NLRP7基因突变葡萄胎患者的NLRP7蛋白，caspase-1蛋白及IL-1b因子表达研究
摘要
背景
通过对家族性复发性葡萄胎的研究发现导致葡萄胎的基因为NLRP7基因,其可能通过对炎症通路的影响导致其发发生,但具体机制尚未明确。
研究目的
本研究通过对NLPR7基因突变患者和正常对照妇女的外周血淋巴细胞培养,检测其NLRP7蛋白,caspase-1蛋白和IL-1β细胞因子的表达情况,间接检测NLRP7基因突变者是否会引起功能功能障碍导致Casepase-1依赖的IL-1β的分泌异常。
材料与方法
通过大样本的NLRP7基因定位测序,选取基因突变患者4例,同时选择生育结局良好并且无不良生育史的妇女4例作为对照。抽取其外周血15-20ml,分离淋巴细胞经行培养,分别对患者组和对照组经行LPS刺激,收集培养上液经行IL-1p测定,收集细胞采用Western Blotting方法测定NLRP7蛋白及caspase-1蛋白表达情况,并对2组经行比较。
结果
(1)实验组(A组)：在LPS刺激下,除A4外,NLRP7蛋白和caspase-1蛋白表达成反向趋势,IL-1β因子表达量均降低。
(2)对照组(B组)：在LPS刺激下,除B2外,NLRP7蛋白和caspase-1蛋白表达成正向趋势,IL-1β因子表达量均升高。
(3)实验组(A组)出现40kd的Caspase-1蛋白表达,对照组(B组)未见明显表达。
结论
NLRP7基因突变者可能引起NLRP7蛋白表达异常,进而引起Caspase-1蛋白表达异常，进一步对Caspase-1依赖的IL-1β因子的分泌引起异常,可能导致是葡萄胎的产生的一个途径。
Background
It is presume that the cause of recurrent hydatidiform mole is NLRP7 gene by studying familial hydatidiform mole. Its possible mechanism is thought as inflammation pathway but it is not sure yet.
Objective
This research was detecting NLRP7 protein, caspase-1 and expression of cytokines IL-1βby peripheral blood lymphocytes culture of the NLPR7 gene mutations women and normal control of women.
Materials and methods
Through large sample NLRP7 gene sequencing, select 4 cases of gene mutations in patients, as well as choice 4 cases of women as a control whose growth history is normal with no adverse reproductive outcomes. The major process is taking their peripheral blood 15-20ml, lymphocytes culturing respectively by LPS stimulation. collecting culture fluid for the determination of IL-1 beta, collecting cells to Western Blotting for determination of NLRP7 and Caspase-1 protein expression and comparing the 2 groups.
Results
(1) the experimental group (group A):Without A4, stimulated by LPS, NLRP7 protein and Caspase-1 protein in a reverse trend, expression of IL-1 beta factor are reduced.
(2) the control group (Group B):Without B2, stimulated by LPS, NLRP7 and Caspase-1 protein expression of a positive trend, expression of IL-1 beta factor is increased.
(3) 40kd Caspase-1 protein expression occurs in the experimental group (group A), but the control group (Group B) has not been expression.
Conclusions
NLRP7 gene mutations may cause NLRP7, Caspase-1 protein and IL-1βabnormal expression, and may has a certain impact on the hydatidiform mole by immunology mechanism.
通过对家族性复发性葡萄胎的研究发现导致葡萄胎的基因为NLRP7基因,其可能通过对炎症通路的影响导致其发发生,但具体机制尚未明确。
研究目的
本研究通过对NLPR7基因突变患者和正常对照妇女的外周血淋巴细胞培养,检测其NLRP7蛋白,caspase-1蛋白和IL-1β细胞因子的表达情况,间接检测NLRP7基因突变者是否会引起功能功能障碍导致Casepase-1依赖的IL-1β的分泌异常。
材料与方法
通过大样本的NLRP7基因定位测序,选取基因突变患者4例,同时选择生育结局良好并且无不良生育史的妇女4例作为对照。抽取其外周血15-20ml,分离淋巴细胞经行培养,分别对患者组和对照组经行LPS刺激,收集培养上液经行IL-1p测定,收集细胞采用Western Blotting方法测定NLRP7蛋白及caspase-1蛋白表达情况,并对2组经行比较。
结果
(1)实验组(A组)：在LPS刺激下,除A4外,NLRP7蛋白和caspase-1蛋白表达成反向趋势,IL-1β因子表达量均降低。
(2)对照组(B组)：在LPS刺激下,除B2外,NLRP7蛋白和caspase-1蛋白表达成正向趋势,IL-1β因子表达量均升高。
(3)实验组(A组)出现40kd的Caspase-1蛋白表达,对照组(B组)未见明显表达。
结论
NLRP7基因突变者可能引起NLRP7蛋白表达异常,进而引起Caspase-1蛋白表达异常，进一步对Caspase-1依赖的IL-1β因子的分泌引起异常,可能导致是葡萄胎的产生的一个途径。
Background
It is presume that the cause of recurrent hydatidiform mole is NLRP7 gene by studying familial hydatidiform mole. Its possible mechanism is thought as inflammation pathway but it is not sure yet.
Objective
This research was detecting NLRP7 protein, caspase-1 and expression of cytokines IL-1βby peripheral blood lymphocytes culture of the NLPR7 gene mutations women and normal control of women.
Materials and methods
Through large sample NLRP7 gene sequencing, select 4 cases of gene mutations in patients, as well as choice 4 cases of women as a control whose growth history is normal with no adverse reproductive outcomes. The major process is taking their peripheral blood 15-20ml, lymphocytes culturing respectively by LPS stimulation. collecting culture fluid for the determination of IL-1 beta, collecting cells to Western Blotting for determination of NLRP7 and Caspase-1 protein expression and comparing the 2 groups.
Results
(1) the experimental group (group A):Without A4, stimulated by LPS, NLRP7 protein and Caspase-1 protein in a reverse trend, expression of IL-1 beta factor are reduced.
(2) the control group (Group B):Without B2, stimulated by LPS, NLRP7 and Caspase-1 protein expression of a positive trend, expression of IL-1 beta factor is increased.
(3) 40kd Caspase-1 protein expression occurs in the experimental group (group A), but the control group (Group B) has not been expression.
Conclusions
NLRP7 gene mutations may cause NLRP7, Caspase-1 protein and IL-1βabnormal expression, and may has a certain impact on the hydatidiform mole by immunology mechanism.
引文
[1]Grimes DA. Epidemiology of gestational trophoblastic disease [J]. Am J Obstet Gynecol 1984,150(3):309-318.
[2]Berkowitz RS, Im SS, Bernstein MR, et al. Gestational trophoblastic disease. Subsequent pregnancy outcome, including repeat molar pregnancy [J]. J Reprod Med,1998,43(1):81-86.
[3]Horn LC, Kowalzik J, Bilek K, et al. Clinicopathologic characteristics and subsequent pregnancy outcome in 139 complete hydatidiform moles [J]. Eur J Obstet Gynecol Reprod Biol,2006,128(1/2):10-14.
[4]El-Maarri O, Slim R. Familial hydatidiform molar pregnancy:the germline imprinting defect hypothesis? [J]. Curr Top Microbiol Immunol,2006,301: 229-241.
[5]Moglabey YB, Kircheisen R, Seoud M, et al. Genetic mapping of a maternal locus responsible for familial hydatidiform moles [J]. Hum Mol Genet,1999,8(4): 667-671.
[6]Murdoch S, Djuric U, Mazhar B, et al. Mutations, in NALP7 cause recurrent hydatidiform moles and reproductive wastage in humans [J]. Nat Genet,2006, 38(3):300-302.
[7]Djuric U, El-Maarri O, Lamb B, et al. Familial molar tissues due to mutations in the inflammatory gene, NALP7, have normal postzygotic DNA methylation [J]. Hum Genet,2006,120(3):390-395.
[8]Zhang P, Dixon M, Zucchelli M, et al. Expression analysis of the NLRP gene family suggests a role in human preimplantation development [J]. PLoS ONE, 2008,3(7):e2755.
[9]Okada K, Hirota E, Mizutani Y, et al. Oncogenic role of NALP7 in testicular seminomas[J]. Cancer Sci,2004,95(12):949-954.
[10]Ohno S, Kinoshita T, Ohno Y, et al. Expression of NLRP7 (PYPAF3, NALP7) protein in endometrial. cancer tissues[J]. Anticancer Res,2008,28(4C): 2493-2497.
[11]Slavova N, Drescher A, Visekruna A, et al. NALP expression in Paneth cells provides a novel track in IBD signaling[J]. Langenbecks Arch Surg,2010,395(4): 351-357.
[12]Kinoshita T, Wang Y, Hasegawa M, et al. PYPAF3, a PYRIN-containing APAF-1-like protein, is a feedback regulator of caspase-1-dependent interleukin-lbeta secretion[J]. J Biol Chem,2005,280(23):21720-21725.
[13]Martinon F, Tschopp J. NLRs join TLRs as innate sensors of pathogens [J]. Trends Immunol,2005,26(8):447-454.
[14]Koonin EV, Aravind L. The NACHT family—a new group of predicted NTPases implicated in apoptosis and MHC transcription activation [J]. Trends Biochem Sci, 2000,25(5):223-224.
[15]Tschopp J, Martinon F, Burns K. NALPs:a novel protein family involved in inflammation[J]. Nat Rev Mol Cell Biol,2003,4(2):95-104.
[16]Pinheiro AS, Proell M, Eibl C, et al. The 3-dimensional structure of the NLRP7 pyrin domain-insight into pyrin:pyrin mediated effector domain signaling in innate immunity [J]. J Biol Chem,2010 Jun 11. [Epub ahead of print]
[17]Zhang P, Dixon M, Zucchelli M, et al. Expression analysis of the NLRP gene family suggests a role in human preimplantation development[J]. PLoS ONE, 2008,3(7):e2755.
[18]Gumucio DL, Diaz A, Schaner P, Richards N, et al. Fire and ICE:the role of pyrin domain-containing proteins in inflammation and apoptosis [J]. Clin Exp Rheumatol.2002 Jul-Aug;20(4 Suppl 26):S45-53.
[19]Tian X, Pascal G, Monget P. Evolution and functional divergence of NLRP genes in mammalian reproductive systems [J]. BMC Evol Biol,2009.9:202.
[20]Deveault C, Qian JH, Chebaro W, et al. NLRP7 mutations in women with diploid androgenetic and triploid moles:a proposed mechanism for mole formation [J]. Hum Mol Genet 2009,18(5):888-897.
[21]Hussein MR, Abd-Elwahed AR, Abodeif ES, et al. Decidual immune cell infiltrate in hydatidiform mole [J]. Cancer Invest,2009,27(1):60-66.
[22]Nagymanyoki Z, Callahan MJ, Parast MM, et al. Immune cell profiling in normal pregnancy, partial and complete molar pregnancy [J]. Gynecol Oncol,2007, 107(2):292-297.
[23]Baasanjav B, Usui H, Kihara M, et al. The risk of post-molar gestational trophoblastic neoplasia is higher in heterozygous than in homozygous complete hydatidiform moles [J]. Hum Reprod,2010,25(5):1183-1191.
[24]Qian J, Deveault C, Bagga R, et al. Women heterozygous for NALP7/NLRP7 mutations are at risk for reproductive wastage:report of two novel mutations [J]. Hum Mutat,2007,28(7):741.
[25]Wang C, Dixon P, Decordova S, et al. Identification of 13 novel NLRP7 mutations in 20 families with recurrent hydatidiform mole; missense mutations cluster in the leucine rich region [J]. J Med Genet,2009,46(8):569-575.
[26]Franchi L, Eigenbrod T, Munoz-Planillo R, et al. The inflammasome:a aspase-1-activation platform that regulates immune responses and disease pathogenesis. Nat Immunol.2009 Mar;10(3):241-7.
[2]Berkowitz RS, Im SS, Bernstein MR, et al. Gestational trophoblastic disease. Subsequent pregnancy outcome, including repeat molar pregnancy [J]. J Reprod Med,1998,43(1):81-86.
[3]Horn LC, Kowalzik J, Bilek K, et al. Clinicopathologic characteristics and subsequent pregnancy outcome in 139 complete hydatidiform moles [J]. Eur J Obstet Gynecol Reprod Biol,2006,128(1/2):10-14.
[4]El-Maarri O, Slim R. Familial hydatidiform molar pregnancy:the germline imprinting defect hypothesis? [J]. Curr Top Microbiol Immunol,2006,301: 229-241.
[5]Moglabey YB, Kircheisen R, Seoud M, et al. Genetic mapping of a maternal locus responsible for familial hydatidiform moles [J]. Hum Mol Genet,1999,8(4): 667-671.
[6]Murdoch S, Djuric U, Mazhar B, et al. Mutations, in NALP7 cause recurrent hydatidiform moles and reproductive wastage in humans [J]. Nat Genet,2006, 38(3):300-302.
[7]Djuric U, El-Maarri O, Lamb B, et al. Familial molar tissues due to mutations in the inflammatory gene, NALP7, have normal postzygotic DNA methylation [J]. Hum Genet,2006,120(3):390-395.
[8]Zhang P, Dixon M, Zucchelli M, et al. Expression analysis of the NLRP gene family suggests a role in human preimplantation development [J]. PLoS ONE, 2008,3(7):e2755.
[9]Okada K, Hirota E, Mizutani Y, et al. Oncogenic role of NALP7 in testicular seminomas[J]. Cancer Sci,2004,95(12):949-954.
[10]Ohno S, Kinoshita T, Ohno Y, et al. Expression of NLRP7 (PYPAF3, NALP7) protein in endometrial. cancer tissues[J]. Anticancer Res,2008,28(4C): 2493-2497.
[11]Slavova N, Drescher A, Visekruna A, et al. NALP expression in Paneth cells provides a novel track in IBD signaling[J]. Langenbecks Arch Surg,2010,395(4): 351-357.
[12]Kinoshita T, Wang Y, Hasegawa M, et al. PYPAF3, a PYRIN-containing APAF-1-like protein, is a feedback regulator of caspase-1-dependent interleukin-lbeta secretion[J]. J Biol Chem,2005,280(23):21720-21725.
[13]Martinon F, Tschopp J. NLRs join TLRs as innate sensors of pathogens [J]. Trends Immunol,2005,26(8):447-454.
[14]Koonin EV, Aravind L. The NACHT family—a new group of predicted NTPases implicated in apoptosis and MHC transcription activation [J]. Trends Biochem Sci, 2000,25(5):223-224.
[15]Tschopp J, Martinon F, Burns K. NALPs:a novel protein family involved in inflammation[J]. Nat Rev Mol Cell Biol,2003,4(2):95-104.
[16]Pinheiro AS, Proell M, Eibl C, et al. The 3-dimensional structure of the NLRP7 pyrin domain-insight into pyrin:pyrin mediated effector domain signaling in innate immunity [J]. J Biol Chem,2010 Jun 11. [Epub ahead of print]
[17]Zhang P, Dixon M, Zucchelli M, et al. Expression analysis of the NLRP gene family suggests a role in human preimplantation development[J]. PLoS ONE, 2008,3(7):e2755.
[18]Gumucio DL, Diaz A, Schaner P, Richards N, et al. Fire and ICE:the role of pyrin domain-containing proteins in inflammation and apoptosis [J]. Clin Exp Rheumatol.2002 Jul-Aug;20(4 Suppl 26):S45-53.
[19]Tian X, Pascal G, Monget P. Evolution and functional divergence of NLRP genes in mammalian reproductive systems [J]. BMC Evol Biol,2009.9:202.
[20]Deveault C, Qian JH, Chebaro W, et al. NLRP7 mutations in women with diploid androgenetic and triploid moles:a proposed mechanism for mole formation [J]. Hum Mol Genet 2009,18(5):888-897.
[21]Hussein MR, Abd-Elwahed AR, Abodeif ES, et al. Decidual immune cell infiltrate in hydatidiform mole [J]. Cancer Invest,2009,27(1):60-66.
[22]Nagymanyoki Z, Callahan MJ, Parast MM, et al. Immune cell profiling in normal pregnancy, partial and complete molar pregnancy [J]. Gynecol Oncol,2007, 107(2):292-297.
[23]Baasanjav B, Usui H, Kihara M, et al. The risk of post-molar gestational trophoblastic neoplasia is higher in heterozygous than in homozygous complete hydatidiform moles [J]. Hum Reprod,2010,25(5):1183-1191.
[24]Qian J, Deveault C, Bagga R, et al. Women heterozygous for NALP7/NLRP7 mutations are at risk for reproductive wastage:report of two novel mutations [J]. Hum Mutat,2007,28(7):741.
[25]Wang C, Dixon P, Decordova S, et al. Identification of 13 novel NLRP7 mutations in 20 families with recurrent hydatidiform mole; missense mutations cluster in the leucine rich region [J]. J Med Genet,2009,46(8):569-575.
[26]Franchi L, Eigenbrod T, Munoz-Planillo R, et al. The inflammasome:a aspase-1-activation platform that regulates immune responses and disease pathogenesis. Nat Immunol.2009 Mar;10(3):241-7.