摘要
目的探讨肝细胞与液态Ⅰ型胶原复合构建可植入的工程化肝组织的可行性。方法SD 大鼠肝细胞与液态Ⅰ型胶原及 DMEM 复合,形成肝细胞/胶原凝胶复合物。该复合物被接种在培养板中培养,采用相差显微镜和 HE 染色方法对培养肝细胞形态特征进行观察,并采用 MTT 法和免疫组化染色方法分别对肝细胞的活性和功能进行检测。肝细胞/胶原复合物同时被植入皮下腔中观察肝细胞的分化及工程化肝组织的形成情况,采用 HE 染色和免疫组织化学染色方法对植入的工程化肝组织进行评价。结果肝细胞与液态Ⅰ型胶原复合后形成凝胶状复合物,可见肝细胞均匀分布在整个复合物中,呈三维立体生长。在整个体外培养过程中,肝细胞始终保持圆形的形态;肝细胞在培养初期活性稍有下降,直至第7天时仍然保持活性的87%,之后随培养时间延长而活性逐渐降低。经过2周培养,肝细胞仍具有白蛋白的合成功能。皮下植入后1周,肝细胞/胶原复合物形成工程化灶性肝组织,免疫组化染色证实这种工程化的肝组织具有白蛋白的合成功能。结论采用胶原凝胶作为肝细胞生长和分化的支架可在体内构建类肝样的工程化肝组织。这种可植入的工程化肝组织提供了一种基于肝细胞治疗的新途径,并有望用于损伤肝组织修复和重建。
Objective To construct implantable engineered liver tissue （ELT） using type Ⅰ collagen gel as scaffold. Methods Type Ⅰ collagen was obtained from the tail of a rat. Hepatocytes were collected from a Sprague-Dawley rat, mixed with liquid type Ⅰ collagen and Dulbecco＇s modified Eagle＇s medium to create hepatocyte/collagen gel construct. The construct was inoculated in a 96-well plate. 0, 3, 5, 7, 9, 11, 13, and 15 days after the inoculation the viability of hepatocytes in vitro was measured by MTT assay. Phase contrast microscopy was used to observe the morphology of the hepatocyte/collagen gel construct. Three SD rats underwent injection of the hepatocyte/collagen gel construct into the subcutaneous space. One week later the implant was taken out. The morphology was conducted by routine H.E. staining and immunohistochemical staining. The morphology and function of hepatocytes was investigated by inverted microscopy, routine H.E. staining and immunohistochemical staining. The constructs were also implanted into subcutaneous space, and the differentiation of hepatocytes and the formation of engineered liver tissue were observed by routine H. E. staining and immunohistochemical staining. Results Phase contrast microscopy showed that the hepatocytes were distributed evenly in the construct and remained round-shape throughout the in vitro culture. MTT assay demonstrated that the high viability of hepatocytes （87%） was maintained up to 7 days, and then decreased gradually. Albumin, the specific marker of hepatocytes remained positive by immunohistochemical staining after 15-day culture. One week after implantation into subcutaneous space, the implanted hepatocytes retained its hepatocyte-specific morphology, i. e. round shape, large nuclear/cytoplasm ratio as well as binuclear cells, and formed small engineered liver tissue containing blood vessels within and surrounding the tissue. Conclusion A novel approach to construct implantable engineered liver tissue using collagen gel as scaffold for growth an
出处
《中华医学杂志》
CAS
CSCD
北大核心
2007年第29期2065-2068,共4页
National Medical Journal of China
基金
国家自然科学基金(50573091)
关键词
肝细胞
胶原Ⅰ型
皮下植入
Hepatocytes
Collagen type Ⅰ
Subcutaneous implantation
