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Gastrointestinal tissue regeneration using small intestinal submucosa (SIS) with mesenchymal stem cell

使用小肠粘膜下层（SIS）和间充质干细胞进行胃肠组织再生

基本信息

批准号：
17591405
负责人：
UENO Tomio
金额：
$ 2.18万
依托单位：
Yamaguchi University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

项目摘要

in vivo and in vitro Functional Evaluation of the Grafted Wall with SIS.A full-thickness defect was created in a rodent stomach. SIS was secured to the gastric wall. The motility of the regenerated tissue was evaluated using force transducers in vivo. Muscle strips were harvested from within the grafted area to make sure both a histological and a functional recovery in vitro. Dose response curves were obtained with carbachol (CCH) or sodium nitroprusside (SNP). Activation of intrinsic nerves was achieved by electrical field stimulation. Tissue specimens were stained with hematoxylin and eosin and were also examined by immunohistochemistry using antibodies against S100 protein, a-smooth muscle, and proton pump a-subunit. As a result, we identified a different type of wound healing with similar contraction and relaxation response to normal stomach, both in vivo and in vitro. However, the in vitro organ bath studies showed significantly lower contraction and relaxation in the defect compared to normal stomach. There was, therefore, little normal muscle function, and thus likely poor motility, in the defect. The clinical implications of such findings warranted further investigation of SIS with stem cell.Stomach Wall Defect Regeneration using SIS with Mesenchymal Stem Cell.The femora were excised aseptically from 7-week-old male Fisher 344 rats, and both ends of the femora were cut at the epiphyses. The bone marrow was flushed out using 10 ml of culture medium expelled from a syringe through a 20-gauge needle. The released cells were collected and cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Maintaining the cells in a CO2 incubator, marrow stromal cells were detached from the flask using 0.25% trypsin after 10 days of primary culture and seeded on a SIS matrix. A full-thickness defect was created in a rodent stomach and the SIS with mesenchymal stem cell was secured to the gastric wall. Long-term evaluation is now in process.

具有SIS的移植壁的体内和体外功能评价在啮齿动物胃中产生全层缺损。将SIS固定在胃壁上。使用力传感器在体内评价再生组织的运动性。从移植区域内收获肌条，以确保体外组织学和功能恢复。用氨甲酰胆碱（CCH）或硝普钠（SNP）获得剂量反应曲线。通过电场刺激实现内源性神经的激活。组织标本用苏木素和伊红染色，并使用针对S100蛋白、a-平滑肌和质子泵a-亚基的抗体进行免疫组织化学检查。因此，我们确定了一种不同类型的伤口愈合，在体内和体外具有与正常胃相似的收缩和舒张反应。然而，体外器官浴研究显示，与正常胃相比，缺损处的收缩和舒张显著降低。因此，在缺陷中几乎没有正常的肌肉功能，因此可能运动性差。这些发现的临床意义保证了SIS与干细胞的进一步研究。胃壁缺损再生使用SIS与间充质干细胞。股骨从7周龄雄性Fisher 344大鼠无菌切除，并在骺处切割股骨的两端。使用10 ml培养基通过20号针头从注射器中排出，冲洗骨髓。收集释放的细胞并在含有10%胎牛血清的Dulbecco改良Eagle培养基中培养。将细胞保持在CO2培养箱中，在原代培养10天后使用0.25%胰蛋白酶将骨髓基质细胞从烧瓶中分离，并接种在SIS基质上。在啮齿动物胃中产生全层缺损，并将具有间充质干细胞的SIS固定到胃壁。目前正在进行长期评估。