【作者】 袁松涛；

【导师】 赵堪兴；

【作者基本信息】 天津医科大学 ， 眼科学， 2006， 博士

【摘要】 视网膜色素变性(retinitis pigmentosa,RP)是一种视网膜光感受器细胞和色素上皮细胞(retinal pigment epithelial cells,RPEs)发生进行性退行性变的视网膜变性疾病,具有显著的遗传倾向。其临床特点是双眼视野进行性缩小,进而累及中心视力,最终导致视力丧失。到目前为止视网膜色素变性仍被WHO划归为不可治疗盲,没有一个理想的治疗方法,因此视网膜色素变性的治疗于成为眼科领域的研究热点。目前临床可以推荐的治疗手段仅仅是维生素A和中药治疗,不能完全阻止病程的进展,而且药效非常有限,甚至可以说是“聊胜于无”。神经营养因子是一类对中枢和周围神经系统具有多种生物学活性的细胞因子,一系列干预视网膜变性类疾病的动物研究显示:神经营养因子具有神经元保护作用,神经营养因子有望成为治疗RP的有效手段。 睫状神经营养因子(Ciliary Neurotrophic Factor,CNTF)是一种广泛存在于中枢和外周神经系统,对神经系统的分化、发育,对神经元损伤后的存活,轴突再生都有重要作用的细胞因子。本研究通过CNTF玻璃体腔注射观察其对视网膜色素变性小鼠模型(rd mouse)的治疗作用,以及在ARPE-19细胞中表达后的生物学效应,探讨其作用机理。 研究方法: 第一部分:将出生10天(P10)rd小鼠随机分成三组,CNTF治疗组、伪治疗组和空白对照组,治疗组和伪治疗组的玻璃体腔分别注射鼠CNTF和磷酸缓冲液PBS,分别在出生后14、18、22、30天麻醉小鼠后行视网膜电图(Electroretinograph,ERG)检查,过量麻醉处死小鼠,摘取眼球行光、电镜检查、视网膜外核层厚度测定和TUNEL凋亡检测。 第二部分:通过逆转录聚合酶链反应(RT-PCR)扩增小鼠CNTF野生型全长编码序列,体外定点突变获取截短型CNTF的互补DNA(complementary DNA,cDNA)编码序列,将上述两序列分别克隆至pTracer-CMV真核表达载体,转染更多还原

【Abstract】 Retinitis pigmentosa (RP) is the kind of disease that photoreceptors and retinal pigment epithelial cells degenerate gradually. The clinical sign of RP is progressive visual field defect, and leaves only tiny island of central visual field which will be disappeared at last. Until now, RP is classified as an unpreventable blindness disease by WHO, and there is no remedy for it. The only medicines could be recommended are VitA and Chinese traditional medicine on clinical, and can not stop the progress of RP, and there is limited effect. Neurotrophic factor is a kind of cytokines that have multi biological activities to central peripheral nervous system. Lots of animal experiments showed that neurotrophic factors have neuroprotective effect, and these factors are promising medicals for RP.CNTF can be found in wide range of central and peripheral nervous system, and play a key role on surviving of traumatic neuron and regeneration of axon. In this research, we observed the therapeutic effect of CNTF vitreous cavity injection on rd mice and biological effect of CNTF gene expressing in ARPE-19 cells.Methods:Section I : postnatal 10 days (P10) rd mice were randomized into three groups, CNTF treated group, sham-surgery group and blank control group. CNTF and PBS were injected into the vitreous of the treatment group and the sham-surgery group respectively on P10. After anesthesia, ERG of rd mice were examined on postnatal 14、 18、 22 and 30days, and then the mice were killed by over dosage anesthetic. Transmission electron microscope, light microscope, measurement of thickness of the retina outer nuclear layer and TUNEL assay were carried out.Section II: RT-PCR was used to amplify the cDNA of mouse CNTF gene, and truncated CNTF cDNA was obtained by site-directed mutagenesis. The two types of CNTF gene were cloned into plasmid pTracer-CMV and transfected ARPE-19 cells by transfectamine 2000. Dot blotting was used to detect the expression of CNTF. MTT and flow cytometry apoptosis assay were performed to observe the biologicaleffect of CNTF expression in ARPE-19 cells. Results:1. It was observed that the retina outer nuclear layer of the treatment group was thicker than that of the other two groups under light microscope in P14, P18, P22 and P30. There were statistical significance (p<0.05).2. TUNEL positive cells could be found in the outer nuclear layer of retina of the rd mice.3. Transmission result showed that apoptosis was a major pathway of pathologic changes in rd mouse retina, and inflammation was also involved in, and the CNTF treated group retina ultra structure of rd mouse was better than PBS treated group.4. ERG examination implicated that the retina function of CNTF treated group is better than control group.5. Wild type and truncated CNTF gene were amplified by RT-PCR, and their eukaryotic expression plasmids were successfully constructed.6. After ARPE-19 cells transfected with two types of recombinant plasmids, the CNTF had been detected in the cells culturing supernatant.7. Though Secretome 2.0 prediction, it is possible that CNTF is a non-classical secretory protein.8. MTT showed that the numbers of the cells transfected with two types of CNTF gene are more than cells transfected with blank pTracer, and the number of cells transfected with truncated CNTF gene is more than transfected with wild type.9. Quantitive apoptosis assay implicated that CNTF could partially inhibit the apoptosis that induced by the cells culturing with serum free culture.Conclusion:Vitreous injection of CNTF could slow photoreceptors’ reduction of the rd mice, and may have therapeutic action on the retinitis pigmentosa of rd mouse. The therapeutic effect may be taken effect via anti-apoptosis. The duration of therapeutic is relatively short compared with the course of RP, and which is because of CNTF degradation and reduction of its concentration. Incorporation with molecular biologytechniques, it is a promising remedy for RP that to construct a drug delivery system of long period and delayed releasing CNTF. The truncated CNTF may have more powerful therapeutic effect than wild type. Expression of two types of CNTF in ARPE-19 cells gets prepared for gene therapy research of retinitis pigmentosa.更多还原