【作者】 董兴刚；

【导师】 陈江华；

【作者基本信息】 浙江大学 ， 内科学， 2009， 博士

【摘要】 研究背景生物人工肾小管辅助装置（bioartificial renal tubule assist device,RAD）是目前治疗终末期肾脏病的有效方法,RAD是肾脏组织工程研究的重点之一,目前国外RAD已经完成由美国食品药品管理局（FAD）批准的Ⅰ/Ⅱ期临床实验,并取得了令人鼓舞的结果.尽管RAD研究取得了很大的进展,然而,在RAD构建的许多关键问题仍未解决.首先,构建RAD的支架是肾脏组织工程种子细胞生长和增殖的载体,因此如何把支架构建好,是肾脏组织工程研究工作的关键问题之一.其二,中空纤维聚砜膜与种子细胞的更好地黏附,以及其后的生长、增殖、分化等是作好肾脏组织工程研究工作的关键问题之二.构建RAD的支架的问题:RAD的支架可以用血液透析器,例如F60等,但是如果采用F60,其内的中空纤维的滤过有效面积1.2m²,假如在1.2m²的纤维上置入的种子细胞并生长,显然面积过大.在本研究的第一章,针对RAD的支架问题,我们研制了一种生物反应器,以此来克服RAD的支架的缺陷提供了一个有效的办法.中空纤维聚砜膜与种子细胞的黏附不能满足需要的问题:目前种子细胞种植在生物反应器的中空纤维聚砜膜上,由于聚砜膜表面的疏水性和粗糙度不佳,致使中空纤维聚砜膜与种子细胞的黏附不佳,针对这一缺陷,我们考虑采用如下方法进行改进:对材料表面进行纳米修饰,材料表面特性对细胞的黏附、增殖、分化和凋亡起重要作用.将材料表面进行纳米修饰是材料改性研究的热点之一.方法1.研制生物反应器,中空纤维管呈同心圆排列,内植1000根聚砜膜中空纤维,有效表面积0.1m²,将纳米金组装到中空纤维聚砜膜表面,根据纳米金溶液浸泡纤维的时间分别为0h、2h、5h、14h,共四组,得到4种中空纤维聚砜膜/纳米金仿生支架,观察各组中空纤维聚砜膜的粗糙度、吸水性以及材料的表征等情况。2.植入非洲恒河绿猴肾细胞细胞株（CV₁细胞）、鼠肾小管上皮细胞株（NRK-52E）等细胞在制备的4种生物反应器内的中空纤维聚砜膜/纳米金仿生支架的中空纤维外腔面,继续培养2周,构建生物人工肾小管,通过采用免疫荧光染色、原子力显微镜和扫描电镜等方法观察中空纤维聚砜膜表面贴壁生长细胞的黏附、铺展、增殖、细胞数目和形貌等变化,以及检测RAD对Na⁺、葡萄糖转运功能,观察哇巴因、根皮苷对转运的选择性地抑制作用,并与未植入细胞的空白生物反应器内中空纤维做对照。3.我们自行改良的扫描电镜观察的方法,对样品制备中乙醇脱水进行了改进,用改良的方法,观察材料表面的成片细胞的图形完整性。结果1.我们研制的生物反应器,这种壳体装1000根中空纤维膜,封装的壳体里的中空纤维膜通过试压测试,全通;壳体内的中空纤维膜能做成平板状。2.我们自行改良的方法,结果图片可见,材料表面成片细胞连接,无断裂,图形完整,从而避免了原来常规方法的图片失真的缺陷。3.三组纳米化与一组未纳米化的材料相比,三组中空纤维聚砜膜/纳米金的吸水率与未纳米化相比明显增加（P＜0.01）,三组纳米化之间比较,5h组的中空纤维聚砜膜/纳米金的吸水率与2h组、14h组相比明显增加（P＜0.05）.4.纳米化与一组未纳米化的材料相比,2h组、5h组中空纤维聚砜膜/纳米金的粗糙度与未纳米化相比明显增加（P＜0.05）,三组纳米化之间比较,5h组的中空纤维聚砜膜/纳米金的粗糙度与2h组、14h组相比明显增加（P＞0.05）..5.三组纳米化与一组未纳米化的材料相比,三组中空纤维聚砜膜/纳米金的的细胞数目与未纳米化相比明显增加（P＜0.01）,能显著提高种子细胞贴壁及增殖,三组纳米化之间比较,5h组的中空纤维聚砜膜/纳米金的粗糙度与14h组相比明显增加（P＜0.05）.。6.培育14天后的RAD组具有对Na⁺和葡萄糖的转运功能,转运率在哇巴因、根皮苷作用后明显降低;去除药物抑制因素后,Na⁺、葡萄糖转运率又能恢复,与对照组比较差异有显著性意义（P＜0.01）。结论1.成功地研制生物反应器,并用于构建RAD的支架。2.我们研制了一种扫描电镜的样本制备的新方法,这种方法适合观察中空纤维聚砜/纳米金表面生长的细胞,能真实的观察到成片生长细胞的形貌,避免了原来的常规方法对样品的形态的破坏.3.我们研制了中空纤维聚砜膜/纳米金仿生支架,全部三组纳米化与一组未纳米化的材料相比能显著提高种子细胞贴壁及增殖,其中5小时组的中空纤维聚砜膜/纳米金的生物学性能最佳,中空纤维聚砜膜/纳米金的表面修饰方法对于中空纤维聚砜膜表面改性有着重要意义。4.初步成功建立了一种体外构建生物人工肾小管的新方法.我们体外构建了生物人工肾小管,其具备RAD的Na⁺、葡萄糖选择性转运功能,本研究为RAD的临床应用提供了一定的实验证据。更多还原

【Abstract】 BackgroundBioartificial renal tubule assist device（RAD） is an efficient therapy for end-stage renal disease.With the progress in study on engineering kidney,there are several problems to be solved,such as how to obtain a bioartificial kidney and how to make seeding cells growing on the surface of the scaffold?Materials and methods1.The nano-sized gold colloid was prepared by chemical reduction.Polysulfone hollow fiber membrane/nano-sized gold colloid scaffold was fabricated by self-assembly technology.Africa green monkey kidney cell line CV₁ cells were cultured on the surface of the scaffold.Inoculation time of the membrane and nano-sized gold colloid was 0h,2h,5h and 14h respectively.Cell attachment and proliferation were examined with scanning electron microscopy.2.The construction of RAD.Rat kidney proximal renal tubule cell line NRK-52E cells were seeded onto the outer space of the bioreactor hollow fiber cartridges and incubated for two weeks.And then,the transport rates of sodium and glucose were measured.Meanwhile,the inhibitory effects of oubaine and phlorizin on the transport of sodium and glucose were determined and compared with control device.Results1.Biological features of the polysulfone hollow fiber membrane/nano-sized gold colloid scaffold:Surface roughness（Ra values） of four kinds of scaffolds（0 h,2h,5h, 14h） were 29.43±12.38nm,53.02±23.52 nm,61.57±16.08 nm and 51.42±12.22 nm respectively.Water absorption ratios of them were 40.71±2.26%,61.96±3.85%, 70.26±4.24%and 63.21±4.67%respectively,and the highest ratio was seen at 5 hours.2.Growth of seeding cells on the outer surface of the polysulfone hollow fiber membranes of RAD:Cell attachment and proliferation on the 2h,5h 14h scaffolds were better than that of the Oh scaffold（P＜0.05）,and the highest cell number was also seen at 5 hours.In the scaffolds（2h,5h,and 14h） groups,growth of seeded cells with many microvillus was seen on the hollow fibre surface but not the hollow fibre in control group.Transport functions of the RAD:After 14 days of incubation,cells on the RAD had functions of transporting sodium and glucose,which could be inhibited significantly by oubaine and phlorizin,and could be recovered after agents were removed.ConclusionsA bioartificial renal tubule assist device was successfully made with the polysulfone hollow fiber membrane/nano-sized gold colloid scaffold.It had increased water absorption,cell adhesion and proliferation,and had the function of transporting sodium and glucose.更多还原