【作者】 胡宝洋；

【导师】 周国民；

【作者基本信息】 复旦大学 ， 人体解剖学与组织胚胎学， 2004， 博士

【摘要】 早产儿视网膜病(Retinopathy of prematurity, ROP)是发生于早产和低体重儿的一种致盲性视网膜病变,其病理特征为视网膜血管异常增生,严重者可引起视网膜变性、视网膜脱离等多种并发症。上世纪 80 年代以来,由于医疗技术的快速发展,早产儿的成活率也显著提高,ROP 的发病率也随之呈上升趋势,在体重≦1000g 的超低体重儿,发病率可高达 80%以上。ROP 已引起人们的普遍重视。防治 ROP 以改善早产儿的生存质量已成为全球关注的焦点。已经明确 ROP与吸氧治疗高度相关,早产、高氧和神经发育相关是发病过程中最为关键的因素,但其发病确切机制至今仍不明确。本课题将在建立氧诱导视网膜病模型(Oxygeninduced retinopathy, OIR)的基础上,应用基因芯片技术研究 ROP 发生过程中基因表达谱的变化规律及意义,用体外视网膜内皮细胞培养和其他细胞分子技术研究雌激素和神经营养因子等在 ROP 发生中的可能作用及机制,并结合骨髓基质细胞(BMSC)对 OIR 模型的治疗作用探讨 ROP 的发病机制。 课题的主要研究方法和内容有:(1)应用 ADP 酶法染色显示了 C57BL/6J小鼠从出生当天(P0)至生后 18 天(P18)视网膜铺片中的血管,并结合观察相同时期的视网膜连续切片,对正常小鼠视网膜血管的发育过程及规律进行研究。(2)建立高氧诱导的小鼠视网膜病(Oxygen induced reinopathy, OIR)模型,应用TRizol 法分别抽提 P17、P21 及 P27 小鼠 OIR 模型及同龄对照组的视网膜总 RNA,纯化 mRNA,逆转录并用 Cy3、Cy5 标记,与小鼠 Oligo 表达谱芯片杂交,经扫描获取基因表达信息。对所得信息进行差异分析、聚类及基因分类等生物信息学分析。(3)体外研究雌二醇和神经生长因子对视网膜内皮细胞增殖、迁移及管腔形成的影响,并采用受体阻断法研究雌二醇对神经生长因子信号的调节作用。(4)应用 RT-PCR 及免疫组织化学方法观察体外培养的 BMSC 中 VEGF、IGF 等因子的表达。,研究 BMSC 分泌因子对视网膜血管内皮细胞的作用(5)用荧光染料CM-DiI 对体外培养的 BMSC 进行标记,在高氧期注射入 OIR 小鼠玻璃体腔内。制作 ADP 酶法染色的视网膜铺片和连续切片,观察移植 BMSC 在 OIR 模型眼内的分布,并通过计数视网膜前异常增生的血管内皮细胞核数量,分析 BMSC 对OIR 的治疗效果。(6)购建 VEGF RNAi 干扰质粒,通过脂质体 Lipofectamin 2000介导转染 BMSC。ELISA、RT-PCR 检测干扰效果。体外检测经 VEGF RNA 干扰 3<WP=6>胡宝洋博士学位论文 OIR 基因表达谱及机制 摘要后的 BMSC 培养上清对内皮细胞作用的变化。进一步将转染后的 BMSC 移植入OIR 模型小鼠眼的玻璃体内,研究其抗血管新生的机制。 研究的主要结果有: 1.ADP 酶法能使内皮细胞着色,呈黑色或棕褐色。正常小鼠刚出生时,结合铺片和切片观察,发现视网膜中暂无血管分布,但可见散在分布的 ADP 酶阳性细胞。此时,在视网膜表面可见密集的玻璃体血管网。之后,视网膜血管以自中心向周边和自内层向外层的规律逐渐发生。在血管从视网膜内层向外层发生的过程中,约在 P12,先从内层向外发出较大的螺旋形血管,再进一步形成外层血管网。至 P18,视网膜血管发育基本成熟,而玻璃体血管则退化。 2.从 P7 开始,将 C57BL/6J 小鼠置于 75%的高氧环境中饲养 5 天后,至 P18可见异常新生的血管突破视网膜内界膜进入玻璃体,表明小鼠 OIR 模型制备成功。 3.基因芯片杂交结果显示,1402 个基因至少一个时间点呈差异表达。基因树聚类、条件聚类将基因良好的聚为四个区两大类。SOM 聚类显示 12 类不同的表达差异改变模式。基因本体学(GO)分析显示发育相关基因及 G 蛋白信号通路相关基因改变明显。 4.RT-PCR 显示视网膜血管内皮细胞株 RF/6A 有 ER、NGF 及 TrkA 等表达。一定剂量的 E2、NGF 均可促进 RF/6A 细胞的增殖及划痕修复,且呈剂量—效应关系。E2、NGF 处理的细胞有相似的凋亡率。应用 K252a(NGF 受体 TrkA 的拮抗剂)既可阻断 NGF 的作用,也可部分抑制 E2的作用。对于内皮细胞的管腔化形成实验,E2和 NGF 的作用则不一致。 5.免疫组化与 ELISA 显示,体外培养的 BMSC 表达多种生长因子,如 VEGF、IGF 及 NGF 等。BMSC 的培养上清可促进 RF/6A 细胞的增殖、迁移及管腔化形成。 6.CM-DiI 可有效的标记 BMSC。将标记的 BMSC 注移植入 OIR 小鼠的玻璃体腔内,发现部分 BMSC 可迁移至视网膜神经纤维层,并表达 GFAP。根据视网膜铺片 ADP 酶染色观察及连续切片视网膜前内皮细胞核计数,均显示 BMSC可抑制 OIR 小鼠视网膜的血管新生。 7.成功构建大鼠 VEGF 的干扰质粒 pSuper/VEGFi,经 Blastn 检索具有特异性。经重组质粒酶切、测序鉴定正确后,抽提超纯质粒转染第 5 代左右的 BMSC,转染效率为 25%。转染后 BMSC 培养上清中的 VEGF 也较对照组下降相似比例。干扰 VEGF 的表达后,其上清对 RF/6A 细胞的作用较对照减低。将转染质粒的BMSC 注入 OIR 模型小鼠的玻璃体腔内,其作用也较未转染制质粒的 BMSC 有所下降。 4<WP=7>胡宝洋博更多还原

【Abstract】 Purpose: Retinopathy of prematurity (ROP) is an ischemia-induced proliferativeretinopathy, which affects premature infants with low birth weight. It is a leadingcause of visual impairment and blindness in children, and shares pathophysiologicalcharacteristics with other common ocular diseases such as diabetic retinopathy,central vein occlusion, and age-related macular degeneration. Much attention has beenpaid on the prevention and treatment of this preventable disorder in the last few years.Among the many features of ROP, neovascularization in nervous system, sufferedin preterm babies, and receiving hyperxia treatment take the 3 most unique ones. TheROP has been explained as the hypoxia induced neovascularization following highdose oxygen delivery. Despite the much information got from an mouse model ofoxygen induced retinopathy(OIR), the detailed mechanism is still leading the interestsof researchers. In this study, we study the development of mouse vasculature, and thegene expression profile in the oxygen induced OIR model, with the intention to findgenes account for the neovascularization. To investigate the regulatory cascade of E2and NGF in retinal vascular endothelial cell, we evaluate the effects of the two,respectively or combined with K252a, one of the Trk A inhibitor,s on RF/6A retinalendothelial cell line. Also on the OIR model, we study the effects of bone marrowstromal cells(BMSCs), by intravitreal transplantation before oxygen exposure. Wealso knock down the BMSC expression of VEGF with a RNA interference plasmid,pSuper-EGFP to study the possible involvement of growth factors, such as VEGF, inROP.Methods: 1. Retina whole mounts of 8 developmental stages selected from postnatal day zero (P0) to P18 mouse were stained with ADPase histo-chemistrical method. Retina serial sections of the same day were used to assess the developmental growth of the retina vasculature. 2. Oxygen induced mouse model of ROP was developed in C57BL mice by a 5-day exposure to 75% oxygen from P 7 trough P12. Pooled total RNA was preparieded with a Trizol method from retinas of the follow groups respectively, P17, P21 and P27 ROP mice together with age corresponded control. Purified mRNA was reverse transcripted to cDNA, and abeled with 6<WP=9>胡宝洋博士学位论文 OIR 基因表达谱及机制 摘要 Cy3 and Cy5 conjuncted neucletide by PCR. Each group of the labeled cDNA was hybridized with an expression profile microarray of oligonucleotides of 16,000 mouse genes. Signal were extracted from the scaned images of chips, followed by background extraction and normalization, and subjected for further analysis. Genes altered 2 fold with statistically significance, from either of the the groups were included in our Gene tree, Condition and SOM cluster anlysis. 3. The estrogen-NGF regulatory cascade was studied on RF/6A retinal vascular endothelial cell line. MTT assay, wound healing assay and ECM matrix gel based tubogenesis assay were carried to study the proliferation, migration and angiogenesis of RF/6A following the administration of estrodial, NGF and inhibitor of Trk A receptor. 4. Bone marrow stromal cells (BMSCs) were separated from the whole marrow cells according to its much more potent adhenrence to the plastic ware. Immunoohistology were used to examine the expression of growth factors, such as VEGF、IGF、bFGF and NGF. The conditioned medium of BMSC, which was enrichment in growth factors, was added to the RF/6A culture medium. Effects on RF/6A were evaluated via the MTT, wound healing and bubogenesis assay after a 24h-incubation. 5. BMSCs of passage 3 to 5 were labeled with CM-DiI 12 hours before transplantation. 1×105 CM-DiI labeled BMSCs in 0.5 ul PBS were intravitreal injected into P7 mi更多还原