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醫(yī)學(xué)論文范文:體外非接觸共培養(yǎng)誘導(dǎo)骨髓間充質(zhì)干細(xì)胞向肺泡上皮細(xì)胞的分化
【摘要】 目的 建立體外非接觸共培養(yǎng)模型，誘導(dǎo)小鼠骨髓間充質(zhì)干細(xì)胞(MSCs)向肺泡上皮細(xì)胞的分化。方法 分三組，每組6例。對照組：小鼠MSCs單獨培養(yǎng)組;實驗組A：正常肺組織單細(xì)胞懸液+MSCs;實驗組B：損傷肺組織單細(xì)胞懸液+MSCs。共同培養(yǎng)8d，激光共聚焦和RT-PCR檢測共培養(yǎng)體系下室中的SP-C和AQP5的表達(dá)情況。結(jié)果 對照組和實驗組A都只檢測到AQP5;而實驗組B可同時檢測到AQP5和SP-C。實驗組B的AQP5 mRNA的表達(dá)量較對照組明顯增多(P<0.01)，而與實驗組A比較，其AQP5 mRNA的表達(dá)量也有統(tǒng)計學(xué)差異(P<0.01)。但是，實驗組A與對照組比較則無明顯統(tǒng)計學(xué)差異(P>0.05)。結(jié)論 本實驗室成功建立了體外非接觸誘導(dǎo)小鼠MSCs分化為肺泡上皮細(xì)胞的實驗?zāi)Ｐ汀?/P>
【關(guān)鍵詞】 骨髓間充質(zhì)干細(xì)胞;肺泡上皮細(xì)胞;激光共聚焦
Establishment of co-culture model in vitro to induce bone marrow mesenchymal stem cells differentiate into lung epithelial cellsWANG Yan， YANG Zhi-jun, HE Xi-yu, FENG Zhi-chunDepartment of Pediatrics, Southern Medical University Affiliated Zhujiang Hospital,Guangzhou 510080; Department of Neonatology, Beijing Military General Hospital Affiliated Bayi Childrens Hospital, Beijing 100700, ChinaABSTRACT: Objective To establish the co-culture model in vitro and induce bone marrow mesenchymal stem cells (MSCs) to differentiate into lung alveolar epithelial cells. Methods Each group had 6 samples, control group was MSCs alone; Group A was the MSCs cultured with the cells from normal lung; and Group B was the MSCs with the cells from injuried lung. Each group was cultured for 8 days and the two markers of lung alveolar epithelial cells including AQP5 and SP-C were tested by laser confocal microscopy and RT-PCR. Results Only AQP5 was detected in the control group and Group A, both AQP5 and SP-C were detected in Group B, the AQP5 mRNA expression in Group B was significantly increased compared with that in the control group(P<0.01). The AQP5 mRNA expression in Group B was also significantly increased compared with that in Group A (P<0.01). But there was no significant difference in AQP5 mRNA expression between Group A and control group. Conclusion We have successfully established the co-culture model in vitro to induce bone marrow mesenchymal stem cells to differentiate into lung epithelial cells醫(yī)學(xué)全.在線www.gydjdsj.org.cn.
KEY WORDS: mesenchymal stem cells; lung alveolar epithelial cell; laser confocal microscopy
骨髓間充質(zhì)干細(xì)胞(mesenchymal stem cells, MSCs)是一種具有多分化潛能的干細(xì)胞，在不同的培養(yǎng)條件下可分化為一系列的細(xì)胞類型。肺損傷體內(nèi)實驗研究表明，肺損傷小鼠移植MSCs后，MSCs可轉(zhuǎn)化為肺泡Ⅱ型(type Ⅱ alveolar epithelial cell, AECⅡ)和Ⅰ型上皮細(xì)胞(type Ⅰ alveolar epithelial cell, AECI)等，以修復(fù)損傷肺組織[1]。ROJAS等[2]研究發(fā)現(xiàn)，在體外損傷肺組織分泌的因子可以誘導(dǎo)MSCs增殖并向損傷肺遷移，而正常肺組織細(xì)胞并沒有此功能。POPOV等[3]采用MSCs與肺上皮細(xì)胞(A549)非接觸共同培養(yǎng)，成功誘導(dǎo)MSCs向肺上皮細(xì)胞分化。本研究采用MSCs與肺組織單細(xì)胞懸液非接觸共培養(yǎng)模型，觀察能否在體外利用損傷肺組織誘導(dǎo)MSCs向肺泡上皮細(xì)胞分化,為進(jìn)一步研究骨髓源性的間充質(zhì)干細(xì)胞治療肺損傷奠定基礎(chǔ)。
1 材料與方法
1.1 材料
出生4周的健康昆明小鼠，體重為10.0～11.5g，共25只，均為雄性(中國醫(yī)學(xué)科學(xué)院實驗動物中心提供)。DMEM完全培養(yǎng)基和標(biāo)準(zhǔn)胎牛血清(GIBCO公司，美國)，胰蛋白酶(Sigma公司，美國)。Taq DNA聚合酶(TaKaRa公司，日本)，RT-PCR試劑盒(QIAGEN，德國)。 倒置顯微鏡(TE2000, Nikon公司，日本)，高分辨率成像系統(tǒng)(DXM1200C，尼康，日本)，激光共聚焦顯微鏡(C1-SHS，尼康公司，日本)，二氧化碳細(xì)胞培養(yǎng)箱(SKP-02B，湖北省黃石市恒豐醫(yī)療公司)，24孔板(Hanging Cell Culture Insert PET 0.4μm，MILLPORE公司，德國)。
1.2 小鼠骨髓間充質(zhì)干細(xì)胞(MSCs)的體外分離、擴(kuò)增和鑒定
