慢性肾功能衰竭患者线粒体基因组D-loop区突变研究

目的通过研究慢性肾功能衰竭（CRF）患者及家系成员中线粒体D-loop区的基因变异情况,探讨其与CRF发病之间可能的联系。方法收集42例CRF患者（CRF组）和95名家系成员（UAPM组）,以及随机选取的122例正常人（对照组）的外周血,提取线粒体DNA,通过PCR扩增D-loop区并对产物纯化,然后进行基因测序来检测线粒体基因组D-Loop区域基因变异情况,并进行比较,分析该区域基因变异与CRF发病的可能相关性。结果通过与对照组及标准序列比较,共发现79个核苷酸改变,3个高频变异位点,其中总的碱基突变率在CRF组及UAPM组明显增加,两组与对照组碱基变异率差异均有统计学意义（均P＜0.01）。CRF组及UAPM组在D310区线粒体微卫星不稳定（mtMSl）及碱基变异率与对照组比较差异均有统计学意义（均P＜0.01）。结论线粒体基因组D-Loop区突变可能与CRF的发生、发展有一定的关系。     

腹针对多囊卵巢综合征(PCOS)促排卵的临床疗效

目的分析在多囊卵巢综合征患者实施促排卵治疗时使用腹针的效果。方法将80例多囊卵巢综合征患者分为对照组和观察组,分别使用药物治疗和腹针治疗。结果相比对照组,观察组患者的排卵率明显更高,差异有统计学意义(P0.05)。而两组患者的并发症发生率差异无统计学意义(P0.05)。结论相比常规药物治疗,腹针治疗可对多囊卵巢综合征患者起到更加有效的效果,同时安全性也可得到保证,有较高应用价值。     

自身免疫性甲状腺病与多囊卵巢综合征之间相关性研究

目的探讨自身免疫性甲状腺病与多囊卵巢综合征之间的相关性。方法选取2016年5月～2017年5月我院收治的多囊卵巢综合征伴糖脂代谢紊乱患者45例为研究组,选取同期我院收治的多囊卵巢综合征未伴糖脂代谢紊乱患者45例为对照组。结果研究组FSH低于对照组,差异有统计学意义(P0.05),研究组LH、雌二醇、催乳素以及睾酮高于对照组,差异有统计学意义(P0.05);研究组FT3、FT4、TSH以及TSH2.5μIU/mL例数与对照组比较,差异无统计学意义(P0.05);研究组TGAb、TPOAb高于对照组,差异有统计学意义(P0.05),研究组TGAb60 U/mL例数、TPOAb60 U/mL例数高于对照组,差异有统计学意义(P0.05)。结论自身免疫性甲状腺病可能会参与患者多囊卵巢综合征的发病并使其代谢表型恶化。     

Klinefelter综合征及双亲X染色体着丝粒区α-卫星DNA变异研究

目的:本文从DNA分子水平对Klinifelter综合征患者及双亲X染色体着丝粒区域的α-卫星DNA变异进行研究,探讨Klinefelter综合征患者X染色体不分离形成的原因.方法:采用(representative sampling of multiple repetitive units,rep)PCR方法,通过设计适当的引物,对-卫星DNA多个拷贝串联重复序列同时进行扩增,检测患者、双亲和正常人重复序列中变异发生.结果:(1)扩增产物中分别出现1774bp、570bp(由缺失产生)片段和1410bp、583bp(由缺失产生)、220bp(由缺失产生)片段,(2)患者组570bp和583bp产生率高于正常组,x2统计有显著性意义(P＜0.05).(3)患者与患者双亲比较差异不显著.结论:Klinefelter综合征患者及双亲的X染色体着丝粒区α-卫星DNA表现出较正常人具更多的变异.提示通过孕前或产前对双亲外周血X染色体着丝粒区域的α-卫星DNA变异分析,对Klinefelter综合征发生风险进行估计,为产前绒毛或羊水染色体检查提供重要的依据.     

中药苍附导痰丸对多囊卵巢综合征病人的治疗效果观察

目的探究中药苍附导痰丸对多囊卵巢综合征病人的治疗效果。方法选取102例多囊卵巢综合征患者,对照组采用克罗米芬治疗,观察组采用中药苍附导痰丸治疗,考察患者的治疗效果、血清性激素指标、排卵率和妊娠率。结果观察组的总有效率(90.20%)显著高于对照组(70.59%),治疗前两组血清性激素指标均无统计学差异(P 0.05),治疗后观察组的FSH、LH、T显著低于对照组(P0.05),两组患者的排卵率和妊娠率不存在统计学差异(P 0.05)。结论中药苍附导痰丸治疗多囊卵巢综合征的效果良好。     

Klinefelter综合征X染色体着丝粒区α-卫星DNA变异

目的从DNA分子水平建立研究X染色体着丝粒区域的α-卫星DNA变异的方法,发现Klinefelt综合征患者的X染色体着丝粒区域的α-卫星DNA串联重复序列结构变化与染色体不分离的关系.方法采用(representative sampling of multiple repetitive units,rep)PCR方法,通过设计适当的引物,对α-卫星DNA多个单拷贝串联重复序列同时进行扩增,检测重复序列中结构变化的种类、频率及重组位点.结果首次发现由缺失产生的562 bp和220 bp变异片段和重组位点,异常组的570、562、220 bp构成显著高于正常组(P＜0.05).Logistic回归分析显示:570、562bp和220 bp 3个短片段同时出现和任两个短片段同时出现均是疾病相关的危险因素.结论Klinefelt综合征的X染色体着丝粒区α-卫星DNA过多的重组可能是造成X染色体不分离的重要原因之一.     

中医周期疗法对多囊卵巢综合征患者卵泡发育及血流动力学的影响

目的:分析中医周期疗法对多囊卵巢综合征患者卵泡发育及血流动力学的影响。方法:将80例多囊卵巢综合征患者随机分为对照组与试验组,每组各40例,对照组采取常规西药治疗,试验组在对照组基础上行中医周期疗法治疗,比较两组患者临床疗效、卵泡发育及子宫、卵巢血流动力学情况。结果:试验组治疗有效率、排卵率、基础体温双相率分别为85.0%、65.0%、75.0%,较对照组的55.0%、40.0%、52.5%,差异有统计学意义(P0.05)。试验组平均窦卵泡数(6.01±0.42)个,成熟卵泡数(1.52±0.41)个,对照组平均窦卵泡数(8.79±0.65)个、成熟卵泡数(1.20±0.48)个,两组比较,差异有统计学意义(P0.05)。两组患者子宫、卵巢血流动力学参数比较,差异有统计学意义(P0.05)。结论:中医周期疗法能明显促进多囊卵巢综合征患者卵泡发育及排卵,显著改善子宫、卵巢血流动力学指标,疗效确切。     

补肾调经汤治疗多囊卵巢综合征所致不孕临床研究

目的:观察补肾调经汤治疗多囊卵巢综合征肾虚证所致不孕的临床疗效。方法:将89例多囊卵巢综合征肾虚证所致不孕患者按照随机分组法分为治疗组46例和对照组43例。对照组给予常规西药治疗,治疗组在对照组基础上加补肾调经汤治疗。两组患者疗程均为3个月经周期。比较两组患者临床疗效、激素水平、排卵情况、妊娠情况、卵巢体积、子宫内膜厚度和不良反应情况。结果:治疗组有效率为93.48%,对照组有效率为69.77%,两组比较,差异有统计学意义(P0.05);两组患者雌二醇(estradiol,E_2)、卵泡刺激素(follicle-stimulating hormone,FSH)治疗后水平较治疗前无明显变化,差异无统计学意义(P0.05),而促黄体生成素(luteinizing hormone,LH)治疗后较治疗前显著减少,差异有统计学意义(P0.05),治疗组LH治疗后显著低于对照组,而E_2、FSH治疗后水平较对照组无明显变化,差异无统计学意义(P0.05);治疗组排卵率为89.13%,对照组排卵率为72.09%,两组比较,差异有统计学意义(P0.05);治疗组妊娠率为86.96%,对照组妊娠率为55.81%,两组比较,差异有统计学意义(P0.05);两组卵巢体积治疗后较治疗前显著减少,差异有统计学意义(P0.05);子宫内膜厚度较治疗前显著增加,差异有统计学意义(P0.05);治疗组卵巢体积显著小于对照组,差异有统计学意义(P0.05);子宫内膜厚度显著高于对照组,差异有统计学意义(P0.05)。结论:补肾调经汤治疗多囊卵巢综合征肾虚证所致不孕临床疗效显著,补肾调经汤能够明显提高治疗疗效,降低LH水平,提高患者排卵和妊娠,增加子宫内膜厚度,减小卵巢体积,且未见明显不良反应。     

穴位埋线结合耳针对肾虚痰湿型多囊卵巢综合征月经过少患者临床疗效研究

目的研究穴位埋线疗法结合耳针治疗肾虚痰湿型多囊卵巢综合征月经过少的临床疗效。方法将86例肾虚痰湿型多囊卵巢综合征月经过少门诊患者随机分为对照组43例和观察组43例。对照组给予耳穴疗法,观察组在对照组的基础上采用穴位埋线疗法,观察2组患者治疗1个疗程(三次埋线治疗)后的临床疗效。结果两组患者临床疗效比较,对照组有效率为72.09%,观察组的有效率为86.05%,2两组差异比较有统计学意义,P0.05。两组患者中医证候疗效比较,观察组有效率90.68%,对照组有效率72.09%,两组差异比较有统计学意义,P0.05。结论穴位埋线疗法结合耳针是治疗肾虚痰湿型多囊卵巢综合征月经过少患者的有效方法。     

经阴道三维超声在多囊卵巢综合征超声评价中的应用价值

目的评价经阴道三维超声在多囊卵巢综合征超声评价中的应用价值。方法将25例多囊卵巢综合征患者设为观察组,25例因其他因素引发不孕的患者设为对照组,经阴道三维超声检查获得两组患者卵巢间质的血管化指数(VI)、血流指数(FI)、血管化-血流指数(VFI)、卵巢体积及卵泡数目等指标,同时测定两组患者内分泌指标。结果观察组患者VI、FI、VFI、卵巢体积及卵泡数目均高于对照组,差异有统计学意义(P<0.05)。观察组患者T、FSH、E2、PRL、P等内分泌指标水平均高于对照组,差异有统计学意义(P<0.05)。结论经阴道三维超声在多囊卵巢综合征超声评价中的应用价值较高。     

Relationship between mutations of mitochondrial DNA ND1 gene and type 2 diabetes

Background Recent studies have indicated that many mutations in mitochondrial (mt)DNA NDI gene region are related to diabetes mellitus. In this study we explored the relationship between various mtDNA ND1 gene mutations and type 2 diabetes mellitus (DM) among Chinese. Methods Using PCR restriction fragment length polymorphism (PCR-RFLP) analysis and gene sequencing, 4 spots of mtDNA (nt3243, nt3316, nt3394, nt3426) were screened in 478 diabetics and 430 non-diabetic subjects.Results In diabetic group, there were 13 carriers (2.72%)of 3316 G→A mutation,12 (2.51%) of 3394 T→C mutation and 2 (0.42%) of 3426A→G mutation. In controls, only 3394 T→C mutation was observed in 2 subjects (0.47%). There was significant difference in the frequency of 3316 and 3394 mutation between two groups (P&lt;0.05, respectively). More subjects with mitochondrial DNA ND1 gene mutations had DM family history and greater tendency of maternal inheritance when compared to those patients without mutation in diabetic group（P&lt;0.01）. A 3426 mutation diabetic pedigree was studied, and we found 12 maternal members in the family had the same mutation. Conclusion mtDNA ND1 gene mutations at nt3316 (G→A), nt3394 (T→C) and 3426 (A→G) might contribute to the pathogenesis of DM with other genetic factors and environment factors.     

Point mutations of muscle mitochondrial DNA from patients with mitoch ondrial encephalomyopathies

Objective To study the relation between point mutations at nt3243 and nt8344 of muscle mit ochondrial DNA from patients with mitochondrial encephalomyopathies and phenotyp es. Methods DNA was extracted from muscle specimens from 5 patients with mitochondrial encep halomyopathies and amplified by PCR method, using corresponding oligonucleotide primers. DNA fragments were digested with restriction enzymes BglⅠ and ApaⅠ, then the digested DNA fragments were analyzed with an electrophoresis method. Results The point mutation at nt3243 of mtDNA was found in 2 patients, one with mitochon drial encephalomyopathy, lactic acidosis and stroke- like episodes (MELAS) and a nother with myoclonic epilepsy with ragged red fibers (MERRF). The point mutati on at nt8344 was found in 2 patients with MERRF, including the one with point mu tation at nt3243. Conclusion The point mutation of DNA at nt3243 correlated with MELAS and nt8344 correlated with MERRF. In addition, the detection of point mutations at both nt3243 and nt 8344 in a patient with MERRF shows the association of mutation with diversity i n clinical manifestations of mitochondrial encephalomyopathies.     

Mass genetics study of rhodopsin point mutations in retinitis pigmentosa

Objective: To evaluate the incidence and pattern of rhodopsin (RHO) mutations in Chinese patients with retinitis pigmentosa (RP). Methods: Conformation sensitive gel electrophoresis (CSGE) and direct DNA sequencing were apphed to detect point mutations that occurred in the five coding exons and splice sites of RHO gene in 98 index patients with RP. Results: Four patients of one ADRP family were found to have a missense mutation at codon 347, Pro347Leu. One late-onset RP patient and her daughter, without clinical expression at present, were discovered to have a novel frameshift mutation at codon 327, Pm327 (1-bp del). Neither of the two mutations was found in 100 normal controls. Ma299Ser was found in one RP patient. Two control subjects also had Ma299Ser, suggesting its nonpathogenicity and just single nucleotide polymorphism (SNP). Conclusion: Two RP patients had rhodopsin mutations, thus the expected frequency of RHO mutations in RP is about 2.0% (95% confidence interval: 0.3%-4.4%). A highly conserved C-terminal sequence QVS（A) PA was altered due to Pro347Leu and thereby misdirecting rhodopsin to incorrect subcellular location. Loss of all phosphorylation sites at the C-tenninns and a highly conserved sequence QVS(A) PA may occur because of Pm327(1-bp del). To elucidate the predominant biochemical defects in such mutant, transgenic mice and transfected culture cells carrying Pm327(1-bp del) would be of great value.     

银染PCR-SSCP方法检测大肠癌组织线粒体DNA的突变

目的: 研究大肠癌组织线粒体DNA的突变情况. 方法: 用银染PCR-SSCP技术结合测序的方法检测40例大肠癌组织及其癌旁正常组织的线粒体DNA D-环区的点突变情况. 结果: 40例大肠癌组织中检测到7例点突变改变,突变率为18%(7/40),在检测的9个突变位点,3个突变位点存在于Dukes D期的1例大肠癌中,其他6个突变位点分别存在于Dukes C期的6例大肠癌组织中,其中498位C→ T, 16298位C→T两个突变位点在检测到点突变的7例大肠癌组织中均检测到. 结论:大肠癌组织线粒体DNA D-环区存在一定程度的点突变,突变的发生可能与大肠癌的易感性和恶性程度有一定相关性, 但是否是大肠癌的发病机制尚有待进一步研究.     

Screening for mt-DNA mutations in optic neuritis of unknown cause

Objective To investigate mitochondrial DNA (mt-DNA) mutations in optic neuritis of unknown cause（ONUC）and to assess the practical value of mt-DNA mutation detection in etiologically and differentially diagnosing ONUC. Methods Thirty patients with ONUC were screened for mt-DNA mutations of nt11778, nt3460 and nt15257 by using SSCP, mutation-specific primer PCR and sequencing.Results mt-DNA mutations were found in twelve of thirty ONUC patients.All of the mutations were at nt11778 position, but no one at nt3460 and nt15257. Conclusions Forty percent (12/30) of ONUC patients were caused by an mt-DNA mutation.Combined with other routine measures, screening for mt-DNA mutations in ONUC patients is of great significance in diagnosing ONUC etiologically and differentially.     

三个封闭群实验兔线粒体DNA D-loop区多态性分析

目的 通过对三个封闭群实验兔线粒体DNA控制区序列进行分析,从分子水平探讨其群体的多样性和均一性.方法 利用特异引物对新西兰白兔、日本大耳白兔、青紫蓝兔三个封闭群共71份血液样品线粒体D-loop区部分序列进行扩增,纯化,测序,用Mega4.0和DNA SP软件对变异位点数、简约信息位点数、单倍型、单倍型多样度、核苷酸多样度等遗传信息进行分析.结果 在71份样品中,共检测出了6个多态性位点,多为C→G突变,定义了9种单倍型,且三个种群分别有各自单倍型,互不重叠.三个种群D-loop区多态性及种群多样性均偏低.结论 本研究为兔线粒体D-Loop区序列特征和国内常用三个封闭群实验兔种群结构提供了更多数据,并为遗传育种提供指导.     

Mitochondrial D-loop variation in leber hereditary neuropathy patients harboring primary G11778A, G3460A, T14484C mutations: J and W haplogroups as high-risk factors

BACKGROUND: Leber hereditary optic neuropathy (LHON) is a maternally inherited form of retinal ganglion cell degeneration leading to optic atrophy in young adults. It is caused by three primary point mutations including G11778A, G3460A, and T14484C in the mitochondrial genome. These three mutations account for the majority of LHON cases and affect genes that encode for different subunits of mitochondrial complex I. Mitochondrial DNA (mtDNA) has a non-coding region at the displacement loop (D-loop) that contains two hypervariable segments (HVS-I and HVS-II) with high polymorphism. METHODS: To investigate any possible association between LHON primary mutations and mtDNA haplogroups (hg), the nucleotide sequence of the HVS-I region of mtDNA was determined in 30 unrelated Iranian patients with LHON harboring one of the primary mutations and 100 normal controls with the same ethnicity. DNA was extracted from the peripheral blood after having obtained informed consent. The nucleotide sequence of HVS-I (np 16,024-16,383) was directly determined. RESULTS: Our analysis revealed a relatively high proportion of haplogroup J in LHON patients (53.3%) compared to normal controls (20%). In addition, a slightly significant increase of normal controls of haplogroup L has been confirmed (14% in normal controls vs. 0% in LHON patients at p = 0.03), whereas other haplogroups did not show contribution to LHON contingency. CONCLUSIONS: The analysis presented here provides evidence that there is an association between G11778A and G3460A with haplogroup J (including J1 and J2) and W, respectively. Therefore, we hypothesize that mtDNA haplogroups J (J1 and J2) and W might act as predisposing haplotypes, increasing penetrance of LHON disease.     

SCREENING FOR MUTATIONS IN A NOVEL RETIAL—SPECIFIC GENE AMONG CHINESE PATIENTS WITH RETINTITS PIGMENTOSA

Objective.To identify and evaluate mutations in the RP1 gene among Chinese patients with retinitis pigmentosa(RP).Methods.Leukocyte DNA of 92 RP patients were collected in Hong Kong.Sequence changes of the entire coding region of the RP1 gene were examined using PCR,conformation sensitive gel electrophoresis and DNA sequencing.Results.In total,1 nonsense mutation and 1 nonsense variant as well as 10 missense alterations were identified in the RP1 gene,among which,Arg 677 Ter was found in 1 RP patient and another nonsense variant,Arg 1933Ter ,was identified in 3 normal individuals and 1 patient with Stargardt‘s disease,suggesting its nonpatogenicity,Arg667 Ter is expected to lead to large disruptions of the encoded protein.Couclusions.The nonpathogenicity of Arg 1933 Ter indicates that the C-terminal 224 residues of RP1 protein may be not critical for RP1.The most C-terminal truncation previously reported was due to Tyr1053(1-bp del) and occurred in RP patients.Thus RP can be caused by reduction in the level of the region of RP1 protein after condon 1052 but before 1933.T o ascertain such a proposition,genotypes of more RP patients may reveal more RP cousative mutations and more sequence alterations different than those of other ethnic groups.     

MMP-2、MMP-9及TIMP-1在多囊卵巢综合征病人种植窗口期内膜的表达及其临床意义

目的 检测基质金属蛋白酶-9(MMP-9)、基质金属蛋白酶-2(MMP-2)及组织金属蛋白酶抑制物-1(TIMP-1)在多囊卵巢综合征(PCOS)病人种植窗口期子宫内膜组织中的表达情况,并探讨其临床意义。 方法 选择接受促排卵后治疗的PCOS病人(PCOS组)12例和因男方因素不孕且月经正常前来就诊病人(对照组)15例,检测2组排卵后第7天子宫内膜组织中MMP-9、MMP-2和TIMP-1蛋白表达情况,利用病理图像分析技术,对实验结果进行半定量分析。 结果 MMP-9、MMP-2和TIMP-1在2组子宫内膜组织中均呈阳性表达,在间质细胞和腺上皮细胞的细胞质内均有棕黄色颗粒物沉着,以腺上皮表达为主。PCOS组子宫内膜腺上皮MMP-2、MMP-9及TIMP-1表达均明显低于对照组(P < 0.01),子宫内膜间质MMP-9表达亦低于对照组(P < 0.05)。 结论 MMP-9、MMP-2和TIMP-1在PCOS组病人的种植窗口期子宫内膜中的表达水平较低,这可能是导致PCOS病人采用促排卵治疗后妊娠率低、流产率高的原因之一。     

A 63 kDa VSP9B10A-like protein expressed in a C-8 Giardia duodenalis Mexican clone

BACKGROUND: It is well documented that Giardia duodenalis undergoes surface antigenic variation both in vivo and in vitro. Proteins involved have been characterized and referred to as VSP (variable surface protein). METHODS: Two cloned cDNA inserts of 0.45 and 1.95 kb were obtained from G. duodenalis expression library and sequenced. Comparison sequence analyses were made against Genbank. PCR analysis was performed on G. duodenalis isolates to identify isolates bearing genes encoding such a peptide. Specific antiserum was prepared against 450-bp encoded peptide and tested by Western blot, immunofluorescence, and inhibition of adhesion of G. duodenalis to target cells. RESULTS: We cloned and characterized a G. duodenalis 450-bp DNA fragment; its DNA sequence analysis revealed that this fragment displayed 99% identity with vsp9B10A gene. Predicted amino acid sequence for this fragment also had significant (99%) identity to VSP9B10A. A second 1.95-kb insert, which encompassed the 450-bp cDNA fragment, was also isolated; its DNA and amino acid sequence displayed 99.5% identity with vsp9B10A gene and 99.2% with the corresponding inferred protein, respectively. This inferred protein contained 24 Cys-X-X-Cys motifs and long ORF of 642 aminoacids. PCR analysis showed that DNA sequence encoding a fragment of this gene was present in P1, CIEA:0487:2-C-8 clone and in INP:180800-B2 G. duodenalis human isolates, while it was absent in sheep isolate of G. duodenalis INP:150593-J10. CONCLUSIONS: Immunofluorescence analysis using antibodies raised against the peptide encoded by 450-bp fragment showed that expression of this epitope varies on trophozoite surface of the C-8 Mexican clone and is involved in parasite adhesion to target epithelial cells.