MMP9、TIMP1基因在原发性肝癌中的表达

目的 探讨MMP9、TIMP1基因在原发性肝癌中的表达状况及其对肝癌侵袭转移的影响.方法 应用逆转录-聚合酶链反应(RT-PCR) 检测34例肝癌和相应癌旁组织及12例正常肝组织中MMP9、TIMP1 mRNA表达水平.结果 正常肝组织中MMP9仅有少量表达,与癌旁组织相比无显著差异;肝癌组织中MMP9表达明显升高,而TIMP1表达显著下降,两者表达状况呈负相关(P＜0.01),且两者在转移肝癌较未转移肝癌中表达水平亦有显著性差异(P＜0.05).结论 MMP9、TIMP1表达改变可能与肝癌侵袭转移的发生发展有一定关系.     

24 大肠癌组织中EphA2，VEGF和MMP9蛋白的表达及其相关性研究

为探讨大肠癌组织中EphA2，VEGF和MMP9蛋白的表达及其与大肠癌生物学行为的关系。笔者应用免疫组织化学S-P法检测69例大肠癌组织及30例远癌肠黏膜中EphA2，VEGF，MMP9蛋白的表达。结果示,大肠癌组织中EphA2蛋白（100%）表达显著高于远癌肠黏膜（80.0%）（P<0.01）,且与Dukes分期、肿瘤组织分化程度及肉眼类型有关（P<0.05），而与年龄无关（P>0.05）。VEGF蛋白及MMP9蛋白在大肠癌中的表达均显著高于远癌肠黏膜(均P<0.01)，与Dukes分期有关（P<0.05），但与年龄、肿瘤组织分化程度及肉眼类型等无关（均P>0.05）。EphA2蛋白在大肠癌中的表达与VEGF，MMP9呈正相关（r1=0.404，P1=0.000;r2=0.282，P2=0.000）。提示联合检测EphA2,VEGF和MMP9有助于对大肠癌侵袭及转移能力的评估，对大肠癌的预后判断可能具有一定的临床意义。     

Association of the c.‐9C>T and c.368A>G transitions in H2BFWT gene with male infertility in an Iranian population

The H2BFWT (H2B family, member W, testis specific) gene is a testis‐specific histone which is involved in the spermatogenesis process. This study aimed to investigate the association of H2BFWT gene c.‐9C>T and c.368A>G polymorphisms with male infertility in an Iranian population. The 232 independent individuals, including 109 infertile men and 123 healthy controls, were recruited from IVF centre (Kashan, Iran). The allele types of c.‐9C>T and c.368A>G polymorphisms were detected by using PCR‐RFLP method. In overall analysis, we found that the c.‐9T (OR: 1.75, 95% CI: 1.04–2.95, p = .035) and c.368G (OR: 1.71, 95% CI: 1.02–2.89, p = .042) alleles are associated with male infertility. The c.‐9T allele was also associated with nonobstructive azoospermia (OR: 2.08, 95% CI: 1.01–4.25, p = .046), while c.368G allele was associated with oligozoospermia (OR: 2.10, 95% CI: 1.15–3.85, p = .016). It is concluded that H2BFWT gene c.‐9C>T and c.368A>G polymorphisms might be genetic risk factors for idiopathic male infertility.     

Genetic polymorphisms in glutathione S-transferase T1 affect the surgical outcome of varicocelectomies in infertile patients

Glutathione S-transferases （GSTs）, superoxide dismutase 2 （SOD2） and NAD（P）H：quinone oxidoreductase 1 （NQO1） are anti-oxidant enzyme genes. Polymorphisms of GSTs, SOD2 and NQO1 have been reported to influence individual susceptibility to various diseases. In an earlier study, we obtained preliminary findings that a subset of glutathione S-transferase 7：1 （GSTT1）-wt patients with varicocele may exhibit good response to varicocelectomy. In this study, we extended the earlier study to determine the distribution of genotype of each gene in the infertile population and to evaluate whether polymorphism of these genes affects the results of surgical treatment of varicocele. We analyzed 72 infertile varicocele patients, 202 infertile patients without varicocele and 101 male controls. Genotypes of GSTs were determined by polymerase chain reaction （PCR）. Genotyping of SOD2 and NQO1 was performed using the PCR-restriction fragment length polymorphism （PCR-RFLP） method. A significantly better response to varicocelectomy was found in patients with the GSTTI-wt genotype （63.2%） and NQO1-Ser/Ser genotype （80.0%） than in those with GSTTI-null genotype （35.3%） and NQO1-Pro/Pro or NQO1- Pro/Ser genotype （45.2%）, respectively. The frequencies of glutathione S-transferase M1/T1, SOD2 and NQO1 genotypes did not differ significantly among the varicocele patients, idiopathic infertile patients and male controls. GSTT1 genotype is associated with improvement of semen parameters after varicocelectomy. As the number of patients with NQO1-Ser/Ser genotype was not sufficient to reach definite conclusions, the association of NQO1 genotype with varicocelectomy requires further investigation.     

基质金属蛋白酶(MMP)9、MMP2 mRNA及基质金属蛋白酶抑制剂2 mRNA在人膀胱移行细胞癌中的表达及意义

目的探讨膀胱移行细胞癌中基质金属蛋白酶（MMP）9、MMP2及基质金属蛋白酶抑制剂2（TIMP2）mRNA表达与膀胱肿瘤恶性程度和侵袭性之间的关系及其临床意义。方法采用逆转录-聚合酶链反应（RT-PCR）方法检测36例膀胱移行细胞癌及5例正常膀胱组织MMP9、MMP2、TIMP2mRNA的表达。结果MMP9、MMP2mRNA随着肿瘤分级分期的增高，表达明显增强[x^-值（MMP9）G1：1．218，G2：1．681，G3：1．811；T2～T4：1．840，Tis～T1：1．399；P〈0．01；（MMP2）G1：1．323，G2：1．694，G3：2．060；T2～T4：1．950，Tis-T1：1．505；P〈0．01]，且明显高于正常膀胱组织中的表达[互值（MMP9）0．455，（MMP2）0．461；P〈0．01]。TIMP2mRNA随肿瘤分级的增高而明显减少（x^-值：G1：1．489，G2：1．391，G3；0．580；P〈0．01）。结论（1）MMP9、MMP2mRNA的高表达与BTCC的恶性程度呈正相关；（2）TIMP2mRNA的表达与BTCC的恶性程度呈负相关。     

Association of polymorphisms in glutathione S-transferase genes (GSTM1, GSTT1, GSTP1) with idiopathic azoospermia or oligospermia in Sichuan,China

The reported effects of the glutathione S-transferase (GSTs) genes (GSTM1, GSTT1, and GSTP1) on male factor infertility have been inconsistent and even contradictory. Here, we conducted a case-control study to investigate the association between functionally important polymorphisms in GST genes and idiopathic male infertility. The study group consisted of 361 men with idiopathic azoospermia, 118 men with idiopathic oligospermia, and 234 age-matched healthy fertile male controls. Genomic DNA was extracted from the peripheral blood, and analyzed by polymerase chain reaction and restriction fragment length polymorphism analysis. There was a significant association between the GSTP1 variant genotype (Ile/Val + Val/Val) with idiopathic infertility risk (odds ratio [OR]: 1.53; 95% confidence interval [CI]: 1.11–2.11; P = 0.009). Similarly, a higher risk of infertility was noted in individuals carrying a genotype combination of GSTT1-null and GSTP1 (Ile/Val + Val/Val) (OR: 2.17; 95% CI: 1.43–3.31; P = 0.0002). These results suggest an increased risk of the GSTP1 variant genotype (Ile/Val + Val/Val) for developing male factor infertility. Our findings also underrate the significance of the effect of GSTM1 and/or GSTT1 (especially the former) in modulating the risk of male infertility in males from Sichuan, southwest China.     

Aquaporin-9 immunohistochemistry in varicocele testes as a consequence of hypoxia in the sperm production site

Aquaporin-9 (AQP-9) regulates tissue hydration by promoting transmembrane exchanges of both water and solutes, such as lactate. The latter is a key metabolite of primary spermatocytes and of maturing haploid germ cells (h-GCs). The present investigation was aimed at immunolocalising human AQP-9 in both normal and varicocele testes. Histology and immmunocytochemistry were investigated in archival biopsies from 20 varicocele testes and in eight unaffected ones. AQP-9 immunostaining was performed using a rabbit antibody, and either focal or diffuse cell membrane labelling was recorded. Varicocele testes showed disarranged tubular compartments, with sloughing h-GCs, tissue hyperhydration, spermiogenesis failure and fibrosis. AQP-9 immunohistology of the control testes showed a diffuse cell membrane staining of the primary spermatocytes and h-GCs, without any positive reaction of spermatogonia and Sertoli cells. AQP-9 cell expression in the varicocele testes was focal or lacking in both adluminal and sloughing GCs. AQP-9 expression occurs in normal human testis, at cell membrane of primary spermatocytes and h-GCs, suggesting a possible role of AQP-9 in the water and lactate transport from Sertoli cells to GCs. AQP-9 is focal or lacking in adolescent varicocele testes, and this suggests AQP-9 to be downregulated in such testicular disorder, leading to lactate deprivation with subsequent hypospermatogenesis.     

Leptin levels in infertile male patients are correlated with inhibin B, testosterone and SHBG but not with sperm characteristics

In the present study, differences in leptin levels between different groups of male patients presenting with infertility problems and possible correlations between leptin levels and clinical, spermiological, histological and hormonal characteristics were examined. Two hundred and ten male partners from infertile couples were included in the study. Based on clinical examination, spermiogram and testicular histology results, patients were divided into four groups: 42 men with non-obstructive azoospermia, 15 men with obstructive azoospermia, 68 men with oligoasthenoteratozoospermia and 85 men with normozoospermia. Serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), inhibin B, testosterone, sex hormone binding globulin (SHBG) and leptin were measured. After adjustment for body mass index, there was a negative correlation between serum levels of leptin and inhibin B, total testosterone and SHBG (r = -0.189, p = 0.009, r = -0.250, p = 0.001 and r =-0.221, p = 0.003 respectively) but there was no correlation between leptin and classical sperm characteristics. Our results therefore demonstrate a link between leptin and testicular function, independently of FSH and LH, possibly involving testosterone and SHBG through a regulation of Leydig cell function.     

The micronutrient supplements,zinc sulphate and folic acid,did not ameliorate sperm functional parameters in oligoasthenoteratozoospermic men

We investigated the effects of folic acid and zinc sulphate supplementation on the improvement of sperm function in subfertile oligoasthenoteratozoospermic (OAT) men. Eighty‐three OAT men participated in a 16‐week intervention randomised, double‐blind clinical trial with daily treatment of folic acid (5 mg day^?1) and zinc sulphate (220 mg day^?1), or placebo. Before and after treatment, semen and blood samples were obtained for determining sperm concentration, motility, and morphology, sperm viability, sperm mitochondrial function, sperm chromatin status using toluidine blue, aniline blue, acridine orange and chromomycin A₃ staining; and semen and blood folate, zinc, B₁₂, total antioxidant capacity ( TAC) and malondialdehyde (MDA) concentrations. Sperm concentration (×10⁶ ml^?1) increased in subfertile men receiving the combined treatment of folic acid and zinc sulphate and also in the group receiving only folic acid treatment; however, it was not statistically significant (P = 0.056 and P = 0.05, respectively). Sperm chromatin integrity (%) increased significantly in subfertile men receiving only zinc sulphate treatment (P = 0.048). However, this improvement in sperm quality was not significant after adjusting placebo effect. This study showed that zinc sulphate and folic acid supplementation did not ameliorate sperm quality in infertile men with severely compromised sperm parameters, OAT. Male infertility is a multifactorial disorder, and also nutritional factors play an important role in results of administration of supplementation on sperm parameters. However, these results should be confirmed by multiple studies in larger populations of OAT men.     

不育男性精浆抗心磷脂抗体的检测与意义

应用间接ＥＬＩＳＡ法检测男性不育病人精浆的抗心磷脂抗体（ＡＣＡ），并对其与精子密度、精浆抗精子抗体及血清ＡＣＡ的关系进行了研究。结果表明，不孕、流产组精浆ＡＣＡ总检出率分别为２０．０％和１２．５％，前者较对照组显著增高（Ｐ＜０．０５），ＡＣＡ阳性率有随精子数的减少而逐渐上升趋势，但无统计学意义。精浆中ＡＣＡ与ＡｓＡｂ及与血清ＡＣＡ无关联性。     

精子染色质扩散实验检测精索静脉曲张及不明原因不育患者精子DNA碎片

目的了解精索静脉曲张(VC)及不明原因不育患者精子DNA碎片的发生比例。方法改进的精子染色质扩散(SCD)实验分析精子DNA碎片。检测VC不育患者39例,不明原因不育患者57例。以生育健康成年男性32例为对照组。结果VC不育患者SCD小光晕和无光晕精子(精子DNA碎片)比值平均为(36.6±18.9)%,VC不育组明显高于对照组(12.1±5.2)%(P<0.001),而大光晕和中光晕精子比值VC不育组明显低于对照组(P<0.01);不明原因不育患者精子DNA碎片比值平均为(26.8±10.2)%,与对照组[(12.1±5.2)%]比较有显著性差异(P<0.001)。结论SCD实验表明,VC及不明原因不育患者精子DNA碎片比值增高。     

CA repeat and RsaI polymorphisms in ERbeta gene are not associated with infertility in Indian men

Oestrogen Receptor β (ERβ) gene plays an important role in the regulation of fertility in both males and females. Polymorphism in CA repeat located in the flanking region of ERβ has been shown to be associated with several diseases, but its association with male infertility has not been analysed so far. However, Rsa I polymorphism (rs1256049) in exon 5 of ERβ has been shown to be associated with male infertility in Caucasian patients. Hence, we have analysed 695 Indian men, including 443 infertile and 252 fertile men to evaluate the association of CA repeat length and Rsa I polymorphisms in male infertility. Our results revealed no significant difference in the distribution of CA repeat length between infertile (mean ± SD 23.24 ± 2.06, median 24) and fertile men (mean ± SD 23.16 ± 2.27, median 24). The analysis of dosage effect by classifying samples into SS (short/short), SL (short/long) and LL (long/long) groups also did not show any significant difference between infertile and fertile men. Similarly, Rsa I polymorphism also did not show any significant difference between infertile and fertile men. Furthermore, the combined analysis of CA repeat and Rsa I polymorphisms by haplotyping showed that the distribution of haplotypes was not significantly different between fertile and infertile men. Our results suggest that CA repeat length and Rsa I polymorphisms in ERβ are not associated with infertility in Indian men.     

Urinary levels of matrix metalloproteinases and their tissue inhibitors in nephrotic children

The aim of this study was to determine the effects of cyclosporine A (CyA) on urinary levels of matrix metalloproteinase 2 and 9 (MMP2, MMP9) and their tissue inhibitors 1 and 2 (TIMP1, TIMP2) in steroid-dependent nephrotic syndrome (SDNS). The study group (1) consisted of 18 children SDNS aged 3.5-17.0 years treated with CyA. All NS children were examined three times: (A) at proteinuria relapse, before CyA treatment, (B) after 6 months, and (C) after 12 months of CyA administration. The control group (2) consisted of 18 healthy children. Serum CyA level was assessed by immunofluorescence. Enzyme-linked immunosorbent assay kits for total human MMP2 and 9 and TIMP1 and 2 were obtained from R&D Systems. Compared with healthy controls, urinary MMP9/Cr in NS children before CyA was on the same level and increased during CyA treatment, and urinary TIMP1/Cr was twice as high and increased significantly during CyA treatment. MMP9/TIMP1 in NS children treated with CyA increased, but the difference was not statistically significant. Urinary MMP2/Cr was similar, and urinary TIMP2/Cr was significantly higher in children treated with CyA (p < 0.01). The MMP2/TIMP2 ratio in NS children treated with CyA was significantly lower in comparison with healthy controls (p < 0.01). A negative correlation was noted between urinary MMP2/TIMP2 ratio and serum CyA in NS children (r = -0.541, p < 0.01). An imbalance within the MMP2 and TIMP2 and MMP9 and TIMP1 system may play a role in the pathogenesis CyA nephropathy.     

Detection and clinical significance of phospholipase A2 in semen of infertile males

Objective: To explore the clinical significance of anti-human seminal plasma phospholipase A2 (PLA2) monoclonal antibody (McAb) in male infertility. Methods: Enzyme-linked immunoabsorbent assay (ELISA), immunocytochemistry (ICC), and flow cytometry (FCM) analysis were employed using two strains of anti-human seminal plasma PLA2 McAb prepared by this laboratory to detect the PLA2 content in the seminal plasma and the anterior head region of spermatozoa in infertile patients and normal fertile controls. Seminal routine observation was performed with computer-assisted semen analysis (CASA). Results: The PLA2 level of the infertile groups was (31.13±14.49) ng/mL in azoospermic patients, (17.71±12.45) ng/mL in oligozoospermic patients and (16.46±11.31) ng/mL in patients with normal sperm density, which were all higher than that of normal controls [(8.09±3.15) ng/mL, P <0.01]. There was a significant negative correlation between the PLA2 content in seminal plasma and the sperm density (r=-0.602, P<0.05), w     

Mutational screening of the INSL3 gene in azoospermic males with a history of cryptorchidism

The insulin‐like factor 3 (INSL₃) gene encodes 131 amino acids, consisting of two exons. In human beings, mutations of this gene may lead to bilateral cryptorchidism and infertility. However, the role of INSL₃ in male spermatogenesis still remains controversial. We have analysed the coding sequence of INSL₃ by PCR and DNA sequencing in 97 azoospermic patients with a history of bilateral cryptorchidism (patient group) versus 49 males with obstructive azoospermia (control group). The G178A mutation, which were predicted to alter the protein sequence (alanine to threonine), was detected in the patient group but not in the control group. While synonymous mutations G27A and G126A were detected in the control group, each occurred only in a single sample. When the patient group were divided into two subgroups according to the testicular biopsy result: sperm+ subgroup (51 cases with spermatozoa can be detected) or sperm‐ subgroup (46 cases with spermatozoa could not be detected). The INSL₃ G178A polymorphism was not significantly associated with spermatozoa or no spermatozoa in the testes of males with a history of bilateral cryptorchidism. In conclusion, the evidence suggests that mutations of INSL₃ may not directly contribute to the damage of spermatogenesis in patients with bilateral cryptorchidism history.     

基质金属蛋白酶9及组织型基质金属蛋白酶抑制因子1在大鼠外伤性脑水肿中的表达及意义

目的 探讨大鼠脑损伤中基质金属蛋白酶9(MMP9)及组织型基质金属蛋白酶抑制因子1(TIMP1)的表达变化规律及意义.方法 采用Feeney自由落体法制作大鼠轻度脑损伤模型,采用干湿重法测定脑组织含水量;采用逆转录-聚合酶链反应(RT-PCR)法对大鼠脑损伤模型中MMP9及TIMP1 mRNA进行检测并分析两者比值.结果 实验组脑组织含水量(BWC),MMP9mRNA及TIMP1 mRNA表达量及两者比值均明显高于对照组,P值分别为0.013、0.000、0.000及0.011,差异有统计学意义(P<0.05),MMP9 mRNA与TIMP1mRNA表达量及两者比值与BWC均呈正相关,r值分别为0.654、0.355(P<0.05).结论 MMP9/TIMP1的比例失衡可能在外伤性脑水肿的发生发展过程中起重要作用.     

Association of G/T(rs222859) polymorphism in Exon 1 of YBX2 gene with azoospermia,among Iranian infertile males

Animal model studies have shown that MSY2 gene has a potential role in spermatogenesis. Some mutations on this gene have been proposed to be associated with human male infertility. In this study, polymorphisms of exon 1 of YBX2 gene have been investigated. A total of 276 men were evaluated. They included 96 men with normal spermatogenesis, 60 men with nonobstructive azoospermia, 60 men with oligospermia and 60 men with asthenospermia. We extracted DNA from blood and testis tissues of samples, and analysed polymorphisms of exon 1 by sequencing method. Moreover, YBX2 gene expression was studied by real‐time PCR on blood and testis tissue of samples. Sequencing results showed that among the studied polymorphisms, frequency of TT genotype in rs222859 polymorphism was significantly higher in azoospermic patients compared to control group (P < 0.001). Azoospermic men exhibited significant underexpression of YBX2 gene in blood and testis samples in comparison with controls, oligosperm and asthenosperm samples (P < 0.001), but there was no significant difference in gene expression of YBX2 gene in blood and testis tissues of azoospermic men, with and without mutation (P > 0.05). According to our results, the alterations of this gene might be involved in azoospermia among Iranian population.     

MMP2、TIMP2在人肾透明细胞癌中的表达及意义

目的　研究基质金属蛋白酶 -2(MMP2)、基质金属蛋白酶组织抑制剂- 2(TIMP2)在肾透明细胞癌(RCCC)中的表达及意义。　方法　采用免疫组织化学SAB法测定 44例RCCC及20例正常肾组织中MMP2与TIMP2蛋白的表达。44例RCCC中病理分级G1 14例,G2 28例,G3 2例,病理分期pT1 24例,pT2 8例,pT3 12例。　结果　MMP2在RCCC中的阳性表达率为 79. 6% (35 /44),高于正常肾组织的 15% ( 3 /20 ),MMP2表达与RCCC病理分级、分期呈正相关 (r=0. 6316,P<0. 01;r=0.5096,P<0. 01);TIMP2在RCCC中的阳性表达率为 100% (44 /44),高于正常肾组织的 35% (7 /20),但TIMP2表达与RCCC病理分期、分级无相关性(r=0.2404,P>0. 05;r=-0.1236,P>0. 05)。在RCCC中MMP2与TIMP2阳性表达率无相关性 (r=0.2015,P>0. 05 )。　结论　RCCC中MMP2表达与RCCC病理分期、分级呈正相关,提示MMP2免疫活动增高不良预后有关系,而TIMP2的反常表达以及作用值得进一步深入研究。MMP2可以作为判断RCCC预后的分子指标。