CAJ | 학술논문

目的研究重组hTGF-β1腺病毒(adeno-hTGF-β1)转染BMSCs对其向软骨分化的作用.方法重组adeno-hTGF-β1转染第一代猪BMSCs,对照组转染adeno-LacZ,腺病毒的量以200pfu/细胞计算,转染后1 d,消化收集重组腺病毒转染后的BMSCs,置于无菌15 ml聚丙烯试管中,体外细胞团聚集连续诱导培养21 d,然后分别从大体观察、组织学和Ⅱ型胶原蛋白免疫组化的检测对形成组织进行评价. 结果实验组细胞团收缩成近似小球形的组织块,外观成乳白色,触之有一定的弹性.苏木素-伊红染色观察可见细胞团外周为由数层扁平状成纤维样细胞组成的纤维软骨膜,下层和中部区域有巢状软骨形成,软骨陷窝明显可见,软骨细胞较均匀分布,包埋在软骨陷窝内.Safranin'O染色显示,形成的软骨组织区域有大量被染成桔红色蛋白多糖类基质分泌,Ⅱ型胶原免疫组化染色检测显示细胞团中央出现较明显的阳性染色区域,可见棕黄色的颗粒分布于胞浆内.而对照组形成的组织块略小,苏木素-伊红染色观察见无软骨样组织形成,主要为较致密无结构特征的纤维样组织,Safranin'O染色也无被染成桔红色蛋白多糖类基质分泌,Ⅱ型胶原免疫组化染色检测显示也无明显的阳性染色区. 结论应用重组hTGF-β1腺病毒转染的BMSCs进行细胞团聚集诱导培养,可诱导BMSCs向软骨细胞表型分化而形成软骨,为hTGF-β1基因转染的BMSCs在软骨组织工程应用中奠定了基础.
목적 연구중조hTGF-β1선병독(adeno-hTGF-β1)전염BMSCs대기향연골분화적작용.방법 중조adeno-hTGF-β1전염제일대저BMSCs,대조조전염adeno-LacZ,선병독적량이200pfu/세포계산,전염후1 d,소화수집중조선병독전염후적BMSCs,치우무균15 ml취병희시관중,체외세포단취집련속유도배양21 d,연후분별종대체관찰、조직학화Ⅱ형효원단백면역조화적검측대형성조직진행평개. 결과 실험조세포단수축성근사소구형적조직괴,외관성유백색,촉지유일정적탄성.소목소-이홍염색관찰가견세포단외주위유수층편평상성섬유양세포조성적섬유연골막,하층화중부구역유소상연골형성,연골함와명현가견,연골세포교균균분포,포매재연골함와내.Safranin'O염색현시,형성적연골조직구역유대량피염성길홍색단백다당류기질분비,Ⅱ형효원면역조화염색검측현시세포단중앙출현교명현적양성염색구역,가견종황색적과립분포우포장내.이대조조형성적조직괴략소,소목소-이홍염색관찰견무연골양조직형성,주요위교치밀무결구특정적섬유양조직,Safranin'O염색야무피염성길홍색단백다당류기질분비,Ⅱ형효원면역조화염색검측현시야무명현적양성염색구. 결론 응용중조hTGF-β1선병독전염적BMSCs진행세포단취집유도배양,가유도BMSCs향연골세포표형분화이형성연골,위hTGF-β1기인전염적BMSCs재연골조직공정응용중전정료기출.
Objective To investigate the action of chondrogenesis differentiation of bone marrow stromal cells (BMSCs) transfected with adeno-hTGF-β1. Methods In the experiment group, replication-deficient a denoviruses carrying human hTGF-β1 complementary DNA (adeno-hTGF-β1 was constructed and applied to transfect to the first generation BMSCs. As a control, each BMSCs was transduced with 200 pfu of adeno-LacZ gene. One day after transfer, BMSCs were trypsinized, counted, and 5×105 cells aliuots were spun down at 500 rpm per minute in 15 ml polypropylene conical tubes and then cultured in a defined medium in an incubator at 37℃ for 21 days. The aggregates were harvested at time points to 21 days and assessed by gross observation, histological analyses and immunohistochemical localization of type Ⅱ collagen. Results When harvested at 21 days, each pellet shrinked to spheroid tissue with apearly opalescence in gross morphology and found to be relatively firm. H.E staining showed elongate dlining cells appeared as perichon drium-like cells at the surface. Some nests of cartilage were observed at the substrate of the tissue. Mature chon drocytes were embeded in the lacuna in the experiment group. In addition, Safranin'O staining confirmed the presence of sulfated proteoglycans in the ECM of chondrogenesis region. Immunohistochemical staining revealed the presence of type Ⅱ collagen in chondrogenesis region. By contrast, HE staining showed no evidence of cartilage formation in the control group. They were fibrous tissue with no architectural feature. Safranin'O staining and Immunohistochemical staining showed no evidence of sulfated proteoglycans or typeⅡ collagen expression. Conclusion BMSCs transfected with adeno-hTGF-β1 could induce its chondro-genesis when aggregate cultured in a defined medium in vitro, laying a foundation for the application of hTGFβ1 gene-transfected BMSCs in cartilage tissue engineering.