课题背景：本课题受国家自然科学基金支持，主要研究肝脏细胞和不同材料之间的相互作用。重点是研究肝脏细胞和生物可降解支架复合构建可植入工程化肝组织。目前已经用液态生物支架如胶原和纤维蛋白胶构建了可注射的工程化肝组织，分别植入皮下、腹腔和肝脏原位，证实肝细胞能够存活并表达功能。

应用要点：因为肝脏构造及功能极其复杂，工程化肝组织的构建在国内很少涉及。国外有部分学者进行了工程化肝组织的构建研究，但多使用聚乳酸、聚乳酸-乙醇酸共聚物等硬质材料。作者选用了液态生物材料，从而使得构建的工程化肝组织具有简便、高效的优点。而选用的种子细胞，因其成分、功能的多样性，也为进一步的细胞共移植研究提供了良好的模型。

同行评价：实验采用新生大鼠肝细胞及胶原构建工程化肝组织，一旦在体内实验中获得成功，将为临床治疗肝衰竭等疾病提供新的思路。

摘要
目的：在供体肝短缺的情况下，构建可植入的工程化肝组织具有重要的临床意义。实验尝试以新生大鼠肝细胞为种子细胞体外构建工程化肝组织，以期进一步的体内植入。
方法：实验于2007-04/08在解放军总医院普通外科研究所完成。①实验材料：SPF级、出生24 h以内的雄性SD新生大鼠。②实验过程：采用胰酶消化法获取新生大鼠肝细胞；以2×L-DMEM液（添加地塞米松10 μg/L、表皮生长因子20 μg/L、肝细胞生长因子40 μg/L及胰岛素0.04 U/mL）与液态鼠尾胶原等比例混合；再将肝细胞与胶原凝胶复合构建细胞/凝胶复合物，接种于培养板进行培养。③实验评估：培养后第1，3，5，7，9天，采用相差显微镜观察、四甲基偶氮唑盐比色法、苏木精-伊红染色和免疫组织化学染色分别对工程化组织的生长情况及组织形态特征进行观察，并对工程化组织的白蛋白合成功能及尿素的代谢水平进行评价。
结果：①肝细胞与胶原凝胶复合后，细胞均匀的分布在复合物中。在整个培养过程中，肝细胞保持着稳定的细胞形态。②四甲基偶氮唑盐检测显示肝细胞活性在生长初期平缓下降，直至第5天时仍然保持75%的细胞活性，之后快速下降。③体外培养5 d后，相差显微镜下观察显示肝细胞在胶原凝胶中呈三维立体生长，并保持肝细胞胞体圆形，核大而圆的特异形态；免疫组织化学证实这些肝细胞抗白蛋白抗体染色呈强阳性。④对培养上清中白蛋白和尿素的含量测定表明胶原凝胶中的肝细胞在培养初期保持着稳定的代谢合成功能。
结论：用新生大鼠肝细胞及胶原构建出一种有功能的工程化肝组织模型，这种模型可以应用于今后的工程化肝组织研究。
关键词：组织工程，胶原；凝胶类，大鼠，肝细胞

张博峰，赵云山，刘巨超，张兰，李荣，徐迎新．用新生大鼠肝细胞体外构建工程化肝组织[J].中国组织工程研究与临床康复，2008，12(2):201-204 [www.zglckf.com/zglckf/ejournal/upfiles/08-2/2k-201(ps).pdf]

In vitro liver tissue construction using neonatal rat hepatocytes

Abstract

AIM：Donator liver is insufficient, so it is clinically significant to construct implantable engineered liver tissue. This study investigated the feasibility of constructing engineered liver tissue using neonate rat hepatocytes as seed cells in vitro.
METHODS: The experiment was performed at the Institute of General Surgery, General Hospital of Chinese PLA from April to August 2007. ①The livers of male neonate SPF rats (within 24 hours) were digested by trypsin to isolate hepatocytes. 2×L-DMEM solution containing 10μg/L dexamethasone, 20μg/L epidermal growth factor, 40μg/L hepatocyte growth factor, and 0.04 U/mL insulin was mixed with liquid collagen from rat tail at a ratio of 1∶ 1. A collagen-based gel matrix as carrier for neonate rat hepatocytes was constructed in vitro. Collagen-immobilized hepatocytes were seeded onto plastic culture dishes. ②On the first, third, fifth, seventh, and ninth days, phase contrast microscopy was used to observe the morphology of the hepatocytes/collagen gel construct; Cell proliferation was estimated using MTT method. Hepatocyte histology was evaluated by hematoxylin and eosin (HE) staining, and differentiation was evaluated by immunohistology for albumin.
RESULTS: The hepatocytes distributed evenly in the composite and remained round-shape throughout the in vitro culture. ②MTT assay demonstrated that the highly viability of hepatocytes (75%) was maintained up to 5 days, and then decreased rapidly. ③Phase contrast microscopy showed the culture in a collagen matrix allowed stable cell numbers and specific morphous, and permitted three-dimensional neotissue formation. Immunohistology showed preservation of liver-specific markers albumin in vitro. ④The levels of albumin and urea from the supernatant of the culture showed collagen-immobilized hepatocytes maintained liver function stably.
CONCLUSION:A novel approach to construct functional engineered liver tissue using neonate rat hepatocytes as seed cells has been developed. This engineered liver tissue is an attractive tool for the development of liver tissue engineering.

Zhang BF, Zhao YS, Liu JC, Zhang L, Li R, Xu YX. In vitro liver tissue construction using neonatal rat hepatocytes.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(2):201-204(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-2/2k-201(ps).pdf]