【作者】 陈君毅；

【导师】 孙兴怀； 孟樊荣； 钱韶红； 莫晓芬；

【作者基本信息】 复旦大学 ， 眼科学， 2006， 博士

【摘要】 前言青光眼滤过性手术仍然是目前治疗青光眼的主要手段。创伤修复过程决定了手术的成败。手术失败的主要原因是滤过口处成纤维细胞增殖、瘢痕形成而使滤过泡功能减退。目前临床上普遍使用丝裂霉素（Mitomycin C，MMC）和5-氟尿嘧啶（5-fluorouracil，5-FU）来抑制滤过术后结膜下成纤维细胞的过度增殖，虽然取得一定的临床疗效，但是这些药物也能够造成多种严重并发症，因此探索新的抗瘢痕化治疗途径对青光眼滤过性手术具有重要的意义。研究表明在机体创伤修复进程中转化生长因子β（transforming growth factor beta, TGF-β）被认为在其中起关键作用。在眼部TGF-β同样是促进瘢痕形成的重要刺激因素。近期的研究表明TGF-β的信号转导主要通过激活Smad通路实现，Smad复合物与靶基因启动子结合完成对基因转录的调控作用。迄今为止已发现9种不同的Smad蛋白，这些蛋白可分为3个不同的亚族：（1）R-Smad，包括Smad1、2、3、5、8；（2）Co-Smad，Smad4；（3）I-Smad包括Smad6、7。Smad7与磷酸化型受体的直接结合抑制了R-Smads的磷酸化，同时也诱导了受体的降解。Smad7对TGF-β超家族的成员都具有抑制作用，被认为是TGF-β超家族信号转导自我调节的一种负反馈信号。既往研究发现，对皮肤成纤维细胞瞬时转染Smad 7基因后，能够降低Ⅰ型前胶原蛋白α2亚单位（α2-typeⅠprocollagen，COL1A2）基因的基础表达水平。在小鼠体内试验中，腺病毒介导的Smad 7基因转染能够通过减少Ⅰ型前胶原蛋白mRNA的表达，来拮抗博来霉素诱导的肺组织纤维化。本试验通过对体外培养人Tenon囊成纤维细胞（human Tenon’s capsule fibroblasts，HTFs）进行Smad7基因转染，获得过表达Smad7的阳性细胞克隆。观察Smad7基因转染对HTFs细胞Ⅰ型胶原蛋白、纤维连接蛋白表达，细胞增殖，细胞周期，细胞迁移等的作用。并观察Smad7基因对Smad2表达及磷酸化的影响。第一部分过表达Smad7的阳性HTFs细胞克隆的建立第一节HTFs细胞的培养及鉴定目的体外培养HTFs细胞，并鉴定方法于白内障手术中取患者的Tenon’s囊组织，采用组织块贴壁培养法行原代培养。5％CO₂、37℃条件下用含20％FBS的DMEM传代培养。采用波形蛋白抗体、角蛋白抗体、成纤维细胞抗体，应用细胞免疫荧光染色方法对培养的细胞进行鉴定。结果培养的细胞生长活跃、形状表现稳定，波形蛋白染色阳性、角蛋白染色阴性、成纤维细胞抗体染色阳性，确认是HTFs细胞。小结成功体外培养HTFs细胞。第二节过表达Smad7的阳性HTFs克隆的建立目的建立过表达Smad7基因的HTFs细胞阳性克隆方法pCMV5-HA-Smad7真核表达质粒纯化、鉴定，使用Nucleofector^TM核酸转染仪将其转染HTFs细胞，用G418进行筛选，Realtime RT-PCR、Western印迹法对阳性克隆进行鉴定。结果纯化的pCMV5-HA-Smad7真核表达质粒经过EcoRI和Xbal双酶切后得到4.7kb的pCMV5条带和2.8kb的HA-Smad7目的基因条带。经测序鉴定与GeneBank中Smad7两端接口序列完全一致。目的质粒与pmaxGFP 4：1共转染HTFs细胞，筛选出U-23转染程序。Realtime RT-PCR检测Smad7 mRNA表达，无论是否有外源性TGF-β₂刺激，pCMV5-HA-Smad7-HTFs与HTFs、pCMV5-HA-HTFs相比Smad7 mRNA的表达均明显升高；Western印迹法结果显示，pCMV5-HA-Smad7-HTFs中检测到HA-tagged Smad7表达，并且TGF-β₂对HA-tagged Smad7的表达无明显作用。小结采用Nucleofector^TM核酸转染仪将pCMV5-HA-Smad7真核表达质粒成功转染HTFs细胞；成功筛选过表达Smad7阳性克隆株，其Smad7 mRNA和HA-tagged Smad7蛋白表达明显增强。第二部分Smad7基因对HTFsⅠ型胶原蛋白、纤维连接蛋白表达的影响及可能的作用机制目的探讨转染Smad7基因对HTFs细胞Ⅰ型胶原蛋白和纤维连接蛋白（fibronectin，FN）表达的影响，并探讨其可能的作用机制。方法经外源性TGF-β₂刺激培养的HTFs细胞，采用Realtime RT-PCR法检测Smad7基因对HTFs COL 1A2、FN及Smad2 mRNA表达的影响；收集培养液的上清，采用放射免疫测定法检测Smad7基因对HTFs培养液中PICP浓度的影响；采用Western印迹法检测Smad7基因对HTFs Smad2、磷酸化Smad2（phosphorylated Smad2，p-Smad2）蛋白表达的影响；采用细胞免疫荧光法检测Smad7基因对HTFs p-Smad2蛋白表达的影响。以未转染的HTFs和转染空载质粒的pCMV5-HA-HTFs为对照组。结果外源性TGF-β2能够促进HTFs和pCMV5-HA-HTFs细胞COL1A2 mRNA的表达，且呈时间依赖性，分别是未经外源性TGF-β2作用时的1.6倍和1.3倍。pCMV5-HA-Smad7-HTFs与对照组相比COL1A2 mRNA表达在各时段均明显受到抑制；在外源性TGF-β2作用各个时间段，pCMV5-HA-Smad7-HTFs COL1A2 mRNA表达无明显改变；外源性TGF-β2作用能够使三组细胞FN mRNA表达增强，而且呈时间依赖性；TGF-β2作用不同时段内，三组细胞FN mRNA表达差异无统计学意义；外源性TGF-β2作用72h能够提高三组细胞培养液中PICP浓度，分别是未经作用组的2.1倍、2.0倍和1.6倍，pCMV5-HA-Smad7-HTFs组的升高幅度明显小于两个对照组；无论有无外源性TGF-β2作用，pCMV5-HA-Smad7-HTFs培养液中PICP浓度均明显小于对照组；外源性TGF-β2作用能够使三组细胞Smad2 mRNA表达增强，而且呈时间依赖性，分别是未经TGF-β2作用时的2.7倍、2.4倍和2.3倍；三组细胞间Smad2 mRNA表达差异无统计学意义；TGF-β2作用0及30min时三组细胞Smad2蛋白表达水平组间对比差异均无统计学意义；未经TGF-β2作用时，p-Smad2在三组细胞中几乎不能被检出。TGF-β2作用30min后，三组细胞p-Smad2表达明显升高。pCMV5-Smad7-HTFs组p-Smad2表达水平分别是对照组的56.01％和53.48％；未经TGF-β2作用时，HTFs、pCMV5-HA-HTFs、pCMV5-HA-Smad7-HTFs三组细胞p-Smad2阳性细胞比例分别为：4.01±2.35％、3.12±1.78％、3.45±2.33％，三组比较差异无统计学意义；TGF-β2作用30min后，三组细胞p-Smad2阳性细胞比例分别为：85.97±6.34％、90.67±8.25％、26.67±8.42％。小结TGF-β2能够促进HTFs细胞COL1A2、FN mRNA的表达；Smad7基因转染可以降低HTFs细胞COL1A2 mRNA及PICP的表达，并能够拮抗外源性TGF-β2引起的两者表达升高；但对FN mRNA的表达无明显作用；TGF-β2能够促进Smad2 mRNA的表达；TGF-β2作用早期Smad2蛋白表达无明显改变，p-Smad2的表达明显升高；Smad7基因转染对Smad2蛋白表达无明显作用，但是可以明显拮抗外源性TGF-β2引起p-Smad2的表达明显升高。第三部分Smad7对HTFs细胞增殖、细胞周期、迁移作用的研究目的采用BrdU掺入法、流式细胞仪及Transwells法检测转染Smad7基因对HTFs细胞增殖、细胞周期和迁移的作用。方法经外源性TGF-β2刺激培养的HTFs细胞，细胞培养液中加入含BrdU（10μM）标记的10％FBS DMEM培养液，继续培养1h，免疫荧光染色检测BrdU阳性细胞比例；收集细胞，PI染色，采用流式细胞仪检测各周期的细胞比例；取克隆细胞，10⁵个接种于Transwells上层小室，培养16h后，于显微镜下随机计数5个20×倍视野下迁移细胞数。结果外源性TGF-β2作用后，HTFs及pCMV5-HA-HTFs组BrdU阳性细胞比例明显较未作用时减少，分别是原来的73.8％和73.1％（P＜0.01）；pCMV5-HA-Smad7-HTFs组，BrdU阳性细胞比例无明显差异（P＞0.05）；pCMV5-HA-Smad7-HTFs组BrdU阳性细胞数明显较两个对照组多，分别是两个对照组的133％和142％；TGF-β2作用后，HTFs和pCMV5-HA-HTFs组G0／G1期比例差异无统计学意义（P＞0.05）；S期比例下降，差异具有显著统计学差异（P＜0.05），细胞增殖明显受到抑制；pCMV5-HA-Smad7-HTFs组G0／G1期比例、S期比例无统计学意义（P＞0.05）；TGF-β2作用后，pCMV5-HA-Smad7-HTFs组细胞生长并未受到明显抑制，S期细胞比例明显较对照组多，分别是HTFs和pCMV5-HA-HTFs组的1.8倍和1.5倍（P＜0.01）；G0／G1期细胞比例明显低于对照组，差异同样具有非常显著统计学意义（P＜0.01）。；Transwells检测，外源性TGF-β2作用能够轻度提高三组细胞迁移性，但差异无统计学意义（P＞0.05）；三组细胞间相互比较，迁移的细胞数差异无明显统计学意义。小结外源性TGF-β2作用能够明显减少HTFs细胞BrdU阳性细胞数，降低S期比例，从而抑制HTFs细胞的增殖；转染Smad7基因能够拮抗外源性TGF-β2的增殖抑制作用，BrdU阳性细胞数，G0／G1期、S期细胞比例，与作用前相比无明显差异；Smad7基因对HTFs细胞迁移无明显作用。结论1．将pCMV5-HA-Smad7真核表达质粒经电转染法成功转染HTFs细胞，并获得过表达的Smad7阳性细胞克隆；2．外源性TGF-β2作用可上调HTFs细胞Ⅰ型胶原蛋白、FN的表达；Smad7基因转染可以抑制HTFs细胞对Ⅰ型胶原蛋白的表达，同时可以拮抗外源性TGF-β2作用引起的表达上调；Smad7基因对FN的表达无明显作用；3．外源性TGF-β₂作用可迅速导致HTFs细胞Smad2的磷酸化，持续作用可上调Smad2表达；Smad7基因可显著抑制外源性TGF-β2作用引起的Smad2磷酸化，这也可能是其抑制HTFs细胞对Ⅰ型胶原蛋白的表达的机制；Smad7基因对外源性TGF-β2持续作用引起的Smad2表达上调无明显作用；4．外源性TGF-β2作用可减少BrdU阳性细胞数、S期细胞比例，从而抑制HTFs细胞增殖；HTFs细胞转染Smad7基因能够拮抗外源性TGF-β2的抑制增殖作用。5．转染Smad7基因对HTFs细胞迁移无明显作用。更多还原

【Abstract】 PrefaceGlaucoma filtration surgery is still the main therapy method for glaucoma. The healing process after glaucoma filtration is the main determinant of surgical failure and, even more important, the final intraocular pressure. Excessive scarring in the sclerostomy site or bleb connective tissue may reduce the efficiency of the aqueous draining, resulting in a renewed increase in intraocular pressure. Mitomycin C and 5-fluorouracil are now being widely used to suppress over-proliferation of subconjuntival fibroblasts during healing following filtration surgery, resulting in a satisfactory clinical outcome. However, administration of these drugs at the time of filtration surgery can cause late-onset post-operative complications. Therefore, continued research in the area of healing modification is necessary, which is leading to further advances in our understanding of healing processes and ultimately better outcomes for patients after glaucoma surgery.The transforming growth factor beta （TGF-β） superfamily, in particular, has been shown to play a pivotal role in scarring through the body, where it is believed to be a potent stimulator of scarring. TGF-β has also been implicated as a potent stimulant of the scarring process in the eye. Recently researches showed that the signal transduction of TGF-β was conducted mainly by Smad proteins. Smads relay the signal from the cell membrane to the nucleus, where they affect the transcription of target genes.Until now, nine Smad proteins have been found, and they were divided into 3 groups. ① Receptor-regulated （R-） Smad proteins including Smad1, Smad2, Smad5 and Smad8. ② Common-partner （Co-） Smad protein including Smad4. ③ Inhibitory （I-） Smad proteins including Smad6 and Smad7.Previous researchs showed that the transient transfection of Smad7 to skin fibroblasts could inhibit the expression of α 2 - type I procollagen （COL1A2） gene. Transient gene transfer and expression of Smad7 prevents bleomycin-induced lungfibrosis in mice.The main purpose of the present study is to transfect the Smad7 cDNA to human Tenon’s capsule fibroblasts （HTFs）, to construct HTFs transfectant overexpressed Smad7 gene, to observe the effect of Smad7 gene to the expression of type I collagen, fibronectin, cellular proliferation, cell cycle and cell migration, and to observe the effect of Smad7 to the expression of Smad2 and phosphorylated Smad2.Part I Construction of HTFs Transfectants OverexpressingSmad7 GeneSection I Culture and Identification of human Tenon’s capsule fibroblasts Objectives In vitro Culture and Identification of human Tenon’s capsule fibroblasts.Methods The Tenon’s capsule tissue was obtained during cataract surgery and cultured using method of explanting. DMEM contain 20% fetal bovine serum （FBS） was used for subculture in 5% CO₂ 37℃. Antibodies to Vimentin, cytokeration and fibroblast were used in immunocytofluorescense staining for identification of fibroblasts.Results The cultured cells were active growing, stable in manifestation, and confirmed to be fibroblast by positive staining with vimentin and fibroblast antibodies, but negative staining with cytokeratin antibody.Summary In vitro cultured HTFs cells successfully.Section II The Construstion of HTFs Transfectants Overexpressing Smad7 GeneObjectives To construct HTFs Transfectants Overexpressing Smad7 GeneMethods The plasmid was purified and identified. Cultured rat HTFs were transfected stably with recombinant plasmids of pCMV5-HA-Smad7 by Nucleofector? device. Screened by G418, the positive clones were constructed anddetermined by realtime RT-PCR, Western blot analysis respectively.Results After treatment with the enzymes of EcoRI and XbaI, the recombinant plasmid of pCMV5-HA-Smad7 was cut into two fragment, carrier and aim gene, whose lengths were 4.7kb and 2.8kb. Sequencing results showed complete coincidence to Smad7 in GeneBank. U-23 program was selected by 4:1 co-transfection of pCMV5-HA-Smad7 and pmaxGFP to HTFs. Realtime RT-PCR results showed that the expression of Smad7 mRNA in pCMV5-HA -Smad7- HTFs increased significantly compared with HTFs and pCMV5-HA -HTFs whether there was exogenous TGF-β₂ stimulation or not. Western blot results revealed that HA-tagged Smad7 could be detected in pCMV5-HA -Smad7- HTFs and not affected by exogenous TGF-β₂ stimulation.Summary Successfully transfecting plasmid containing Smad7 to HTFs by Nucleofector^TMdevice . Cell clones overexpressing Smad7 mRNA and HA-tagged Smad7 were established by G418 screening.Part II The Changes of Type I Collagen and Fibronectin Expression on HTFs by Smad7 Gene Transfection and Its Possible MechanismObjectives To investigate the influence of Smad7 gene transfection on the expression of Type I Collagen and Fibronectin on HTFs and the possible mechanism.Methods Realtime RT-PCR analysis was adopted to detect the expressions of mRNA of COL 1A2, fibronectin and Smad2 on normal and tranfected HTFs with or without exogenous TGF-β₂ stimulation. Radio immunoassay was used to detect the influence of Smad7 gene on concentration of carboxyterminal propeptide of type I procollagen （PICP） in culture medium. Western blot was used to detect the expression of Smad2 and p-Smad2 protein. Immunocytofluorscense was used to detect the p-Smad2 expression.Results Exogenous TGF-β₂ could time-dependently promote the expression of COL1 A2 mRNA in HTFs and pCMV5-HA-HTFs. In pCMV5-HA-Smad7- HTFs, the COL1 A2 mRNA expression was significantly inhibited compared to control groups inall time intervals. However, it was not changed by exogenous TGF-β₂ in pCMV5-HA-Smad7- HTFs. Exogenous TGF-β₂ could time-dependently increase fibronectin mRNA expression. But the inter-groups variation did not exist in every time intervals. The PICP concentrations in the medium were increased to 2.1, 2.0 and 1.6 folds respectively after stimulating for 72h by exogenous TGF-β₂. In pCMV5-HA -Smad7-HTFs, the extent of increasing was significantly less than the control groups, whether stimulated by TGF-β₂ or not. The expressions of Smad2 mRNA were increased to 2.7,2.4 and 2.3 folds respectively after stimulating by exogenous TGF-β₂. There was not difference in three groups. The p-Smad2 protein couldn’t be detected without exogenous TGF-β₂. After 30min of TGF-β₂ stimulating the p-Smad2 expression elevated significantly in all groups. The p-Smad2 expression in pCMV5-HA-Smad7-HTFs took 56.01% and 53.48% of two control groups. In immunocytofluorescense assay, the p-Smad2 positive cells were 4.01 ±2.35%, 3.12± 1.78% and 3.45 ± 2.33% respectively in HTFs, pCMV5-HA-HTFs and pCMV5-HA-Smad7-HTFs groups. After the stimulation of exogenous TGF-β₂ for 30min, the p-Smad2 positive cells became 85.97±6.34%, 90.67±8.25% and 26.67± 8.42%.Summary Exogenous TGF-β₂ can promote the expression of Coll A2、 FN mRNA. Smad7 gene can inhibit the expression of Coll A2 mRNA and PICP and antagonize the increasing effect of Exogenous TGF-β₂ but has no effect on fibronectin expression. Exogenous TGF-β₂ can promote the expression of Smad2 mRNA. In the early phase of TGF-β₂ stimulation, there is no significant increscent in Smad2 protein expression but the p-Smad2 protein expression increased. Smad7 gene has no effect on Smad2 expression and can antagonize the increasing effect of Exogenous TGF-β₂ on p-Smad2 protein.Part III The Effects of Smad7 on Cell Proliferation, Cell cycle and Migration of HTFsObjectives BrdU assay, FACS and Transwells chamber were adopted to determinate the Smad7 gene transfection on cell proliferation, cell cycle and migration of HTFs.Methods DMEM contain 10μM of BrdU was added to HTFs, with or withoutexogenous TGF-β₂. After 1h, immunocytofluorscense was used to detect the BrdU positive cells. Collecting HTFs, staining with PI, using FACS to determine the cell cycle. 10⁵ HTFs were seeded into upper chamber of Transwells, cultured for 16h, to determine the migrated cells.Results Exogenous TGF-β₂ could decrease the BrdU positive cells to 73.8% and 73.1% in HTFs and pCMV5-HA-HTFs. In pCMV5-HA-Smad7- HTFs, there is no difference in BrdU positive cells. The BrdU positive cells of pCMV5-HA-Smad7-HTFs were significantly more than that of HTFs and pCMV5-HA-HTFs. After stimulated by TGF-β₂, the G0/G1 phase didn’t change in HTFs and pCMV5-HA-HTFs but significantly decreased in S phase cells. In pCMV5-HA-Smad7-HTFs, cell proliferation was not inhibited by TGF-β₂ and S phase cells increase clearly compared to control groups. GO/G1 phase cells decreased clearly. Transwells showed that exogenous TGF-β₂ could slightly enhance the migration, but not statistics significant.Summary Exogenous TGF-β₂ can decrease the BrdU positive cells and S phase cells to inhibit HTFs proliferation. Smad7 gene can antagonize the proliferation inhibiting effect of exogenous TGF-β₂ but has no effects on cell migration.Conclusions1. Successfully establish HTFs transfectants overexpressed Smad7 mRNA and protein.2. Exogenous TGF-β₂ can up-regulate the expression of type I collagen and fibronectin. Smad7 gene can inhibit the expression of type I collagen, and antagonize the increscent effect of TGF-β₂. Smad7 has no effect on fibronectin expression.3. Exogenous TGF-β₂ can phosphorate Smad2 rapidly, and increase the Smad2 protein expression. Smad7 gene can inhibit the Smad2 phosphoration induced by TGF-β₂. It is possibly the mechanism of inhibitory effect on type I collagen. Smad7 gene has on effect on Smad2 expression up-regulating induced by TGF-β₂.4. Exogenous TGF-β₂ can decrease the BrdU positive cells and S phase cells to inhibit HTFs proliferation. Smad7 gene can antagonize the proliferation inhibitingeffect of exogenous TGF-β₂. 5. Smad7 gene has no effects on cell migration.更多还原