Objective To develop a chemiluminescence ( CL ) imaging DNA microarray method for simultaneous detection of seven rickettsiae.Methods Primers and probes were designed based on the specific sequence of seven rickettsia genomes.The probes were immobilized on the aldehyde modified glass surface to prepare DNA microarray for rickettsiae.The nucleic acids of the selected rickettsiae were amplified and labelled by multiplex PCR method, and then hybridized with microarray that was scanned after washing and chemiluminescence coloration, before the results were analyzed.Facilitated by the optimization of the multiplex PCR system, hybridization, and chemiluminescence imagination, we evaluated the specificity,sensitivity and reproducibility of the chip.The serial diluted nucleic acid of Rickettsia mooseri was detected using microarray and real-time PCR approach to compare the sensitivity of these two methods.Double-blind simulated samples were prepared to further evaluate the accuracy of the detection methods.Results One universal primer, four specific primers, one universal probe, and nine specific probes were selected.This DNA microarray demonstrated high specificity and sensitivity.The detection sensitivity of plasmid DNA and double-blind simulated sample DNA was 1.5 ×102 -3 ×103 copies per reaction and 103 -104 copies/μl.The detection results of real-time PCR method was consistent with the microarray, and the microarray possessed 10 fold lower detection sensitivities than the real-time PCR method.The coincidence rate of double-blind simulated sample detection was 100%.Conclusion A chemiluminescence imaging DNA microarray method for simultaneous detection of seven rickettsiae is established successfully,which can serve as a new high throuthput detection method for clinical diagnosis and epidemiological investigation of rickettsiae.

Objective To develop a chemiluminescence imaging DNA microarray method for simultaneous,fast and accurate detection of nine rash-and fever-causing pathogens,namely,Measles virus,Rubella virus,Enterovirus type 71, Varicella zoster virus,Dengue fever virus,Human small FDNA virus B19,Coxsackie virus type A16,A-βStreptococcus pyogenes (hemolytic streptococcus)and Salmonella typhi.Methods Primers and probes were designed based on the specific sequence in the conserved region of genomes of the nine pathogens.The nucleic acids of the nine pathogens were amplified and labelled by multiplex PCR method.The multiplex PCR amplification products were hybridized with specific probes of microarray that was scanned after washing and chemiluminescence coloration to identify the nine pathogens.After the optimization of the multiplex PCR system,hybridization and chemiluminescence imaging,the specificity,sensitivity and reproducibility of the chip were evaluated.The serial diluted nucleic acid of Enterovirus type 71 was detected using microarray and real-time PCR approach to compare the sensitivity of these two methods.Results Nine specific primers and eleven specific probes were selected.The microarray demonstrated high sensitivity and specificity.The minimum detection limit of plasmid DNA and in vitro transcribed RNAs was 3 ×103 copies per reaction.The detection sensitivity of this microarray was 10 percent of that by the real-time PCR method.The rate of sensitivity and specificity of clinical sample detection was 95% and 85.7% respectively,and the rate of accuracy was 93.2%.Conclusion A chemiluminescence imaging DNA microarray method for simultaneous,fast and accurate detection of nine pathogens that cause rash and fever illnesses is established successfully,which can serve as a new high throuthput screening method for clinical diagnosis and epidemiological investigation of rash and fever illnesses.

Objective To develop a chemiluminescence imaging DNA microarray method for simultaneous,quick and accurate detection of serotypes of human adenovirus (HAdV ),namely,HAdV3,HAdV7,HAdV11,HAdV14 and HAdV55.Methods Based on the specific gene sequences in the conserved region of adenovirus from GenBank, oligonucleotide primers and probes were designed and synthesized to prepare the oligonucleotide microarray.The specific genomic sequences were amplified by multiplex PCR method.The multiplex PCR amplification products were hybridized with the specific probes of microarray that was scanned after washing and chemiluminescenceb before the result was analyzed.After optimization of the multiple PCR system,hybridization reactions and conditions of chemiluminescence,the specificity,sensitivity and reproducibility of the chip were evaluated.Results The microarray displayed high sensitivity, specificity and reproducibility.The minimum detection limit of plasmidg DNA was 3 ×103 copies/reaction.The microarray detection results of 38 clinical samples were approximately consistent with those using the direct sequencing method(37 /38).Conclusion A chemiluminescence imaging DNA microarray method for quick,sensitive and specific detection of five serotypes of HAdV is established,which can provide a new means for detecting serotypes of HAdV.

Objective To develop a detection method based on the technology of gene chips which can quickly distinguish genes of Enterococcus faecalis, E.faecium and vancomycin resistance.Methods Based on the specific gene ( ddl) sequences of two types of Enterococcus from GenBank, oligonucleotide probes which could detect and distinguish special genes and drug resistance genes ( vanA,vanB) of Enterococcus were designed and compounded.Then,the probes were dotted to modified slide.The target DNA fragments of vancomycin-resistant Enterococcus ( VRE) were labeled with biotin by multiple PCR amplification, and then hybridized with oligonucleotide probes on slide.The results were analyzed by portable imager.The multiple PCR system, hybridization reaction and condition of the chemiluminescence method were optimized before the specificity, sensitivity and reproducibility of the chip were evaluated.Results One universal primer, four specific primers, one universal probe and four specific probes were selected.This gene chip was demonstrated of high specificity and repeatability.The detection sensitivity was 103 CFU/ml.The gene chip detection results of 10 clinical samples were basically consistent with the drug sensitivity test ( 8/10 ) .Conclusion A gene chip technique for the detection of VRE is established successfully.It is possible to distinguish the type of VRE and detect the genetic phenotypes of drug resistance by gene chip technique.

Objective To evaluate antimicrobial effect and mechanism of meropenem in the model of PA infection by C.elegans.Methods To evaluate drug effects of PA infection with caenorhabditis elegans by different concentrations of culture medium, determinate the lethal rate of C.elegans.Western blot detected mitogen activated protein kinase ( Mitogen-activated protein kinase MAPK ) activity change, and PCR detected antimicrobial peptide genes expression in C.elegans after PA infection,the effect of meropenem on MAPK activity change and antimicrobial peptide genes expression.Results Compared with the control group (OP-50), the death rate of C.elegans in PA infection group changed significantly (P<0.01). Meropenem showed protective effect after C.elegans infection ( P <0.01 ) .Detection of MAPK kinase activity showed that PA infection caused PMK-1 kinase activation, further study showed that antibiotics meropenem did not affect the activation of PMK-1 kinase (no significant difference).C.elegans antimicrobial peptide gene Lys-1, clec-85, F55G11.7, K08D8.5 activity increased in PA infection (P<0.01).Meropenem promoted the expression of the antimicrobial peptide gene increased (P<0.01),with synergistic effects.Conclusion Our results show that a C.elegans pathogenicity model can be applied screening drug susceptible to pathogens infection quickly and easily.

Objective: To investigate the effects of Fufang Jiangzhi No. 3, a compound traditional Chinese herbal medicine, on cholesterol-bile acid metabolism in rabbits with hypercholesterolemia and to explore the mechanism. Methods: Twenty-four male New Zealand white rabbits were randomly assigned into normal control group, untreated group and Fufang Jiangzhi No. 3 group, with 8 rabbits in each group. Rabbits in the untreated group and Fufang Jiangzhi No. 3 group were fed high cholesterol diet to induce hypercholesterolemia. After 4-week treatment, serum total cholesterol and bile acid contents were assessed. Activity of cholesterol 7alpha-hydroxylase (CYP7A1) in liver tissues was measured by enzyme-linked immunosorbent assay. The expressions of CYP7A1, bile salt export pump (BSEP) and small heterodimer partner (SHP) mRNAs in liver tissues were observed by real-time fluorescent quantitative polymerase chain reaction. Results: Compared with the normal control group, serum total cholesterol and bile acid contents in the untreated group were increased (P<0.01). Activity of CYP7A1 and expression of CYP7A1 mRNA were decreased and expressions of BSEP and SHP mRNAs were increased in liver tissues in the untreated group as compared with the normal control group (P<0.01). Serum total cholesterol level, and expressions of BSEP and SHP mRNAs in the Fufang Jiangzhi No. 3 group were lower than those in the untreated group (P<0.01). The CYP7A1 activity and expression of CYP7A1 mRNA in the Fufang Jiangzhi No. 3 group were increased as compared with the untreated group (P<0.01), however, there was no significant difference in bile acid between the Fufang Jiangzhi No. 3 group and the untreated group. Conclusion: Fufang Jiangzhi No. 3 can up-regulate the expression of CYP7A1 mRNA, raise the activity of CYP7A1, and inhibit the expressions of BSEP and SHP mRNAs to regulate the metabolism of total cholesterol in rabbits.

Objective: To observe hypoglycemic effects of Yunu Decoction, Zuogui Pill and Shenqi Pill, three compound traditional Chinese herbal medicines, in treatment of diabetes mellitus induced by alloxan in rats, and to compare the therapeutic effects among the three recipes for nourishing yin, clearing away heat, and nourishing yin and warming yang. Methods: Diabetes mellitus was induced in rats with alloxan at a dose of 60 mg/kg via tail vain injection. The diabetic rats were randomly divided into four groups: alloxan model group, Yunu Decoction-treated group, Zuogui Pill-treated group and Shenqi Pill-treated group. Rats in the three recipe groups were administered intragastrically with water extraction of Yunu Decoction, Zuogui Pill, and Shenqi Pill accordingly for 10 days. Then the level of blood glucose was measured by glucose oxidase method and the glucose tolerance was determined. Results: Compared with the normal rats, blood glucose level in the alloxan model group was obviously increased (P<0.05). Glucose levels in the three recipe groups were obviously decreased as compared with the alloxan model group (P<0.05), and glucose level in the Yunu Decoction-treated group after treatment was significant lower than before treatment (P<0.05). The glucose tolerance test indicated that rats in the alloxan model and three recipe groups revealed impaired glucose tolerance as compared with the normal rats, and there were no significant differences between the alloxan model group and the three recipe groups. Conclusion: Yunu Decoction, Zuogui Pill and Shenqi Pill can effectively decrease the glucose level of the rats with diabetes mellitus induced by alloxan, and Yunu Decoction showed the best therapeutic effects. The glucose tolerance test shows that the three recipes cannot correct the abnormal metabolism of glucose.

Acupoint compatibility is a basic element for acupuncture prescription and an important link for clinical acupuncture workers to obtain a therapeutic effect. Studying factors influencing the effect of acupoint combination and ascertaining its concept and connotation are of important significance for studying the mechanism of acupuncture action. By sorting out, summing up and analyzing relevant modern literature, it is found that factors influencing the effect of acupoint combination are those affecting the therapeutic effects of combination of two or more than two acupoints. Its contents include five aspects: combination mode, combination effect, stimulation method, time factor and body status. Analysis and induction show that acupoint selection is the primary task in studying factors influencing the effect of acupoint combination.

Primary insomnia (PI) is now a nonorganic sleep disorder that seriously affects the quality of life of people around the world and attracts the attention of medical circles at home and abroad. Western medicine treatment has side effects such as drug dependence and withdrawal rebound and cannot obviously improve the quality of sleep and life, while acupuncture has a good therapeutic effect on PI. This article explores the latest research status of acupuncture treatment for PI. Chinese Journal Net (CNKI, VIP and Wanfang) was mainly searched to retrieve and sum up recent five years’ literature about the study of acupuncture treatment for PI. The results show that acupuncture has an advantage in producing a short-term marked therapeutic effect on PI and being of a high total efficacy rate and can effectively treat primary insomnia.

Objective To study the role of glucagon-like peptide-1 (GLP-1) analogue liraglutide played in the proliferation of CD4+CD25 T cells in normal people and newly-onset type 1 diabetic patients,and to evaluate the possible immune regulatory role of liraglutide in the therapy of type 1 diabetes.Methods CD4+ CD25-T cells of 10 normal people and 10 newly-onset type 1 diabetic patients were separated from peripheral blood by MACS immunomagnetic beads and stimulated by Human T-Activator CD3/CD28 Dynabeads to proliferate.CFSE labeling technique was used to evaluate the proliferation of CD4+ CD25-T cells by flow cytometry.Liraglutide of different concentrations(0,25,50,and 100 nmol/ml) was added to the proliferation system,then the proliferation of CD4+CD25-T cell was measured.Results (1) Liraglutide suppressed the proliferation of CD4+ CD25-T cells from either normal people or type 1 diahetic patients with dose-dependent manner (P ＜ 0.05).(2) Under the different concentrationsofliraglutide,the proliferation ofCD4+CD25 T cells from diabetic patients was mueh more robust than that of normal people (P＜0.01).(3) The inhibitory effects of liraglutide on CD4+ CD25-T cells proliferation in normal people and diabetic patients were similar (P＞0.05).Conclusion The proliferation of CD4+ CD25 T cells in type 1 diabetic patients was more robust than normal people,which indicated cellular immune dysfunction in type 1diabetes.Liraglutide inhibits the proliferation of CD4+ CD25-T cells of type 1 diabetic patients in vitro.The immunosuppression effect of liraglutide may have potential value in the treatment of type 1 diabetes.