本文選題：結(jié)締組織生長因子 + 單鏈抗體(scFv)二聚體　； 參考：《東南大學(xué)》2016年博士論文

【摘要】：化療藥物引起的肺纖維化,是一種不可逆性肺間質(zhì)損傷,以肺組織中成纖維細胞的異常增殖、細胞外基質(zhì)的過度沉積、彌漫性或局限性成纖維細胞灶的形成為主要病理特征,傳統(tǒng)藥物治療效果差。結(jié)締組織生長因子(connective tissue growth factor, CTGF)是轉(zhuǎn)化因子-β(transforming growth factor-β, TGF-β)下游效應(yīng)因子,具有介導(dǎo)細胞增殖遷移、誘導(dǎo)細胞轉(zhuǎn)分化、促進ECM的產(chǎn)生等多種生物學(xué)功能,是導(dǎo)致肺纖維的主要關(guān)鍵性因子,阻斷CTGF的表達可能為肺纖維化的治療新的靶點。第一章重組人源抗-CTGF單鏈抗體二聚體的制備目的：制備純度較高、具有生物學(xué)活性的抗-CTGF單鏈抗體二聚體。方法：1.構(gòu)建pET28a-scFv-matrilin表達質(zhì)粒；2.將質(zhì)粒轉(zhuǎn)化到E.coli.BL21(DE3)細胞中,IPTG誘導(dǎo)表達,經(jīng)鎳柱純化得到抗-CTGF單鏈抗體二聚體；3.通過SDS-PAGE、Native-PAGE電泳和蛋白分子雜交進行蛋白及純度鑒定；4. ELISA法檢驗其免疫學(xué)活性。結(jié)果：1. IPTG誘導(dǎo)后目的蛋白的表達量顯著增加,為可溶性表達；2.經(jīng)SDS-PAGE、Native-PAGE電泳和免疫印跡證實分離純化得到的融合蛋白為二聚體,純化后純度在90%以上；3. scFv二聚體對CTGF具有較好的免疫學(xué)活性。結(jié)論：我們通過可溶性表達制備了純度較高,具有較好免疫學(xué)活性的抗-CTGF單鏈抗體二聚體。第二章 重組人源抗-CTGF單鏈抗體二聚體對人氣管平滑肌細胞增殖作用的影響及其機制研究目的：研究抗-CTGF單鏈抗體二聚體對CTGF誘導(dǎo)的氣管平滑肌細胞(ASM)增殖作用的影響及其機制。方法：1.以不同濃度的CTGF (0、5、10、20、30、40、50ng/ml)刺激體外培養(yǎng)的ASM細胞1天、3天和5天,通過MTT檢測ASM細胞的增殖能力；2.選取20 ng/ml的CTGF作用3天為藥物誘導(dǎo)條件,MTT法檢測scFv單鏈抗體或二聚體、抗CTGF單抗、以及通路抑制劑LY294002對CTGF誘導(dǎo)的ASM細胞增殖活性的影響；3. Western blot分別檢測空白組、CTGF組、scFv二聚體組、抗-CTGF單克隆抗體組和通路抑制劑LY294002組細胞中p-Akt和p-mTOR蛋白表達水平。結(jié)果：1.20 ng/ml的CTGF作用3天能夠顯著誘導(dǎo)ASM細胞增殖(P0.05)；2.scFv二聚體能夠抑制CTGF誘導(dǎo)ASM細胞的增殖效應(yīng)(P0.05); 3. CTGF誘導(dǎo)的ASM細胞Akt和mTOR蛋白的磷酸化程度升高(P0.05),加入scFv二聚體、抗-CTGF mAb和LY294002干預(yù)后,p-Akt和p-mTOR蛋白的表達量顯著降低；結(jié)論：scFv二聚體能夠抑制CTGF誘導(dǎo)的氣管平滑肌細胞的增殖效應(yīng)；scFv三聚體通過抑制PI3K/Akt/mTOR信號通路的活性來抑制CTGF誘導(dǎo)的ASM細胞增殖。第三章重組人源抗-CTGF單鏈抗體二聚體在CTGF誘導(dǎo)的肺成纖維細胞轉(zhuǎn)分化中的作用目的：探討抗-CTGF單鏈抗體二聚體在CTGF誘導(dǎo)的肺成纖維細胞轉(zhuǎn)分化中的作用。方法：1.分別采用0、10、20、30、40、50、60 ng/ml的CTGF刺激體外培養(yǎng)的HLEF細胞12 h、24 h和36 h, CCK8檢測HLEF細胞的增殖能力；2.選取50 ng/ml的CTGF作用12 h為誘導(dǎo)條件,CCK8法檢測scFv二聚體對CTGF誘導(dǎo)的HLEF細胞增殖活性的影響；3. RT-PCR分別檢測CTGF誘導(dǎo)后CTGF組、scFv單鏈抗體組、scFv二聚體組、抗CTGF單克隆抗體組HLEF細胞中α-SMA和FNmRNA的轉(zhuǎn)錄水平；4. Western Blot檢測scFv二聚體對以上各組HLEF細胞中α-SMA蛋白表達水平的影響。結(jié)果：1.50 ng/ml的CTGF作用12 h能夠顯著誘導(dǎo)ASM細胞增殖(P0.05)；2.scFv二聚體能夠抑制CTGF誘導(dǎo)的HLEF細胞的增殖效應(yīng)(P0.05);3.CTGF誘導(dǎo)后HLEF細胞中α-SMA和FN mRNA的轉(zhuǎn)錄水平顯著升高(P0.05),加入scFv二聚體和抗CTGF mAb干預(yù)后,α-SMA和FN mRNA的轉(zhuǎn)錄水平明顯下調(diào)(P0.05), scFv單鏈效果弱于二聚體；4. CTGF誘導(dǎo)后HLEF細胞中α-SMA蛋白表達量顯著增多(P0.05),加入scFv二聚體和抗CTGF mAb干預(yù)后α-SMA蛋白表達量明顯降低(P0.05), scFv單鏈抗體的效果同樣弱于二聚體組。結(jié)論：scFv二聚體可以通過下調(diào)a-SMA蛋白從而抑制CTGF誘導(dǎo)的肺成纖維細胞HLEF細胞向肌成纖維細胞的表型轉(zhuǎn)化。第四章重組人源抗-CTGF單鏈抗體二聚體在化療藥物引起肺纖維化作用中的研究目的：探討scFv二聚體在抑制肺纖維化中的作用及相關(guān)機制。方法：1.氣管內(nèi)滴注博萊霉素構(gòu)建小鼠肺纖維化模型,尾靜脈注射抗-CTGF scFv單鏈抗體和scFv二聚體進行干預(yù)。實驗分為四組：A組(NS+NS組)、B組(BLM+NS組)、C組(BLM+scFv單鏈抗體組)和D組(BLM+scFv二聚體組)；2.分別收集造模后第7天、14天和28天時小鼠肺組織支氣管肺泡灌洗液,進行細胞計數(shù)和分類計數(shù)；3.觀察第7天、14天和28天的小鼠肺組織羥脯氨酸和TGF-β1含量的動態(tài)變化；4.各組小鼠肺組織進行HE染色與Masson染色觀察肺泡炎和肺纖維化程度并評分；5. RT-PCR分別檢測各組肺纖維化小鼠肺組織CTGF和α-SMA mRNA轉(zhuǎn)錄水平；6. Western blot分析各組小鼠肺組織中P13K蛋白表達、Akt蛋白和mTOR蛋白磷酸化程度的差異。結(jié)果：1.成功構(gòu)建小鼠肺纖維化模型；2.BLM組小鼠BALF中的細胞總數(shù)顯著高于其它各組(P0.05),以中性粒細胞、淋巴細胞和巨噬細胞增多為主(P0.05),嗜酸性細胞未見明顯增加(P0.05),BLM+scFv單鏈抗體組次之,BLM+scFv二聚體組BALF細胞計數(shù)和分類計數(shù)均明顯低于單鏈抗體組(P0.05)；3.肺病理切片HE染色和Masson染色顯示BLM+scFv二聚體組肺泡炎和纖維化程度較BLM組和BLM+scFv單鏈抗體組降低(P0.05);4.BLM+scFv二聚體組小鼠肺組織羥脯氨酸和TGF-β1的含量顯著降低(P0.05)；5.BLM組小鼠肺組織中CTGF和α-SMA mRNA的轉(zhuǎn)錄水平最高,BLM+scFv二聚體組的轉(zhuǎn)錄水平顯著降低(P0.05);6.BLM組P13K蛋白表達量顯著增加(P0.05),Akt和mTOR蛋白的磷酸化程度最高(P0.05),BLM+scFv二聚體組的P13K.p-Akt和p-mTOR蛋白的表達量均顯著降低(P0.05)。結(jié)論：抗-CTGF scFv二聚體可能通過抑制P13K/Akt/mTOR信號通路減輕博萊霉素誘導(dǎo)的小鼠肺纖維化。
[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 偽 - 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 偽 - 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 偽 - SMA and FN mRNA in HLEF cells increased significantly ( P0.05 ) , and the transcription level of 偽 - SMA and FN mRNA decreased significantly ( P0.05 ) .
4 . The expression of 偽 - 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 偽 - 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 ) .

【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R730.5

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