OBJECTIVE To develop a preparation technique of sample of one-time PCR with common primers based on ribotyping which was combined with the detection system of nanogold-based gene chip to detect clinical bacterial pathogens.METHODS According to the highly conserved regions of rDNA,the common primers were designed and used to amplify each target bacterial ISRs by one-time PCR,and the specific oligonucleotide probes for each target ISRs were designed,utilized to establish the new nanogold-based gene chips.After the characteristics of the chip such as sensitivity,specificity and reliability were determined,the chip was used to detect clinical samples.RESULTS The designed common primers could amplify the 12 target bacteria successfully by one-time PCR.All selected probes were of strong specificity and great reliability.The chip had high sensitivity,specificity and reliability,reaching 50 fmol/L of detection sensibility.Clinical detection results showed the chip had a great accuracy.CONCLUSIONS Compared to multi-PCR chip detection,the detection procedure and complexity of the chip are decreased significantly,and have more practical value in clinical pathogens detection.

Objective For realizing the simultaneous detection of multiple pathogens,by looking for a method with a combination of the new gene chip detection system based on nano-gold with the technology of restriction endonuclease without PCR.Methods Helicobacter pylori,Mycoplasma pneumoniae,Chlamydia trachomatis,Candida albicans,Ureaplasma urealyticum,and EB virus were selected as the experimental targets.Endonuclease Hha Ⅰ was selected as tool enzyme.After bering digested by Hha Ⅰ,the digested fragments of samples were tailed with poly-A.The samples were then detected by the gene chip detection system based on nano-gold.Both specific and common probes were used in the hybridization.The coincidence rate of the detection results between the new constructed chip test and the fluorescence quantitative PCR test in 168 clinical samples was examined.The stability and sensitivity of chips detection were also checked.Results The new constructed nano-gold-baesd gene chip combined with restrictive enzyme digestion without PCR could be used to detect the target pathogens.The coincidence rate of the chip detection test and fluorescence quantitative PCR test in 168 clinical samples was 89.2%.Chip detection results showed that the stability of chips detection was 100% and the sensitivity was 50pmol/L.Conclusion The newly constructed nano-gold-baesd gene chip combined with restrictive enzyme digestion without PCR can be widely applied in the simultaneous detection of Mycoplasma,Chlamydia,fungus,virus and bacteria.It shows a bright prospect in increasing the throughput of identifieation of pathogene.

OBJECTIVE A practical gene chip which aimed to detect and identify pathogens rapidly and exactly is developed on the basis of patent technology of nano-enlargement-detection. METHODS Oligonucleotide probes for the specific gene fragments of target pathogens were designed and immobilized on gene chip.Target sequences were labeled by nanogold as reporter materials.After hybridization,its results were recorded by the interaction between nanogold and silver which amplified the hybridization signal to form brown particles,which could be detected by naked eyes. RESULTS The probes designed were all of strong specificity and great reliability possessing identity of hybridization conditions.The reaction time for marking could be decreased by properly raising the ratio of nanogold and nucleic acid and the speed of labeling reaction could be fastened significantly by gentle agitation.A better hybridization results could be obtained when the samples were hybridized for 8 hours at 45℃ with 0.8 mol/L ionic strength,and then strictly rinsed.Furthermore,the hybridization efficiency could be increased remarkably by slight circumgyratation.A better chromatic effect resulted from the reaction way in 3min?3 at 37℃.The sensitivity of gene chip assays in this test could reach to 100 fmol/L.Compared with traditional detection approach,detection by the chip displayed such advantages as speediness and simplicity and the detection results could be easily recognized by naked eyes. CONCLUSIONS The chip detection technology has met the demand of design exhibiting high sensitivity,strong specificity,and easy operation without special device and showing a promising prospect.

Objective To evaluate the therapeutic effects of endoscopic biliary double stents for advanced malignant hilar biliary obstruction.Methods From January 2007 to December 2010,double stents was attempted in 28 patients (15 men and 13 women,median age 66.4 years (44-88 years),including 9 with Bismuth Ⅱ,8 with type Ⅲa,5 with type Ⅲb and 6 with type Ⅳ.A total of 23 consecutive patients ( 11 men and 12 women,median age 65.8 years (42-83 years) with malignant hilar obstruction undergoing a therapy with single stent were recruited as the control group,including 7 with Bismuth Ⅱ,5 with Ⅲa,6 with Ⅲlb and 5 with Ⅳ.The rates of successful drainage,complications,mean survival time of patients and the average duration of biliary stent patency were compared between the two groups.Results Successful rate of cannulation was both 100％ in the two groups.Successful rate of drainage and complications of double stent group were 96.4％ (27/28) and 17.9％ (5/28),and these two variables of single stent group were 87.0％ (20/23) and 13.0％ (3/23),which were not significantly different (P ＞0.05).23 patients (82.1％ ) in double stent and 19 ( 82.6％ ) in single stent group were followed up.The average duration of stent patency and mean survival time of double stent group were ( 129 ±48.5) d and ( 187 ±94.5) d,which were superior to those of the single stent group,i.e.( 102 ±37.8) d and ( 103 ±98.5) d.Conclusion Double stenting is an effective therapy for malignant hilar obstruction of Bismuth Ⅱ and above.It is superior to single stent method in the mean duration of patency and mean survival time.

Objective To investigate the relationship between ?3 adrenergic receptor(?3AR) gene Trp~(64)Arg mutation and insulin resistance(IR) in Chinese.Methods The 120 patients with type 2 diabetes mellitus(group DM) and 80 nondiabetic subjects as normal controls(group N)were involved in the study.Polymerase chain reaction(PCR) and the restriction fragment length polymorphism analysis(PCR-RFLP) were used to determine the ?3AR genotype.The indexes such as body weight,height,waist circumstance,hip circumstance,blood pressure,lipids,fast plasma glucose and insulin were detected in all subjects.The IR was estimated by homeostasis model assessment(HOMA-IR).The patients in the diabetes group were divided into relative insulin sensitive group and relative insulin resistant group by the 75th percentile HOMA-IR.Results The BMI of the patients with the ?3AR Trp~(64)Arg mutation was significantly higher than that of those without the mutation(P

Objective To observe the pathological changes of tissues of the injured bile duct, and to provide theoretical basis for bile duct repair. Methods Dog models of obstructive biliary injury were established.Sixty dogs were equally divided into five groups according to the duration of biliary obstruction: biliary obstruction for 5 days (BDL5 group), 10 days (BDL10 group), 15 days (BDL15 group), 20 days (BDL20 group) and 30 days (BDL30 group). The morphological and pathological changes of bile duct and local tissues were observed, and biliary-enteric Roux-en-Y anastomosis was applied to repair the injured bile duct and postoperative complications were observed. All data were analyzed by LSD test, independent sample t test, one-way analysis of variance and chi-square test. Results Proximal bile duct rapidly expanded as the pressure increased in the early stage, and the bile duct expanded to ( 15.6 ± 1.8)mm in the BDL10 group. The expansion rate decreased in the later stage,and the bile duct expanded to (18.9 ± 1.9)mm in the B DL15 group. Acute inflammation was observed in injured local tissues. The acute inflammation was severe in the BDL5 group with white blood cell count of 54 ± 6, and the acute inflammation was relatively mild in the BDL15 group with white blood cell count of 42 ± 7. There was a significant difference between the BDL5 group and BDL15 group in the degree of acute inflammation (t =4. 688,P ＜ 0. 05). The content of the collagen was increased in the injured bile duct as time passed by. Bile duct repair was successfully performed on 57 dogs. Ten dogs ( three in the BDL5 group, four in the BDL10 group, one in the BDL15 group, one in the BDL20 group and one in the BDL30 group) died of bile leakage after the operation. The incidences of bile leakage was 30% (7/23) within 10 days and 9% (3/34) beyond 10 days, with a significant difference between the two groups (x2 =4.429, P＜0.05). Conclusion Ten days after obstructive biliary injury,an obvious reduction of bile duct expansion and edema of the bile duct is observed, the difficulty of the operation is reduced and the incidence of bile leakage is low, so 10 days after the incidence of obstructive biliary injury is the proper timing for the surgical repair.

ObjectiveTo explore diagnosis and surgical treatment of pancreatic duct stone.MethodsClinical data of 21 patients with pancreatic duct stone was analyzed retrospectively. All patients were diagnosed definitely by B-ultrasonography,CT and MRI, the positive rates were 90.5％( 19/21 ),66.7％(14/21) and 55.6％ (5/9) respectively, 15 cases did transpancreatic duct lithotomy and pancreatico-intestinal anastomosis, 1 case excised the tail of pancreas simultaneously, 1 case did pancreaticoduodenectomy, 4 cases treated by endoscopy. ResultsAll operations succeeded, 1 case occurred with pancreatic leakage after a small amount of discharge tube, 1 case appeared acute pancreatitis after endoscopic treatment,who was discharged after conservative treatment. Following up 19 cases from 4 to 72 months, the symptom was released, no stone relapsed,6 cases with mild catarrhal dysentry. ConclusionB-ultrasonography, CT can basically make definite diagnosis for pancreatic duct stone, and B-ultrasonography has higher rate of diagnosis, transpancreatic duct lithotomy and pancreatico-intestinal anastomosis are the main surgical treatments, endoscopy is the method to treat pancreatic duct stone.

Objective To investigate the expression of Aire,Foxp3,AchR and other immune factors in human thymoma tissue and plasma and explore their role in myasthenia gravis with thymoma.Methods T lymphocyte subsets,immunoglobulin and other immune factors in plasma were compared,and the Expression of Aire,Foxp3 and AchR were examined in thymoma by reverse transcriptional polymerase chain reaction and immunohistochemical staining,and the results were analyzed by SPSS statistics software.Results The ratio of CD4 + to CD8 + T lymphocyte was much higher in plasma,while the expressions of Aire,Foxp3 and AchR at mRNA and protein level were much lower in thymoma patients with myasthenia gravis,and related to Ossermann subtype,WHO subgroup and Masaoka stage.The differences were statistically significant (P ＜ 0.05).Conclusion The ratio of CD4 + to CD8 + T lymphocyte and the abnormal expressions of Aire and Foxp3could used as an indicator of immune state in thymoma patient with myasthenia gravis and play an important role in the development of thymoma with myasthenia gravis,but the mechanism is indefinite.

Objective To explore the effects and safety of endoscopic parallel placement of double metal stents on unresectable hilar malignant obstruction.Methods The clinical data of 11 patients with malignant hilar obstructive jaundice due to advanced carcinoma who were treated with parallel placement of double biliary stents from January 2011 to September 2012 were retrospectively analyzed.Results Out of 11 patients,10(90.9％) were successfully embedded with double biliary stents and 4 were dead during the follow-up.There was no sign of stent occlusion during the follow-up period.The survival time ranges from 128 to 185 days.One case was lost during the follow-up and 5 others are still alive.Conclusion The endoscopic parallel placement of double biliary stents is effective and safe for patients with unresectable malignant hilar obstruction.

Objective To investigate the effect of the multimodal analgesia on postoperative pain after renal transplantation and the cytokines .Methods 40 cases of allogaft renal transplantation due to chronic renal failure were randomly divided into two groups (n=20) .The group D received the multimodal analgesia :preemptive analgesia plus patient controlled epidural analgesia(PCEA) and the group C(control) received analgesic drugs by intermittent intramuscular injection .The visual analogue scale(VAS) scores , the Ramsay sedation scores ,HR ,MAP and SPO2 at postoperative 2 ,6 ,12 ,24 ,48 h were recorded .Blood interleukin-2(IL-2) ,in-terleukin-6(IL-6) and interleukin-10(IL-10) levels were measured before anesthesia ,at the end of operation and postoperative 6 , 24 ,48 h .Results Postoperative MAP and SPO2 had no obvious change in the two groups ,no statistical differences in the various time points existed between the two groups (P>0 .05) .HR was significantly increased at 6 ,24 h after operation in the group C , which had statistical difference compared with that at the same time points in the group D (P<0 .05) .The VAS scores at postoper-ative 6 ,12 ,24 h in the group D were significantly lower than those in the group C ,the difference showed statistical significance (P<0 .05) .The sedation scores at various time points had no statistical difference between the two groups (P>0 .05) .The levels of IL-2 and IL-10 at postoperative 6 ,24 ,48 h in the two groups were significantly higher than those before anesthesia and at the end of operation (P<0 .05) .The levels of IL-2 and IL-6 at postoperative 6 ,24 ,48 h in the group D were significantly lower than those in the group C(P<0 .05) .Conclusion Multimodal analgesia can reach the effective analgesic effect ,down-regulate the pro-inflam-matory cytokines and up-regulate anti-inflammatory cytokines for maintaining postaperative serum cytokines balance .