实时荧光定量聚合酶链式反应检测瘢痕疙瘩中核心蛋白多糖的表达

目的探讨瘢痕疙瘩成纤维细胞中核心蛋白多糖的表达、含量，及其在瘢痕疙瘩形成中的作用和机制。方法对瘢痕疙瘩、正常瘢痕以及正常皮肤成纤维细胞进行体外培养，采用光镜、透射电镜观察成纤维细胞形态、活性及凋亡；应用实时荧光定量聚合酶链式反应（FQ-PCR）对核心蛋白多糖以及B1转化生长因子（TGF-β1）的mRNA表达进行检测、分析。结果瘢痕疙瘩成纤维细胞形态不规则、排列紊乱，线粒体增多，粗面内质网扩张呈囊，细胞核常染色质丰富，表明其合成蛋白的功能活跃；瘢痕疙瘩成纤维细胞中核心蛋白多糖mRNA含量较正常瘢痕或正常皮肤成纤维细胞降低，而TGF-β1 mRNA表达则较正常皮肤及瘢痕组织成纤维细胞升高。结论核心蛋白多糖在瘢痕疙瘩成纤维细胞内含量较正常皮肤明显减少，提示其对成纤维细胞增殖、合成的抑制作用随之减弱，同时使TGF-β1表达上调，导致成纤维细胞的大量增生、迁移，合成过量胶原。表明核心蛋白多糖是抑制瘢痕疙瘩形成的重要因子。

甘薯丛枝病植原体的PCR检测

以报道的植原体 (Phytoplasma) 1 6SrDNA基因保守序列为依据 ,设计合成了两对引物对R1 6mF2 /R1 6mR2 和R1 6F2 /R1 6R2 ,以甘薯丛枝病 (SPWB)带病植株的叶脉中提取的DNA为模板 ,应用聚合酶链式反应(PCR)技术和巢式PCR(Nested_PCR)技术对甘薯丛枝病病原进行分子检测。结果表明PCR扩增出了 1 .5kb的特异片段 ,在PCR基础上的巢式PCR扩增出了 1 .2kb的特异片段。灵敏度实验显示该方法所需PCR模板DNA量为 0 .1 0 73ng/μl,在PCR基础上的巢式PCR可以将灵敏度提高约 1 0 0 0 0倍 ,所需模板DNA仅为 0 .0 1 0 73pg/μl,在甘薯丛枝病的检测中是一种快速、灵敏、可靠的方法。

大明胶囊对糖尿病大鼠心肌L型Ca^(2+)通道蛋白mRNA表达的影响

目的探讨大明胶囊对2型糖尿病大鼠心肌L型Ca2+通道蛋白mRNA表达的影响。方法建立2型糖尿病大鼠模型,筛选空腹血糖值大于16.7mmol·L-1的大鼠随机分组:大明胶囊大(200mg·kg-1·d-1)、中(100mg·kg-1·d-1)、小(50mg·kg-1·d-1)剂量组、糖尿病模型组、苯乙双胍(75mg·kg-1·d-1)组,连续用药14d,用快速血糖仪测空腹血糖值。提取心肌总RNA,采用逆转录聚合酶链式反应(RTPCR)的方法,观察大明胶囊治疗后2型糖尿病大鼠心肌L型Ca2+通道蛋白mRNA水平的变化。结果2型糖尿病组大鼠心肌L型Ca2+通道蛋白mRNA表达高于正常组大鼠(P<0.01),经大明胶囊治疗后的心肌L型Ca2+通道蛋白mRNA表达降低(P<0.05),空腹血糖也明显下降。结论大明胶囊对2型糖尿病大鼠有明显的降糖作用,并且可以降低2型糖尿病大鼠心肌L型Ca2+通道蛋白mRNA的表达。

禽流感病毒和新城疫病毒二联RT-PCR检测方法的建立

参照国内外已发表的禽流感病毒(AIV)和新城疫病毒(NDV)的基因序列及其相关的RT-PCR检测方法,根据禽流感病毒M蛋白基因和新城疫病毒NP基因各设计一套特异性通用引物,扩增目的带分别为600bp和340 bp。通过对相关病毒检测,建立了AIV和NDV通用型二联RT-PCR检测方法。该方法具有快速、敏感、特异等优点,可为AIV和NDV的检测、流行病学调查及疫苗使用等奠定基础。

MT01对实验性牙移动大鼠牙周组织中TLR9、TRAF6和IL-6表达水平的影响

目的:探讨Toll样受体9(TLR9)及其下游炎症因子白细胞介素6(IL-6)在实验性大鼠牙移动过程中牙周组织中表达水平的变化,以及MT01对牙周组织TLR9、肿瘤坏死因子受体相关因子6(TRAF6)和IL-6表达水平的影响,阐明其相关机制。方法:30只雄性Wistar大鼠分为无MT01干预组(n=24)和MT01干预组(n=6)。其中,无MT01干预大鼠右侧加力为实验组,左侧不加力为对照组;MT01干预大鼠左侧上颌第一磨牙颊侧黏膜注射MT01+加力为实验组,右侧上颌第一磨牙颊侧黏膜注射PBS+加力作为对照组。0.49N力近中移动大鼠上颌第一磨牙,无MT01干预大鼠于加力后第3、7、14和21天处死,MT01干预组于加力后第7天处死。分别获取各组大鼠上颌第一磨牙近远中侧牙槽骨,采用实时荧光定量PCR(RT-PCR)法检测张力侧和压力侧牙周组织中TLR9、TRAF6和IL-6mRNA的表达水平。结果:无MT01干预大鼠中,实验组张力侧和压力侧牙周组织中IL-6和TLR9表达水平均明显高于对照组(P<0.05),且加力7d后两者表达水平达高峰;MT01干预大鼠中,加力7d后实验组张力侧及压力侧牙周组织中TLR9表达水平较对照组降低(P<0.01),实验组压力侧牙周组织中TRAF6表达水平较对照组降低(P<0.01)。结论:MT01能够抑制实验性牙移动大鼠牙周组织中TLR9及下游相关因子TRAF6的表达,进而产生抑制牙移动过程中牙周组织炎症反应的作用。

Bioleaching of Pb-Zn-Sn chalcopyrite concentrate in tank bioreactor and microbial community succession analysis

The variation of microbial community structure was investigated for the tank bioleaching process of Pb-Zn-Sn chalcopyrite concentrate in the presence of mixed moderately thermophilic bacteria. The parameters, such as pH value, solution potential and concentrations of metal ions, were determined by the method of polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） to analyze the succession of microbial community. The results showed that a final copper extraction rate of 85.6% could be obtained after tank bioleaching for 30 d. The Acidithiobacillus caldus was the dominant population with abundance of about 73.80%in the initial stage, then Sulfobacillus thermosulfidooxidans dominated from the 18th day to the end of bioleaching, while the abundance of Leptospirillum ferriphilum changed slightly. A higher solution potential within a certain range and appropriate concentration of ferric ions were essential for this tank bioleaching of chalcopyrite.

A Molecular Approach to Identification of the Chinese Drug“pu Gong Ying”(Herba Taraxaci)and Six Adulterants byDNA Fingerprinting Using Random Primed PolymeraseChain Resaction(PCR)

DNA fingerprinting among members of the Chinese drug Pu Gong Ying(Taraxacum mongolicum Hand,-Mazz.)and six adulterants of Tu Gong Ying were demonstrated with random-primed polymerase chain reaction(PCR)including arbitrarily primed polymerase chain reaction(AP-PCR)and random amplified polymorphic DNA(RAPD).Distinctive,reproducible genomic fingerprints from DNA from 7 species belonged to Compositae were generated with two long(20 and 24 mer)and one short(10 mer)randomly chosen primers.The Pu Gong Ying can be differentiated from six species of Tu Gong Ying according to the banding pattems of their amplified DNA on agarose gels.The results showed that AP-PCR and RAPD methods can be used for identifying Chinese drugs.Moreover,the Similarity Indexes of the genomic DNA fingerprints showed that Pu Gong Ying and its adulterants are unrelated.Therefore,AP-PCR and RAPD methods can be used for identifying Chinese drugs.

Comparison of Different Extraction Methods of Genomic DNA of Raw Soybean Milk

[Objective] The paper was too explore and compare methods of DNA extraction from raw soybean milk.[Method] Taken the soybean milk purchased from market as the material,pyrolysis method,isopropanol precipitation method,CTAB method,SDS method,high-salt low-pH and guanidine isothiocyanate method,as well as their improved methods were used to extract genomic DNA,and the extraction effects of these methods were compared by detecting the DNA using optical density,agarosegel electrophoresis and polymerase chain reaction（PCR）methods.[Result] The genomic DNA extracted by all methods except isopropanol precipitation method could be used in PCR reaction.Meanwhile,the high DNA concentration and purity will be gained by different methods in the order of high-salt low-pH method,high-salt low-pH method,improved CTAB method,improved isopropanol precipitation method,guanidine isothiocyanate method and improved pyrolysis method.[Conclusion] These methods are simply to operate,fast to gain results,and suitable for the extraction of total DNA from raw soybean milk.

Selection of reference genes for qRT-PCR analysis of gene expression in sea cucumber Apostichopus japonicus during aestivation

Quantitative real-time reverse transcription-polymerase chain reaction （qRT-PCR） is a technique that is widely used for gene expression analysis, and its accuracy depends on the expression stability of the internal reference genes used as normalization factors. However, many applications of qRT-PCR used housekeeping genes as internal controls without validation. In this study, the expression stability of eight candidate reference genes in three tissues （intestine, respiratory tree, and muscle） of the sea cucumber Apostichopus japonicus was assessed during normal growth and aestivation using the geNorm, NormFinder, delta CT, and RefFinder algorithms. The results indicate that the reference genes exhibited significantly different expression patterns among the three tissues during aestivation. In general, the β-tubulin （TUBB） gene was relatively stable in the intestine and respiratory tree tissues. The optimal reference gene combination for intestine was 40S ribosomal protein S18 （RPSI 8）, TUBB, and NADH dehydrogenase （NADH）; for respiratory tree, it was β-actin （ACTB）, TUBB, and succinate dehydrogenase cytochrome B small subunit （SDHC）; and for muscle it was α-tubulin （TUBA） and NADH dehydrogenase [ubiquinone] 1α subcomplex subunit 13 （NDUFA13）. These combinations of internal control genes should be considered for use in further studies of gene expression in A.japonicus during aestivation.

Helicobacter species and gut bacterial DNA in Meckel's diverticulum and the appendix

AIM:To analyse the possible association of various Helicobacter species and certain common gut bacteria in patients with Meckel's diverticulum and appendicitis.METHODS:A nested-polymerase chain reaction (PCR),specific to 16S rRNA of the Helicobacter genus,was performed on paraffin embedded samples,50 with acute appendicitis,50 normal appendixes,and 33 Meckel's diverticulum with gastric heterotopia and/or ulcer.Helicobacter genus positive samples were sequenced for species identification.All samples were also analysed for certain gut bacteria by PCR.RESULTS:Helicobacter pullorum DNA was found in one out of 33 cases and Enterobacteria in two cases of Meckel's diverticulum.Helicobacter pylori (H.pylori) was found in three,Enterobacter in 18,and Bacteroides in 19 out of 100 appendix samples by PCR.Enterococcus was not found in any MD or appendix samples.All H.pylori positive cases were from normal appendixes.CONCLUSION:Helicobacter is not an etiological agent in the pathogenesis of symptomatic Meckel's diverticulum or in acute appendicitis.

Effect of Temperature on Gene Expression in the Pearl Oyster Pinctada fucata

In this study, we examined the effect of elevated temperature on the expression patterns of genes, i.e., nacrein, irr, n16, n19, and hsp70 in the pearl oyster Pinctada fucata. The experiment was carried out at 4 temperatures, i.e., 20℃(ambient, control), 24, 28℃, and 32℃. The expression levels of target genes in P. fucata were assayed at 0, 6, 24, 48, and 96 h via real-time polymerase chain reaction. Results showed that the expression levels of nacrein and irr had no significant variations among different time points below 28℃, but significantly increased over time at 32℃. The expression levels of n16 and n19 did not change markedly at 20℃. The former increased significantly at 6 h and 24 h while the latter substantially decreased during 6–96 h at 24, 28 and 32℃. Among different temperatures, the level of n16 was significantly lower at 20℃ than at other temperatures during 6–96 h, and the level of n19 significantly varied among different temperatures at 48 h and 96 h. The expression level of hsp70 was significantly higher at 32℃ than at 20, 24 and 28℃ at 24 h. These results demonstrated that elevated temperature impacted the physiological processes of P. fucata and potentially influenced its adaptability to thermal stress.

Scrub typhus hepatitis confirmed by immunohistochemical staining

Scrub typhus is an acute febrile disease caused by Orientia tsutsugamushi （0. tsutsugamushi）. We report herein the case of a woman who presented with fever and elevated serum levels of liver enzymes and who was definitively diagnosed with scrub typhus by his- topathological examination of liver biopsy specimens, serological tests and nested polymerase chain reaction. Immunohistochemical staining using a monoclonal an- ti-O. tsutsugamushi antibody showed focally scattered positive immunoreactions in the cytoplasm of some hepatocytes. This case suggests that scrub typhus hepatitis causes mild focal inflammation due to direct liver damage without causing piecemeal necrosis or interface hepatitis. Thus, scrub typhus hepatitis differs from acute viral hepatitis secondary to liver damage due to host immune responses, which causes severe Iobular disarray with diffuse hepatocytic degeneration, necrosis and apoptosis as well as findings indicative of hepatic cholestasis, such as hepatic bile plugs or brown pigmentation of hepatocytes.

Xenogeneic hepatic progenitor cell transplantation ameliorate CCl_4 /partial hepatectomy-induced rat acute liver failure

Objective： Hepatic progenitor cell transplantation has shed light on the treatment of liver failure. The present study was designed to evaluate whether xenogeneic liver epithelial progenitor cells （LEPCs） transplantation could promote liver recovery in a rat model of acute liver failure. The engraftment and hepatocytic differentiation of transplanted hepatic progenitor cells in the rat spleen was also investigated. Methods： LEPCs were propagated in vitro for long and transduced with lentiviral vector carrying mCherry gene. Intraperitoneal injection of CC14 followed by 2/3 partial hepatectomy three days later were used to establish rat models of acute liver failure. Rats were intrasplenically injected with mCherry modified LEPCs （n=20, 1× 107 cells/0.5 mL） or the same volume of medium （n=20）. Serum liver enzymes （ALT, AST） and liver histology were evaluated for 21 days after transplantation. The engraftment of transplanted LEPCs in the spleens was tested by polymerase chain reaction （PCR） amplification targeting mCherry gene. The differentiation into hepatocytic lineage of transplanted LEPCs was investigated usingimmunohistochemistry staining against Alb. Results： LEPCs were effectively transduced with lentiviral vector showing a transduction efficiency of 90%. Compared with control, cell-injected group displayed significantly lower levels of ALT and AST （P〈0.05） and better histological features including less swelling change and hepatocyte death. PCR amplification of mCherry sequences confirmed the engraftment of LEPCs in the spleens. Alb-positive cells first appeared 5 days after cell transplantation and the number of Alb-positive cells increased substantially （P〈0.05）, which revealed the hepatocytic differentiation process Conclusion： Xenogeneic hepatic progenitor cells can engraft and differentiate into hepatocytes in the splenic parenchyma. Intrasplenic delivery of hepatic progenitor cells ameliorates CCh/partial hepatectomy-induced liver injury in rats

A Few Questions that Should be Clarified in the Polymerase Chain Reaction

The polymerase chain reaction is one of the most useful technical ad- vance and inventions in modern molecular biology. Developed in 1983 by Kary Mullis, PCR is now a common and indispensable technique used in medical and bi- ology research labs for a variety of applications. A large number of articles relat- ed to PCR are available on the internet and other places. People know well about the basic principle and are very familiar with the procedures of the PCR. But, some details were neglected on the numbers of the target sequence and other DNA strands number after 30 to 35 cycles of the PCR. In most papers, the number of newly synthesized DNA strands including target DNA and non target DNA is am- biguous and even wrong. In this paper, highlights were given to the theoretical number of target DNA number in details and the exact number of the target DNA number can be concluded by analysis.

Contamination of Groundwater Sources with Common Waterborne Pathogens and Heficobacter pylori in the West Bank： Case Study of Wadi AI Arroub, Tulkarem and Jericho

Detection of waterborne pathogens in drinking water via rapid DNA amplification assays is an important and crucial public health method. The aim of this study is to investigate the presence of waterborne pathogens in groundwater resources in Al Arroub, Tulkarem and Jericho areas using direct PCR （polymerase chain reaction） analysis. Forty-six groundwater samples were collected. The total DNA was extracted from each sample and subjected to PCR analysis directed to specific genes of enteric bacteria, β-lactamases producing bacteria, L. pneumophila （Legionella pneumophila） and H. pylori （Helicobacter pylori）. Enteric bacteria were detected with high frequency in Palestinian water sources followed by 13% occurrence of β-lactamases producing bacteria, 9% of H. pylori and 4% of L. pneumophila. The study shows that shallow groundwater wells and water taped from karstic aquifer is potential for contamination and could not be reliable sources of potable water.

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＂.

The role of sexual related Y gene detection in the diagnosis of patients with gonadal dysgenesis

Objective To clarify the role of sexual related Y (SRY) gene detection in the diagnosis of gonadal dysgenesis.Methods Sixteen cases of gonadal dysgenesis were included in this study: 5 with androgen insensitivity syndrome, 1 with 17-α-hydroxylase deficiency, 4 with true hermaphrodite, 2 with 45,X/46,XY gonadal dysgenesis, 1 with 45,X gonadal dysgenesis, 1 with XY pure gonadal dysgenesis, 1 with testicular regression, and 1 XY female who gave birth to a normal baby. SRY gene was detected by using polymerase chain reaction (PCR) in blood and gonad samples and by direct sequencing of the SRY motif.Results Among the 16 cases, 15 were blood SRY positive, among which 13 (86.7%) showed the presence of testicular tissue, and 2 showed ovaries without testicular tissue. One SRY negative case showed the presence of testicular tissue. In 3 cases, SRY detection in gonadal tissue correlated with pathological findings but not with blood karyotype. The correlation between peripheral blood SRY and the pathology of the gonads was 81.25% and the correlation between the presence of peripheral blood Y chromosome and pathology of the gonads was 68.75%. Sequencing of the SRY motif in an XY female who gave birth to a normal baby showed no mutation.Conclusions SRY detection is more sensitive and specific than blood karyotype in the prediction of the presence of testicular tissue. Peripheral blood karyotype does not necessarily reflect gonadal type. There may be testicular related factors other than the SRY gene.

Assessment of polymerase chain reaction and serology for detection of chlamydia pneumoniae in patients with acute respiratory tract infection

OBJECTIVE: To study Chlamydia pneumoniae (C. pneumoniae) infection in 110 patients with respiratory tract infection admitted to our hospital from January to December 1995 in Nanjing. METHODS: Sputum and throat swab specimens were taken and C. pneumoniae DNA was detected by using polymerase chain reaction (PCR) with the HM-1-HR-1 primer pair. At the same time, serum samples were taken and immunoglobulin G and M (IgG and IgM) fractions of antibodies to C. pneumoniae were studied by microimmunofluorescence test. RESULTS: Prevalence of specific IgG was 70% in patients with respiratory tract infection. Seventeen patients (15.5%) were serologically diagnosed as having recent C. pneumoniae infections and 12 patients (10.9%) had positive PCR in sputum and/or swab specimens. The total positive rate was 22.7% (25/110) detected by PCR combined with serological tests. Acute infection of C. pneumoniae was common in patients with asthma (57.1%), pneumonia (35.0%), COPD (25.9%) and bronchitis (25.0%). Clinical features between C. pneumoniae infection and non-C. pneumonia infection showed no significant differences. CONCLUSIONS: Chlamydia pneumoniae is an important pathogen that causes infection of the human respiratory tract and attention should be drawn to this special illness.

New Primers for Denaturing Gradient Gel Electrophoresis Analysis of Nitrate-Reducing Bacterial Community in Soil

The narG gene is frequently used as a molecular marker for bacterial nitrate-reducing community analysis. In this study, a new set of primers targeting the narG gene was designed and applied to semi-nested polymerase chain reaction-denaturing gradient gel electrophoresis （PCR-DGGE） assay. The potential of the new primers was verified on DNA directly extracted from soils from five different experimental sites distributed in Central and Southern Italy. Specificity of the primers was determined by excision, amplification, and sequencing of bands resolved by DGGE. A phylogenetic analysis showed the correlation between the sequences retrieved from the soils studied and the narG sequences from βand y-Proteobacteria. These primers expanded the existing molecular tools for ecological study on the size and diversity of nitrate-reducing bacterial community in soil.

Molecular identification and interaction assay of the gene(OsUbc13) encoding a ubiquitin-conjugating enzyme in rice

The ubiquitin （Ub）-conjugating enzyme, Ubc13, has been known to be involved in error-free DNA damage tolerance （or post-replication repair） via catalyzing Lys63-linked polyubiquitin chains formation together with a Ubc variant. However, its functions remain largely unknown in plant species, especial y in monocotyledons. In this study, we cloned a Ub-conjugating enzyme, OsUbc13, that shares the conserved domain of Ubc with AtUBC13B in Oryza sativa L., which encodes a protein of 153 amino acids; the deduced sequence shares high similarities with other homologs. Real-time quantitative polymerase chain reaction （PCR） indicated that OsUbc13 transcripts could be de-tected in al tissues examined, and the expression level was higher in palea, pistil, stamen, and leaf, and lower in root, stem, and lemma;the expression of OsUbc13 was induced by low temperature, methylmethane sulfate （MMS）, and H2O2, but repressed by mannitol, abscisic acid （ABA）, and NaCl. OsUbc13 was probably localized in the plasma and nuclear membranes. About 20 proteins, which are responsible for the positive yeast two-hybrid interaction of OsUbc13, were identified. These include the confirmed OsVDAC （correlated with apoptosis）, OsMADS1 （important for development of floral organs）, OsB22EL8 （related to reactive oxygen species （ROS） scavenging and DNA protection）, and OsCROC-1 （required for formation of Lys63 polyubiquitylation and error-free DNA damage tolerance）. The molecular characterization provides a foundation for the functional study of OsUbc13.