• 1 同濟(jì)大學(xué)附屬上海市肺科醫(yī)院呼吸科（上海 200433）;;
  • 2 復(fù)旦大學(xué)附屬華東醫(yī)院呼吸科（上海 200040）;;
  • 3 復(fù)旦大學(xué)附屬中山醫(yī)院呼吸科（上海 200032）;;
  • 4 復(fù)旦大學(xué)遺傳工程國家重點(diǎn)實(shí)驗(yàn)室（上海 200433）;

目的  將間充質(zhì)干細(xì)胞( MSCs) 作為基因載體, 利用基因轉(zhuǎn)染技術(shù)構(gòu)建高表達(dá)促血管生成素1( Ang1) 基因的MSCs, 回輸體內(nèi)治療炎癥性肺損傷模型, 觀察其肺內(nèi)定位和修復(fù)作用。方法  分離、培養(yǎng)和擴(kuò)增MSCs 至第四代, 經(jīng)流式細(xì)胞儀鑒定, 得到純度較高的MSCs。同時(shí)以三質(zhì)粒共轉(zhuǎn)染法在293T 細(xì)胞中制備病毒顆粒Lenti-GFP-Ang1, 并轉(zhuǎn)染MSCs, 通過實(shí)驗(yàn)確定轉(zhuǎn)染的最佳MOI 和最佳時(shí)間, 通過RT-PCR 檢測轉(zhuǎn)染后MSCs 中Ang1 的基因表達(dá), 確定轉(zhuǎn)染成功。以脂多糖霧化吸入的方式誘導(dǎo)小鼠炎癥性肺損傷模型, 設(shè)未處理組為對照, 設(shè)三組干預(yù)組, 包括攜帶Ang1 的MSCs 組( MSC-Ang1 組) 、單純Ang1 組( Ang1 組) 和單純MSCs 組( MSCs 組) 。觀察并記錄各組生存天數(shù), 計(jì)算生存率并進(jìn)行生存分析; 免疫組化確定外源MSCs 源性細(xì)胞在肺部的表現(xiàn)。結(jié)果  經(jīng)多重純化后,流式細(xì)胞儀鑒定獲得的干細(xì)胞為CD44( + ) 、Sca-1( + ) 、CD31( - ) 和CD45( - ) 的MSCs, 并具有分化潛能。病毒載體構(gòu)建亦通過鑒定成功。MSCs 株經(jīng)轉(zhuǎn)染后高表達(dá)Ang1 基因, 當(dāng)MOI = 20 時(shí), MSCs 的細(xì)胞活性及轉(zhuǎn)染達(dá)到最佳效果, 經(jīng)熒光檢測在第5 d 達(dá)表達(dá)高峰。生存率分析顯示MSC-Ang1 組生存率稍高于對照組, 但差異無統(tǒng)計(jì)學(xué)意義( P = 0. 066) 。移植后受損肺組織內(nèi)可見表達(dá)綠色熒光蛋白的肺泡上皮樣細(xì)胞和肺血管內(nèi)皮樣細(xì)胞。結(jié)論  高表達(dá)目的基因Ang1 的MSCs 可通過基因技術(shù)有效構(gòu)建, 移植體內(nèi)后, 可在肺內(nèi)檢測到MSCs 的轉(zhuǎn)化修復(fù)。MSCs 可作為治療肺部疾病的基因運(yùn)載工具

引用本文： 徐金富 ,瞿介明,宋琳,何禮賢,賽音,余龍,李惠萍. 高表達(dá)促血管生成素1 間充質(zhì)干細(xì)胞的構(gòu)建及其在肺損傷中的應(yīng)用. 中國呼吸與危重監(jiān)護(hù)雜志, 2009, 09(6): 575-579. doi: 復(fù)制

1. Loebinger MR, Janes SM. Stemcells for lung disease. Chest, 2007 ,132: 279-285.
2. McCarter SD, Mei SH, Lai PF, et al. Cell-based angiopoietin-1 gene therapy for acute lung injury. Am J Respir Crit Care Med, 2007 ,175: 1014-1026.
3. Witzenbichler B, Westermann D, Knueppel S, et al. Protective role of angiopoietin-1 in endotoxic shock. Circulation, 2005, 111: 97-105.
4. Karmpaliotis D, Kosmidou I, Ingenito EP, et al. Angiogenic growth factors in the pathophysiology of a murine model of acute lung injury. Am J Physiol Lung Cell Mol Physiol, 2002, 283: L585 -L595.
5. 郭強(qiáng), 黃建安, 金鈞, 等. 血管生成素-1 對小鼠早期急性肺損傷的保護(hù)作用, 中華結(jié)核和呼吸雜志, 2007, 30: 926-931.
6. Peister A, Mellad JA, Larson BL, et al. Adult stem cells from bone marrow ( MSCs) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential. Blood, 2004, 103: 1662-1668.
7. Jeyaseelan S, Chu HW, Young SK, et al. Transcriptional Profiling of Lipopolysaccharide-Induced Acute Lung Injury. Infect Immun,2004, 72: 7247-7256.
8. Rojas M, Xu J, Woods CR, et al. Bone marrow-derived mesenchymal stem cells in repair of the injured lung. Am J Respir Cell Mol Biol, 2005, 33: 145-152.
9. Snyder JC, Teisanu RM, Stripp BR. Endogenous lung stem cells and contribution to disease. J Pathol, 2009 , 217: 254-264 .
10. Loebinger MR, Sage EK, Janes SM. Mesenchymal stem cells as vectors for lung disease. Proc Am Thorac Soc, 2008, 5: 711-716.
11. Gao J, Dennis JE, Muzic RF, et al. The Dynamic in vivo Distribution of Bone Marrow-Derived Mesenchymal Stem Cells after Infusion. Cells Tissues Organs, 2001, 169: 12-20.
12. Reiser J, Zhang XY, Hemenway CS, et al. Potential of mesenchymal stem cells in gene therapy approaches for inherited and acquired diseases. Expert Opin Biol Ther, 2005, 5: 1571-1584.
13. Van Damme A, Thorrez L, Ma L, et al. Efficient lentiviral transduction and improved engraftment of human bone marrow mesenchymal cells. StemCells, 2006, 24: 896-907.
14. Harris RG, Herzog EL, Bruscia EM, et al. Lack of a fusion requirement for development of bone marrow-derived epithelia.Science, 2004, 305: 90-93.
  1. 1. Loebinger MR, Janes SM. Stemcells for lung disease. Chest, 2007 ,132: 279-285.
  2. 2. McCarter SD, Mei SH, Lai PF, et al. Cell-based angiopoietin-1 gene therapy for acute lung injury. Am J Respir Crit Care Med, 2007 ,175: 1014-1026.
  3. 3. Witzenbichler B, Westermann D, Knueppel S, et al. Protective role of angiopoietin-1 in endotoxic shock. Circulation, 2005, 111: 97-105.
  4. 4. Karmpaliotis D, Kosmidou I, Ingenito EP, et al. Angiogenic growth factors in the pathophysiology of a murine model of acute lung injury. Am J Physiol Lung Cell Mol Physiol, 2002, 283: L585 -L595.
  5. 5. 郭強(qiáng), 黃建安, 金鈞, 等. 血管生成素-1 對小鼠早期急性肺損傷的保護(hù)作用, 中華結(jié)核和呼吸雜志, 2007, 30: 926-931.
  6. 6. Peister A, Mellad JA, Larson BL, et al. Adult stem cells from bone marrow ( MSCs) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential. Blood, 2004, 103: 1662-1668.
  7. 7. Jeyaseelan S, Chu HW, Young SK, et al. Transcriptional Profiling of Lipopolysaccharide-Induced Acute Lung Injury. Infect Immun,2004, 72: 7247-7256.
  8. 8. Rojas M, Xu J, Woods CR, et al. Bone marrow-derived mesenchymal stem cells in repair of the injured lung. Am J Respir Cell Mol Biol, 2005, 33: 145-152.
  9. 9. Snyder JC, Teisanu RM, Stripp BR. Endogenous lung stem cells and contribution to disease. J Pathol, 2009 , 217: 254-264 .
  10. 10. Loebinger MR, Sage EK, Janes SM. Mesenchymal stem cells as vectors for lung disease. Proc Am Thorac Soc, 2008, 5: 711-716.
  11. 11. Gao J, Dennis JE, Muzic RF, et al. The Dynamic in vivo Distribution of Bone Marrow-Derived Mesenchymal Stem Cells after Infusion. Cells Tissues Organs, 2001, 169: 12-20.
  12. 12. Reiser J, Zhang XY, Hemenway CS, et al. Potential of mesenchymal stem cells in gene therapy approaches for inherited and acquired diseases. Expert Opin Biol Ther, 2005, 5: 1571-1584.
  13. 13. Van Damme A, Thorrez L, Ma L, et al. Efficient lentiviral transduction and improved engraftment of human bone marrow mesenchymal cells. StemCells, 2006, 24: 896-907.
  14. 14. Harris RG, Herzog EL, Bruscia EM, et al. Lack of a fusion requirement for development of bone marrow-derived epithelia.Science, 2004, 305: 90-93.

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