由人脂肪来源的经骨形态发生蛋白-2（BMP-2）转导的干细胞进行的骨诱导

Bone induction by BMP-2 transduced stem cells derived from human fat.

作者信息

Dragoo Jason L, Choi Joon Y, Lieberman Jay R, Huang Jerry, Zuk Patricia A, Zhang Jeffery, Hedrick Marc H, Benhaim Prosper

机构信息

Laboratory for Regenerative Bioengineering and Repair, Department of Surgery, UCLA School of Medicine, 90095-6092, USA.

出版信息

J Orthop Res. 2003 Jul;21(4):622-9. doi: 10.1016/S0736-0266(02)00238-3.

DOI:10.1016/S0736-0266(02)00238-3

PMID:12798061

Abstract

PURPOSE

We have isolated pluripotent mesenchymal progenitor cells in large numbers from liposuction aspirates (processed lipoaspirate cells or PLAs). This study examines the osteogenic potential of PLAs and bone marrow aspirate cells (BMAs), when exposed to either recombinant human bone morphogenetic protein (BMP)-2 (rh-BMP-2) or adenovirus containing BMP-2 cDNA (Ad-BMP-2).

METHODS

Liposuction aspirates underwent proteolytic digestion to obtain PLAs. After exposure to exogenous rh-BMP-2 or Ad-BMP-2 for four or seven days, PLAs and BMAs were assessed by histochemistry, spectrophotometry and RT-PCR. Western blotting and ELISA confirmed BMP gene transduction. Results were compared to osteoblasts and cells in osteogenic media only. PLA-Ad-BMP-2 cells were seeded on matrices and implanted in the hind limbs of SCID mice.

RESULTS

Analysis of quantified bone precursor assays including extracellular ALP histomorphometry, intracellular ALP spectrophotometry, and calcified extracellular matrix (von Kossa) histomorphometry revealed that PLAs treated with exogenous rh-BMP-2 or transduced with a BMP-2 containing adenovirus (PLA-Ad-BMP-2) produced more bone precursors than osteoblasts (p=0.001). PLAs treated with exogenous rh-BMP-2 or PLA-Ad-BMP-2 also produced more bone precursors than BMAs (p=0.001), except for day 7 ALP histomorphometry (p=0.343). ELISA confirmed successful BMP-2 production by both progenitor cell groups transduced with Ad-BMP-2. H&E sections from collagen I matrices seeded with PLA-Ad-BMP-2 cells confirmed bone formation at six weeks.

CONCLUSIONS

Liposuction aspirates contain PLAs that can be transfected with the BMP-2 gene, with rapid induction into the osteoblast phenotype at a rate comparable to rh-BMP-2 and osteoblast groups. Transduced PLAs produce more bone precursors with faster onset of calcified extracellular matrix than transduced BMAs. PLAs may be an ideal source of mesenchyme-lineage stem cells for gene therapy and tissue engineering.

摘要

目的

我们已从抽脂吸出物（处理后的脂肪抽吸细胞或PLA）中大量分离出多能间充质祖细胞。本研究检测了PLA和骨髓抽吸细胞（BMA）在暴露于重组人骨形态发生蛋白（BMP）-2（rh-BMP-2）或含BMP-2 cDNA的腺病毒（Ad-BMP-2）时的成骨潜能。

方法

对抽脂吸出物进行蛋白水解消化以获得PLA。在暴露于外源性rh-BMP-2或Ad-BMP-2四天或七天后，通过组织化学、分光光度法和逆转录聚合酶链反应（RT-PCR）对PLA和BMA进行评估。蛋白质印迹法和酶联免疫吸附测定（ELISA）证实了BMP基因转导。将结果与仅在成骨培养基中的成骨细胞和细胞进行比较。将PLA-Ad-BMP-2细胞接种在基质上并植入重症联合免疫缺陷（SCID）小鼠的后肢。

结果

对包括细胞外碱性磷酸酶（ALP）组织形态计量学、细胞内ALP分光光度法和钙化细胞外基质（冯·科萨染色）组织形态计量学在内的定量骨前体细胞分析显示，用外源性rh-BMP-2处理或用含BMP-2的腺病毒转导的PLA（PLA-Ad-BMP-2）比成骨细胞产生更多的骨前体细胞（p = 0.001）。用外源性rh-BMP-2处理的PLA或PLA-Ad-BMP-2也比BMA产生更多的骨前体细胞（p = 0.001），除了第7天的ALP组织形态计量学（p = 0.343）。ELISA证实了用Ad-BMP-2转导的两个祖细胞组均成功产生BMP-2。接种PLA-Ad-BMP-2细胞的I型胶原基质的苏木精-伊红（H&E）切片证实六周时形成了骨。