先天性巨结肠患儿肠壁中转录因子NKX2-1表达的研究
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摘要
背景和目的
先天性巨结肠症又称为赫希施普龙病(Hirschsprung's disease,HD)是一种比较常见的小儿消化道发育畸形,属于一种先天性疾病。发病率约为1/5000,存在一定的性别差异,男女比例约为4：1。HD是造成结肠肠管增粗的原因之一可发生在近端结肠,主要位于乙状结肠和部分降结肠；也可发生在直肠和远端乙状结肠；HD患儿结肠狭窄段和扩张段两部分结肠组织之间有一过渡或移形区,往往呈漏斗形,长约3cm-8cm不等。已有实验证实了转录因子NKX2-1基因蛋白在整个肠神经系统的发育中存在。近年来NKX2-1的研究主要集中在甲状腺、脑及肺组织等有关神经系统发育疾病过程的研究中,在肠神经系统的研究比较少。先天性巨结肠患儿的肠蠕动功能可能与转录因子NKX2-1基因蛋白在HD患儿肠管管壁中的表达存在一定的相关性,本次实验的目的是应用免疫组化SP法和逆转录聚合酶链反应(RT-PCR)技术定性和定量测定转录因子NKX2-1基因在HD患儿结肠壁中的表达,以便更加深入的了解HD在分子基础上的确切发病机制,为HD疾病的临床诊断提供可靠的参考。
材料和方法
1应用免疫组化SP法观察30例HD手术切除标本(分别取HD患儿狭窄段、移行段、扩张段结肠壁全层组织)同时取30例肠套叠患儿手术切除并具备神经节细胞的全层结肠组织做为对照组,观察各组织标本中转录因子NKX2-1基因在HD患儿结肠壁中的表达情况；
2应用逆转录聚合酶链反应方法检测30例新鲜的HD手术切除标本(包括HD患儿狭窄段、移行段、扩张段全层结肠组织)和30例肠套叠患儿手术切除的存在神经节细胞的全层结肠组织做对照组,观察转录因子NKX2-1基因在HD患儿结肠组织中的表达情况；
3统计学处理：数据应用SPSS17.0软件处理,HD患儿各组结肠组织和正常对照组结肠组织中转录因子NKX2-1基因蛋白表达的比较采用两个独立样本的秩和检验；HD患儿各组结肠组织和正常对照组结肠组织中转录因子NKX2-1mRNA表达的比较采用两个独立样本的t检验或校正t检验。检验水准a=0.05。
结果
1转录因子NKX2-1在巨结肠患儿窄段结肠组织中表达明显减少,在扩张段结肠组织中表达正常,在移行段中的表达处于二者之间。
2HD患儿狭窄、移行段结肠组织中转录因子NKX2-1基因蛋白和nRNA表达与正常对照组结肠组织中转录因子NKX2-1基因蛋白的mRNA表达进行比较,差异有统计学意义(Z=5.236和2.926,P<0.008；t’=19.475和14.429,P<0.05),HD患儿扩张段结肠组织和对照正常结肠组织中NKX2-1蛋白和mRNA的表达比较,差异无统计学意义(Z=2.336,P=0.019>0.008；t=0.985,P=0.329>0.05)。
结论
1HD患儿狭窄段结肠壁组织中缺少转录因子NKX2-1基因的表达是HD发病的基本病理改变之一。
2转录因子NKX2-1基因在HD患儿结肠组织中表达减少是导致HD发病的原因之一。
Background and Objective
Congenital megacolon (Hirschsprung's disease, HD)is a relatively common digestive tract malformation and a congenital disease.Incidence of the disease is approximately1/5000. Colon bowel thickening is one of the reasons. It may occur in the proximal colon, sigmoid colon and general as part of the descending colon and may also in expand the distal intestine, rectum and sigmoid colon is the part; part two is arranged between the transition zone or transitional zone, often forms a funnel shape,3cm-8cm long., It has been confirmed that NKX2-1throughout the development of the nervous system in existence.The gene is located on chromosome14q13,its encoded protein products containing371amino acid residues, a molecular weight of38000, contains3exons and introns, is a containing helix turn helix structure of transcription factors. In recent years, NKX2-1's research is mainly about the thyroid, brain and lung disease related to nervous system development process research, in the enteric nervous system research is less. Congenital giant colon intestinal peristalsis may be associated with transcription factor NKX2-1in children with HD expression in intestinal wall have a certain degree of relevance, this experiment is using immunohistochemistry and RT-PCR technique for detection of NKX2-1in HD colon wall in expression, further understanding of the HD in the molecular basis of pathogenesis to provide reference for the clinical diagnosis.
Materials and Methods
1To observe the expression of Transcription factor NKX2-1in surgical specimens of30case of HD (narrow segments、transition segments and Dilation segments in separate) and of30cases of normal colon tissue with the method of immunohistochemistry.
2To observe the expression of Transcription factor NKX2-1in surgical specimens of30case of HD (narrow segments、transition segments and Dilation segments in separate) and of30cases of normal colon tissue with the method of RT-PCR
3Statistical analysis:data applications SPSS17.0software processing, transcription factors in colon tissue of the colon in each group and normal control group, the HD children of NKX2-1protein expression was used to compare two independent samples rank sum test; HD children colon in each groupand normal colon tissue of the control group transcription factor of NKX2-1mRNA expression was used to compare two independent samples t-test or calibration t-test. Inspection level a=0.05.
Results
1Transcription factor NKX2-1is apparently expressed less in narrow segments of HD and normally in dilation segments, the expression of the Transcription factor NKX2-1in transition segments between the two.
2The protein expression of Transcription factor NKX2-1of narrow and transition segments in HD colon tissue and normal colon tissue were singnificantly different(Z=5.236and2.926,P<0.008; t'=19.475and14.429,P<0.05),The protein expression of Transcription factor NKX2-1of dilationsegments in HD colon tissue and normal colon tissue were no singnificantly different(Z=2.336,P=0.019>0.008; t=0.985,F=0.329>0.05)。
Conclusion
1Like the absence of ganglion cells,the less expression of the Transcription factor NKX2-1contributes to the pathological changes of HD.
2The reduction of transcription factor of NKX2-1expression in the intestinal wall of Hirschsprung is possibly one of the reasons that lead to HD onset.
先天性巨结肠症又称为赫希施普龙病(Hirschsprung's disease,HD)是一种比较常见的小儿消化道发育畸形,属于一种先天性疾病。发病率约为1/5000,存在一定的性别差异,男女比例约为4：1。HD是造成结肠肠管增粗的原因之一可发生在近端结肠,主要位于乙状结肠和部分降结肠；也可发生在直肠和远端乙状结肠；HD患儿结肠狭窄段和扩张段两部分结肠组织之间有一过渡或移形区,往往呈漏斗形,长约3cm-8cm不等。已有实验证实了转录因子NKX2-1基因蛋白在整个肠神经系统的发育中存在。近年来NKX2-1的研究主要集中在甲状腺、脑及肺组织等有关神经系统发育疾病过程的研究中,在肠神经系统的研究比较少。先天性巨结肠患儿的肠蠕动功能可能与转录因子NKX2-1基因蛋白在HD患儿肠管管壁中的表达存在一定的相关性,本次实验的目的是应用免疫组化SP法和逆转录聚合酶链反应(RT-PCR)技术定性和定量测定转录因子NKX2-1基因在HD患儿结肠壁中的表达,以便更加深入的了解HD在分子基础上的确切发病机制,为HD疾病的临床诊断提供可靠的参考。
材料和方法
1应用免疫组化SP法观察30例HD手术切除标本(分别取HD患儿狭窄段、移行段、扩张段结肠壁全层组织)同时取30例肠套叠患儿手术切除并具备神经节细胞的全层结肠组织做为对照组,观察各组织标本中转录因子NKX2-1基因在HD患儿结肠壁中的表达情况；
2应用逆转录聚合酶链反应方法检测30例新鲜的HD手术切除标本(包括HD患儿狭窄段、移行段、扩张段全层结肠组织)和30例肠套叠患儿手术切除的存在神经节细胞的全层结肠组织做对照组,观察转录因子NKX2-1基因在HD患儿结肠组织中的表达情况；
3统计学处理：数据应用SPSS17.0软件处理,HD患儿各组结肠组织和正常对照组结肠组织中转录因子NKX2-1基因蛋白表达的比较采用两个独立样本的秩和检验；HD患儿各组结肠组织和正常对照组结肠组织中转录因子NKX2-1mRNA表达的比较采用两个独立样本的t检验或校正t检验。检验水准a=0.05。
结果
1转录因子NKX2-1在巨结肠患儿窄段结肠组织中表达明显减少,在扩张段结肠组织中表达正常,在移行段中的表达处于二者之间。
2HD患儿狭窄、移行段结肠组织中转录因子NKX2-1基因蛋白和nRNA表达与正常对照组结肠组织中转录因子NKX2-1基因蛋白的mRNA表达进行比较,差异有统计学意义(Z=5.236和2.926,P<0.008；t’=19.475和14.429,P<0.05),HD患儿扩张段结肠组织和对照正常结肠组织中NKX2-1蛋白和mRNA的表达比较,差异无统计学意义(Z=2.336,P=0.019>0.008；t=0.985,P=0.329>0.05)。
结论
1HD患儿狭窄段结肠壁组织中缺少转录因子NKX2-1基因的表达是HD发病的基本病理改变之一。
2转录因子NKX2-1基因在HD患儿结肠组织中表达减少是导致HD发病的原因之一。
Background and Objective
Congenital megacolon (Hirschsprung's disease, HD)is a relatively common digestive tract malformation and a congenital disease.Incidence of the disease is approximately1/5000. Colon bowel thickening is one of the reasons. It may occur in the proximal colon, sigmoid colon and general as part of the descending colon and may also in expand the distal intestine, rectum and sigmoid colon is the part; part two is arranged between the transition zone or transitional zone, often forms a funnel shape,3cm-8cm long., It has been confirmed that NKX2-1throughout the development of the nervous system in existence.The gene is located on chromosome14q13,its encoded protein products containing371amino acid residues, a molecular weight of38000, contains3exons and introns, is a containing helix turn helix structure of transcription factors. In recent years, NKX2-1's research is mainly about the thyroid, brain and lung disease related to nervous system development process research, in the enteric nervous system research is less. Congenital giant colon intestinal peristalsis may be associated with transcription factor NKX2-1in children with HD expression in intestinal wall have a certain degree of relevance, this experiment is using immunohistochemistry and RT-PCR technique for detection of NKX2-1in HD colon wall in expression, further understanding of the HD in the molecular basis of pathogenesis to provide reference for the clinical diagnosis.
Materials and Methods
1To observe the expression of Transcription factor NKX2-1in surgical specimens of30case of HD (narrow segments、transition segments and Dilation segments in separate) and of30cases of normal colon tissue with the method of immunohistochemistry.
2To observe the expression of Transcription factor NKX2-1in surgical specimens of30case of HD (narrow segments、transition segments and Dilation segments in separate) and of30cases of normal colon tissue with the method of RT-PCR
3Statistical analysis:data applications SPSS17.0software processing, transcription factors in colon tissue of the colon in each group and normal control group, the HD children of NKX2-1protein expression was used to compare two independent samples rank sum test; HD children colon in each groupand normal colon tissue of the control group transcription factor of NKX2-1mRNA expression was used to compare two independent samples t-test or calibration t-test. Inspection level a=0.05.
Results
1Transcription factor NKX2-1is apparently expressed less in narrow segments of HD and normally in dilation segments, the expression of the Transcription factor NKX2-1in transition segments between the two.
2The protein expression of Transcription factor NKX2-1of narrow and transition segments in HD colon tissue and normal colon tissue were singnificantly different(Z=5.236and2.926,P<0.008; t'=19.475and14.429,P<0.05),The protein expression of Transcription factor NKX2-1of dilationsegments in HD colon tissue and normal colon tissue were no singnificantly different(Z=2.336,P=0.019>0.008; t=0.985,F=0.329>0.05)。
Conclusion
1Like the absence of ganglion cells,the less expression of the Transcription factor NKX2-1contributes to the pathological changes of HD.
2The reduction of transcription factor of NKX2-1expression in the intestinal wall of Hirschsprung is possibly one of the reasons that lead to HD onset.
引文
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[1]赵金超,张瑞,汪蓓蕾.甲状腺素对甲状腺功能减低大鼠子代脑组织中同源盒基因Nkx2.2表达的影响[J].天津医药,2011,39(6)：539-541
[2]李建榕.甲状腺刺激抗体对甲状腺细胞TTF-1和PAX-8表达的影响[J].中华内分泌代谢志,2006,22(2)：1
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[9]WeiQ,M isk im in sWK,M isk im in s R.S tage-specific expression of myelin basic protein in oligodendrocytes involves Nkx2.2-m ediated repression that is relieved by the Sptran scrip tion factor[J].BiolChem,2005,280(16):16284-16294
[10]Takahashi M,Buma Y,Iwamoto T,et al.Cloning and expression of the RET protoon cogene encoding at yrosine kinase with two potential transmembrane domains[J].Oncogene, 1988,3:571
[11]Ordonez NG.Value of thyroid transcript ion factor-1 immunostaining in distinguishing small cell lung carcinomas from other small cell carcinomas[J].Am J Surg Pathol,2000, 24:1212～1223
[12]庞智,曹锴,魏文祥.中国汉族人群NKX2-3基因上游区域rs10883365多态性与克罗恩的相关性[J].胃肠病学,2010,15(9)：532-535
[13]李霞,王琦光,朱鲜阳.心脏特异性同源盒基因变异与法洛四联症[J].心血.管病学展,2009,30(3)：479-482
[14]Jude Kendall,Qing Liu,Amy Bakleh,et al.Oncogenic cooperation and coamplification of developmental transcription factor genes in lung cancer[J].ProcNatl Acad Sci USA,2007, 104:16663-16668
[15]Pang Y,von Turkovich M,Wu H,et al.The binding of thyroid transcription factor21 and hepatocyte paraffin 1 to mitochondrial proteins in hepatocytes:a molecular and immunoelectron microscopic study[J].Am J Chin Pathol,2006,125(5):722-726
[16]Kakinuma A.Nagayama Y.Multiple messenger ribonucleic acid transcripts and revised gene organization of human TSH receptor[J].Endocr J,2002,49 (2):175-180
[17]Bishop JA,Sharma R,Llei PB. Transcription factor-1 expression in carcinomas of the lung, breast, pancreas, colon,kidney, thyroid, and malignant mesothelioma[J].Hum Pathol,2010, 41(1):20-25
[18]Kelly SE,Bachurski CJ,Burhans MS,et al.Transcription of the lung specific surfactant protein C gene is mediated by thyroid transcription factor 1[J].J Biol Chem,1996,271: 6881-6888
[19]Edery P,Lyonnet S,Mu lligan LM,et al.Mutations of the RET protooncogene in Hirsch sprung's d isease[J].Nature,1994, (6461):367-378
[20]Perrone L,Zannini M,Di Lauro R,et al.The thyroid transription factor-2 (TTF-2) is a promoter specific DNA binding independent transcriptional repeessor[J].Biochem Biophys Res Common,2000,275:203-208
[21]Rouseau A, Nutt CL, Be ten sky RA,et al.Expression of oligod endroglial and astrocytic 1 ineage markers in diffuse gliom as:use of YKL-40, A poE, ASCL1, and Nkx2-2[J].J Neuropathol ExpN euro,2006,65(12):1149-1156
[22]Tanaka M,C hen Z,Bartun kova S,et al.The cardiacho meobox gene Csx/Nk x2.5 lies genet icallyupst ream of mutiple genesess entialfor heart development[J]. Development,1 999,126(6):1269-1280
[23]Yoon SO,Kim YT,Jung KC,et al.TTF-1 mRNA-positive circulating tumor cells in the peripheral blood predict poor prognosis in surgically resected non-small cell lung cancer patients[J].Lung Cancer,2011,71(2):206-209
[24]Bartlett H,Sutherland L,Kolker S,et al.Transient early embryonic expression of Nkx2.5 mutations linked to congenital heart defects in human causes heart defects in Xenopus laevis[J].Dev Dyn,2007,236(9):2475-2484
[25]Yoon SO,Kim YT,Jung KC,et al.TTF-1 mRNA-positive circulating tumor cells in the peripheral blood predict poor prognosis in surgically resected non-small cell lung cancer patients[J].Lung Cancer,2011,71(2):206-209
[26]E.M.Hol,F-W.Schwaiger.Regulation of the LIM-type homeobox gene islet-1 during neuronal regeneration [J].Neuroscience 1999,88(3):917-925
[27]Fancy SP,Zhao C,Frank lin RJ.In creased expression of Nkx2.2 and Olig2 identifies reactive oligod endrocyte progenitor cells responding to demyelination in the adult CNS[J].Mol Cell Neu rosc,2004,27(3):247-254
[28]尹光浩,刘伟,吴勇等.甲状腺转录因子1在原发性肺腺癌中的表达[J].广东学,2009,30(4)：565-567
[29]Amendola E,Sanges R,Galvan A,et al.Alocus on mouse chromosome 2 is involved in susceptibility to congenital hypothyroidism and contains an essential gene expressed in thyroid[J]. Endocrinology,2010,151(4):1948-1958
[30]Sinclair AH,Betra P,Palmer MS,et al.Agene from the human sex determing region encodes a Protein with homology to a consered DNA-binding motif[J].Nature,1990,346:240-244
[31]Kwei KA,Kim YH,Girard L,et al.Genomic profiling identifies TITF1 as a lineagespecific oncogene amplified in lung cancer[J].Oncogene,2008,27:3635-3640
[32]Narumi S,Muroya K,Asakura Y,et al.Transcription factor mutations and congenital hypothyroidism:systematic genetic screening of a populationvased cohort of Japanese patients[J].J Clin Endocrinol Metab,2010,95:1981-1985
[33]Zhu W,Shiojim a I,Hiroi Y,et al.Functional analyses of three Csx/Nkx 2.5 mutations that cause human congenit alheart disease[J].J Biol C hem,2000,275 (45):35291-35296
[34]Bejarano PA,Nikiforov YE,Swenson ES,et al.Thyroid transcription factor 21, thyroglobulin, cytokeratin 7 and cytokeratin 20 in thyroid neop lasms[J]. Applimmunohistochem MolMorphol,2000,8 (3):189-194
[35]刘兴元,杨奕清,杨颖.NKX2-5基因与先天性心脏病的关系[J].国际心血管杂,2009,36(4)：232-253
[36]De Felice M,Ovitt C,Biffali E,et al.A mouse model for hereditary thyroid dysgenesis and cleft palate[J].Nat Genet,1998;19:395-398
[37]N. Thong,D. Tan,T. Khoury, Expression of thyroid transcription factor-1 in colorectal carcinoma, Appl[J]. Immunohistochem. Mol. Morphol.2010 (18):244-249
[38]Martins SJ/Takagaki TY,Silva AGP,et al.Prognostic relevance of TTF-1 and MMP-9 expression in advanced lung adenocarcinoma[J]. Lung Cancer,2009,64:105-109
[39]Bai XY,Shen H.Mutational analysis of thyroid transcription factor-1 gene(TTF-1) in lung carcinomas[J].In Vitro Cell Dev Biol Anim,2008,44(1):17-25
[40]Doff B, McElh inney, Elizabeth G,et al. NKX 2.5 mutations in patients with congenital heart disease[J].J Am Coll Cardiol,2003,42:1650-1655
[41]Maeshima AM,Omatsu M,Tsuta K,et al.Immunohistochemical expression of TTF-1 in various cytological subtypes of primary lung adenocarcinoma, with special reference to intratumoral heterogeneity[J].Pathol Int,2008,58:31-37
[42]RaffinM,LeongLM,RonesMS,et al. Subdivision of the cardiac Nkx2.5 exp ression domain into myogenic and nonmyogenic compartments [J].DevBiol,2000,218(2):32623-32640
[43]Krumlauf R.Hox genes in vertebratedevelopment [J].Cell,1994,78(2):191-201.
[44]Jay PY, HarrisBS,Maguire CT,et al.Nkx2.5 mutation causes anatomichypop lasia of the cardiac conduction system[J]. Clin Invest,2004,113:1130