Trx-1重组载体转染hucMSCs及肺纤维化模型的初步探索

发布时间：2018-01-08 19:17

本文关键词：Trx-1重组载体转染hucMSCs及肺纤维化模型的初步探索　出处：《中国人民解放军军事医学科学院》2016年硕士论文　论文类型：学位论文

【摘要】：背景:特发性肺纤维化(IPF)的防治一直是各国重点开展研究的领域。目前应用糖皮质激素和细胞毒性药物仍是其临床上治疗的主要措施,但并不能有效抑制肺纤维化的进展,改善其肺功能或者延长其生存期限。随着对该病的发病机制的理解加深,许多新型药物不断被研制出来,但其应用于临床方面仍然存在着诸多问题。因此,找寻出新的预防或治疗方法迫在眉睫。间充质干细胞(MSC)能够向组织损伤处趋化迁移,参与修复作用,同时容易被外源基因转染和表达,在特发性肺纤维化的治疗方面已显现出巨大的应用前景。由于涉及氧化应激,N-乙酰半胱氨酸、超氧化物歧化酶(SOD)等抗氧化剂也逐渐出成为特发性肺纤维化的新型治疗措施。而硫氧还蛋白(Trx)作为一种抗氧化剂,除了能够清除自由基,在抑制细胞凋亡和调节细胞周期等方面也具有非常重要的作用。所以,通过慢病毒转染,我们构建出目的基因Trx-1修饰的MSC,同时建立了博来霉素诱导的肺纤维化小鼠模型,为基因修饰的MSC治疗特发性肺纤维化奠定了一定的实验基础。方法:从NCBI网站查得Trx-1的CDS区序列,构建针对CDS区的基因特异性引物,并在引物两头加上可以和慢病毒载体同源重组的15bp左右的序列。在载体酶切线性化后,进行载体与基因片段的重组,将片段构建到载体上,最后通过荧光直接观察与Western Blot实验验证质粒的表达效果并通过荧光观察法计算得到病毒在293T细胞中的滴度。制备携带目的基因Trx-1的重组慢病毒Lentivirus-Trx-1,并测定病毒的滴度;设置一定的感染复数(MOI)梯度:0、5、10、30、50,用不同MOI值感染人脐带MSC,通过观察其荧光蛋白的表达确定其最佳MOI值,随后以空载体转染的人脐带MSC和未转染的人脐带MSC作为对照,利用重组慢病毒以最佳MOI值感染人脐带MSC,应用Western Blot检测细胞中目的蛋白Trx-1蛋白的表达情况;流式细胞术检测细胞的细胞周期、免疫表型;CCK-8法测定并绘制出细胞的生长曲线;以博来霉素作为诱导剂,以C57BL/6J小鼠作为实验动物,分别按照5mg/kg的用量经气管内注入博来霉素,或者按照用量约6mg/kg的用量经鼻滴入博来霉素,建立两种肺纤维化动物模型,通过对其一般情况、体重的观察和肺组织HE染色和Masson染色等病理染色,观察并比较两种造模方法的效果。结果:成功构建了pGC-LV-Ubi-TRX-1-CMV-EGFP慢病毒载体质粒,并通过Western Blot发现此质粒在293T中成功表达,并发生翻译后修饰,同时利用293T细胞测得了病毒的滴度为1×108 TU/ml;当MOI值为10及以上时,MSC的感染效率均能达到80%以上,由于慢病毒可能具有细胞毒性,最佳MOI值确定为10。Western Blot检测显示各组细胞均能不同程度地表达Trx-1蛋白,并且实验组目的蛋白Trx-1的表达明显增加;流式细胞术检测发现,三组MSC均高表达CD105、CD73,不表达CD34、CD80等,符合MSC的表型特点;细胞周期检测显示,慢病毒感染前后,人脐带MSC均有80%以上的细胞处于G0/G1期,提示转染慢病毒后的人脐带MSC与转染前类似,同样具有典型的干细胞特性;而通过对其生长曲线的观察对比则发现,与转染前不同,转染后实验组人脐带MSC生长受到抑制;造模结果发现,经气管内注入和经鼻滴入博来霉素均能成功建立肺纤维化模型。但通过对进行其病理纤维化评分后发现,与气管内给药相比,经鼻给药造模后小鼠肺组织病理改变较轻,提示气管内给药更容易造模成功。结论:成功构建了携带有人Trx-1基因的重组慢病毒载体,通过其介导获得修饰的人脐带MSC并能稳定高表达目的蛋白Trx-1,且其细胞学特性无明显改变;在本实验中,手术经气管内注入博来霉素后第28天诱导建立的C57小鼠肺纤维化模型成模最好,是一种复制肺纤维化的较好模型;本研究将基因治疗与细胞治疗相结合,为治疗特发性肺纤维化奠定了一定的实验基础,提供了新的可能手段。
[Abstract]:Background: idiopathic pulmonary fibrosis (IPF) prevention is always the key research field. The main measures are currently using glucocorticoid and cytotoxic drugs is the treatment of the progress, but it does not inhibit pulmonary fibrosis, improve lung function or prolong the survival period. With the pathogenesis of the disease understanding, many new drugs have been developed, but its clinical application still exist many problems. Therefore, to find new methods for preventing or treating imminent. Mesenchymal stem cells (MSC) can damage to tissue chemotactic migration, participate in the repair, and at the same time easily the expression of exogenous gene transfection, in the treatment of idiopathic pulmonary fibrosis has shown great application foreground. Due to oxidative stress, N- acetylcysteine, superoxide dismutase (SOD) and other antioxidants Gradually become a new treatment for idiopathic pulmonary fibrosis. And thioredoxin (Trx) as an antioxidant, in addition to the removal of free radicals, but also has a very important role in inhibiting apoptosis and regulating cell cycle. Therefore, the lentiviral transfection, we constructed the Trx-1 gene modified MSC also established a mouse model of pulmonary fibrosis induced by bleomycin, MSC gene modified for treatment of idiopathic pulmonary fibrosis provides a certain experimental basis. Methods: CDS sequences from the NCBI website Richard Trx-1, construct the gene specific primers for the CDS region, and sequence and lentiviral vectors homologous recombination of about 15bp in the two primers added. In vector linearized vector, and the recombinant gene fragment, the fragment into vector, finally by direct fluorescence observation and Western Blot experiment Effect of expression plasmid and verified by fluorescence observation method is used to calculate the viral titer in 293T cells. The recombinant lentiviral Lentivirus-Trx-1 preparation with Trx-1 gene, and the titer of virus was determined by setting a multiplicity of infection; (MOI) gradient: 0,5,10,30,50, with different values of MOI infected human umbilical cord MSC, the best MOI value to observe the expression determined by fluorescent protein, followed by transfected human umbilical cord MSC and untreated human umbilical cord MSC as control, the use of recombinant lentivirus to optimal MOI infection of human umbilical cord MSC, expression of target protein Trx-1 protein by Western Blot detection cell; cell cycle, flow cytometry detection cell with the immunophenotype; and determination to draw cell growth curve by CCK-8 method; bleomycin as inducers in C57BL/6J mice as experimental animal, respectively according to the amount of 5mg/kg intratracheal injection In bleomycin, or according to the amount of about 6mg/kg the dosage of intranasal instillation of bleomycin, established two kinds of animal models of pulmonary fibrosis, based on the general situation, and lung tissue HE staining and Masson staining pathological weight, observe and compare the two models the effect. PGC-LV-Ubi-TRX-1-CMV-EGFP was successfully constructed lentiviral vector plasmid, and by Western Blot found that the successful expression of the plasmid in 293T, and the occurrence of post-translational modifications, and 293T cells were measured by the titer of virus was 1 * 108 TU/ml; when the MOI value is 10 and above, the infection efficiency of MSC could reach more than 80%, because slow virus may have cell toxicity, the optimum MOI value was determined by 10.Western Blot assay showed that the cells in each group were different level of expression of Trx-1 protein and Trx-1 protein expression in the experimental group increased significantly; flow cytometry detection Now, three groups of MSC had high expression of CD105, CD73, the expression of CD34 CD80, consistent with the phenotype characteristics of MSC; cell cycle analysis showed that before and after lentivirus infection, human umbilical cord MSC cells were more than 80% in G0/G1 phase, suggesting that human umbilical cord MSC transfected with lentivirus after similar, with the same the typical characteristics of stem cells; by comparative observation on the growth curve of the found that, unlike before transfection, the experimental group transfected human umbilical cord MSC inhibited growth; modeling results show that intratracheal injection and intranasal instillation of bleomycin can induce pulmonary fibrosis. But according to the author the pathological fibrosis score, compared with intratracheal administration, intranasal administration after the model of mouse lung tissue pathological change is lighter, suggesting that intratracheal administration easier to model success. Conclusion: constructed the recombinant lentiviral vector of human Trx-1 gene, through The mediated modification of human umbilical cord MSC and overexpression of Trx-1 protein, and the cytological characteristics had no significant change; in this experiment, the operation by intratracheal injection of C57 mice model of pulmonary fibrosis in bleomycin induced twenty-eighth days after establishment of model best, is a good model of pulmonary fibrosis in this study; gene therapy and cell therapy combined, which provided the experimental basis for the treatment of idiopathic pulmonary fibrosis, may provide a new means.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R563

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