聚合酶链反应产物特异性的阳性证实

以聚合酶链反应(PCR)法在mRNA水平检测T淋巴细胞受体α链可变区基因表达为例,介绍用~(32)P标记的人工合成寡核苷酸探针对PCR产物特异性作阳性证实的方法。该法以干琼脂糖凝胶作为支持物、相对较为简便和省财。用Ca探针以干凝胶作支持物的杂交结果,证实29个Vα基因的PCR扩增中物均为特异性的,放射自显影的带型与位置和溴乙锭染色所示完全吻合。     

反义寡核苷酸抑制肿瘤细胞表达VEGF的研究

研究用反义寡核苷酸（ＯＤＮ）抑制肿瘤细胞ＶＥＧＦ的表达，探讨发展新的抗肿瘤药物的可能性。方法Ｓ１８０细胞培养上清液有促进牛脐血管内皮细胞生长的作用。用ＶＥＧＦ的反义ＯＤＮｓ分别加入Ｓ１８０细胞培养液，检查各上清液刺激内皮细胞生长的受抑情况，即可了解ＯＤＮｓ抑制肿瘤细胞表达ＶＥＧＦ的情况。结果证明ＶＥＧＦ反义ＯＤＮｓ确有抑制ＶＥＧＦ表达的作用。结论反义ＶＥＧＦ的ＯＤＮｓ有望成为新一代的抗肿瘤药物。     

Light and electron microscopic study of the effect of L-carnitine on the sperm morphology among sub fertile men

ObjectiveThis work was conducted to evaluate the effect of L-carnitine on sperm morphology in sub fertile patients who need enhance for intra cytoplasmic sperm injection (ICSI) as a method of infertility treatment.SettingAssisted Reproduction Unit, at the International Islamic Centre for Population Studies and Researchs (IICPSR), Al-Azhar University and Zoology Department Faculty of Science (Girl's Branch), Al-Azhar University.Materials and methodsAccording to the routine semen analysis, 85 patients were divided into:Group 1: Ten normal fertile men.Group 2: 25 oligozoospermic cases with sperm count less than 20 million/ml.Group 3: 25 athenozoospermic cases with reduced sperm motility <40%.Group 4: 25 teratozoospermic cases with more than >40% abnormal forms.L. carnitine therapy in the form of Carnivita forte 1gm /tab. b.i.d. for three months were given to groups 2, 3 and 4. All the patients underwent semen analysis before and 90 days after therapy. Group 1 was subjected to two semen analysis 3 months apart as a control. Smears were made on slides, fixed in absolute ethyl alcohol and stained with H&E and methyl green pyronin for light microscopic study. A second semen sample was processed for electron microscopic study.ResultsIn the fertile control group, there have not been any statistically significant differences among the patients at the beginning of the study and after 3 months regarding all the studied parameters. In Oligo group, highly significant decrease in the mean percent of head defects, cytoplasmic droplet and mitochondrial sheath defects were observed after treatment. In both atheno and terato groups, highly significant decrease in the mean percent of head defects, midpiece defects, cytoplasmic droplet and mitochondrial sheath defects were observed after treatment. The mean of DNA content of sperm heads demonstrated highly significant increase in oligo, atheno and terato-zoospermia groups as compared to the same groups before treatment.ConclusionThese results clearly show that L-carnitine treatment has ameliorative impact on the quality of spermatozoa in the infertile men, resulting in a decrease number in morphologically abnormal spermatozoa. These data indicated that this agent affects the quality of spermatozoa in infertile patients who need intra cytoplasmic sperm injection (ICSI) as a method for infertility treatment.     

体外培养大鼠骨髓间充质干细胞成骨基因表达谱的检测

目的:探讨体外培养大鼠骨髓间充质干细胞(MSC)成骨相关基因的表达谱,明确体外培养大鼠骨髓MSC的成骨潜能。方法:体外培养SD大鼠骨髓MSC,取第3代细胞提取总核糖核酸(RNA),制备杂交探针,并运用SuperArray公司提供的大鼠Oligo GEArray骨再生基因芯片(每张芯片可测113种基因)进行杂交,化学发光检测,通过X线胶片或台式扫描获得化学发光的芯片图像,使用网上提供的综合型GEArry表达分析配套软件(GEArryExpression Analysis Suite)进行完整的芯片数据分析。结果:检测体外培养大鼠骨髓MSC的113种成骨相关的基因,其中有31种基因表达强度较低,45种基因呈中等强度表达,37种基因表达强度较高。结论:体外培养大鼠骨髓MSC具有部分成骨细胞基因表型特征,但成熟成骨细胞的特征基因表达量较低,提示大鼠MSC是具有较强成骨细胞分化潜能的成纤维样细胞。     

病毒性肝炎联合诊断寡核苷酸芯片的临床应用性研究

目的：制备联合诊断乙型（HBV）、丙型（HCV）、丁型（HDV）肝炎病毒寡核苷酸（Oligo）芯片,并进行系列优化及临床应用性研究。方法：首先利用软件Array designer 3.0对BLAST检索所得的HBV、HCV、HDV种属保守序列逐一分析,设计出60mer Oligo探针。用PixSys5500型基因芯片打印仪将设计好的探针打印到氨基化玻片上制备成基因芯片,并对杂交条件进行系列优化以适应临床血清标本的检测。结果：芯片杂交条件进行优化后,芯片检测的敏感性、特异性都大大增强,还缩短了检测时间;杂交结果显示,Oligo芯片检测HBV、HCV、HDV的敏感性、特异性分别为92.2%,85.7%,91.3%和96.0%,94.5%,98.0%。结论：制备的长链Oligo集合肝炎病毒检测芯片检测敏感性、特异性均佳,为下一阶段进行中试规模的临床血清样品检测做好了准备,同时也为集合更多种、不同亚型肝炎病毒联合检测芯片的制备打下了坚实的基础。     

抗氧化剂保护少弱精子离心过程氧化应激损伤的实验研究

目的：探讨抗氧化剂谷胱甘肽、左卡尼汀对少弱精子体外离心过程中抗氧化应激损伤的作用.方法：按WHO标准选取38份少弱精子样本,每份样本各取1350μL,分为对照组、谷胱甘肽组和左卡尼汀组,各450μL.对照组仅加入EBSS平衡液,谷胱甘肽组加入含有一定水平谷胱甘肽的EBSS平衡液,左卡尼汀组加入左卡尼汀的EBSS平衡液,检测三组活性氧（ROS）、丙二醛（MDA）及精子DNA断裂指数（DFI）并进行比较、分析.结果：三组组间比较均有统计学意义（F活性氧=9.45、P=0.000;F丙二醛=15.79,P=0.000;FDFI=13.56,P=0.000,P均〈0.05）;两两比较示谷胱甘肽组与左卡尼汀组均较对照组间的活性氧、丙二醛及DFI水平明显降低,差异有统计学意义（P均〈0.05））;而谷胱甘肽组及左卡尼汀组间的比较均无统计学意义（P均〉0.05）.结论：在精液离心前添加一定浓度的左卡尼汀或谷胱甘肽可减少离心过程中产生的过量活性氧对精子的氧化应激性损伤,从而提高精子质量.     

人乳头状瘤病毒感染型别与子宫颈上皮内瘤变的关系

目的采用聚合酶链反应(PCR)-寡核苷酸微流芯片进行人乳头状瘤病毒(HPV)分型,并研究子宫颈上皮内瘤变(CIN)与HPV感染型别的关系。方法对335例临床病例进行筛查、细胞学分级并分组,活检标本通过寡核苷酸微流芯片上特异性探针进行HPV分型。结果335例患者中,HPV阳性率为22．1%。宫颈良性疾病组的HPV阳性率为10．9%,CINⅠ、CINⅡ、CINⅢ和宫颈鳞癌组分别为47．2%、66．7%、68．4%和75．0%,均显著高于宫颈良性疾病组(P值均＜0．05)。结论随着CIN级别的升高,高危HPV型感染比例增加,寡核苷酸微流芯片检测能区分多种HPV型别,可用于临床宫颈上皮内瘤变的HPV筛查。     

Inhibition by nitroso-chloramphenicol of the proton translocation in mitochondria

We have found recently that, unlike chloramphenicol (CAP), its nitroreduction derivative nitroso-chloramphenicol (NO-CAP) behaved as a potent inhibitor of the energy-conserving mechanism in mitochondria [Abou-Khalil et al. Biochem. Pharmac.29, 2605 (1980)]. Concentrations of 75 and 250 μM NO-CAP were required to inhibit ATP formation with glutamate and succinate, respectively, whereas similar CAP concentrations were without effect. Testing several key reactions associated with the biosynthesis of ATP, inhibitory concentrations of NO-CAP were found to interfere as follows: (a) the transport of an NAD-linked substrate (e.g. glutamate) into mitochondria was only partially inhibited, whereas that of an FAD-linked substrate (e.g. succinate) was not inhibited but was rather slightly activated; (b) the transport of P_i was only inhibited at about 50%; (c) mitochondrial ADP transport was not affected at all; (d) the ATPase activity, measured either by Pi release in the presence of an uncoupler or by H⁺ ejection, was only slightly affected; and (e) under either phosphorylation or no phosphorylation conditions and in the absence of P_i, NO-CAP was found to completely block mitochondrial H⁺ extrusion resulting from the oxidation of either succinate or glutamate; however, under similar conditions the oxidation of the two substrates was not totally inhibited. The possibility of interference by NO-CAP with reactive mitochondrial thiols groups is discussed in the light of previous data and current experiments showing protection by P_i against NO-CAP effects on respiration. Moreover, NO-CAP as compared to conventional inhibitors of oxidative phosphorylation (e.g. rotenone, antimycin A, oligomycin, mersalyl and others) appeared to have a distinct mode of action on that process. The results demonstrate that the inhibitory effect of NO-CAP is primarily located at the respiratory chain level where the proton translocation activity is fully blocked.     

High selenium impairs hepatic insulin sensitivity through opposite regulation of ROS

Insulin resistance is the hallmark of type 2 diabetes. As an essential trace element, selenium (Se) is recommended worldwide for supplementation to prevent Se-deficient pathological conditions, including diabetes and insulin resistance. However, recent evidence has shown that supra-nutritional Se intake is positively associated with the prevalence of diabetes. In the present research, we examined the effect of high Se on insulin sensitivity, and studied possible mechanisms in rats and in rat hepatocytes. Insulin sensitivity and glucose/lipid metabolism were determined by glucose/insulin tolerance test, western blot, immunofluorescence, specific probes and other biochemical assays. We show that high Se activates selenoproteins, including glutathione peroxidase and selenoprotein P, and depletes chromium, leading to a common metabolic intersection—lipolysis in adipose tissue and influx of fatty acids in liver. Fatty acid β-oxidation generates acetyl-CoA, which is metabolized in trichloroacetic acid cycle, supplying excessive electrons for mitochondrial oxidative phosphorylation and leading to increased “bad” reactive oxygen species (ROS) production in mitochondria and final disturbance of insulin signaling. Furthermore, high Se-activated selenoproteins also weaken insulin-stimulated “good” ROS signal generated by NAD(P)H oxidase, leading to attenuation of insulin signaling. Taken together, these data suggest that excessive intake of Se induces hepatic insulin resistance through opposite regulation of ROS.