转化生长因子-β1基因修饰的人视网膜色素上皮细胞移植治疗老年性黄斑变性的实验研究
摘要
人转化生长因子-β1(hTGF-β1),在多种组织细胞的增殖分化、间质形成、组织损伤修复、骨质再生、胚胎发育以及免疫调控中起着重要的作用。目前已经证实TGF-β1 是一种强效免疫抑制因子,其功能比环孢素A 还要强大约104-106倍。
本研究以hTGF-β1 为目的基因,构建了TGF-β1 真核表达载体,并成功地转染到体外培养的人视网膜色素上皮(RPE)细胞,建立了稳定表达TGF-β1的基因工程化RPE 细胞。将TGF-β1 的基因工程化RPE 细胞移植到兔视网膜下腔,移植术后通过眼底照相、眼底荧光造影、组织病理等方法对移植细胞的存活状态、TGF-β1 表达情况及宿主对移植细胞的排异情况进行观察。结果显示,与实验对照组比较,TGF-β1 基因修饰的RPE 细胞在兔视网膜下腔可以较长期存活,无明显荧光渗漏,移植区域脉络膜层无明显的组织细胞浸润。上述结果表明hTGF-β1 基因修饰的RPE 细胞异体移植是可行的,并有望成为老年性黄斑变性等RPE 细胞变性性疾病的一种有效的治疗手段。
With the development of mental and material civilization, age-related maculardegeneration (ARMD) has been one of the most common ophthalmopathies causingblindness among the olds. Especially to exudative ARMD, neovascular membraneunder fovea centralis exudes, hemorrhages and scars repeatedly, which would effectthe visual function seriously. Since neovascular membrane of fovea centralis or scarsare resected by operating, the serious loss and lesion of RPE cells are inevitable. Thedefect of RPE would affect visual cells and choroid capillary plate once more. It notonly couldn,t improve the visual function, but also be in danger of visual activitydescent.
In 1983 Gouras was the first to report the transplantation of cultured human RPEinto the retinal cavity of monkey. Since then, many researchers try to transplant RPEcells from healthy donor into the retinal cavity. Twenty years later, manyachievements in animal and clinic experiments have been acquired. RPE cellstransplantation has now been utilized to rebuilt normal anatomic structure andphysiological function of macula at the subretinal space in exudative ARMD withserious visual disturbance. It has also been used to provide suitable support andnutrition for the photoreceptors of fovea centralis, as well as prevent the atrophy ofchoroid capilliary.
However, transplantation immunologic rejection is an inevitable importantproblem in RPE cells transplantation, the same as other cells transplantation.Although retinal cavity is considered as the immune privilege region for blood-retinalbarrier and chorioretinal barrier, it has been verified that the immune privilege isrelative. Especially, the immune privilege behavior of retinal cavity could bedestructed, following the retinal barriers destruction. Once immunological rejectionoccurrence, it would not only effect the long-term living of donor RPE cells at thesubretinal space, but also destruct the blood-retinal barrier, even lead to hemorrhage,exudation or edema of the transplant region and visual activity descent.
Recently, the immune rejection has been prevented with environment intervention.Particularly, the tissue and organ transplantation are successful in the application ofthe new type immuno-suppressive drugs. But they are harmful to the body. We applyCiclosporin A (CsA) in clinic, that could prevent T cells activating and lymphokinesreleasing. It would lead to liver-kidney toxicity, hypertension and retinal toxicity inlong-term use. Gene therapy is another important progress to block transplantationimmunological rejection with environment intervention. At present, the establishmentof engineered transgenic cells and the transplantation of RPE cells have rathermaturated. We should make use of the immunosupression factor gene to modify thecultured RPE cells in vitro, which could express products in RPE cells stably andregulate the xenograft reject reaction in the subretinal space. It would be helpful tosolve the short of cell source and the ill effect of immuno-supressive drugs. Moreover,it could replace the RPE cells completely and break a new pathway to treat ARMD. The objective gene TGF-β1 in our research is a powerful immunosupressionfactor, whose function is 104-106 times stronger than CsA. TGF-β1 could take part inthe transplantation immunity with downregulation the non-specificimmunosuppressive action of lymphocytes and macrophage. Even more, it couldinhibit the expression of MHC and co-sitmulating factor on the surface of DC,decrease antigen presentation function of DC and confuse DC differentiation to inductspecific donor transplantation tolerance. We extracted TGF-β1 from human blood andamplified by clone. Then eukaryotic expression plasmid of pcDNA3-hTGF-β1 wasconstructed successfully and it was transferred into cultured RPE cells in vitro withlipofectamine. At last, the RPE cells of expression TGF-β1 stably were transplantedinto the subretinal space of rabbit and the transplantation immunological rejection wasobserved. PartⅠConstruction and identification of eukaryotic expression plasmid of hTGF-β1 The hTGF-β1 cDNA (1200bp) was RT-PCR amplified, purified and connectedwith pGEM-T by TA clone. The positive recombinant plasmid was renamed pGEM-T-hTGF-β1.It was digested with double enzyme EcoR and Xhol I. The plasmidpcDNA3-hTGF-β1 was constructed successfully with inserting the objective genefragment of pGEM-T-hTGF-β1 into the eukaryotic expression plasmid pcDNA3. Thestudy would be applied to study engineered RPE cells with expression the hTGF-β1stably and the subretinal transplantation of hTGF-β1 gene modified RPE cells to treatRPE cells degenerative disease.
PartⅡCulture and identification of RPE cells Accidental death eyes were studied. The death time is no more than 4 hours. Theeye bulbs were washed externally with D-Hanks. The anterior segment with cornea,iris, lens and vitreous was removed. The posterior segment was fixed into whiterubber tray, and RPE patches were separated gently from neural retina under adissection microscope. After washing in D-Hanks, 0.25% trypsin and 0.01% EDTAwere added, bathing the RPE cells for a 30 min digestion cycle. Loosened RPE cellswere boasted and transferred to centrifuge tubes containing DMEM supplementedwith 10% fetal calf serum. The cellular suspension centrifuged at 1000 rpm for 10 min,was resuspended in DMEM/F12/10% FCS, plated in culture bottles and incubated in95% O2 and 5% CO2 atmosphere at 37 °C. The initial cell seeding density was80,000-100,000 cells/ml. The primary cultured RPE cells were round in shape. Theydrifted in the culture medium and contained large amount of pigments. We could notwatch nucleus by invert microscope. After attaching to culture bottles, RPE cellsstretched out pseudopodium, then they changed into irregular multiangle shapes. Thecentral round hyalomere was nucleus. RPE cells expressed cytokeratin, which ischaracteristic of epithelial cells. The cells were around and maldistribution aftercryopeservating the third passages, cells. By 24 hours, they were attached and beganto proliferate. 72 hours later, the RPE cells contacted each other. The cryopeservatedcells were positive with anti-cytokeratin staining too. The hRPE cells were cultured successfully and cryopeservation of RPE couldmaintain biological activities of cells after thawing. It would provide target cells forexperiments in vitro and benefit on cells transplantation. Part ⅢEstablishment and evaluation of gene modified RPE cells with stably expressing the TGF-β1 The gene introduction system is the core of gene therapy. Eukaryotic expressionplasmid pcDNA3-hTGF-β1 was transferred into RPE cells with the help oflipofectamine. The positive cells clones were selected with G418. The cells werecultured in media of least fatal dose G418. In 2 weeks, most of the cells were dead,except for the resistant cells transferred pcDNA3-hTGF-β1. G418-resistant colonieswere picked up and grown in medium in the presence of G418 for 4 weeks. Theexpression of TGF-β1 in RPE cells was detected by Western blot andimmunocytochemical analysis. It was resulted that TGF-β1 mRNA and TGF-β1protein were stably expressed in RPE cells transferred pcDNA3-hTGF-β1. Geneticengineered RPE cells with stably expressing the hTGF-β1 are established and it wasuseful to subretinal transplantation.
本研究以hTGF-β1 为目的基因,构建了TGF-β1 真核表达载体,并成功地转染到体外培养的人视网膜色素上皮(RPE)细胞,建立了稳定表达TGF-β1的基因工程化RPE 细胞。将TGF-β1 的基因工程化RPE 细胞移植到兔视网膜下腔,移植术后通过眼底照相、眼底荧光造影、组织病理等方法对移植细胞的存活状态、TGF-β1 表达情况及宿主对移植细胞的排异情况进行观察。结果显示,与实验对照组比较,TGF-β1 基因修饰的RPE 细胞在兔视网膜下腔可以较长期存活,无明显荧光渗漏,移植区域脉络膜层无明显的组织细胞浸润。上述结果表明hTGF-β1 基因修饰的RPE 细胞异体移植是可行的,并有望成为老年性黄斑变性等RPE 细胞变性性疾病的一种有效的治疗手段。
With the development of mental and material civilization, age-related maculardegeneration (ARMD) has been one of the most common ophthalmopathies causingblindness among the olds. Especially to exudative ARMD, neovascular membraneunder fovea centralis exudes, hemorrhages and scars repeatedly, which would effectthe visual function seriously. Since neovascular membrane of fovea centralis or scarsare resected by operating, the serious loss and lesion of RPE cells are inevitable. Thedefect of RPE would affect visual cells and choroid capillary plate once more. It notonly couldn,t improve the visual function, but also be in danger of visual activitydescent.
In 1983 Gouras was the first to report the transplantation of cultured human RPEinto the retinal cavity of monkey. Since then, many researchers try to transplant RPEcells from healthy donor into the retinal cavity. Twenty years later, manyachievements in animal and clinic experiments have been acquired. RPE cellstransplantation has now been utilized to rebuilt normal anatomic structure andphysiological function of macula at the subretinal space in exudative ARMD withserious visual disturbance. It has also been used to provide suitable support andnutrition for the photoreceptors of fovea centralis, as well as prevent the atrophy ofchoroid capilliary.
However, transplantation immunologic rejection is an inevitable importantproblem in RPE cells transplantation, the same as other cells transplantation.Although retinal cavity is considered as the immune privilege region for blood-retinalbarrier and chorioretinal barrier, it has been verified that the immune privilege isrelative. Especially, the immune privilege behavior of retinal cavity could bedestructed, following the retinal barriers destruction. Once immunological rejectionoccurrence, it would not only effect the long-term living of donor RPE cells at thesubretinal space, but also destruct the blood-retinal barrier, even lead to hemorrhage,exudation or edema of the transplant region and visual activity descent.
Recently, the immune rejection has been prevented with environment intervention.Particularly, the tissue and organ transplantation are successful in the application ofthe new type immuno-suppressive drugs. But they are harmful to the body. We applyCiclosporin A (CsA) in clinic, that could prevent T cells activating and lymphokinesreleasing. It would lead to liver-kidney toxicity, hypertension and retinal toxicity inlong-term use. Gene therapy is another important progress to block transplantationimmunological rejection with environment intervention. At present, the establishmentof engineered transgenic cells and the transplantation of RPE cells have rathermaturated. We should make use of the immunosupression factor gene to modify thecultured RPE cells in vitro, which could express products in RPE cells stably andregulate the xenograft reject reaction in the subretinal space. It would be helpful tosolve the short of cell source and the ill effect of immuno-supressive drugs. Moreover,it could replace the RPE cells completely and break a new pathway to treat ARMD. The objective gene TGF-β1 in our research is a powerful immunosupressionfactor, whose function is 104-106 times stronger than CsA. TGF-β1 could take part inthe transplantation immunity with downregulation the non-specificimmunosuppressive action of lymphocytes and macrophage. Even more, it couldinhibit the expression of MHC and co-sitmulating factor on the surface of DC,decrease antigen presentation function of DC and confuse DC differentiation to inductspecific donor transplantation tolerance. We extracted TGF-β1 from human blood andamplified by clone. Then eukaryotic expression plasmid of pcDNA3-hTGF-β1 wasconstructed successfully and it was transferred into cultured RPE cells in vitro withlipofectamine. At last, the RPE cells of expression TGF-β1 stably were transplantedinto the subretinal space of rabbit and the transplantation immunological rejection wasobserved. PartⅠConstruction and identification of eukaryotic expression plasmid of hTGF-β1 The hTGF-β1 cDNA (1200bp) was RT-PCR amplified, purified and connectedwith pGEM-T by TA clone. The positive recombinant plasmid was renamed pGEM-T-hTGF-β1.It was digested with double enzyme EcoR and Xhol I. The plasmidpcDNA3-hTGF-β1 was constructed successfully with inserting the objective genefragment of pGEM-T-hTGF-β1 into the eukaryotic expression plasmid pcDNA3. Thestudy would be applied to study engineered RPE cells with expression the hTGF-β1stably and the subretinal transplantation of hTGF-β1 gene modified RPE cells to treatRPE cells degenerative disease.
PartⅡCulture and identification of RPE cells Accidental death eyes were studied. The death time is no more than 4 hours. Theeye bulbs were washed externally with D-Hanks. The anterior segment with cornea,iris, lens and vitreous was removed. The posterior segment was fixed into whiterubber tray, and RPE patches were separated gently from neural retina under adissection microscope. After washing in D-Hanks, 0.25% trypsin and 0.01% EDTAwere added, bathing the RPE cells for a 30 min digestion cycle. Loosened RPE cellswere boasted and transferred to centrifuge tubes containing DMEM supplementedwith 10% fetal calf serum. The cellular suspension centrifuged at 1000 rpm for 10 min,was resuspended in DMEM/F12/10% FCS, plated in culture bottles and incubated in95% O2 and 5% CO2 atmosphere at 37 °C. The initial cell seeding density was80,000-100,000 cells/ml. The primary cultured RPE cells were round in shape. Theydrifted in the culture medium and contained large amount of pigments. We could notwatch nucleus by invert microscope. After attaching to culture bottles, RPE cellsstretched out pseudopodium, then they changed into irregular multiangle shapes. Thecentral round hyalomere was nucleus. RPE cells expressed cytokeratin, which ischaracteristic of epithelial cells. The cells were around and maldistribution aftercryopeservating the third passages, cells. By 24 hours, they were attached and beganto proliferate. 72 hours later, the RPE cells contacted each other. The cryopeservatedcells were positive with anti-cytokeratin staining too. The hRPE cells were cultured successfully and cryopeservation of RPE couldmaintain biological activities of cells after thawing. It would provide target cells forexperiments in vitro and benefit on cells transplantation. Part ⅢEstablishment and evaluation of gene modified RPE cells with stably expressing the TGF-β1 The gene introduction system is the core of gene therapy. Eukaryotic expressionplasmid pcDNA3-hTGF-β1 was transferred into RPE cells with the help oflipofectamine. The positive cells clones were selected with G418. The cells werecultured in media of least fatal dose G418. In 2 weeks, most of the cells were dead,except for the resistant cells transferred pcDNA3-hTGF-β1. G418-resistant colonieswere picked up and grown in medium in the presence of G418 for 4 weeks. Theexpression of TGF-β1 in RPE cells was detected by Western blot andimmunocytochemical analysis. It was resulted that TGF-β1 mRNA and TGF-β1protein were stably expressed in RPE cells transferred pcDNA3-hTGF-β1. Geneticengineered RPE cells with stably expressing the hTGF-β1 are established and it wasuseful to subretinal transplantation.
引文
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