何美鄉金傳春臺灣大學：黃美椋Huang, Mei-LiangMei-LiangHuang2007-11-272018-06-292007-11-272018-06-292007http://ntur.lib.ntu.edu.tw//handle/246246/56213序言：干擾素-γ誘發蛋白-10（IP-10）/ CXCL10在過去許多研究中顯示為嚴重呼吸道症候群（SARS）的病程指標；在疾病初期時，血漿中含有高量干擾素-γ誘發蛋白-10的嚴重呼吸道症候群病人會有較嚴重的臨床結局。然而對於嚴重呼吸道症候群冠狀病毒感染時誘發干擾素-γ誘發蛋白-10機轉至今仍未被釐清。 方法：我們以基因流行病學的研究方法，確認干擾素-γ誘發蛋白-10基因上可能與嚴重呼吸道症候群的嚴重臨床結局有關的單一核苷酸多型性。以螢火蟲冷光酶分析法（Luciferase assay）以及電泳遷移率改變實驗 (EMSA, electrophoretic mobility shift assay)在體外針對干擾素-γ誘發蛋白-10起動子上單一核苷酸多型性的對偶基因的功能進行分析，並嘗試找出干擾素-γ誘發蛋白-10表現時與此單一核苷酸多型性相關的調控因子。 結果：由108位嚴重呼吸道症候群病人以及242位健康控制組血球中萃取的DNA，5個干擾素-γ誘發蛋白-10基因上的單一核苷酸多型性被正確的分型，其中位於起動子上位置-938的基因型TT在統計上顯著地與嚴重呼吸道症候群病人的疾病嚴重程度相關，尤其是可以在鼻咽喉中偵測到嚴重呼吸道症候群冠狀病毒的病人中，位置-938的基因型TT與嚴重呼吸道症候群病人的疾病嚴重程度的相關更顯著。 把干擾素-γ誘發蛋白-10的DNA片段，自轉錄起始點上游的996鹼基對（base pair），包含位置-938的單一核苷酸多型性的對偶基因型T或是C，以及其他自5’端縮短的DNA片段，插入表現螢火蟲冷光酶的質體（pGL3-basic）內，作為調控螢火蟲冷光酶表現的起動子，進而探討干擾素-γ誘發蛋白-10的起動子。當這些帶有不同長度的干擾素-γ誘發蛋白-10起動子基因的螢火蟲冷光酶表現質體轉殖至A549和HMEC-1細胞，24小時後利用干擾素-γ刺激之後，包含自轉錄起始點上游的704鹼基對以及413鹼基對的起動子DNA片段呈現最高的活性，並且隨著起動子DNA長度增加而減少活性。但若在轉殖24小時之後利用干擾素-γ和腫瘤壞死因子-α共同刺激細胞，顯示這些DNA片段的活性更高，然而起動子的活性隨著DNA片段長度的減少而降低，而413鹼基對的起動子DNA片段仍呈現較高的活性。根據這些結果可以推測，在干擾素-γ誘發蛋白-10起動子位置-996與-412之間可能存在著干擾素-γ相關負調控因子辨識位置和腫瘤壞死因子-α相關正調控因子辨識位置。而干擾素-γ誘發蛋白-10起動子上位置-938的對偶基因型T具有較高的活性，然而在經由干擾素-γ或干擾素-γ以及腫瘤壞死因子-α共同刺激之後增加的幅度卻沒有差異。 我們也利用干擾素-γ刺激厚的THP-1細胞核萃取蛋白，32P標示的干擾素-γ誘發蛋白-10（-928∼-948）探子以及抗轉譯調控因子的抗體，進行電泳遷移率改變實驗。探子包含-938C基因型相較于探子包含-938T可以和較多的細胞核萃取蛋白結合，而加入抗轉譯調控因子-YY1，MZF，Pax6的抗體之後，雖然沒有抗體-轉譯調控因子-探子複合物的電泳帶出現，但是與未加入轉譯調控因子-探子複合物的電泳帶比較電泳帶變淡了，表示這些轉譯調控因子可能會與干擾素-γ誘發蛋白-10（-928∼-948）探子結合。但是位置-938基因型C或是T都可以與這些轉譯調控因子結合。 轉譯調控因子YY-1與MZF對於干擾素-γ誘發蛋白-10起動子的影響再次研究中也藉由螢火蟲冷光酶分析法探討，而結果也顯示，YY-1和MZF都可以活化干擾素-γ誘發蛋白-10起動子，但是活化的程度在996鹼基對（base pair）DNA片段，包含位置-938的單一核苷酸多型性的對偶基因型T或是C位置-938基因型C或是T並沒有差別。 結論：干擾素-γ誘發蛋白-10起動子位置-938的單一核苷酸多型性的基因型TT和嚴重呼吸道症候群的疾病嚴重程度相關，而此位置可能被轉譯調控因子YY1，MZF以及Pax辨識，然而，對於這二個基因型的功能上差異經由本研究卻無法區分。Introduction - Interferon-γ inducible protein 10 (IP-10)/CXCLl10 was shown to be an indicator of disease progress for severe acute respiratory syndrome (SARS); a high plasma level in the early clinical stage was associated with subsequent adverse outcome. The mechanism that triggers CXCL10 expression in SARS-CoV infection is still unknown. Method - We conducted a genetic epidemiological study to identify the single nucleotide polymorphism (SNP) of CXCL10 that might be associated with severe SARS clinical outcomes. With luciferase assay and electromobility shift assay (EMSA), we conducted in vitro functional study of the polymorphic alleles of CXCL10 promoter with the attempt to identify the regulatory factors for CXCL10 expression. Results - Five SNPs of CXCL10 were typed for 108 SARS patients along with 242 healthy control DNAs. A genotype TT at the CXCL10(-938) SNP locus was identified to correlate with severity of SARS-CoV infected patients, especially among SARS patients with a detectably higher nasopharyngeal virus load. DNA fragment of the 996 bp upstream of the CXCL10 start codon containing either (-938C) or (-938T) SNP was cloned into the luciferase reporter pGL3 vector along with a series of 5’ end truncated CXCL10 promoter DNA fragments. With IFN-γ stimulation in A549 cell and HMEC-1 cells, the shortest two fragments (-704, and -413) showed a high luciferase activity, which dropped with each increment of the 5’ end DNA length; stimulation with IFN-γ and TNF-α in combination induced a higher luciferase activity, but the drop of activity was reversed with the fragment of -704 and -996, suggesting possibly IFN-γ associated negative regulation factors and TNF-α associated positive regulation factors could bind to this region. The difference of luciferase activity between the two alleles of CXCL10(-996C) and CXCL10(-996T) could not be consistently demonstrated, however. We used nuclear extracts from IFN-γ induced THP-1 cells and the 32P-labeled probes of CXCL10(-928~-948) promoter sequence containing (-938C) or (-938T) and antibodies against a number of TFs antibodies to perform EMSA. The (-938C) probe consistently binds to more nuclear proteins than the (-938T) probe, and three putative binding proteins, YY-1, MZF and Pax-6, of CXCL10 (-938) were found to reduce the shifted band in EMSA and supershift assay. The activation functions of YY-1 and MZF on CXCL10 expression were demonstrated by luciferase assay and the results showed YY-1 and MZF could trigger the activation of CXCL10, however, YY-1 and MZF induced activity were not different between the two alleles. Conclusion - The genotype TT of CXCL10 (-938) SNP was associated with adverse outcome of SARS patient. The DNA sequence flanking the CXCL10 (-938) SNP locus possibly contain binding motifs of YY-1, MZF and Pax-6. However, the functional difference between these two alleles of CXCL10 (-938) could not be demonstrated in vitro by luciferase assay and EMSA in the study.Content English Abstract…………………………………………………………………..1 Chapter 1 Literature Review 1.1 The characteristics of CXCL10 1.1.1 IP-10/CXCL10 and the chemokine receptors…………………..3 1.1.2 Receptors of IP-10/CXCL1………………………………………..3 1.2 The function of IP-10/CXCL10 1.2.1 Chemotaxis and Migration of leukocyte…………………………5 1.2.2 Modulator of T cell development and function………………….6 1.3 The regulation of IP-10/CXCL10 expression 1.3.1 The gene of CXCL10……………………………………………...7 1.3.2 The regulation of CXCL10 in promoter……………………….....7 1.4 The association of IP-10/CXCL10 and SARS 1.4.1 CXCL10 expression in SARS patients…………………………..9 1.4.2 CXCL10 expression in ex-vivo infection of SARS-CoV………..10 Chapter 2 Material & methods 2.1 Cell culture and cytokines…………………………………………….12 2.2 Luciferase Assay 2.2.1 Promoter-reporter plasmid construction and mutagenesis (A) pGL3-IP10clones…………………………………………………13 (B) Mutagenesis………………………………………………………14 (C) PGL3-IP10 (-928 to -948) repeats clones……………………..14 2.2.2 Transient transfection and co-transfection………………………15 2.3 Electrophoretic Mobility Shift Assay (EMSA) 2.3.1 Preparation of 32P-labeled primer pairs (A) Labeling probes with 32P by PCR……………………………….16 (B) 5’ end labeling of probes…………………………………………16 2.3.2 Nuclear proteins preparation (A) Nuclear extract preparation……………………………………..17 (B) In-vitro transcription/translation…………………………………18 2.3.3 Binding Reaction…………………………………………………..18 2.3.4 Competition & supershift of EMSA……………………………....19 2.4 Enzyme-link immunosorbent assay(ELISA) ………………………19 2.5 Prediction of transcriptional factors binding motifs in DNA……20 2.6 Isolation of transcriptional factors 2.6.1 Design of DNA bait…………………………………………………21 2.6.2 Nuclear Extract preparation……………………………………….22 2.6.3 Isolation of transcriptional factors…………………………………22 2.6.4 Two-dimensional electrophoresis…………………………..…….23 2.6.5 Preparation proteins for MALDI-TOF/MS………………………..24 2.7 Genotyping……………………………………………………………….25 Chapter 3 Results 3.1 SNPs on IP-10/CXCL10 promoter associated with clinical severity of SARS……………………………………………………………………27 3.2 CXCL10 level in the culture medium of EBV transformed B cell after IFN-γ or IFN-γ/TNF-α stimulation……………………………....30 3.3 Prediction of transcription factor-binding motifs…………………31 3.4 Functional analysis of CXCL10 promoter………………………….32 3.5 CXCL10 (-928~-948) containing transcriptional protein binding Site……………………………………………………………………….36 3.6 Identification of transcription factor 3.6.1 Characterization of the DNA-protein complex………………......38 3.6.2 Identification of TFs………………………………………………...39 Chapter 4 Discussion 4.1 Genotype of CXCL10 (C-938T) and the phenotype of CXCL10 Level……………………………………………………………………..41 4.2 The IP-10 Expression level and CXCL10 (C-938T)………………..43 4.3 The putative transcriptional factor of CXCL10 (-938)…………….45 4.4 Conclusion and Future…………………………………………………45 Chapter 5 Future direction 5.1 Short-term goal………………………………………………………….47 5.2 Long-term goal………………………………………………………….47 References…………………………………………………………………..…49 Tables Figures Appendix Table content Table 1. Distribution of allele frequency of CXCL10 SNPs in SARS patients.57 Table 2. Distribution of genotypes of the CXCL10(-938) polymorphic locus in SARS patients by clinical severity and0.2 healthy Taiwanese reference group………………………………………………………..…58 Table 3. Genotypes of the CXCL10(-938) polymorphic locus in SARS patients grouped by clinical severity and nasopharyngeal virus load….……..59 Table 4. Analysis of the 21 base pairs of oligonucleotides flanking the CXCL10(C-938T) SNP locus by five programs………………….……60 Table 5. Selected nuclear proteinsa captured by the CXCL10(-938C)- or CXCL10(-938T) probeB as predicted by peptide mass fingerprinting………………………………………………………….…...61 Table 6. Distribution of genotypes of the CXCL10(-938) polymorphic locus in SARS patients by clinical severity and healthy Taiwanese reference group (II)…………………………………………………………….….….62 Figure Content Fig 1. The known SNPs of CXCL10 gene and the selected SNPs…………...63 Fig 2. The known functional domains in human CXCL10 promoter…………..64 Fig 3. Construction of pGL3-IP10 clones………………………………………...65 Fig 4. Standardization of probe preparation for EMSA…………………………66 Fig 5. Plasma CXCL10 level of SARS patients during acute phase, by ELISA and grouped by clinical severity…………………………………………..67 Fig6. CXCL10 level in culture medium of EBV Transformed B cells (n=11) after IFN-γ stimulation……………………………………………………………68 Fig 7. Change of CXCL10 level in culture medium of EBV transformed B cells after IFN-γ and TNF-α stimulation………………………………………...69 Fig 8. The representative figures of three experiments of luciferase assay at 24 hours post IFN-γ (200 IU/mL) treatment………………………………….70 Fig 9. The representative figures of 6 experiments of luciferase assay at 24 hours post IFN-γ (500 IU/mL) and TNF-α (50ng/mL) treatment ….……71 Fig 10. Relative CXCL10 promoter activity in (A) A549 and (B) HMEC-1 cells by luciferase assay……………………………………………………….…….72 Fig 11. Relative luciferase activity ….using pGL3 plasmids containing repeated sequence of IP10(-930~-945) …………………………………………….73 Fig 12. Relative luciferase activity …..using pGL3-IP10-230 plasmids containing repeated sequence of IP10(-930~-945) ……………………………..….74 Fig 13. Relative luciferase activities of the two polymorphic alleles of pGL3-IP-10(C-996T) co-transfected with pcDNA3.1 expression constructs of MZF, YY1, mGATA-1……………………………………….75 Fig 14. Relative luciferase activities of pGL3-IP10 plasmids containing various 5’ deletion of human CXCL10 promoter while cotransfected with pcDNA3.1 expression constructs of YY-1………………………………..76 Fig 15. Electrophoretic mobility shift assay with nuclear extract from cell lines and 100-bp probes of CXCL10(-899~ -996) …………………………….77 Fig 16. EMSA analysis of CXCL10(C-938T) SNP with THP-1 cell nuclear proteins………………………………………………………………………78 Fig 17. EMSA analysis of nuclear extracts collected from IFN-γ (200IU/mL) treated THP-1 cells with (A) CXCL10(-938C), (B) CXCL10(-938C) probes and competitors…………………………………………………….……….79 Fig 18. Supershift analysis with antibodies against putative TFs in EMSA…...80 Fig 19. EMSA analysis of YY-1 protein with CXCL10(C-938T)……………….81 Fig 20. Analysis of CXCL10 (-928~-948) probes bound nuclear proteins by ultra violet light cross-linking and SDS-PAGE. ………………………..……….82 Fig 21. EMSA analysis of nuclear extract proteins from THP-1 with CXCL10(C-938T) probes in binding buffer with various concentration of NaCl…………………………………………………………………………..83 Fig 22. EMSA analysis of nuclear proteins THP-1 cells with allele T of CXCL10(-938) probe and competitors…………………………………………..………….84 Fig 23. EMSA analysis of DNA bait (CXCL10-938C or CXCL10-938T) captured nuclear proteins with 32P-labeled CXCL10-938C or CXCL10-938T probes…………………………………………………………..…….……….85 Fig 24. 2D IEF/SDS/PAGE analysis of the probe, CXCL10(-928~-948), bound nuclear protein………………………………………………………………..861597041 bytesapplication/pdfen-US人類干擾素γ誘發蛋白10啟動子多型性嚴重呼吸道症候群IP-10/CXCL10promoterpolymorphismSARS[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/56213/1/ntu-96-D87842002-1.pdf