(yīng)能力差，可能是導(dǎo)致細(xì)胞移植效率低的一個(gè)重要原因。部分研究在體外對(duì)細(xì)胞進(jìn)行低氧預(yù)處理，試圖提高其對(duì)低氧環(huán)境的耐受能力[4,5]。但是，目前關(guān)于低氧預(yù)處理對(duì)臍帶間充質(zhì)干細(xì)胞生物學(xué)行為影響的研究較少。為此，我們于2015年7月～2016年7月進(jìn)行了如下研究。

1 材料與方法

1.1 材料 DMEM/F12完全培養(yǎng)液購于美國Gibco公司，CO2恒溫培養(yǎng)箱購于美國Thermo公司，培養(yǎng)瓶購于美國Corning公司。

1.2 臍帶間充質(zhì)干細(xì)胞分離與培養(yǎng) 收集產(chǎn)婦分娩過程中丟棄的臍帶組織，以含1%青霉素-鏈霉素的PBS沖洗3次。用眼科剪去除血管組織，并將臍帶組織剪成碎片，接種于DMEM/F12完全培養(yǎng)液(含10%FBS、100 IU/L青霉素、100 mg/L鏈霉素)的25 cm2培養(yǎng)瓶中，置于37 ℃、5% CO2恒溫培養(yǎng)箱中進(jìn)行培養(yǎng)。培養(yǎng)72 h后換液，去除懸浮細(xì)胞，細(xì)胞逐漸長出，即為原代臍帶間充質(zhì)干細(xì)胞。待原代細(xì)胞融合至80%時(shí)進(jìn)行細(xì)胞傳代，消化離心后用培養(yǎng)基重懸細(xì)胞，輕柔吹打形成單細(xì)胞懸液，按1∶4進(jìn)行傳代，并接種于新的25 cm2培養(yǎng)瓶中。本研究通過醫(yī)院倫理委員會(huì)審核，產(chǎn)婦及其家屬均簽署知情同意書。

1.3 細(xì)胞分組處理 取傳至第3代的臍帶間充質(zhì)干細(xì)胞，制備細(xì)胞懸液，調(diào)整密度為1×105個(gè)/mL。將細(xì)胞隨機(jī)分為低氧組和常氧組，37 ℃條件下分別置于氧濃度為5%、21%的恒溫培養(yǎng)箱中培養(yǎng)6天。

1.4 細(xì)胞表型相關(guān)指標(biāo)檢測(cè) ①CD44、CD45、CD90、HLA-DR表達(dá)：采用流式細(xì)胞術(shù)。取對(duì)數(shù)生長期的兩組細(xì)胞，消化后調(diào)整細(xì)胞密度為1×106個(gè)/mL，用含0.1%疊氮鈉和0.5% BSA的PBS沖洗2遍，PBS重懸細(xì)胞。加入一抗，4 ℃條件下孵育30 min，PBS沖洗2遍。加入異硫氰酸熒光素或藻紅蛋白標(biāo)記的小鼠抗人IgG二抗，4 ℃條件下孵育30 min，PBS沖洗2遍。用不含BSA的PBS重懸細(xì)胞，上流式細(xì)胞儀檢測(cè)CD44、CD45、CD90、HLA-DR表達(dá)。②CD34及層粘連蛋白、波形蛋白表達(dá)：采用細(xì)胞免疫熒光法。取對(duì)數(shù)生長期的兩組細(xì)胞，吸除培養(yǎng)液，4%多聚甲醛室溫固定30 min，PBS沖洗5 min×3次，0.3% Triton X-100室溫破膜5 min，山羊血清封閉液37 ℃封閉1 h。分別加入血清稀釋液稀釋的一抗兔抗CD34、兔抗層粘連蛋白、鼠抗波形蛋白(稀釋比例分別為1∶100、1∶200、1∶200)，濕盒內(nèi)4 ℃孵育過夜，吸除一抗，PBS沖洗5 min×3次；滴加二抗Alexa Fluor 594標(biāo)記羊抗兔IgG(1∶200)和Alexa Fluor 488標(biāo)記羊抗鼠IgG(1∶200)，室溫孵育1 h；去除二抗后，PBS沖洗5 min×3次。DAPI復(fù)染細(xì)胞核，封片。同時(shí)設(shè)陰性對(duì)照，以抗體稀釋液代替一抗。熒光顯微鏡下隨機(jī)選取5～20個(gè)高倍視野，計(jì)數(shù)DAPI染色細(xì)胞數(shù)，隨后改變觀察波長，計(jì)數(shù)相同視野下CD34、層粘連蛋白、波形蛋白陽性染色的細(xì)胞數(shù)量，計(jì)算陽性表達(dá)率。

1.5 細(xì)胞增殖活性檢測(cè) ①細(xì)胞計(jì)數(shù)：取兩組培養(yǎng)1～6天的細(xì)胞，消化后重懸于2 mL DMEM/F12培養(yǎng)基中，將計(jì)數(shù)板放在低倍鏡下(10×10)觀察并進(jìn)行計(jì)數(shù)。每組取4皿，每皿重復(fù)計(jì)數(shù)3次，取平均值。②細(xì)胞增殖活性：采用CCK-8法。取兩組培養(yǎng)1～6天的細(xì)胞，制成單細(xì)胞懸液，以2×103個(gè)/孔接種于96孔板，共200 μL。每孔加入10 μL CCK-8溶劑，采用酶聯(lián)免疫檢測(cè)儀于450 nm波長下讀取吸光度(OD)值。每組3個(gè)復(fù)孔，取平均值。

1.6 神經(jīng)修復(fù)相關(guān)因子mRNA表達(dá)檢測(cè) 采用實(shí)時(shí)熒光定量PCR法。取對(duì)數(shù)生長期的兩組細(xì)胞，棄去培養(yǎng)液，預(yù)冷PBS洗滌單層細(xì)胞。采用TRIzol試劑提取總RNA，檢測(cè)總RNA濃度和純度合格后，逆轉(zhuǎn)錄合成cDNA。腦源性神經(jīng)營養(yǎng)因子(BDNF)、血管內(nèi)皮生長因子(VEGF)、肝細(xì)胞生長因子(HGF)、睫狀節(jié)神經(jīng)細(xì)胞營養(yǎng)因子(CNTF)及內(nèi)參基因β-actin引物序列見表1，PCR反應(yīng)過程參照前期研究[6]。PCR反應(yīng)體系：Q-PCR Mix 10 μL，ddH2O 4 μL，上下游引物各2 μL，cDNA(1∶5) 2 μL。PCR反應(yīng)條件：95 ℃預(yù)變性5 min、95 ℃變性45 s、56 ℃退火45 s、72 ℃延伸20 s，共40個(gè)循環(huán)。采用2-ΔΔCt法計(jì)算目的基因相對(duì)表達(dá)量。

表1 各基因引物序列及擴(kuò)增長度

2 結(jié)果

2.1 兩組細(xì)胞表型相關(guān)指標(biāo)表達(dá)比較 兩組均高表達(dá)CD44、CD90、層粘連蛋白及波形蛋白，低表達(dá)CD45、HLA-DR、CD34，證實(shí)兩組細(xì)胞均具備間充質(zhì)干細(xì)胞表型特征；兩組CD44、CD45、CD90、HLA-DR、CD34、層粘連蛋白、波形蛋白等細(xì)胞表型相關(guān)指標(biāo)表達(dá)比較P均>0.05。見表2。

2.2 兩組細(xì)胞增殖能力比較 兩組培養(yǎng)1～6天細(xì)胞計(jì)數(shù)及細(xì)胞增殖活性均先升高后降低； 低氧組培養(yǎng)1～6天細(xì)胞計(jì)數(shù)均高于常氧組，培養(yǎng)2～6天細(xì)胞增殖活性均高于常氧組(P均<0.05)。見表3、4。

表2 兩組細(xì)胞表型相關(guān)指標(biāo)表達(dá)

表3 兩組培養(yǎng)1～6天細(xì)胞計(jì)數(shù)比較

注：與常氧組同時(shí)間點(diǎn)比較，*P<0.05。

表4 兩組培養(yǎng)1～6天細(xì)胞增殖活性比較

注：與常氧組同時(shí)間點(diǎn)比較，*P<0.05。

2.3 兩組神經(jīng)修復(fù)相關(guān)因子mRNA表達(dá)比較 低氧組BDNF、VEGF、HGF、CNTF mRNA相對(duì)表達(dá)量均高于常氧組(P均<0.05)。見表5。

表5 兩組細(xì)胞神經(jīng)修復(fù)相關(guān)因子mRNA表達(dá)比較(相對(duì)表達(dá)量

注：與常氧組比較，*P<0.05。

3 討論

間充質(zhì)干細(xì)胞移植治療在神經(jīng)損傷的修復(fù)中發(fā)揮越來越重要的作用，臍帶間充質(zhì)干細(xì)胞移植后可調(diào)節(jié)宿主炎癥反應(yīng)及凋亡等，有助于促進(jìn)神經(jīng)生長[7]。但任何細(xì)胞移植發(fā)揮治療作用均需以一定的細(xì)胞數(shù)量為基礎(chǔ)，目前臍帶間充質(zhì)干細(xì)胞移植領(lǐng)域的一個(gè)瓶頸就是移植后細(xì)胞存活率較低，尤其是神經(jīng)損傷患者。由于血管損傷、組織撕裂、血栓形成等導(dǎo)致局部缺血、炎癥、壞死，組織含氧量進(jìn)一步下降，細(xì)胞生長環(huán)境惡劣，間充質(zhì)干細(xì)胞移植后往往大量死亡，難以發(fā)揮其修復(fù)作用[8]。

生理狀態(tài)下，女性生殖系統(tǒng)組織氧濃度為1.5%～8%，胎兒體內(nèi)循環(huán)氧濃度不超過5%[9]。正常情況下體外培養(yǎng)臍帶間充質(zhì)干細(xì)胞的氧濃度是21%，將其移植到低于1%的損傷局部勢(shì)必會(huì)導(dǎo)致部分細(xì)胞無法適應(yīng)驟然下降的氧濃度而大量死亡。為了解決細(xì)胞移植后存活率低的問題，有學(xué)者提出在移植前對(duì)細(xì)胞進(jìn)行低氧預(yù)處理，以適應(yīng)移植后宿主體內(nèi)的低氧狀態(tài)[10,11]。目前有研究證實(shí)，低氧預(yù)處理能減少細(xì)胞內(nèi)活性氧自由基的生成，低氧預(yù)處理的間充質(zhì)干細(xì)胞對(duì)心肌梗死的修復(fù)效果更強(qiáng)[12]。在脊髓損傷的修復(fù)實(shí)驗(yàn)中，低氧預(yù)處理臍帶間充質(zhì)干細(xì)胞的移植存活率高于常氧細(xì)胞，且能進(jìn)一步下調(diào)損傷局部ED-1、caspase-3陽性細(xì)胞數(shù)量，表明低氧預(yù)處理的臍帶間充質(zhì)干細(xì)胞對(duì)損傷組織炎癥的調(diào)控能力更強(qiáng)[13]。有研究證實(shí)，低氧刺激會(huì)降低樹突狀細(xì)胞的遷移能力，氧分壓越低，其遷移能力越低[14]。但間充質(zhì)干細(xì)胞的生物學(xué)行為與樹突狀細(xì)胞差別較大，低氧是否影響臍帶間充質(zhì)干細(xì)胞的遷移仍需進(jìn)一步研究。本研究選用低氧濃度為5%，該濃度與體內(nèi)生理氧濃度，尤其是女性生殖系統(tǒng)組織氧濃度更接近。結(jié)果顯示，兩組均高表達(dá)干細(xì)胞相關(guān)標(biāo)志物CD90、CD44及間充質(zhì)干細(xì)胞的標(biāo)志性蛋白波形蛋白、層粘連蛋白，低表達(dá)血管內(nèi)皮細(xì)胞相關(guān)標(biāo)志物CD34、CD45及HLA-DR，證實(shí)兩組細(xì)胞均為間充質(zhì)干細(xì)胞；兩組上述表型相關(guān)指標(biāo)表達(dá)比較差異均無統(tǒng)計(jì)學(xué)意義，表明低氧預(yù)處理對(duì)臍帶間充質(zhì)干細(xì)胞的表型特征無明顯影響。

劉林奇等[15]研究顯示，低氧刺激可提高脂肪間充質(zhì)干細(xì)胞的增殖速度。本研究結(jié)果顯示，兩組培養(yǎng)1～6天細(xì)胞計(jì)數(shù)及細(xì)胞增殖活性均先升高后降低，低氧組培養(yǎng)1～6天細(xì)胞計(jì)數(shù)均高于常氧組，培養(yǎng)2～6天細(xì)胞增殖活性均高于常氧組；說明低氧預(yù)處理可提高臍帶間充質(zhì)干細(xì)胞的增殖能力，對(duì)于在短期內(nèi)收集足夠移植所需數(shù)量的細(xì)胞具有重要意義。臍帶間充質(zhì)干細(xì)胞在神經(jīng)修復(fù)中具有重要作用，與其神經(jīng)因子分泌功能密切相關(guān)，其中BDNF、VEGF、HGF、CNTF均為重要的神經(jīng)營養(yǎng)因子。本研究結(jié)果顯示，低氧組BDNF、VEGF、HGF、CNTF mRNA相對(duì)表達(dá)量均高于常氧組，說明低氧預(yù)處理可提高臍帶間充質(zhì)干細(xì)胞的神經(jīng)因子分泌功能，對(duì)于其修復(fù)神經(jīng)損傷具有重要作用。

綜上所述，低氧預(yù)處理對(duì)臍帶間充質(zhì)干細(xì)胞的表型特征無明顯影響，但可提高其增殖能力及神經(jīng)營養(yǎng)因子分泌功能。本研究為提高干細(xì)胞移植效率提供了新的實(shí)驗(yàn)和理論基礎(chǔ)。臍帶間充質(zhì)干細(xì)胞移植修復(fù)神經(jīng)損傷除了對(duì)移植細(xì)胞的存活率、分泌功能有要求外，還涉及宿主體內(nèi)細(xì)胞的增殖、分化等方面。低氧預(yù)處理臍帶間充質(zhì)干細(xì)胞的氧濃度及其對(duì)宿主細(xì)胞炎癥、凋亡、增殖的影響仍需進(jìn)一步研究。

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Effects of hypoxic preconditioning on cell phenotype, proliferation, and secretion of neurotrophic factors of umbilical cord mesenchymal stem cells

YAOShun,JINAnmin,ZHANGHui,MINShaoxiong,HUANGShuai,ZHOUZhilai

(ZhujiangHospitalofSouthernMedicalUniversity,Guangzhou510282,China)

國家自然科學(xué)基金青年科學(xué)基金資助項(xiàng)目(81400996)；廣東省自然科學(xué)基金博士啟動(dòng)項(xiàng)目(2014A030310100)。

姚舜(1990-)，男，住院醫(yī)師，研究方向?yàn)榧怪顾钃p傷的基礎(chǔ)與臨床。E-mail: yaoshun810@163.com

周治來(1983-)，男，主治醫(yī)師，研究方向?yàn)榧怪顾钃p傷的基礎(chǔ)與臨床。E-mail: 273344078@qq.com

10.3969/j.issn.1002-266X.2017.32.007

R329.2

A

1002-266X(2017)32-0025-04

2017-02-15)