CX3CL1基因修饰骨髓间充质干细胞对光损伤视网膜的保护作用

发布时间：2018-05-13 16:07

本文选题：间充质干细胞 + 视网膜变性　；参考：《福建医科大学》2015年博士论文

【摘要】：视网膜变性是一类重要的致盲性眼病,可引起视网膜光感受器或神经节细胞的死亡。当前的治疗策略侧重于针对病因治疗,或通过维持疗法延缓视网膜细胞的死亡过程。随着对干细胞研究的深入,骨髓间充质干细胞(bone marrow-derivedmesenchymal stem cells,MSCs)由于其具有可塑性、神经保护、免疫调控等特性,在治疗视网膜疾病方面获得了越来越多的关注。而作为视网膜上固有的吞噬细胞,小胶质细胞在视网膜变性的发病过程中起到重要作用。MSCs是否与视网膜小胶质细胞相互作用,激活相关信号通路,维持视网膜自我稳定状态,鲜有相关文献报道。趋化因子CX3C模体配体1(Chemokine C-X3-C motif ligand 1,CX3CL1)通过识别其特异性受体CX3CR1,控制着视网膜小胶质细胞的活化程度。本研究通过体外共培养体系,评估CX3CL1基因修饰的MSCs(CX3CL1gene-modified MSCs,CX3CL1-MSCs)对活化的视网膜小胶质细胞功能的影响,并通过体内移植观察其对光损伤视网膜的保护作用,探讨CX3CL1/CX3CR1信号通路的作用。第一部分建立CX3CL1基因修饰的骨髓间充质干细胞目的建立CX3CL1基因修饰的MSCs,并了解其功能。方法全骨髓贴壁法培养SD大鼠MSCs,流式细胞术检测表面标记物,并进行成骨、成脂分化诱导鉴定,免疫荧光检测MSCs表达神经营养因子的情况。酶切含有CX3CL1基因的质粒与载体质粒,线性连接获得的酶切产物,转化后阳性克隆行PCR鉴定并测序、比对。脂质体介导慢病毒三质粒共转染293T细胞,收集上清液,采用离心超滤装置浓缩,获得病毒液,Real-time PCR测定病毒滴度。CX3CL1慢病毒感染MSCs,荧光显微镜下观察其感染效率,ELISA检测细胞上清中CX3CL1水平,Real-time PCR检测细胞CX3CL1 mRNA的表达情况。结果全骨髓贴壁法成功分离培养出MSCs,流式细胞术检测第3代的MSCs表面标记物的表达阳性率分别为cd90:(95.21±2.13)%、cd44:(96.75±3.30)%、cd34:(1.84±0.92)%。mscs能够被诱导分化为成骨细胞和脂肪细胞。mscs可在体外表达cntf、bdnf、gdnf、bfgf。成功构建了过表达cx3cl1基因的慢病毒,滴度至少为2×108tu/ml。cx3cl1慢病毒高效感染mscs,最佳感染复数(moi)为10。慢病毒感染mscs后,其cx3cl1的分泌量与cx3cl1mrna的表达均高于未感染组与空载对照组(f=338.300、640.300,p0.05)。结论cx3cl1慢病毒感染后,cx3cl1基因能够整合至mscs基因组中,并准确转录,cx3cl1基因修饰的mscs能够稳定表达和分泌cx3cl1。第二部分cx3cl1基因修饰的骨髓间充质干细胞对活化的视网膜小胶质细胞生物学功能的影响目的评估cx3cl1/cx3cr1信号通路在mscs和视网膜小胶质细胞相互作用中的影响。方法采用视网膜胶质细胞混合培养和振荡分离的方法分离培养sd大鼠视网膜小胶质细胞,免疫荧光染色鉴定细胞纯度。脂多糖(lps)刺激视网膜小胶质细胞,并与mscs、cx3cl1基因修饰的mscs(cx3cl1-mscs)、中和性抗体封闭cx3cl1的mscs共培养24h,观察共培养后视网膜小胶质细胞增殖、吞噬、迁移能力的变化,griess法检测其一氧化氮(no)释放量,采用real-timepcr、westernblot、elisa等方法分析其表达和分泌细胞因子的变化。结果视网膜胶质细胞混合培养和振荡分离的方法成功分离培养出视网膜小胶质细胞,免疫荧光检测显示,cd11b、iba1阳性率分别为(96.18±3.40)%、(94.05±4.18)%,gs呈阴性表达,说明获得的视网膜小胶质细胞纯度较高。lps能够活化视网膜小胶质细胞。与mscs共培养,使活化的视网膜小胶质细胞的增殖被抑制(f=32.280,p0.05),而吞噬、迁移能力增强(f=72.200、20.020,p0.05),tnf-α、il-1β、inosmrna表达下调(f=20.710、63.530、78.580,p0.05),cntf、bdnf、cx3cr1mrna表达量上调(f=169.400、29.050、79.060,p0.05),bdnf、cntf蛋白表达量增加(f=24.460、20.510,p0.05),细胞上清中no、tnf-α、il-1β分泌量降低(f=23.670、84.960、30.760,p0.05)。与cx3cl1-mscs共培养,能够增强这些反应,而与cx3cl1封闭的mscs共培养,能够抑制这些反应。结论MSCs可能主要通过CX3CL1/CX3CR1信号通路维持视网膜小胶质细胞的稳态。第三部分视网膜下腔移植CX3CL1基因修饰的骨髓间充质干细胞对光损伤视网膜的保护作用观察目的探讨视网膜下腔移植CX3CL1基因修饰的MSCs(CX3CL1-MSCs)对光损伤视网膜结构和功能的保护作用。方法SD大鼠散瞳后持续暴露于(9500±500)Lux的白光24h。HE染色观察强光暴露对视网膜组织结构的影响,末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(TUNEL)测定法检测视网膜细胞凋亡情况。将MSCs和CX3CL1-MSCs移植到光损伤大鼠视网膜下腔,比较其向视网膜内层迁移的能力,采用HE染色观察外核层厚度变化,闪光视网膜电图(F-ERG)和TUNEL法评估其对宿主视网膜的保护作用。ED-1抗体标记视网膜上活化的小胶质细胞。Western blot检测移植后宿主视网膜表达CNTF、BDNF、IL-1β、TNF-α的水平。免疫荧光定位视网膜上CNTF、BDNF的表达。结果光损伤后视网膜外核层厚度较正常组变薄,凋亡细胞增多(t=8.009、8.753,P0.05)。视网膜下腔移植后,CX3CL1-MSCs向视网膜内层的迁移能力强于MSCs(t=3.674,P0.05)。与MSCs移植组、DMEM移植组相比,移植CX3CL1-MSCs对于延缓视网膜外核层变薄,抑制视网膜细胞凋亡,减少活化的视网膜小胶质细胞的数量,具有更好地作用。同时,F-ERG结果显示移植后1w、3w、6w,移植CX3CL1-MSCs能够更好地保护视网膜功能。移植后3w,CX3CL1-MSCs移植组视网膜CNTF、BDNF的蛋白表达量高于MSCs移植组和DMEM移植组(FCNTF=31.490,FBDNF=26.040,P0.05),而IL-1β、TNF-α的蛋白表达量低于其他组(FIL-1β=62.730,FTNF-α=21.940,P0.05)。免疫荧光显示,CX3CL1-MSCs移植后CNTF、BDNF能够表达于视网膜全层和整合入视网膜的MSCs。结论视网膜下腔移植CX3CL1-MSCs可强化对光损伤视网膜结构和功能的保护作用。
[Abstract]:Retinal degeneration is an important type of blindness, which can cause the death of retinal photoreceptors or ganglion cells. The current treatment strategy focuses on etiological treatment, or delays the death process of retinal cells through maintenance therapy. With the further study of stem cells, bone marrow mesenchymal stem cells (bone marrow-derivedmesench) Ymal stem cells, MSCs) has gained more and more attention in the treatment of retinal diseases because of its plasticity, neuroprotection, immune regulation and so on. As an inherent phagocyte on the retina, microglia plays an important role in the pathogenesis of retinal degeneration, whether.MSCs is associated with retinal microglia. Function, activating the related signaling pathway to maintain the self stable state of the retina, rarely reported in the literature. Chemokine ligand 1 (Chemokine C-X3-C motif ligand 1, CX3CL1) controls the degree of activation of retinal microglia by identifying its specific receptor CX3CR1. This study evaluated CX3CL1 based in vitro co culture system. The effect of modified MSCs (CX3CL1gene-modified MSCs, CX3CL1-MSCs) on the function of activated retinal microglia, and to observe the protection of light damaged retina by transplantation in vivo, and explore the role of CX3CL1/CX3CR1 signaling pathway. The first part established the CX3CL1 gene modified bone marrow mesenchymal stem cells for the purpose of establishing the CX3CL1 base. Because of the modified MSCs, and understand its function. Methods all bone marrow adherent method was used to train SD rat MSCs, flow cytometry was used to detect surface markers, osteogenesis, differentiation induction and identification of lipid differentiation, immunofluorescence detection of MSCs expression of neurotrophic factor. The enzyme cut products containing CX3CL1 gene particles and carrier plasmid, linear connection obtained enzyme cut products, turn The positive clones were identified and sequenced by PCR. Compared with the liposome mediated lentivirus three plasmids, 293T cells were co transfected, the supernatant was collected and concentrated by the centrifuge ultrafiltration device to obtain the virus. Real-time PCR was used to determine the virus titer.CX3CL1 lentivirus infection MSCs. The infection efficiency was observed under the fluorescence microscope. The CX3CL1 level in the cell supernatant was detected by ELISA. The level of CX3CL1 was detected by ELISA Real-time PCR was used to detect the expression of CX3CL1 mRNA in cells. Results all bone marrow adherent methods were successfully isolated and cultured for MSCs. The positive rates of MSCs surface markers in third generation by flow cytometry were cd90: (95.21 + 2.13)%, cd44: (96.75 + 3.30)% and cd34: (1.84 + 0.92)%.mscs can be induced into osteoblasts and fatty cells.Mscs In vitro expression of CNTF, BDNF, GDNF, bfgf. successfully constructed the lentivirus that overexpressed cx3cl1 gene, and the titer was at least 2 * 108tu/ml.cx3cl1 lentivirus infection MSCs, and the optimal infection complex number (MOI) was 10. lentivirus infection MSCs, and the secretion of cx3cl1 and cx3cl1mrna were higher than those in the uninfected and no-load control group (f=338.300640.300, P0.05) conclusion after cx3cl1 lentivirus infection, the cx3cl1 gene can be integrated into the MSCs genome and transcribed accurately. The cx3cl1 gene modified MSCs can express and secrete the cx3cl1. second partial cx3cl1 gene modified mesenchymal stem cells on the activated retinal microglia biological function in order to evaluate the purpose of evaluating cx3cl1/cx3cr1. The effect of signal pathway on the interaction of MSCs and retinal microglia. Methods the microglia of SD rat retinal microglia were isolated and cultured by mixed culture of glial cells and oscillatory separation, and the purity of the cells was identified by immunofluorescence staining. LPS stimulated the microglia in the retina membrane and modified with the MSCs, cx3cl1 gene. MSCs (cx3cl1-mscs), neutralizing antibody closed cx3cl1 MSCs co culture 24h, observed the proliferation, phagocytosis and migration of retinal microglia after co culture. Griess method was used to detect the release of nitric oxide (no). The changes of expression and secretion of cytokines were analyzed by real-timepcr, Westernblot, ELISA, etc. results of retinal glia. The retinal microglia cells were successfully isolated and cultured by mixed culture and oscillatory separation. The immunofluorescence detection showed that the positive rates of CD11b, Iba1 were (96.18 + 3.40)%, (94.05 + 4.18)% and GS negative expression, indicating that the obtained retinal microglia with high purity.Lps could activate retinal microglia. Co culture with MSCs, The proliferation of activated retinal microglia was inhibited (f=32.280, P0.05), and phagocytosis, mobility enhancement (f=72.200,20.020, P0.05), tnf- alpha, IL-1 beta, iNOSmRNA expression downregulation (f=20.710,63.530,78.580, P0.05), CNTF, BDNF, cx3cr1mrna expression increased 10, P0.05), the secretion of no, tnf- a, and IL-1 beta in the cell supernatant decreased (f=23.670,84.960,30.760, P0.05). Co culture with cx3cl1-mscs can enhance these reactions, and can inhibit these reactions with cx3cl1 closed MSCs co culture. Conclusion MSCs may maintain the homeostasis of retinal microglia mainly through CX3CL1/ CX3CR1 signal pathway. Third parts can be maintained by CX3CL1/ CX3CR1 signal pathway. Protective effect of bone marrow mesenchymal stem cells modified by CX3CL1 gene on light damage retina by subretinal transplantation to investigate the protective effect of CX3CL1 gene modified MSCs (CX3CL1-MSCs) on retinal structure and function of light damaged retina transplantation. Methods SD rats were exposed to white light 24h. (9500 + 500) Lux after diffuse pupil of SD rats. The effect of strong light exposure on retinal tissue structure was observed by HE staining. Apoptosis of retinal cells was detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay. MSCs and CX3CL1-MSCs were transplanted into the inferior retinal cavity of rats with light damage, and the ability to migrate to the inner layer of the retina was compared. HE staining was used to observe the outer layer of retina. Nuclear layer thickness change, flash electroretinogram (F-ERG) and TUNEL method to evaluate the protection of the retina,.ED-1 antibody labeled retinal activation microglia.Western blot was used to detect the expression of CNTF, BDNF, IL-1 beta, TNF- alpha in the host retina after transplantation. The immunofluorescent localization of CNTF, BDNF on the retina. Results light damage The thickness of the outer retinal nucleus was thinner and apoptotic cells increased (t=8.009,8.753, P0.05). After the subretinal transplantation, the migration of CX3CL1-MSCs to the inner retina was stronger than MSCs (t=3.674, P0.05). Compared with the MSCs transplantation group and DMEM transplantation group, the transplantation CX3CL1-MSCs was used to delay the thinning of the outer retinal nuclei and inhibit the apoptosis of the retina cells. To reduce the number of activated retinal microglia, it has a better effect. At the same time, F-ERG results show that 1W, 3W, 6W, and transplanted CX3CL1-MSCs can better protect the retinal function. 3W, CX3CL1-MSCs transplantation group after transplantation, the retinal CNTF, the protein expression of BDNF is higher than that of the MSCs transplantation group and the DMEM transplantation group. 0.05), while the protein expression of IL-1 beta, TNF- alpha was lower than that of other groups (FIL-1 beta =62.730, FTNF- alpha =21.940, P0.05). Immunofluorescence showed that after CX3CL1-MSCs transplantation CNTF, BDNF could be expressed in the whole retina and integrated into the retina, and the MSCs. conclusion retina transplantation CX3CL1-MSCs could strengthen the protection of the structure and function of light damaged retina.

【学位授予单位】：福建医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R774.1

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