左归丸药物血清对骨髓间质细胞转化为肝细胞的作用  

Effect of Zuogui Wan drug serum on the differentiation of bone marrow mesenchymal stem cells into hepatocytes

作　　者：李瀚旻[1] 晏雪生[1] 罗建君[1] 李晶津[2] 高翔[1] 明安萍[1] 彭亚琴[1] 

机构地区：[1]湖北中医学院附属医院肝病研究所,湖北省武汉市430061 [2]武汉大学医学院

出　　处：《中国组织工程研究与临床康复》2007年第28期5465-5468,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金资助(30672590;30271562;30371787);国家科技部重大基础研究前期研究专项资助(2002CCC00300);湖北省自然科学基金资助(2001ABB171)~~

摘　　要：目的:一些实验已证实骨髓干细胞可以向肝细胞横向分化,观察补肾中药左归丸含药血清对骨髓间质细胞分化为肝细胞的影响。方法:实验于2002-01/2004-12在湖北中医学院附属医院肝病研究所完成。①实验动物:选取SPF级纯系Wistar大鼠46只,1只用于骨髓间质细胞的分离及培养,剩余45只用于含药血清的制备。另取2d龄Wistar乳鼠20只,用于肝脏细胞的培养。②实验方法:以体外肝细胞培养上清为诱导条件培养液,采用贴壁分离法纯化大鼠骨髓间质细胞。45只Wistar大鼠依次取15只用生理盐水、中药左归丸(由熟地、淮山药、枸杞子、山茱萸、菟丝子、川牛膝、鹿角胶、龟版胶组成)浓缩煎剂、中药八珍汤(由人参10g,白术10g,茯苓10g,甘草6g,当归15g,川芎10g,白芍15g,熟地15g组成)浓缩煎剂按10mL/kg灌胃,灌胃量以生药10g/kg计算,1次/d。第15天灌胃后2h,腹腔麻醉颈动脉插管取血,分管盛装含药血清,放置备用。大鼠骨髓间质细胞传至6代,分6组诱导培养:常规培养组使用常规培养液(DMEM培养液+体积分数为0.2的胎牛血清+2mmol/L谷氨酸胺)进行培养;肝细胞生长因子诱导组以常规培养液+25μg/L肝细胞生长因子+10-7mol/L地塞米松进行培养;肝细胞生长因子加左归丸组以常规培养液+25μg/L肝细胞生长因子+10-7mol/L地塞米松+10%左归丸含药血清进行培养;条件培养液加对照血清组以常规培养液+50%条件培养液+10%正常大鼠血清进行培养;条件培养液加八珍汤组以常规培养液+50%条件培养液+10%八珍汤含药血清进行培养;条件培养液加左归丸组以常规培养液+50%条件培养液+10%左归丸含药血清进行培养。③实验评估:诱导28d,每组10个复孔,分别于诱导第0,7,14,21,28天免疫细胞化学法检测肝细胞标志物甲胎蛋白、细胞角蛋白18及白蛋白阳性细胞率,PAS染色检测糖原阳性细胞率。结果:①诱导第0天,各组甲胎蛋白、白蛋白阳�AIM： Some experiments have proved that bone marrow mesenchymal stem cells （MSCs） could transdifferentiate into hepatocyte. In this study, we observed the influence of Chinese herb Zuogui Wan serum on the differentiation of hepatocyte from MSCs. METHODS： The experiment was carried out at Research Institute of Liver Disease, Affiliated Hospital of Hubei College of Traditional Chinese Medicine from January 2002 to December 2004. ①Forty-six Wistar rats （SPF） were selected. Except one was used for the separation and culture of MSCs, the others were used to prepare drug serum. And another 20 Wistar rats of 2 days old were selected for the culture of hepatocytes. ②MSCs of Wistar rat were cultured and purified by attachment method, then the supernatant of the hepatic tissue cultured in vitro served as conditioned medium. Forty-five Wistar rats were divided into 3 groups with 15 rats in each group, and wore respectively treated by physiological saline, Chinese herb Zuogui Wan （composed of prepared rhizome of rehmannia, Rhizoma Diescoreae, Fructus Lycii, Fructus Corni, Semen Cuscutae, Radix Achyranthis Bidentatae, Colla Comus Cervi and colla plastri testudinis）, Bazhen Tang （composed of 10 g Radix Ginseng, 10 g Rhizoma Atractylodis Macrocephalae, 10 g Poria cocos Woff, 6 g Radix Glycyrrhizae, 15 g Radix Angelicae Sinensis, 10 g Rhizoma, 15 g Radix Paeoniae AIba, 15 g Radix Rehmanniae Praeparata） with 10 mL/kg apozeme or 10 g crude drug/kg, once daily. Two hours after administration on the 15^th day, the rats were coeliac anaesthetized and common carotid artery punctured to collect drug serum. Rat MSCs passaged to 6 generation were divided into 6 groups： Common culture group was cultured in common medium （DMEM medium, 0.2 volume fraction fetal bovine serum, and 2 mmol/L glutamine）; hepatocyte growth factor （HGF） inducing group was cultured with common medium, 25μg/L HGF and 10^-7 mol/L dexamethasone （DXM）; HGF plus Zuogui Wan group was cultured with common medium, 25μg/L HGF, 10^-7 mo

关 键 词：左归丸 含药血清 肝细胞 骨髓间质细胞 

分 类 号：R575[医药卫生—消化系统]