重组人源抗-CTGF scFv二聚体在ASM细胞增殖及化疗药物引起肺纤维化作用中的研究
本文选题:结缔组织生长因子 + 单链抗体(scFv)二聚体 ; 参考:《东南大学》2016年博士论文
【摘要】:化疗药物引起的肺纤维化,是一种不可逆性肺间质损伤,以肺组织中成纤维细胞的异常增殖、细胞外基质的过度沉积、弥漫性或局限性成纤维细胞灶的形成为主要病理特征,传统药物治疗效果差。结缔组织生长因子(connective tissue growth factor, CTGF)是转化因子-β(transforming growth factor-β, TGF-β)下游效应因子,具有介导细胞增殖迁移、诱导细胞转分化、促进ECM的产生等多种生物学功能,是导致肺纤维的主要关键性因子,阻断CTGF的表达可能为肺纤维化的治疗新的靶点。第一章重组人源抗-CTGF单链抗体二聚体的制备目的:制备纯度较高、具有生物学活性的抗-CTGF单链抗体二聚体。方法:1.构建pET28a-scFv-matrilin表达质粒;2.将质粒转化到E.coli.BL21(DE3)细胞中,IPTG诱导表达,经镍柱纯化得到抗-CTGF单链抗体二聚体;3.通过SDS-PAGE、Native-PAGE电泳和蛋白分子杂交进行蛋白及纯度鉴定;4. ELISA法检验其免疫学活性。结果:1. IPTG诱导后目的蛋白的表达量显著增加,为可溶性表达;2.经SDS-PAGE、Native-PAGE电泳和免疫印迹证实分离纯化得到的融合蛋白为二聚体,纯化后纯度在90%以上;3. scFv二聚体对CTGF具有较好的免疫学活性。结论:我们通过可溶性表达制备了纯度较高,具有较好免疫学活性的抗-CTGF单链抗体二聚体。第二章 重组人源抗-CTGF单链抗体二聚体对人气管平滑肌细胞增殖作用的影响及其机制研究目的:研究抗-CTGF单链抗体二聚体对CTGF诱导的气管平滑肌细胞(ASM)增殖作用的影响及其机制。方法:1.以不同浓度的CTGF (0、5、10、20、30、40、50ng/ml)刺激体外培养的ASM细胞1天、3天和5天,通过MTT检测ASM细胞的增殖能力;2.选取20 ng/ml的CTGF作用3天为药物诱导条件,MTT法检测scFv单链抗体或二聚体、抗CTGF单抗、以及通路抑制剂LY294002对CTGF诱导的ASM细胞增殖活性的影响;3. Western blot分别检测空白组、CTGF组、scFv二聚体组、抗-CTGF单克隆抗体组和通路抑制剂LY294002组细胞中p-Akt和p-mTOR蛋白表达水平。结果:1.20 ng/ml的CTGF作用3天能够显著诱导ASM细胞增殖(P0.05);2.scFv二聚体能够抑制CTGF诱导ASM细胞的增殖效应(P0.05); 3. CTGF诱导的ASM细胞Akt和mTOR蛋白的磷酸化程度升高(P0.05),加入scFv二聚体、抗-CTGF mAb和LY294002干预后,p-Akt和p-mTOR蛋白的表达量显著降低;结论:scFv二聚体能够抑制CTGF诱导的气管平滑肌细胞的增殖效应;scFv三聚体通过抑制PI3K/Akt/mTOR信号通路的活性来抑制CTGF诱导的ASM细胞增殖。第三章重组人源抗-CTGF单链抗体二聚体在CTGF诱导的肺成纤维细胞转分化中的作用目的:探讨抗-CTGF单链抗体二聚体在CTGF诱导的肺成纤维细胞转分化中的作用。方法:1.分别采用0、10、20、30、40、50、60 ng/ml的CTGF刺激体外培养的HLEF细胞12 h、24 h和36 h, CCK8检测HLEF细胞的增殖能力;2.选取50 ng/ml的CTGF作用12 h为诱导条件,CCK8法检测scFv二聚体对CTGF诱导的HLEF细胞增殖活性的影响;3. RT-PCR分别检测CTGF诱导后CTGF组、scFv单链抗体组、scFv二聚体组、抗CTGF单克隆抗体组HLEF细胞中α-SMA和FNmRNA的转录水平;4. Western Blot检测scFv二聚体对以上各组HLEF细胞中α-SMA蛋白表达水平的影响。结果:1.50 ng/ml的CTGF作用12 h能够显著诱导ASM细胞增殖(P0.05);2.scFv二聚体能够抑制CTGF诱导的HLEF细胞的增殖效应(P0.05);3.CTGF诱导后HLEF细胞中α-SMA和FN mRNA的转录水平显著升高(P0.05),加入scFv二聚体和抗CTGF mAb干预后,α-SMA和FN mRNA的转录水平明显下调(P0.05), scFv单链效果弱于二聚体;4. CTGF诱导后HLEF细胞中α-SMA蛋白表达量显著增多(P0.05),加入scFv二聚体和抗CTGF mAb干预后α-SMA蛋白表达量明显降低(P0.05), scFv单链抗体的效果同样弱于二聚体组。结论:scFv二聚体可以通过下调a-SMA蛋白从而抑制CTGF诱导的肺成纤维细胞HLEF细胞向肌成纤维细胞的表型转化。第四章重组人源抗-CTGF单链抗体二聚体在化疗药物引起肺纤维化作用中的研究目的:探讨scFv二聚体在抑制肺纤维化中的作用及相关机制。方法:1.气管内滴注博莱霉素构建小鼠肺纤维化模型,尾静脉注射抗-CTGF scFv单链抗体和scFv二聚体进行干预。实验分为四组:A组(NS+NS组)、B组(BLM+NS组)、C组(BLM+scFv单链抗体组)和D组(BLM+scFv二聚体组);2.分别收集造模后第7天、14天和28天时小鼠肺组织支气管肺泡灌洗液,进行细胞计数和分类计数;3.观察第7天、14天和28天的小鼠肺组织羟脯氨酸和TGF-β1含量的动态变化;4.各组小鼠肺组织进行HE染色与Masson染色观察肺泡炎和肺纤维化程度并评分;5. RT-PCR分别检测各组肺纤维化小鼠肺组织CTGF和α-SMA mRNA转录水平;6. Western blot分析各组小鼠肺组织中P13K蛋白表达、Akt蛋白和mTOR蛋白磷酸化程度的差异。结果:1.成功构建小鼠肺纤维化模型;2.BLM组小鼠BALF中的细胞总数显著高于其它各组(P0.05),以中性粒细胞、淋巴细胞和巨噬细胞增多为主(P0.05),嗜酸性细胞未见明显增加(P0.05),BLM+scFv单链抗体组次之,BLM+scFv二聚体组BALF细胞计数和分类计数均明显低于单链抗体组(P0.05);3.肺病理切片HE染色和Masson染色显示BLM+scFv二聚体组肺泡炎和纤维化程度较BLM组和BLM+scFv单链抗体组降低(P0.05);4.BLM+scFv二聚体组小鼠肺组织羟脯氨酸和TGF-β1的含量显著降低(P0.05);5.BLM组小鼠肺组织中CTGF和α-SMA mRNA的转录水平最高,BLM+scFv二聚体组的转录水平显著降低(P0.05);6.BLM组P13K蛋白表达量显著增加(P0.05),Akt和mTOR蛋白的磷酸化程度最高(P0.05),BLM+scFv二聚体组的P13K.p-Akt和p-mTOR蛋白的表达量均显著降低(P0.05)。结论:抗-CTGF scFv二聚体可能通过抑制P13K/Akt/mTOR信号通路减轻博莱霉素诱导的小鼠肺纤维化。
[Abstract]:In the first chapter , we construct pET28a - scFv - matrilin expression plasmid .
2 . transforming the plasmid into E . coli BL21 ( DE3 ) cells , inducing the expression by IPTG , and purifying by a nickel column to obtain an anti - ctgf single - chain antibody dimer ;
3 , carrying out protein and purity identification by SDS - PAGE , Native - PAGE electrophoresis and protein molecular hybridization ;
4 . The immunological activity was tested by ELISA . The results were as follows : 1 . The expression of the target protein was increased significantly after IPTG induction , which was soluble expression .
2 . SDS - PAGE , Native - PAGE electrophoresis and Western blot prove that the fusion protein obtained by separation and purification is dimer , and the purity is above 90 % after purification ;
Objective : To study the effect of anti - ctgf single chain antibody dimer on the proliferation of human tracheal smooth muscle cells ( ASM ) induced by ctgf and its mechanism . The second chapter is to study the effect of anti - ctgf single chain antibody dimer on proliferation of human tracheal smooth muscle cells ( ASM ) and its mechanism .
2 . The effects of single - chain antibody or dimer , anti - ctgf monoclonal antibody , and pathway inhibitor LY294004 on the proliferation of ASM were detected by MTT assay .
3 . Western blot was used to detect the levels of p - and p - mtor protein in the blank group , the ctgf group , the scFv dimer group , the anti - ctgf monoclonal antibody group and the pathway inhibitor LY294004 , respectively . The results showed that : 1 . 20 ng / ml of ctgf could significantly induce the proliferation of ASM cells ( P0.05 ) .
2 . scFv dimer can inhibit the proliferation of ASM cells induced by ctgf ( P0.05 ) ; The levels of phosphorylation and phosphorylation of MMP - 3 and MMP - 3 were increased ( P0.05 ) , and the expression of p - and p - mtor proteins was significantly decreased after the intervention of scFv dimer , anti - ctgf mAb and LY294004 .
Conclusion : scFv dimer can inhibit the proliferation of tracheal smooth muscle cells induced by ctgf .
The purpose of this study was to investigate the role of anti - ctgf single chain antibody dimer in the differentiation of lung fibroblasts induced by ctgf . Methods : 1 . The proliferation of HLEF cells cultured in vitro was investigated by using 0 , 10 , 20 , 30 , 40 , 50 , 60 ng / ml of ctgf to stimulate the proliferation of HLEF cells in vitro .
2 . The effect of scFv dimer on the proliferation activity of HLEF cells induced by ctgf was detected by c8 method by selecting 50 ng / ml ctgf for 12 h .
3 . RT - PCR was used to detect the levels of 伪 - SMA and FN mRNA in HLEF cells induced by ctgf , single chain antibody group , scFv dimer group and anti - ctgf monoclonal antibody group .
4 . The effect of scFv dimer on the expression level of 伪 - SMA was detected by Western Blot . Results : 1 . 50 ng / ml ctgf could significantly induce the proliferation of ASM cells ( P0.05 ) .
2 . The transcription level of 伪 - SMA and FN mRNA in HLEF cells increased significantly ( P0.05 ) , and the transcription level of 伪 - SMA and FN mRNA decreased significantly ( P0.05 ) .
4 . The expression of 伪 - SMA in HLEF cells was significantly increased ( P0.05 ) .
2 . The bronchoalveolar lavage fluid was collected from the lung tissues of mice at 7 , 14 and 28 days after molding respectively , and the cell count and classification count were carried out .
3 . To observe the dynamic changes of the contents of Hydroxyproline and TGF - 尾1 in the lung tissues of mice at 7 , 14 and 28 days ;
4 . The lung tissues of each group were stained with HE and eosin stain to observe the degree of alveolar inflammation and pulmonary fibrosis and score ;
5 . RT - PCR was used to detect the mRNA transcription level in lung tissue and 伪 - SMA mRNA in each group of pulmonary fibrosis mice .
6 . Western blot analysis the expression of P13K protein in lung tissues of each group , and the difference of phosphorylation degree of protein and protein in lung tissues of mice . Results : 1 . The model of pulmonary fibrosis was successfully constructed .
2 . The total number of cells in BALF was significantly higher than that in other groups ( P0.05 ) .
3 . The lung pathological sections were stained with HE and eosin staining to show that the level of alveolus and fibrosis was lower ( P0.05 ) . 4 . The content of hydroxyproline and TGF - 尾1 in lung tissue was significantly decreased ( P0.05 ) .
5 . There was a significant decrease in the transcription level of the mRNA levels in the lung tissues ( P0.05 ) . The levels of P13K protein expression were significantly increased ( P0.05 ) .
【学位授予单位】:东南大学
【学位级别】:博士
【学位授予年份】:2016
【分类号】:R730.5
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