胎儿肠壁结缔组织诱导骨髓间充质干细胞分化为上皮细胞(英文)

Differentiation of bone marrow mesenchymal stem cells into epithelial cells induced by fetal intestinal connective tissue

背景:间充质干细胞具有多向分化能力,在体外能诱导分化为上皮细胞,而胚胎原始消化管上皮的分化受肠壁间充质的诱导。肠壁结缔组织能否诱导骨髓间充质干细胞分化形成上皮细胞,尚不明确。目的:观察胎儿肠壁结缔组织能否诱导骨髓间充质干细胞分化形成上皮细胞。设计:体外培养,对比观察。单位:实验于2004-07/2006-07在重庆医科大学组织胚胎学教研室完成。材料:表皮生长因子为Sigma公司产品;CK,CK20为中山生物有限公司产品。收集四五个月流产胎儿标本6例的四肢骨髓,Ficoll离心,分离、培养、扩增获取骨髓间充质干细胞。取胎儿十二指肠乳头以下肠段,剥去肌层和外膜,酶消化去除上皮,剪成约15 mm×5 mm的组织块。胎儿标本由临床学院妇产科提供,产妇同意提供胎儿用于实验,实验经医院伦理委员会批准。方法:实验分4组进行:干细胞移植组和干细胞移植+表皮生长因子组分别将DAPI标记的第3代骨髓间充质干细胞(3×104)个移植于肠壁结缔组织块的黏膜下层表面;干细胞组和干细胞+表皮生长因子组为骨髓间充质干细胞爬片;其中干细胞移植+表皮生长因子组和干细胞+表皮生长因子组加入终浓度为10μg/L表皮生长因子;4组均体外培养12 d。主要观察指标:①用荧光显微镜观察DAPI标记的骨髓间充质干细胞的形态及分布。②干细胞移植组和干细胞移植+表皮生长因子组组织块培养12 d后,采用苏木精-伊红染色观察与荧光显微镜观察同一肠壁结缔组织表面细胞形态。③免疫组化染色检测标记细胞CK及CK20的表达,以及肠壁结缔组织是否表达表皮生长因子。④高碘酸-希夫反应(PAS反应)检测DAPI标记细胞与结缔组织间有无基膜样物质产生。结果:①荧光显微镜下干细胞移植组和干细胞移植+表皮生长因子组的组织块表面均有DAPI标记的荧光阳性细胞,苏木精-伊红染色显示该部位细胞呈上皮样形态,而且在培养时经多次换液未被洗去为长在结缔组织表面的细胞。②免疫组织化学染色显示两组组织块表面的细胞均可表达CK及CK20。③免疫荧光染色显示组织块表面的细胞同时有标记细胞核的DAPI蓝色荧光与标记CK的红色荧光,表明DAPI标记的骨髓间充质干细胞已经分化为上皮细胞。④干细胞组细胞CK及CK20阴性,而干细胞+表皮生长因子组则为阳性,提示表皮生长因子有诱导骨髓间充质干细胞分化为上皮细胞的作用。⑤PAS反应显示在标记细胞与结缔组织之间有基膜样结构糖蛋白出现。未经培养的结缔组织组织块内有表皮生长因子表达。结论:表皮生长因子和胎儿肠壁结缔组织在体外均能诱导骨髓间充质干细胞分化为上皮细胞;肠壁结缔组织中的表皮生长因子可能是结缔组织诱导骨髓间充质干细胞分化为上皮细胞的因素之一。

BACKGROUND： Mesenchymal stem cells have ability of multi-directional differentiation, and can be induced to differentiate into epithelial cells in vitro. The differentiation of epithelial cells in fetal primitive gut is induced by mesochymal cells of intestines. The report of bone marrow mesenchymal stem cells （BMSCs） differentiate into epithelial cells induced by intestinal connective tissue has not been identified. OBJECTIVE： To observe the possibility of BMSCs differentiate into epithelial cells induced by fetal intestinal connective tissue. DESIGN： Culture in vitro and comparative observation. SETTING： The experiment was carried out in the Department of Histology and Embryology, Chongqing Medical University from July 2004 to July 2006. MATERIALS： Epidermal growth factor （EGF, Sigma）; CK, CK20 （Zhongshan Bio-Tech, Co.,Ltd）. Bone marrow of limbs was collected from 6 aborted fetus samples aged 4 5 months. Adding Ficoll to centrifugalize, BMSCs were isolated, cultured and proliferated. The intestinal segment about 15 mm×5 mm was obtained stefilely from fetal duodenal papilla to colon, then muscular tunic and adventitia were peeled. Enzymatic digestion was used to remove the epithelial cells on the mucosa surface. The lump of intestinal connective tissue was cut into 15 mm×5 mm. Fetus samples were provided by Department of Gynaecology and Obstetrics in Clinical College, all the parturients agreed to the offer, and the experiment was approved by the hospital ethical committee. METHODS： The experiment was assigned into 4 groups. In groups A and B, the DAPI labelled BMSCs （3×10^4） at the third generation were transplanted on the submucosa of intestinal connective tissue; In groups C and D, the DAPI labelled BMSCs were only cultured on the cover glass; In groups B and D, EGF in final concentration of 10 ng/mL was added. MAIN OUTCOME MEASURES： After cultured for 12 days, the morphous and distribution of DAPI labelled BMSCs were observed under fluorescence microscope; the cell morphous on surface of the same intestinal connective tissue was observed with hematoxylin-eosin stain. Expressions of CK and CK20 as well as whether the intestinal connective tissue express EGF were all detected by immunohistochemical stain. Production of basal lamina between the DAPI labelled cells and connective tissue was assayed with periodic acid-Schiff reaction. RESULTS： On the surface of the tissue lump in groups A and B, the DAPI labelled BMSCs could be seen under fluorescence microscope. In the same section stained by hematoxylin-eosin, there were some epithelioid cells. During culture, the medium were changed several times, the marked cells that were not eluted, grew on the surface of connective tissue. Immunohistochemistry revealed, in groups A and B, cells on the surface of the tissue lump both expressed CK and CK20. Immunofluorescence stain shown that some of cells on the tissue lump had the DAPI marked nucelus （sapphirine fluorescence） and CK （red fluorescence） in cytoplasm simultaneously. It indicated that the 3^nd generation of DAPI labelled BMSCs had differentiated into epithelial cells. Cells in group C were negative for CK and CK20, but those in group D were positive, which indicated that EGF could induce BMSCs differentiate into epithelial cells. There were glycosidoprotein with basal membrane-like structures appeared between the cells and connective tissues in periodic acid-schiff stained section. The uncultured connective tissue expressed EGF. CONCLUSION： Both EGF and fetal intestinal connective tissues can induce BMSCs differentiate to epithelial cells in vitro; the EGF presented in the intestinal connective tissues may be one of factors in this induction process.