SRY基因诊断在临床中的

应用聚合酶链式反应（ＰＣＲ）扩增技术，对１０例染色体核型为４６，ＸＹ和１例为４６，ＸＸ／４６，ＸＹ的性反转、睾丸女性化、两性畸形及男性生殖器发育不良的患者进行了性别决定区（ＳＲＹ）基因检测。结果表明９例４６，ＸＹ和１例，ＸＹ和１例４６，ＸＸ／４６，ＸＹ患者的ＳＲＹ基因存在，１例４６，ＸＹ女性患者的ＳＲＹ基因缺失。该结果表明能够用分子生物学技术对性反转、性发育异常的病因进行分析。     

SRY基因序列分析8例

为寻找性反转及生殖器发育不良患者的病因,对８例患者在染色体核型分析的基础上,利用ＰＣＲ扩增技术对患者的ＳＲＹ基因进行检测和序列分析。结果：８例患者的ＳＲＹ基因全部存在;ＤＮＡ序列分析发现１例４６,ＸＸ女性伴ＳＲＹ基因存在的患者在ＨＭＧ盒内有点突变,即ｃｏｄｏｎ１１２Ｇ→Ｃ。１例４６,ＸＹ女性性反转综合征患者有移码突变和点突变并存。为患者从分子基础上找到了病因,并为研究性反转及生殖器发育不良提供了科学的有价值的资料。     

原发性闭经患者60例染色体分析

我院对原发闭经患者６０例进行了杂色体检查，染色体异常者１４例，占原发闭经患者２３．３％，其申４５，Ｘ８例；４５．Ｘ／４６，ＸＸ嵌合体３例；４６，Ｘ，ｄｅｌ（Ｘｑ）１例；４６，ＸＹ２例．对染色体异常形成原因进行分析．     

带HSV—tk基因的重组腺病毒治疗肿瘤的初步研究

以４种人体肺癌细胞株（Ａ５４９，ＬＡＸ，ＳＰＣ和ＳＫＹ）为实验材料，研究腺病毒介导的单纯疱疹病毒脱氧胸苷激酶基因（ＨＳＶ－ｔｋ）结合更昔洛韦（ＧＣＶ）对肿瘤细胞的杀伤作用。在离体实验的基础上，以Ｓ１８０肿瘤细胞进行活体实验。小鼠腋下接种Ｓ１８０细胞，当能触摸到瘤块时在瘤内注入ＡＤＶ／ＲＳＶ－ｔｋ，并腹腔注射ＧＣＶ连续５ｄ；种瘤后１０ｄ杀死小鼠剥离瘤块称重，治疗组瘤重为对照组的１／１４。实验结果表明，以腺病毒为载体介导ＨＳＶ－ｔｋ基因结合ＧＣＶ作用对肿瘤细胞有强杀伤性，是有应用前景的肿瘤基因治疗的一种疗法     

地奥心血康对无症状性心肌缺血的治疗作用

目的：观察地奥心血康（ＤＡＸＸＫ）对无症状性心肌缺血（ＳＭＩ）的治疗作用。方法：ＳＭＩ的冠心病７６例（男性４７例，女性２９例：年龄５４±ｓ７ａ），采用ＤＡＸＸＫ２００ｍｇ，ｐｏ，ｔｉｄ，共治疗１ｍｏ。结果：ＳＭＩ昼夜发作次数、心肌缺血程度和病人的生活质量均有显著改善（Ｐ＜０．０５）；昼夜中ＳＭＩ发生的时间规律从１ｄ出现２个高峰时间带变为１个，且时间提前（Ｐ＜０．０５）。结论：ＤＡＸＸＫ对ＳＭＩ有较好的疗效。     

地奥心血康对无症状性心肌缺血的治疗作用

目的：观察地奥心血康（ＤＡＸＸＫ）对无症状性心肌缺血（ＳＭＩ）的治疗作用。方法：ＳＭＩ的冠心病７６例（男性４７例，女性２９例：年龄５４±ｓ７ａ），采用ＤＡＸＸＫ２００ｍｇ，ｐｏ，ｔｉｄ，共治疗１ｍｏ。结果：ＳＭＩ昼夜发作次数、心肌缺血程度和病人的生活质量均有显改善（Ｐ〈０．０５）；昼夜中ＳＭＩ发生的时间规律从１ｄ出现２个高峰时间带变为１个，且时间提前（Ｐ〈０．０５）。结论：ＤＡＸＸＫ对ＳＭＩ有较     

97例系统性红斑狼疮伴心脏损害临床分析

本文对１７３例系统性红斑狼疮（ＳＬＥ）患者中伴心脏受损的９７例占（５６．１％）进行分析，诊断为心包炎３２．９％（５７／１７３），心肌炎２６．６％（４６／１７３），心内膜炎１６．７％（２９／１７３），肺动脉高压２．３％（４／１７３）。临床有症状者为３７．１％，提出结合心电图，超声心动图等检查手段，有助于提高其临床检出率。     

半巢式聚合酶链反应快速诊断单纯疱疹病毒性脑炎及病毒分型

目的：进一步提高ＰＣＲ诊断方法的特异性和敏感性。方法：在单纯疱疹病毒（ＨＳＶ）ＴＫ基因区设计了一对外层公用此物和内层分型引物,建立了半巢式ＰＣＲ检测和分型ＨＳＶ的方法。用该方法检测了天津地区６４例病毒性脑炎患者（病脑组）,和４６例其他中枢神经系统疾病患者（对照组）的脑脊液（ＣＳＦ）标本。结果：病例组阳性率为１５．６３％（１０／６４）,其中９例为ＨＳＶ－１感染,１例为ＨＳＶ－２感染。对照组阳性率为２     

接种表达细胞因子基因的重组HIV-1基因组DNA疫苗能增强HIV-1特异性免疫应答

１型人类免疫缺陷症病毒（ＨＩＶ－１）ＤＮＡ疫苗能诱导实验动物产生体液和细胞免疫应答,但这些免疫应答弱且短暂。ＤＮＡ疫苗免疫研究结果证实,白细胞介素２（ＩＬ－２）或粒细胞巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）基因与ＨＩＶ－１基因共表达可增强抗原特异性抗体应答。 作者以ＨＩＶ－１基因组为载体,通过ＤＮＡ重组技术分别构建了缺失ｔａｔ及ｎｅｆ基因（以消除其免疫抑制作用）但含有ＩＬ－２、ＧＭ－ＣＳＦ及γ干扰素（ＩＦＮ－γ）基因的重组质粒,分别称为ｐＴＸ ＧＥ／ＩＬ－２、ｐＴＸ ＧＥ／ＧＭ－ＣＳＦ及 ｐＴＸ ＧＥ…     

缩宫素生物活性测定法的初步研究

缩宫素生物活性测定法的初步研究ＰＲＥＬＩＭＩＮＡＲＹＳＴＵＤＹＯＮＡＳＳＡＹＭＥＴＨＯＤＯＦＢＩＯＬＯＧＩＣＡＬＡＣＴＩＶＩＴＹＯＦＯＸＹＴＯＣＩＮ王春林阚鸿顺（泰州生物化学制药厂，江苏２２５３００）ＷＡＮＧＣｈｕｎ－Ｌｉｎ，ＫＡＮＨｏｎｇ－Ｓｈｕｎ...     

性分化异常患者SRY基因检测及病因分析

目的探讨SRY基因在性别分化和发育中的作用。方法应用染色体核型分析和聚合酶链式反应(PCR)对人类性别决定区域(SRY)基因进行特异性扩增。结果在18例患者中有5例为46,XY,SRY( )睾丸女性化综合征患者;4例为46,XX女性性反转患者,其中3例为SRY( ),1例SRY(-);2例为46,XX,SRY( )两性畸形患者;2例为47,XXY克氏征;其他5例患者SRY基因与染色体性别一致,但都伴有不同程度的性发育异常。结论SRY基因的缺失或易位是导致性分化异常的最主要原因,同时性别决定和分化还有其他相关基因参与。     

Bisphenol A induces a shift in sex differentiation gene expression with testis-ova or sex reversal in Japanese medaka (Oryzias latipes)

Bisphenol A (BPA), a very important raw material in the plastics industry, is an endocrine-disrupting chemical in teleost fish. Although BPA induces testis-ova and sex reversal in teleost fish species, the molecular mechanism remains unclear. We evaluated the effects of BPA (measured concentrations: 45, 92, 326, 1030 and 3406 μg/L) on Japanese medaka (Oryzias latipes) using OECD TG234 (2011, Fish Sexual Development Test, OECD Guidelines for the Testing of Chemicals, Section 2). BPA at 1030 and 3406 μg/L induced testis-ova and sex reversal with female-type secondary sexual characteristics in XY males at 30 and 60 days posthatching (dph). Then we examined the BPA effect on the expression of sex differentiation genes related to the testis-ova and sex reversal in XY medaka. BPA exposure (1030 and 3406 μg/L) suppressed gsdf mRNA expression and increased cyp19a1a mRNA expression in XY individuals at stage 38 and 30 dph, although foxl2 mRNA expression showed no change. Interestingly, the concentration of BPA that suppressed gsdf mRNA expression at the larval stage was consistent with that needed to induce testis-ova and sex reversal. These results suggest that the gsdf gene at the embryonic stage can be used as a useful biomarker for predicting the impact of estrogenic endocrine-disrupting chemicals on sexual differentiation in Japanese medaka.     

男性不育症患者RBM、DAZ基因检测的临床意义

目的 探讨男性不育症与RBM，DAZ基因的关系及意义。方法 应用PCR技术对50例无精子症和严重少数子症患者（其中无精子症38例，严重少精子症12例）的外周血细胞进行RBM，DAZ基因检测。结果 50例中发现6例有DAZ基因微缺失（无精子症4例；严重少精子症2例），其中2例合并RBM基因微缺失（无精子症和严重少精子症各1例）；1例无精子症仅有RBM基因微缺失；SRY基因PCR扩增均为阳性，50例已有生育的正常男性均无RBM及DAZ基因的微缺失。结论 RBM和DAZ为无精子因子（AZF）的重要候选成分，RBM及DAZ微缺失是引起无精子和严重少精子并造成男性不育的重要原因之一。对男性不育症患者进行RBM及DAZ基因检测有一定临床意义。     

46XY女性性反转综合征一例sry基因的错义突变分析

目的 探讨46XY女性性反转综合征患者发病的分子机制. 方法 应用荧光原位杂交技术(FISH)、聚合酶链反应(PCR)扩增、限制性内切酶分析、单链构象多态性分析及基因测序技术对1例46XY女性性反转综合征进行sry基因的错义突变分析. 结果 本例FISH证实为X、Y染色体结构,无嵌合体存在;PCR扩增出现609 bp特...     

Effects of 4-tert-pentylphenol on the gene expression of P450 11beta-hydroxylase in the gonad of medaka (Oryzias latipes)

Alkylphenols including 4-tert-pentylphenol (4-PP) have been shown to alter sexual differentiation in fish due to their estrogenic properties. Medaka (Oryzias latipes) is so sensitive to these substances because morphological sex reversal and testis-ova induction are well developed in the exposed males. However, little work has been done to characterize the molecular effects of estrogenic substances on the morphological and gonadal feminization in male fish. Cytochrome P450 11beta-hydroxylase (P450(11beta)) is a key steroidogenic enzyme in production of 11-ketotestosterone which is the predominant androgen in male fish. In this study, we cloned a cDNA encoding medaka testicular P450(11beta), and then investigated the gene expression of P450(11beta) in the testes of genetically male medaka exposed to 4-PP. The cDNA contains 1740 nucleotides that encode a protein of 543 amino acids, which shares 68.9% and 73.4% homology with testicular P450(11beta)s from Japanese eel (Anguilla japonica) and rainbow trout (Oncorhynchus mykiss), respectively. HeLa cells transfected with an expression vector containing the open reading frame of medaka P450(11beta) cDNA showed 11beta-hydroxylating activity in the presence of exogenous testosterone. Analysis of tissue distribution by RT-PCR showed great abundance of P450(11beta) mRNA in testis. In the partial life-cycle exposure with 4-PP, morphologically sex-reversal was observed in XY medaka exposed to 4-PP concentrations of > or =238 microg/L. Furthermore, exposure to 4-PP completely inhibited P450(11beta) mRNA expression in the gonads of sex-reversed XY fish at 60-day posthatch. These results suggest that xeno-estrogen 4-PP may have inhibitory effects on the synthesis of testicular 11-oxygenated androgens through downregulation of P450(11beta) expression in the genetically male fish.     

Effect of atrazine on metamorphosis and sexual differentiation in Xenopus laevis

There is a growing international concern that commonly used environmental contaminants have the potential to disrupt the development and functioning of the reproductive system in amphibians. One such chemical of interests is the herbicide atrazine. Effects of atrazine on sex differentiation were studied using wild-type Xenopus laevis tadpoles and all-ZZ male cohorts of X. laevis tadpoles, produced by mating wild-type ZZ male to sex-reversed ZZ male (female phenotype). Stage 49 tadpoles were exposed to 0.1-100 ppb atrazine or 0.27 ppb (1 nM) 17beta-estradiol (E(2)) until all larvae completed metamorphosis (stage 66). Metamorphosis, gonadal morphology and histology, CYP19 (P450 aromatase) mRNA induction, and hepatic vitellogenin (VTG) induction were investigated. Effects of atrazine on VTG-induction were also assessed in vitro in primary-cultured X. laevis hepatocytes. Atrazine had no effect on metamorphosis of developing wild-type or all-male X. laevis larvae. Statistical increase in female ratios was observed in 10 and 100 ppb atrazine groups in comparison with control group. While no hermaphroditic froglet was observed in all atrazine groups. In ZZ males, sex reversal was induced by 0.27 ppb E(2), but not by atrazine at concentrations of 0.1 and 1.0 ppb. In addition, neither P450 aromatase mRNA in the gonad nor hepatic VTG were induced by atrazine. Furthermore, VTG was not induced by 1000 ppb atrazine in primary-cultured hepatocytes. Our results indicate that female ratios in developing X. laevis tadpoles were increased by 10 and 100 ppb atrazine under the present experimental conditions. While the other endpoints showed no effect in the range of 0.1-100 ppb atrazine. These results suggest that effect of atrazine on sexual differentiation was not caused by estrogenic action and has no induction ability of P450 aromatase gene in gonad.     

Developmental genetics and physiology of sex differentiation in vertabrates

The involvement of the Y chromosome in sex determination was determined by the development and the application of techniques for karyotyping the mammalian chromosome in 1960s. There were many reports on the particular region of the Y chromosome, such as histocompatibility (H-Y) antigen, bandit krait minor satellite (Bkm) the zinc finger Y gene (ZFY) and the sex-determining region of the Y chromosome (SRY) which were believed to be the testis determining factors. However, converging experimental evidence have indicated that the sex determining region of the Y chromosome (sry) is the testis determining factor (TDF) in mammalian species since sex is determined genetically at the time of fertilization in these species. In non-mammalian vertebrates especially in fishes, amphibians and reptiles, genotypic sex can be overridden by the external application of steroid hormones and temperature. In this review paper, after reviewing the complex literature on the molecular and biochemical mechanisms of sex determination and differentiation in all vertebrates, the potential danger of environmentally induced sex determination will be focused on.     

17-alpha-ethinylestradiol affects reproduction, sexual differentiation and aromatase gene expression of the medaka (Oryzias latipes)

Juvenile medaka (Oryzias latipes) of the d-rR strain were exposed for 2 months to 1, 10 and 100 ng/l of the environmental estrogen 17-alpha-ethinylestradiol (EE(2)). The exposure period was followed by a 6 week recovering period in order to detect long-lasting effects on sexual differentiation. Survival rate, sex ratio, gonadal growth, spawning, fecundity, histology as well as the ovarian gene expression of aromatase were monitored. At 100 ng/l EE(2), all XY medaka were sex reversed and had developed an ovary. At lower EE(2) concentrations, which did not result in sex reversal, no alteration of testicular structure was detected and male fertility appeared to be unchanged. In XX females, reduced production of eggs was reflected in a significantly reduced gonadal weight at 10 and 100 ng/l EE(2). Aromatase, which in gonads is normally only expressed in ovaries, was detectable in testis of XY males exposed to 10 ng/l EE(2). The results indicated that a combination of different biological endpoints provide a suitable set of parameters for the biological evaluation of xenoestrogens, since the expression of molecular marker genes was not always paralleled by morphological deficiencies.     

Male‐specific induction of CYP3A2 in rats by zolmitriptan

We report here a novel observation that zolmitriptan induced CYP3A2 in male but not female rats. As part of our research programme to evaluate sex differences in the response to zolmitriptan, we studied the effects of zolmitriptan on CYP3A activity, protein and gene expression in male and female rats. Zolmitriptan was found to induce CYP3A activity, measured as testosterone and diazepam metabolism in‐vitro, as well as midazolam pharmacokinetics in‐vivo, in male but not female rats. The sex difference in response to zolmitriptan was further evaluated by analysis of CYP3A1/2 mRNA levels using real‐time PCR, and CYP3A1/2 protein levels using immunoblotting. Zolmitriptan preferentially induced CYP3A2 in male but not female rats. No obvious effects on CYP3A1 were observed at any dose in either sex. Thus, we concluded that the observed sex‐dependent induction of CYP3A by zolmitriptan was largely due to induction of CYP3A2 in male rats.