It is important to analyze the epidermal growth factor receptor ( EGFR) mutation before makingstrategyonnon-small cell lung cancer ( NSCLC) patients scheduled to EGFR tyrosine kinase inhibitor ( TKI) therapy .Digital PCR is a new generation of molecular diagnostic technique that provides ultra-highersensitive, specific and absolute nucleic acid quantification based on its unique principle.The application of digital PCR indetecting circulate tumor DNA can be the truly tumorliquid biopsy, helps to acquire the accurate EGFR mutation status from peripheral blood and screen out the most appropriate patients for TKI therapy.This breakthrough technology will also contribute to tumor surveillance and drug resistance monitoring.

Objective To inhibit the expression level of SALI4 in AML cell line THP-1 and investigate its potential effects on pathogenesis of leukemia. Methods AML cell line THP-1 was transfected with plasmids that expressed small interfering RNA targeting SALL4. The samples were divided into 4 groups:(1) blank group: samples with not any treatments; (2) control group: cells with empty pRS vector alone;(3) test1 group:cells with SALL4-shRNA-pRS-1 plasmid transfection complex; (4) test2 group:cells with SALL4-shRNA-pRS-2 plasmid transfection complex. The expression levels of SALL4 mRNA and protein were measured by real time fluorescence quantitative PCR and WB. C-myc, Cyclin D1 and β-catenin were important components of Wnt/β-catenin signaling pathway and their expression levels in SALL4 knockdown THP-1 cells were detected by real-time fluorescence PCR. Furthermore, THP-1 apoptosis was analyzed by flow cytometry after Annexin V-PI staining. Results Real time fluorescent quantitative PCR illustrated that the expression of SALL4 in testl group, test2 group, control group and blank group were ( 36. 0 ± 4. 3 ) %,(32. 0 ± 2. 4) %, ( 102. 0 ± 6.5 ) % and ( 100. 0 ± 2. 6 ) % respectively. There was statistical significance ( F = 226. 3, P < 0. 05 ). The expression of SALL4 in testl and test2 group respectively were significant lower than that in blank group (t = 19.7,19. 1, P<0. 05). The expression of SALL4 had no significant difference between blank group and control group (t = 1.1, P ＞0. 05). Western blot analysis revealed SALL4 protein in testl and test2 group were significantly decreased compared with those of control and blank group. All above data indicated the high efficiency of RNA interference targeting SALL4. Comparing with the blank group, the relative expression of C-myc, Cyclin D1 and β-catenin mRNA in test1, test2 and control group were(44.0 ±6.2)%,(44.0 ±5.1)% and (107.0±13.6)%;(22.0±4.5)%,(25.0±3.5)% and (48.0 ± 7. 6 ) %; ( 42.0 ± 3.5 ) %, ( 59. 0 ± 3.7 ) % and ( 79. 0 ± 5.6 ) %. The expression of C-myc,β-catenin and Cyclin D1 mRNA in testl and test2 group were significant lower than that in blank group (t = 10. 1,9. 5, 23. 3, 22. 9; 17.4, 12. 4; P < 0. 05). The percentage of apoptotic cells in group of test1,test2,control, blank were (57.2 ±9.1)%, (34.4 ±8.6)%, (14.4 ±3.6)% and (14.8 ±4.8)%respectively. There was statistical significance ( F = 42. 5, P < 0. 05 ). After the inhibition of SALL4, the percentages of apoptotic cell in testl and test2 group were significantly increased( t =9. 7, 4. 5 ;P <0. 05).Conclusion The inhibition of SALL4 in leukemia cell line THP-1 downregulates the expression of cell proliferation related genes such as C-myc, Cyclin D1,β-catenin and promoted apoptosis.

ObjectiveTo investigate the efifcacy of alarm treatment in a sample of China monosymptomatic nocturnal enuresis (MNE) children and adolescents with smaller than expected bladder capacity (EBC) for age.Methods Fifteen MNE pa-tients with a smaller than age-expected BC and without nocturia were included. All the patients were treated with enuretic alarm and water restriction 2 hours before sleep. All patients were followed up monthly. A success criterion was deifned as “14 con-secutive dry nights” after successive 2-3 months treatment. A relapse criterion was deifned as “more than two wet nights every two weeks” after therapy discontinuation. The relapsed patients were treated with enuretic alarm and followed up again.Results The patients consisted of 9 boys and 6 girls. The mean age was 9.76±4.24 years (6-15 years). Thirteen patients were successfully cured after three months treatment. Two patients discontinued the treatment and received the treatment of desmopressin. Five patients relapsed and received the enuresis alarm treatment again. Four patients had never relapsed, and one failed. The cure rate was 80%.Conclusions The enuretic alarm device is effective on MNE patients with a smaller than age-expected BC and without nocturia.

BACKGROUND:Hydroxyapatite/gel nano-composite has the same mechanical strength to the natural bone, but its ability to repair bone defects and osteogenic effect need to be confirmed by further studies.
OBJECTIVE:To explore the repair effect of hydroxyapatite/gel bionic composite in skul defects of rabbits.
METHODS: The hole-like calvarium defect models were established in rabbits, and treated with hydroxyapatite/ gel composites (hydroxyapatite/gel group), autologous skul as positive control (autologous bone group) and
nothing as negative control (blank group). The repairing condition in the skul defect areas were observed and analyzed by X-ray and CT at 4, 8, 12 weeks after implantation.
RESULTS AND CONCLUSION: After 8 weeks, X-ray assessment showed that normal-like bone tissue appeared in the defect region of the autologous bone group; in the hydroxyapatite/gel group, dense bone with similar
morphology to normal bone tissue was found in the central site of defect region, and the boundary was slightly blurred. After 12 weeks, the hydroxyapatite/gel showed blurred edge compared with autologous bone, and the center of the composite was disconnected; in the blank group, a clear and regular transmitted shadow was
observed. After 12 weeks, CT examination showed that the hydroxyapatite/gel was connected tightly with the surrounding normal bone tissue. As a new bionic composite, the hydroxyapatite/gel can achieve good effect in repairing skul defects of rabbits.

Objective To evaluate the performance of serum sialic acid detection kit using enzymic method and investigate the clinical diagnosis value of sialic acid.Methods one hundred and fifty healthy adults were enrolled in this case control study to establish serum SA reference interval.The analytical performance (accuracy,precision,linearity) of serum sialic acid detection kit using enzymic method was assessed.Two hundred and forty patients were classified into different malignant tumor groups according to their pathological types.Serum SA level of each tumor group was compared with that of normal control group.In tumor groups with statistical difference,benign disease groups were further collected.Receiver operating characteristic (ROC) curve and area under curve (AUC) were used to evaluate the diagnostic value of SA compared with other tumor markers.t test,one-way ANOVA,Mann-Whitney U test were used as statistical methods.Results The reference interval of SA was 479 to 715 mg/L.The detection result of 2 level controls was 584 and 1 482 mg/L respectively,which were both within the acceptable limits.The within-lot and between-lot variations of three level samples were both below 5％.There was a good linear correlation (Y =0.995X-0.177,R2 =0.999) between theoretical value and actual detection result in range of 0-1 052 mg/L.The serum level of SA was (757 ± 177),(514 ± 86) and (597 ± 60) mg/L in gastric cancer group,benign disease control group and normal control group respectively,which had statistically significant difference(F =55.2,P ＜ 0.01).The serum level of SA was(659 ± 127) and (545 ± 66) mg/L in colorectal cancer group and benign disease control group respectively,which had statistically significant difference(F =42.8,P ＜ 0.01).The serum level of SA was (738 ± 157) and (672 ± 161) mg/L in colorectal cancer group and benign disease control group respectively,which did not have statistically significant difference(F =26.3,P ＞ 0.05).The AUC of SA was 0.804,0.724,0.755 in gastric cancer group,colorectal cancer group and lung cancer group respectively,which was higher than that of CEA and CA72-4.In gastric cancer group,the sensitivity of SA was higher than that of CEA (59.5％,24.3％).The AUC of SA was 0.791,0.687,0.790 in gastric cancer,colorectal cancer and lung cancer patients with normal CEA serum level respectively.Conclusions Experimental results show that serum sialic acid detection kit using enzymic method has good performance in the precision and linearity.Sialic acid has some value in the diagnosis of gastric cancer and colorectal cancer and could be a good supplement of CEA in screening of cancer.

【Objective】To observe the anti-depression effect of Shugan Hewei Granules(SHG) on mice with behavioral despair.【Methods】Kunming mice were randomized into 5 groups: model group,low-,moderate-and high-dose SHG groups(in the dosages of 0.25,0.5 and 1.0 g?kg-1?d-1respectively) and chloropromazine(CP) group.Tail suspension test and forced swimming test were used to investigate immobility time of mice.【Results】Moderate-and high-dose SHG shortened the immobility time obviously during tail suspension test and forced swimming test(P

Objective: To investigate the effects of human tissue inhibitor of matelloproteinase 4 (TIMP 4) on vascular smooth muscle cells (VSMCs) migration and the neointimal formation after balloon injury in rats. Methods: The cultured VSMCs were transfected with an adenoviral vector containing human TIMP 4 gene, AdTIMP 4. Effect of TIMP 4 on VSMC migration was investigated by monolayer cell scrape. AdTIMP 4, control adenoviral vector or PBS was transduced into the rat carotid artery from the adventitial after carotid artery injury. Cell number within the internal elastic lamina 4 days after gene transfer was counted and neointima/media area ratio 28 days after gene transfer was calculated. Results: The migration distance of VSMCs infected with AdTIMP 4 was inhibited markedly. Morphometric analysis demonstrated a statistically significant reduction in the number of cells migrated into neointima compared with controls [(32.5?4.8) cells per section, (33.8?7.0) cells per section and (8.2?2.4) cells per section for uninfected, AdGFP treated and AdTIMP 4 treated arteries, respectively]. There was a reduction of intima/media ratio of TIMP 4 treated group by 66.5% compared with control groups 28 days after gene transfer ( P

0.05). Conclusions The expression of angiotensinⅡincreases in BPH tissues in rats. It is suggested that angiotensinⅡmay affect cell proliferation but not significantly affect cell apoptosis of the rat prostate.

Objective To investigate the appropriate setting up of normal reference ranges of lymphocyte subsets in some flow cytometry laboratories and to study the effects of different flow cytometers and various reagents by different manufacturers on the analysis of peripheral blood lymphocyte subsets. Methods Three FCM labs (named A, B and C) in Beijing region were selected representing 3 commonly used flow cytometers (Beckman Coulter Epics XL, Beckman Coulter Cytomics FC500, BD FACS Calibur). 50 samples from healthy donors were distributed to 3 labs and tested according to individual lab's standard operating procedure to verify whether the normal reference ranges of peripheral blood lymphocyte subsets established were appropriate. The application of internal quality control was also investigated. Commercial blood quality control reagents were given to the 3 FCM labs and tested within 20 working days paralleled with routine samples. In addition, 20 patients' samples were prepared using 4 different combinations of reagents ( a , b , c and d). The results from combination a, which used the Beckman Coulter reagents and instrument, were compared to the results from combination b, c and d, which used reagents from different manufacturers. Then the prepared samples were tested on Beckman Coulter Epics XL to evaluate the effects of different combinations of reagents on the results of peripheral blood lymphocyte subsets analyzed by the same instrument. Furthermore, 24 patients' samples prepared by same reagents from Beckman Coulter company were tested on both Beckman Coulter Epics XL and BD FACS Calibur respectively to assess the effects of different instruments on peripheral blood lymphocyte subsets. 20 patients' samples prepared by same reagents and instruments were analyzed by Beckman Coulter Epics XL analytic system and BD FACS Calibur analytic system respectively to assess the effects of the two analytic systems on the lymphocyte subsets. Results Over 10% of the results for NK and T4/T8 in lab A as well as T4 in labs B and C fell outside of their normal reference ranges. The probabilities exceeding corresponding normal reference ranges were 16% ( 9/50 ), 24% ( 12/50 ), 22% (11/50) and 12% ( 6/50 ), respectively. The results using internal blood quality control in 3 FCM labs within 20 working days were all within the reference ranges of the quality control provided by the kit. The biases from b and c reagent combinations were substantial compared with that of reagent a combination. Among the biases from b and c reagent combinations, the lowest probability of bias exceeding 10% was T8 of combination b, which had probability of 70% (14/20). The highest probabilities of hias exceeding 10% were T3 and T4 of b and c reagent combinations, which reached 100% (20/20) . Furthermore, the biases of T3, T8 and B of d reagent combination compared with that of reagent a combination were also substantial. The probabilities of bias exceeding 10% were 35% (7/20) ,85% (17/20) and 75% (15/20), respectively. Comparing the results of samples prepared and analyzed by reagents and instruments from different manufacturers to that of samples prepared and analyzed by the same company's reagents and instruments showed that there were great discrepancies in T3, T4 , T8 , B and NK. The probabilities of bias exceeding 10% were 71% ( 17/24), 80% (19/24) ,38% (9/24), 33% (8/24) and 92% (22/24), respectively. The biases of T8, NK and B were substantial when compared the results from Beckman Coulter Epics XL analytic systems and BD FACS Calibur analytic systems. The probabilities of bias exceeding 10% were 55% (11/20 ), 70% ( 14/20 ) and 55% (11/20), respectively. Conclusions FCM labs should set up their own normal reference range for peripheral blood lymphocyte subsets. The normal reference range should be verified periodically. It is important to apply internal blood quality control regularly and accumulate the quality control results. The reagents and instrument for preparing peripheral blood samples should be from the same manufacturers.

Objective To evaluate the performance of sST 2 ELISA kit and investigate the clinical application of sST2.Methods This verification study validated the precision , linearity of sST2 ELISA kit according to the CLSI EP-15A, EP-6A protocols.300 healthy adults(aged from 20 to 85, 124 male and 176 female) from 5 different districts of Shanghai were used to establish serum sST 2 reference interval .The correlations between sST2, NT-ProBNP, LVEF and NYHA class were analyzed in 117 patients diagnosed with heart failure who were grouped according to the New York heart association ( NYHA).Receiver operating characteristic (ROC) curve was used to compare the ablity of sST2, NT-ProBNP, LVEF in distinguishing heart failure patients .Results The within-lot and between-lot variation of three level samples were below 4% and 10% respectively.There was a good linear correlation ( Y=0.995X+0.005, R2 =0.999) between theoretical value and actual detection result in the range of 0 to 200 μg/ml.The reference interval of sST2 was 10.2 to 41.0μg/ml for males and 8.9 to 28.1μg/ml for females.sST2 was positively correlated with NT-ProBNP and NYHA class but did not correlate with LVEF in heart failure patients . Patients with NYHA class>II (Median:28.3,IQR:19.5-39.2)had higher serum sST2 level than patients with NYHA class≤II (Median:45.1,IQR:34.1 -85.6), P<0.05.The AUC of sST2 in distinguishing heart failure patients from normal people was 0.815(sensitivity :51.2%,specificity:92.7%).The AUC of sST2 ,sST2+NT-ProBNP and sST2+NT-ProBNP+LVEF in distinguishing patients between NYHA class≤II and>II were 0.743, 0.810, 0.831 respectively and the sensitivity of sST 2 +NT-ProBNP+LVEF was 94.7%.Conclusions Experimental results show that this sST 2 ELISA kit has a good performance in the precision, linearity.sST2 correlates with NT-ProBNP and NYHA class but do not correlates with LVEF . Serum sST2 level is not influenced by age , BMI, renal function.sST2 could be a good supplement of NT-ProBNP and LVEF in distinguishing patients between NYHA class≤II and>II.