PCR法快速诊断急性重症胰腺炎早期合并细菌感染

目的探讨以16srRNA基因为基础的细菌聚核酶链反应（PCR）法诊断急性重症胰腺炎早期合并细菌感染的价值。方法收集17例急性重症胰腺炎患者（女13例，男4例，年龄17～79岁）不同时期、不同部位标本45份，其中17份来自细针穿刺（FNA），28份为术中标本。获取胰周渗液后，行细嚣通用引物PCR法检测。并与常规细菌培养结果进行比较。结果10例最终诊断为胰腺感染，并行手术治疗；7例最终诊断为胰腺未感染，5例经保守治疗治愈，2例死亡。常规培养阳性检出率为84．4％；细菌PCR法阳性检出率为80％。PCR、培养诊断胰腺感染的敏感性分别为90％（9／10）、100％（10／10），特异性均为100％（7／7）。两者耗时分别为5h、3d。结论B超引导细针穿刺安全可行；PCR法能快速、准确地诊断急性重症胰腺炎早期合并细菌感染，为手术治疗提供可靠依据。

TRIO的扩增和高表达与膀胱肿瘤细胞的快速增殖有关

目的TRIO基因位于染色体5p扩增区、编码1种在细胞周期调节中起主导作用的蛋白,本研究旨在了解TRIO的扩增和表达是否与膀胱肿瘤细胞的快速增殖有关。方法Ki67标记染色(Ki67LI)、荧光原位杂交(FISH)和定量PCR法(LightCyclerTMPCR)被用于研究。结果TRIO基因扩增与膀胱肿瘤细胞的快速生长相关(P<0.0001)。膀胱肿瘤分化越差、临床分期越高,Ki67LI与TRIO基因扩增数目均越高。含2317例组织微阵列FISH结果显现TRIO扩增仅见于1.8%早期膀胱肿瘤(pTa)(9例/499例),而在膀胱癌(pT1-4)高达12.8%(62例/485例)。定量PCR法进一步证实TRIO在显示5p扩增的膀胱癌组织高表达。结论TRIO扩增与膀胱肿瘤细胞的快速增殖有关,TRIO扩增和高表达可能在膀胱癌的发展过程中发挥重要作用。

Effect of nitric oxide on the expression of apelin receptor mRNA in rat caudate nucleus

Objective To investigate the effect of nitric oxide （NO） on the expression of apelin receptor mRNA, as well as their correlation, in the caudate nucleus of rat. Methods L-Arginine （L-Arg）, N^G-nitro-L-arginine methyl ester （L-NAME） and normal saline （NS） was separately microinjected into rat caudate nucleus. Expressions of neuronal NO synthase （nNOS） mRNA and apelin receptor mRNA were detected by RT-PCR at 4, 8, 12, 24 and 48 h after microinjection, and their correlation was determined. Results The expressions of nNOS mRNA and apelin receptor mRNA were both significantly increased after microinjection of L-Arg, but significantly decreased after microinjection of L-NAME compared with the NS control group. The nNOS mRNA had a positive correlation with the expression of apelin receptor mRNA after microinjection of L-Arg and L-NAME. Conclusion The activity of NOS in the central nervous system, especially in the caudate nucleus, is one of the key factors for NO to exert many kinds of biological actions, such as modulation of central pain, as a neurotransmitter. The neurobiological action of NO in rat caudate nucleus may be associated with apelin receptors.

Acute pancreatitis in aging animals:Loss of pancreatitis-associated protein protection?

AIM:To investigate the effect of age on severity of acute pancreatitis(AP) using biochemical markers,histology and expression of the protective pancreatitisassociated proteins(PAPs).METHODS:AP was induced via intraductal injection of 4% sodium taurocholate in young and old rats.Sera and pancreata were assayed at 24 h for the parameters listed above;we also employed a novel molecular technique to assess bacterial infiltration using polymerase chain reaction to measure bacterial genomic ribosomal RNA.RESULTS:At 24 h after induction of AP,the pancreata of older animals had less edema(mean ± SE histologic score of young vs old:3.11 ± 0.16 vs 2.50 ±-0.11,P < 0.05),decreased local inflammatory response(histologic score of stromal infiltrate:3.11 ± 0.27 vs 2.00 ± 0.17,P < 0.05) and increased bacterial infiltration(174% ± 52% increase from sham vs 377% ± 4%,P < 0.05).A decreased expression of PAP1 and PAP2 was demonstrated by Western blotting analysis and immunohistochemical staining.There were no differences in serum amylase and lipase activity,or tissue myeloperoxidase or monocyte chemotactic protein-1 levels.However,in the most-aged group,serum C-reactive protein levels were higher(young vs old:0.249 ± 0.04 mg/dL vs 2.45 ± 0.68 mg/dL,P < 0.05).CONCLUSION:In older animals,there is depressed PAP expression related to a blunted inflammatory response in AP which is associated with worsened bacterial infiltration and higher C-reactive protein level;this may explain the more aggressive clinical course.

IL28B polymorphisms associated with therapy response in Chilean chronic hepatitis C patients

AIM:To analyze the association of three IL28B single nucleotide polymorphisms with response to therapy in Chilean patients infected with hepatitis C virus CV.METHODS:We studied two groups of patients with chronic CV infection genotype 1,under standard combined treatment with pegylated interferon plus ribavirin.One group consisted of 50 patients with sustained virological response,whereas the second group consisted of 49 null responders.In order to analyze the IL28B single nucleotide polymorphisms rs12979860,rs12980275 and rs8099917,samples were used for polymerase chain reaction amplification,and the genotyping was performed by restriction fragment lengthpolymorphism.RESULTS:The IL28B rs12979860 CC,rs12980275 AA and rs8099917 TT genotypes were much more frequently found in patients with sustained virological response compared to null responders 38%,44% and 50% vs 2%,8.2% and 8.2%,respectively.These differences were highly significant in all three cases(P < 0.0001.CONCLUSION:The three IL28B polymorphisms studied are strongly associated with sustained virological response to therapy in Chilean patients with chronic CV genotype 1.

Multidrug resistance protein 3 R652G may reduce susceptibility to idiopathic infant cholestasis

AIM:To evaluate the role of genetic factors in the pathogenesis of idiopathic infant cholestasis.METHODS:We performed a case-control study,in-cluding 78 infants with idiopathic infant cholestasis and 113 healthy infants as controls.Genomic DNA was extracted from peripheral venous blood leukocytes us-ing phenol chloroform methodology.Polymerase chain reaction was used to amplify the multidrug resistance protein 3(MDR3)R652G fragment,and products were sequenced using the ABI 3100 Sequencer.RESULTS:The R652G single nucleotide polymorphism(SNP)was significantly more frequent in healthy infants(allele frequency 8.0%)than in patients(allele frequency 2.60%)(P < 0.05),odds ratio,0.29;95% confidence interval,0.12-0.84.The conjugated bilirubin in patients with the AG genotype was significantly lower than in those with the AA genotype(44.70 ± 6.15 μmol/L vs 95.52 ± 5.93 μmol/L,P < 0.05).CONCLUSION:MDR3 R652G is negatively correlated with idiopathic infant cholestasis.Children with the R652G SNP in Guangxi of China may have reduced susceptibility to infant intrahepatic cholestasis.

-449 C>G polymorphism of NFKB1 gene,coding nuclear factor-kappa-B,is associated with the susceptibility to ulcerative colitis

AIM:To clarify the association between a polymorphism-449 C>G(rs72696119) in 5'-UTR of NFKB1 with ulcerative colitis(UC).METHODS:The studied population comprised 639 subjects,including patients with UC(UC cases,n = 174) and subjects without UC(controls,n = 465).We employed polymerase chain reaction-single strand conformation polymorphism to detect the gene polymorphism.RESULTS:The rs72696119 G allele frequencies in controls and UC cases were 33.4% and 38.5%,respectively(P = 0.10).Genotype frequency of the GG homozygote in UC cases was significantly higher than that in controls(P = 0.017),and the GG homozygote was significantly associated with susceptibility to UC [odds ratio(OR),1.88;95%CI,1.13-3.14].In male subjects,the GG homozygote was associated with an increased risk for UC(OR,3.10;95%CI,1.47-6.54;P = 0.0053),whereas this association was not found in female subjects.In addition,the GG homozygote was significantly associated with the risk of non-continuous disease(OR,2.06;95%CI,1.12-3.79;P = 0.029),not having total colitis(OR,2.40;95%CI,1.09-3.80,P = 0.040),disease which developed before 20 years of age(OR,2.80;95%CI,1.07-7.32,P = 0.041),no hospitalization(OR,2.28;95%CI,1.29-4.05;P = 0.0090) and with a maximum of 8 or less on the UCDAI score(OR,2.45;95%CI,1.23-4.93;P = 0.022).CONCLUSION:Our results provide evidence that NFKB1 polymorphism rs72696119 was significantly associated with the development of UC.This polymorphism influences the susceptibility to and pathophysiological features of UC.

Na^+/K^+-ATPase α-subunit in swimming crab Portunus trituberculatus: molecular cloning, characterization, and expression under low salinity stress

Na^＋/K^＋-ATPases are membrane-associated enzymes responsible for the active transport of Na^＋ and K^＋ ions across cell membranes, generating chemical and electrical gradients. These enzymes＇ α-subunit provides catalytic function, binding and hydrolyzing ATP, and itself becoming phosphorylated during the transport cycle. In this study, Na^＋/K^＋-ATPase α-subunit eDNA was cloned from gill tissue of the swimming crab Portunus trituberculatus by reverse-transcription polymerase chain reaction （RT-PCR） and rapid amplification of eDNA end methods. Analysis of the nucleotide sequence revealed that the eDNA had a full-length of 3 833 base pairs （bp）, with an open reading frame of 3 120 bp, 5＇ untranslated region （UTR） of 317 bp, and 3＇ UTR of 396 bp. The sequence encoded a 1 039 amino acid protein with a predicted molecular weight of 115.57 kDa and with estimated pI of 5.21. It was predicted here to possess all expected features of Na^＋/K^＋-ATPase members, including eight transmembrane domains, putative ATP-binding site, and phosphorylation site. Comparison of arnino acid sequences showed that the P. tritubereulatus α-subunit possessed an overall identity of 75%-99% to that of other organisms. Phylogenetic analysis revealed that this α-subunit was in the same category as those of crustaceans. Quantitative real-time RT-PCR analysis indicated that this α-subunit＇s transcript were most highly expressed in gill and lowest in muscle. RT-PCR analysis also revealed that α-subunit expression in crab gill decreased after 2 and 6 h, but increased after 12, 24, 48, and 72 h. In addition, α-subunit expression in hepatopancreas of crab decreased after 2-72 h. These facts indicated that the crab＇s Na^＋/K^＋-ATPase α-subunit was potentially involved in the observed acute response to low salinity stress.

Cloning of polyadenylated mRNA fragments of Escherichia coli with restriction display-polymerase chain reaction

Objective:To investigate the polyadenylation of mRNA in E. Coli. Methods: The mRNA of E. Coli was enriched from the total RNA with oligo(dT)-cellulose, prior to reverse transcription using oligo(dT)18as the primer. Double-stranded cDNA was subsequently synthesized, which was subjected to digestion with Sau3A I to produce multiple gene fragments for ligation with the adapters. PCR was carried out in 10 groups according to 10 different pairs of the selective primers, and the PCR products were then cloned into T-vectors. Results: More than 100 gene fragments had been cloned, 30 of which were sequenced. Conclusion:Polyadenylation of E. Coli mRNA may not be a biochemical curiosity but a general attribute of bacterial mRNA.

组氨酸三聚体核苷结合蛋白1在肾透明细胞癌中的表达及意义

目的:通过检测组氨酸三聚体核苷结合蛋白1(histidine triad nucleotide-binding protein1,HINT1)mRNA及其蛋白质在肾透明细胞癌(clear cell renal cell carcinoma,ccRCC)中的表达情况,探讨其与临床病理特征的关系。方法:应用实时荧光定量(real-time fluorescence quantitative,RFQ)-PCR和免疫组织化学分别从mRNA和蛋白质水平检测HINT1在36例新鲜ccRCC组织、37例新鲜正常肾组织(其中29例配对)、30例石蜡包埋ccRCC组织与12例石蜡包埋正常肾组织中的表达水平,并分析其与临床病理特征间的关系。结果:HINT1mRNA在29例配对ccRCC和癌旁正常肾组织中的表达量分别为0.209±0.033和0.733±0.136(P<0.001),在36例ccRCC和37例癌旁正常肾组织中的表达量分别为0.245±0.035和0.694±0.108(P<0.001)。HINT1蛋白在癌旁正常肾组织中的阳性表达率为100%(12/12),弥漫性分布于细胞核及细胞质中,其中近曲小管表达强度高于远曲小管。肾小球基膜、肾小囊壁层上皮细胞可见HINT1蛋白部分表达,其强度低于肾小管,肾间质未见阳性表达。HINT1蛋白在ccRCC中的阳性表达率为60%(18/30),同样分布于细胞核及细胞质,癌组织间质阴性(P<0.01)。HINT1在mRNA和蛋白质水平表达趋势一致,在ccRCC中表达水平低于癌旁正常肾组织,并且在T3-4晚期肿瘤中表达量低于T1-2早期肿瘤中的表达量(P<0.05);但2者在不同Fuhrman核分级、性别、年龄和肿瘤大小中的表达差异无统计学意义(P>0.05)。结论:HINT1可能在ccRCC发病机制中发挥肿瘤抑制作用,其表达异常可能与翻译水平之前的异常调控有关。推测HINT1mRNA和蛋白质的异常表达可作为ccRCC发生和进展的预后指标。

Charcterization of Type Ⅱ Topoisomerase Gene Mutations in Clinical Isolates of Mycoplasma Hominis Resistant to Fluoroquinolones

Objective: To analyze type II topoisomerase genes in clinical isolates of fluoroquinolone-resistant Mycoplasmahominis. Methods: Eight isolates of M.hominis cross resistant to 6fluoroquinolones were selected from 103 clinical strains ofM.hominis using a broth microdilution method. Type IItopoisomerase genes were amplified by PCR and directlysequenced. Nucleotide sequences were compared to sequencesfrom a susceptible strain (M.hominis PG2I). Results: MICs of resistant Mh isolates were 4- to 512-foldhigher than MICs from the susceptible reference strain.Sequence comparison revealed a C to T change at 113nt ingyrA QRDR led to the substitution of Ser83 by Leucine and noamino acid change in gyrB. A change of G to T at 134nt inparC QRDR led to the substitution of Ser80 by Isoleucine anda G to A change at 70nt in ParE QRDR led to the substitutionof Aspartic acid by Asparagine.Conclusion: These results suggest that a C to T change at113nt in gyrA, a G to T change at 134nt in parC and a G to Achange at 70nt i atrE are associated with fluoroquinolone resistance of M.hominis.

Mutations of mitochondrion DNA in mouse tumors

Objective： To ascertain the variations of mitochondrion DNA （mtDNA） in mouse tumors and to inquire into the relationship between mutations of mtDNA and carcinogenesis Methods： The variations of D-loop, ND3 and tRNA^Met＋Glu＋Ile gene fragments of mtDNA from six tumor cell lines of mice were analyzed by PCR technology with restriction fragment length polymorphism analysis （polymerase chain reaction-restriction fragment length polymorphism, PCR-RFLP） and single strand conformation polymorphism analysis （SSCP-PCR） method. Results： ND3 and tRNA^Met＋Glu＋Ile gene fragments ofmtDNA from the tumors showed no variation in 27 endonuclease sites; D-loop ofmtDNA from Hca-F had an additional endonuclease sites of Hinf I in contrast to that of the inbred mouse. Deeply analyzed by PCR-SSCP, the D-loop ofmtDNA was found to possess mutations in 4 of 6 tumors. Conclusion： D-loop is the hot spot of tumor mtDNA mutation which can act as contributors to the carcinogenic

Detection of ＂ Candidatus Phytoplasma asteris＂ in Brussels Sprout and Its Possible Association with Flower Bud Failure in Poland

Severe growth abnormalities including shoot stunting, leaf blade reduction and flower bud failure of Brussels sprout were observed in Poland. The presence of phytoplasma in diseased as well as in healthy looking plants, was demonstrated by nested polymerase chain reaction assay employing phytoplasma universal rRNA primer pairs-P1/P7 followed by R16F2n/R16R2. Products of PCR primed by R 16F2n/R 16R2 primer pair from naturally infected Brussels sprouts were sequenced. Comparison of the obtained 16S rDNAs revealed high nucleotide sequence identity between analyzed phytoplasma isolates （99.8%-100%）. They were also nearly identical with the sequences of other phytoplasmas isolates from sub-group 16SrI-B, and they were classified as members of ＂Candidatus Phytoplasma asteris＂.

Leptin influences estrogen metabolism and increases DNA adduct formation in breast cancer cells

Objective: The elevated incidence of obesity has been paralleled with higher risks of breast cancer. High adiposity increases leptin secretion from adipose tissue, which in turn increases cancer cell proliferation. The interplay between leptin and estrogen is one of the mechanisms through which leptin influences breast carcinogenesis. An unbalanced estrogen metabolism increases the formations of catechol estrogen quinones, DNA adducts, and cancer mutations. This study aims to investigate the effect of leptin on some estrogen metabolic enzymes and DNA adduction in breast cancer cells.Methods: High performance liquid chromatography(HPLC) was performed to analyze the DNA adducts 4-OHE1[E2]-1-N3 adenine and 4-OHE1[E2]-1-N7 guanine. Reporter gene assay, real time reverse transcription polymerase chain reaction(real time RT-PCR), and Western blot were used to assess the expression of estrogen metabolizing genes and enzymes: Cytochrome P-4501B1(CYP1B1), Nicotinamide adenine dinucleotide phosphate-quinone oxidoreductase1(NQO1), and Catechol-O-methyl transferase(COMT).Results: Leptin significantly increased the DNA adducts 4-OHE1[E2]-1-N3 adenine and 4-OHE1[E2]-1-N7 guanine.Furthermore, leptin significantly upregulated CYP1B1 promoter activity and protein expression. The luciferase promoter activities of NQO1 and m RNA levels were significantly reduced. Moreover, leptin greatly reduced the reporter activities of the COMT-P1 and COMT-P2 promoters and diminished the protein expression of COMT.Conclusions: Leptin increases DNA adduct levels in breast cancer cells partly by affecting key genes and enzymes involved in estrogen metabolism. Thus, increased focus should be directed toward leptin and its effects on the estrogen metabolic pathway as an effective approach against breast cancer.

DETECTION OF HUMAN HERPESVIRUS 6(HHV-6) DNA IN SALIVARY GLANDS BY THE POLYMERASE CHAIN REACTION

To assess the presence of HHV-6-Specific DNA in human salivary glands, eighteen specimens of salivary gland tissue were investigated using the polymerase chain reaction. Eight of nine parotid glands, five of seven submandibular glands and one of two sublingual glands were found to have amplification of the HHV-6-specific sequence. The findings suggest that salivary gland tissue is one of the potential sites for HHV-6 persistence following primary infection and that saliva is a vehicle for transmission of the virus.

卵巢上皮性癌组织中结肠癌转移相关基因1的表达及其临床意义

目的:探讨卵巢上皮性癌组织中结肠癌转移相关基因1(metastasis-associated in colon cancer-1,MACC1)的表达及其临床意义。方法:应用RT-PCR和免疫组织化学法检测卵巢上皮性癌组织、卵巢上皮性良性肿瘤组织及正常卵巢组织中MACC1 mRNA及蛋白的表达,分析MACC1蛋白的表达与卵巢上皮性癌患者临床病理特征之间的关系。结果:卵巢上皮性癌组织中MACC1 mRNA和蛋白的表达水平均明显高于卵巢上皮性良性肿瘤组织和正常卵巢组织(P<0.05)。卵巢上皮性良性肿瘤组织与正常卵巢组织之间MACC1 mRNA和蛋白的表达水平无明显差异(P>0.05)。MACC1蛋白的阳性表达率与卵巢上皮性癌患者的临床分期、组织分化程度和淋巴结转移等因素相关(P<0.05)。结论:MACC1蛋白表达与卵巢上皮性癌患者的临床分期、组织分化程度和淋巴结转移相关。MACC1可能参与了卵巢上皮性癌的发生和发展。

组胺H4受体在结直肠癌组织中表达及临床意义

目的观察结直肠癌组织中组胺H4受体的表达变化及其临床意义。方法分别采用免疫组化sP法和RT—PCR法检测80例结直肠癌（观察组）及28例癌旁正常组织（对照组）中的组胺H4受体及mR—NA。结果观察组组胺H4受体阳性9例，对照组为26例，两组H4阳性率差异有统计学意义（χ2=63．0576，P〈0．01）；观察组中组胺H4受体表达在有淋巴结转移患者中的表达阳性率为43．5％（27／62），明显高于无淋巴结转移患者的5．6％（1／18）（χ2=8．8511，P〈0．01）；Ⅰ～Ⅱ期结直肠癌组织中H4表达阳性率为28．6％（8／28），明显高于Ⅲ～Ⅳ期的3．8％（2／52）（χ2=10．1727，P〈0．01）。观察组组胺H4受体mRNA表达量为（1．122±0．046）（A值），明显高于对照组的（0．1544-0．012）（4值）（t=109．7429，P〈0．05）。结论组胺H4受体在结直肠癌组织有表达异常，表达量与病理分期相关。

人疱疹病毒分子生物学诊断的研究进展

人疱疹病毒是免疫功能低下者及儿童感染的常见病原体，其实验室诊断对临床至关重要。近10多年来，随着分子生物学技术的迅速发展，基因诊断方法如核酸分子杂交技术、PCR技术、生物芯片技术等得到不断发展和应用，该文就近年来国内外在人疱疹病毒分子生物学诊断方面的研究现状及进展作一综述。

Difference in DNA sequences in SSU rDNA variable regions among pathogens isola ted from different epidemic foci of visceral leishmaniasis in China

OBJECTIVE: To confirm the existence of point mutations in the SSU rDNA variable regions of 5 Leishmania donovani (L.d.) isolates from different epidemic foci in China. METHODS: Specific SSU rDNA fragments from nuclear DNA of 7 Leishmania species/isolates were amplified by PCR and then cloned into pGEM(R)-T Easy Vectors. After that, the specific fragments were sequenced by an automated DNA sequencer. RESULTS: Sequence analysis showed that the amplified DNA fragments of 7 Leishmania species/isolates were all 392 bp in length. All 5 point mutations were located in two unique sequence blocks (UQ-I and UQ-II), and no insertions or deletions were found. The identities of comparison of Leishmania in GeneBank were more than 98%. CONCLUSION: Five point mutations exist in the SSU rDNA variable region of 5 L.d. isolates from different epidemic foci of visceral leishmaniasis (VL) in China. Sequence differences of the SSU rDNA variable region exist among L.d. isolates from different foci.

Glucocorticoid receptor and heat shock protein 90 in peripheral blood mononuclear cells from asthmatics

Objective To investigate the expression of glucocerticoid receptor (GR) and heat shock protein 90(HSP90) mRNA in peripheral blood mononuclear cells (PBMCs) from steroid-sensitive (SS), steroiddependent (SD) and steroid-resistant (SR) asthmatics patients, and to evaluate the role of GR and HSP90in the pathogenesis of SR.Methods Reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the expressions of GR and HSP90 mRNA in PBMC stimulated with IL-2 and/or IL-4 from 10 normal volunteers,10 SS, 5 SD and 6 SR patients.Results The expressions of GR and HSP90 mRNA were the highest in PBMC from SR patients (0.730±0.171 and 1.122±0.165, respectively) compared with the normals (P＜0.05). The second was from SS patients (0.359±0.350 and 0.885±0.250, respectively). The lowest was from the SD patients (0.017±0.008 and 0.078 ± 0.039, respectively). The ratio of HSP90/GR in SR was significantly lower than that in the others, and it suggested that the expression of HSP90 mRNA was not sufficient in this group of patients. When PBMC from SS, SD and SR was incubated with IL-2 or IL-4 alone, no changes in GR and HSP90 mRNA expression were observed. While incubated with combination of IL-2 and IL-4, a significantly higher expression of GR mRNA was observed in all asthmatics, and a significantly higher expression of HSP90 mRNA was observed only in SS and SD patients.Conclusion The lowering of HSP90/GR ratio may be one of the causes of SR. IL-2 and IL-4 may play roles in the imbalance of HSP90/GR.