吳樹(shù)才，楊永輝，李輝，郭素敏，李秀武，杜杰杰，宋利超，高莉(河北省胸科醫(yī)院，石家莊05004；河北師范大學(xué)生命科學(xué)學(xué)院)

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人胎盤(pán)間充質(zhì)干細(xì)胞對(duì)小鼠肺纖維化的影響及機(jī)制

吳樹(shù)才1，楊永輝1，李輝1，郭素敏1，李秀武2，杜杰杰2，宋利超2，高莉2(1河北省胸科醫(yī)院，石家莊050041；2河北師范大學(xué)生命科學(xué)學(xué)院)

摘要：目的觀察人胎盤(pán)間充質(zhì)干細(xì)胞對(duì)小鼠肺纖維化的影響，并探討其作用機(jī)制。方法采用組織塊貼壁法分離并體外培養(yǎng)人胎盤(pán)間充質(zhì)干細(xì)胞。將30只C57BL/6小鼠隨機(jī)均分為觀察組、對(duì)照組，均給予氣管內(nèi)注入博來(lái)霉素8.5 mg/kg建立肺纖維化模型。造模成功后，觀察組尾靜脈輸注體外培養(yǎng)的人胎盤(pán)間充質(zhì)干細(xì)胞0.3 mL(細(xì)胞數(shù)為1.0×106個(gè))，對(duì)照組注射等量生理鹽水，1次/d，連續(xù)注射3天；處死小鼠，取肺組織，檢測(cè)肺組織羥脯氨酸含量，采用Western blotting法檢測(cè)肺組織血管內(nèi)皮生長(zhǎng)因子(VEGF)、內(nèi)皮素1(ET-1)和血管生成素2(Ang-2)蛋白。結(jié)果觀察組肺組織羥脯氨酸含量為(5.76±0.13)μg/mL，對(duì)照組為(8.13±0.87)μg/mL，兩組比較P<0.01。觀察組肺組織VEGF的相對(duì)表達(dá)量為52.7±4.7、ET-1為68.1±5.4、Ang-2為59.6±2.8，均較對(duì)照組(100)降低(P均<0.05)。結(jié)論人胎盤(pán)間充質(zhì)干細(xì)胞可抑制小鼠肺組織纖維化形成；降低肺組織VEGF、ET-1和Ang-2表達(dá)可能是其作用機(jī)制。

關(guān)鍵詞：肺纖維化；胎盤(pán)間充質(zhì)干細(xì)胞；博來(lái)霉素；血管內(nèi)皮生長(zhǎng)因子；內(nèi)皮素1；血管生成素2；小鼠

肺纖維化是各種內(nèi)外致病原引起慢性肺疾病的共同結(jié)局，是涉及細(xì)胞、細(xì)胞因子、細(xì)胞外基質(zhì)(ECM)等多因素、多環(huán)節(jié)的復(fù)雜疾病[1]，但其發(fā)生的確切機(jī)制仍不明確。激素和非激素免疫抑制劑、抗纖維化、抗細(xì)胞因子及免疫調(diào)節(jié)等是目前臨床常用治療肺纖維化的方法，但效果均不佳，患者病死率高[2，3]。研究發(fā)現(xiàn)，干細(xì)胞移植可以抑制病變器官修復(fù)過(guò)程中的組織重塑與纖維化過(guò)程[4]。胎盤(pán)間充質(zhì)干細(xì)胞是從胎盤(pán)獲取的一類干細(xì)胞，是介于胚胎干細(xì)胞和成體干細(xì)胞之間的新型干細(xì)胞，具有不被受體免疫系統(tǒng)拒絕的優(yōu)點(diǎn)[5]。2014年3月～2015年11月，我們觀察了人胎盤(pán)間充質(zhì)干細(xì)胞對(duì)小鼠肺纖維化的影響，并探討其作用機(jī)制。

1材料與方法

1.1材料C57BL/6小鼠30只，雌性，6～8周齡，平均體質(zhì)量19.8 g，購(gòu)自中國(guó)科學(xué)院上海實(shí)驗(yàn)動(dòng)物中心，飼養(yǎng)于河北師范大學(xué)實(shí)驗(yàn)動(dòng)物中心SPF級(jí)動(dòng)物房。博萊霉素粉針劑購(gòu)于哈爾濱萊博通藥業(yè)有限公司，生理鹽水稀釋，配制成終濃度為1.7 mg/mL的博來(lái)霉素溶液。CD73、CD90、CD105、HLA-DR、CD19、CD34、CD45、鼠抗人IgG2a和IgG1抗體均購(gòu)自BioSciences公司，血管內(nèi)皮生長(zhǎng)因子(VEGF)、內(nèi)皮素1(ET-1)和血管生成素2(Ang-2)抗體購(gòu)自美國(guó)Santa Cruz CA公司。

1.2人胎盤(pán)間充質(zhì)干細(xì)胞分離、培養(yǎng)和鑒定剪取健康新生兒廢棄胎盤(pán)的胎盤(pán)絨毛膜面組織(厚1～2 cm)，剪成1 mm×1 mm×1 mm的碎塊，PBS漂洗至無(wú)色，轉(zhuǎn)入細(xì)胞培養(yǎng)瓶中，加入3 mL間充質(zhì)干細(xì)胞培養(yǎng)液，放入37 ℃、5% CO2培養(yǎng)箱中培養(yǎng)。選取處于對(duì)數(shù)生長(zhǎng)期的第三代胎盤(pán)間充質(zhì)干細(xì)胞，0.25%胰蛋白酶消化，收集細(xì)胞懸液于離心管中，制備成細(xì)胞密度為1×105/mL的細(xì)胞懸液；取9只試管，分別加入150 μL細(xì)胞懸液，再分別加入鼠抗人單克隆抗體CD73、CD90、CD105、HLA-DR、CD19、CD34、CD45，以鼠抗人IgG2a-FITC、IgG1-PE作為陰性對(duì)照，室溫避光放置10 min。流式細(xì)胞儀檢測(cè)結(jié)果顯示，培養(yǎng)的人胎盤(pán)間充質(zhì)干細(xì)胞高表達(dá)間充質(zhì)干細(xì)胞表面標(biāo)志物CD73、CD90、CD105，不表達(dá)HLA-DR、CD19、CD34、CD45，證明其具有間充質(zhì)干細(xì)胞特性，細(xì)胞培養(yǎng)成功。

1.3動(dòng)物分組及處理將小鼠隨機(jī)分為觀察組及對(duì)照組各15只。兩組均制備肺纖維化模型[5,6]：經(jīng)腹腔注射速眠新針麻醉，暴露氣管，氣管內(nèi)注入博來(lái)霉素溶液(8.5 mg/kg)0.1 mL，旋轉(zhuǎn)小鼠變換體位，使博萊霉素均勻分布。造模第4天，對(duì)照組經(jīng)尾靜脈注射生理鹽水0.3 mL，觀察組經(jīng)尾靜脈注射體外培養(yǎng)的人胎盤(pán)間充質(zhì)干細(xì)胞0.3 mL(細(xì)胞數(shù)為1.0×106個(gè))，均每天注射1次，連續(xù)注射3天。X-半乳糖(X-gal)染色結(jié)果顯示，干細(xì)胞經(jīng)靜脈注射進(jìn)入體內(nèi)后能夠在肺臟中定植。處死小鼠，取肺組織。

1.4相關(guān)指標(biāo)觀察

1.4.1肺組織羥脯氨酸含量將左肺組織烘干，研碎成為組織粉末。兩組均稱取7.5 mg肺組織粉末置于水解管中，加入3 mL 6 mol/L鹽酸震蕩懸浮，110 ℃酸解18 h。取1 mL酸解液中和、還原、沸水浴及萃取后，DMBA顯色，采用標(biāo)準(zhǔn)曲線法計(jì)算羥脯氨酸含量。

1.4.2肺組織VEGF、ET-1、Ang-2蛋白相對(duì)表達(dá)量采用Western blotting法。提取右肺組織總蛋白，進(jìn)行聚丙烯酰胺變性凝膠電泳，將特異性條帶電轉(zhuǎn)移至硝酸纖維素膜，脫脂奶粉封閉，分別與VEGF(1∶500)、ET-1(1∶500)、Ang-2(1∶300)特異性抗體孵育，辣根過(guò)氧化物酶標(biāo)記抗體，攝片。將同一張蛋白印跡膜曝光后，采用洗脫抗體再進(jìn)行孵育雜交。以GAPDH作為內(nèi)參照。將對(duì)照組的表達(dá)量計(jì)為100，計(jì)算觀察組相對(duì)表達(dá)量。每項(xiàng)實(shí)驗(yàn)重復(fù)檢測(cè)3批次以上的不同樣本，取平均值。

2結(jié)果

2.1兩組肺組織羥脯氨酸含量比較觀察組肺組織羥脯氨酸含量為(5.76±0.13)μg/mL，對(duì)照組為(8.13±0.87)μg/mL，兩組比較P<0.01。

2.2兩組肺組織VEGF、ET-1和Ang-2蛋白相對(duì)表達(dá)量比較觀察組肺組織VEGF的相對(duì)表達(dá)量為52.7±4.7、ET-1為68.1±5.4、Ang-2為59.6±2.8，均較對(duì)照組(100)降低(P均<0. 05)。

3討論

間充質(zhì)干細(xì)胞具有多向分化、支持造血、促進(jìn)造血干細(xì)胞植入、調(diào)節(jié)免疫以及分離培養(yǎng)操作相對(duì)簡(jiǎn)單等特點(diǎn)。間充質(zhì)干細(xì)胞作為種子細(xì)胞，在臨床上主要用于治療機(jī)體無(wú)法自然修復(fù)的組織細(xì)胞和器官損傷的多種難治性疾病，在修復(fù)器官功能方面具有無(wú)可比擬的優(yōu)勢(shì)[7,8]； 作為免疫調(diào)節(jié)細(xì)胞， 治療免疫排斥和自身免疫性疾病[9]。胎盤(pán)間充質(zhì)干細(xì)胞是從胎盤(pán)獲取的一類干細(xì)胞，與從臍帶血或骨髓中提取的干細(xì)胞相比，其提取干細(xì)胞數(shù)量更多、可分化的組織細(xì)胞更廣泛[10]，且不存在免疫排斥反應(yīng)。胎盤(pán)來(lái)源干細(xì)胞取自母體生產(chǎn)后的胎盤(pán)組織，其提取過(guò)程對(duì)母體和新生兒不會(huì)造成任何損傷，來(lái)源不受倫理學(xué)和臨床醫(yī)學(xué)的限制，是最理想的干細(xì)胞保存和利用資源。胎盤(pán)來(lái)源的間充質(zhì)干細(xì)胞有可能成為骨髓間充質(zhì)干細(xì)胞的理想替代物，并具有更大的應(yīng)用潛能。Lazarus等[11]提取并培養(yǎng)緩解期血液腫瘤患者的自體胎盤(pán)間充質(zhì)干細(xì)胞，在體外擴(kuò)增培養(yǎng)4～7周后靜脈注射入患者體內(nèi)，注射后未觀察到機(jī)體出現(xiàn)不良反應(yīng)。目前自體胎盤(pán)間充質(zhì)干細(xì)胞的臨床應(yīng)用逐漸增多，病種涉及放療及化療后造血重建、移植物抗宿主病、心臟系統(tǒng)疾病等，均證實(shí)經(jīng)靜脈輸注胎盤(pán)間充質(zhì)干細(xì)胞安全可靠。Ninichuk等[12]將原始的成血管細(xì)胞注射進(jìn)入肝臟纖維化模型大鼠的病變肝臟中，發(fā)現(xiàn)肝臟纖維化程度減輕。給予慢性腎損傷模型小鼠每周注射多能間充質(zhì)干細(xì)胞(6～10周)，可減少腎臟的間質(zhì)容量、間質(zhì)膠原含量及表達(dá)平滑肌肌動(dòng)蛋白的間質(zhì)細(xì)胞數(shù)量，從而減輕腎臟纖維化。上述研究結(jié)果提示，干細(xì)胞移植可抑制病變器官修復(fù)過(guò)程中的組織重塑與纖維化過(guò)程，有利于器官功能的修復(fù)。

肺纖維化的病理特征為肺泡壁(肺泡炎)受損，成纖維細(xì)胞/肌成纖維細(xì)胞異常聚集，膠原蛋白和其他ECM過(guò)度沉積，異常組織修復(fù)導(dǎo)致肺功能喪失[13,14]。羥脯氨酸是膠原蛋白的主要組分，其水平反映膠原蛋白含量。本研究給予肺纖維化小鼠連續(xù)3天靜脈注射人胎盤(pán)間充質(zhì)干細(xì)胞，結(jié)果顯示觀察組肺組織羥脯氨酸含量降低，提示人胎盤(pán)間充質(zhì)干細(xì)胞可抑制肺纖維化小鼠肺組織纖維化形成。研究發(fā)現(xiàn)，VEGF、ET-1和Ang-2可促進(jìn)內(nèi)皮細(xì)胞遷移、增殖及血管生成；Ang-2還能抑制脈絡(luò)膜新生血管的生成和成熟，促進(jìn)瘢痕形成，引起肺結(jié)構(gòu)重塑，刺激肺成纖維細(xì)胞增殖，并調(diào)節(jié)成纖維細(xì)胞的趨化、增殖，合成膠原，導(dǎo)致膠原和纖維素沉積增多，造成肺泡壁基底膜增厚和彌漫性上皮下纖維化[15～17]。本研究結(jié)果顯示，觀察組肺組織VEGF、ET-1和Ang-2蛋白表達(dá)明顯降低，提示人胎盤(pán)間充質(zhì)干細(xì)胞可通過(guò)降低以上三種蛋白的表達(dá)進(jìn)而抑制微血管形成、延緩肺組織纖維化進(jìn)程。

總之，胎盤(pán)間充質(zhì)干細(xì)胞可抑制肺組織纖維化形成；降低肺組織VEGF、ET-1和Ang-2蛋白表達(dá)可能是其作用機(jī)制。

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Effect of placenta-derived mesenchymal stem cells on pulmonary fibrosis in mice and its mechanism

WUShucai1,YANGYonghui,LIHui,GUOSumin,LIXiuwu,DUJiejie,SONGLichao,GAOLi

(1HebeiProvinceChestHospital,Shijiazhuang050041,China)

Abstract:ObjectiveTo observe the effect of placenta-derived mesenchymal stem cells on the pulmonary fibrosis in mice and to explore its mechanism. MethodsHuman placenta-derived mesenchymal stem cells were isolated and cultured in vitro by tissue explants adherent method. Thirty C57BL/6 mice were randomly divided into the observation group and the control group, and all mice were injected with 8.5 mg/kg to establish the model of pulmonary fibrosis. After the models were successful established, mice in the observation group received intravenous injection of placenta-derived mesenchymal stem cells 0.3 mL (cell number of 1.0×106), and mice in the control group were injected with the same amount of normal saline, once a day for 3 days. Then, the mice were killed to collect the lung tissues to determine the content of hydroxyproline. The expression of vascular endothelial growth factor (VEGF), endothelin 1 (ET-1) and angiogenin 2 (Ang-2) proteins was determined by Western blotting. ResultsThe hydroxyproline contents in the lung tissue of observation group and control group were respectively (5.76±0.13) μ/mL and (8.13±0.87) μg/mL, and significant difference was found between the two groups (P<0.01). The expression of VEGF, ET-1 and Ang-2 in the lung tissues of the observation group was respectively 52.7±4.7, 68.1±5.4 and 59.6±2.8, which was all lower than that of the control group (all P<0.05). ConclusionPlacenta-derived mesenchymal stem cells can inhibit the formation of pulmonary fibrosis and its mechanism may be related with the decreased expression of VEGF, ET-1 and Ang-2 protein in lung tissue.

Key words:pulmonary fibrosis; placenta-derived mesenchymal stem cells; bleomycin; vascular endothelial growth factor; endothelin 1; angiogenin 2; mice

(收稿日期：2015-12-02)

中圖分類號(hào)：R563

文獻(xiàn)標(biāo)志碼：A

文章編號(hào)：1002-266X(2016)12-0013-03

doi:10.3969/j.issn.1002-266X.2016.12.004

通信作者簡(jiǎn)介：楊永輝(1972-)，男，主任醫(yī)師，主要研究方向?yàn)椴±韺W(xué)診斷及細(xì)胞免疫治療。E-mail： yonghuiyang@vip.163.com

第一作者簡(jiǎn)介：吳樹(shù)才(1963-)，男，主任醫(yī)師，主要研究方向?yàn)榻Y(jié)核及呼吸系統(tǒng)疾病。E-mail： shucaiwu2009@163.com

基金項(xiàng)目：國(guó)家自然科學(xué)基金資助項(xiàng)目(31101638)；河北省省級(jí)重大醫(yī)學(xué)科研課題(ZD2013060)。