Objective To detect the expression level of RKIP in human lung cancer cell lines and to investigate the relationships between RKIP and cell metastasis. Methods The expression level of RKIP in human lung cancer cell lines was analyzed by RT-PCR. To construct and express human RKIP fusion protein and to immunize New Zealand rabbit with purified fusion protein to prepare polyclonal antiserum.The antiserum was titered by ELISA. The specifcity of antiserum and expression level of RKIP in human lung carcinoma cell lines was analyzed by western blotting. Results The fusion protein was successfully expressed. The prepared polyclonal antibody reacted specifically with RKIP in human cells. Downregulation of RKIP was detected in three lung cancer cell lines with metastasis, and it is more significant in A549 with higher metastasis.Conclusion The RKIP antiserum with high titer and good specificity could meet the requirement for studying on RKIP. Downregulation of RKIP has correlation with human lung carcinoma cell metastasis.

Objective To construct a lentivirus vector for RNA interference targeting myosin Va gene and to observe its effect on motility and migration of human pulmonary giant cell carcinoma PG cells. Methods Based on the efficient target sequence for myosin Va RNAi, two pairs of oligo DNA containing myosin Va RNAi target sequence or scramble sequence were synthesized and inserted into pSuper vector, followed by sequence analysis. The expressing cassette H1 promoter-shM5A/shCON from the recombinant pSuper plasmid was then transferred to the lentivirus vector plenti4, and the recombinant lentivirus was packaged. PG cells were transduced with the packaged lentivirus and the positive cells were screened by zeocin selection. RT-PCR was performed to determine the myosin Va RNAi efficiency in zeocin-resistant PG cells, and wounding assay and Boyden chamber assay were utilized to examine the capabilities of motility and migration in myosin Va RNAi PG cells. Results Restriction enzyme digestion and sequencing confirmed the successful construction of the lentivirus vector containing myosin Va RNAi target or scramble sequence. RT-PCR result showed that myosin Va mRNA levels were remarkably reduced in lentivirus-based myosin Va RNAi PG cells. The abilities of motility and migration were also significantly inhibited in lentivirus-based myosin Va RNAi PG cells, as demonstrated in wounding assay and Boyden chamber assay.Conclusion Myosin Va RNAi lentivirus vector was successfully constructed and efficiently repressed myosin Va expression in PG cells. Repression of myosin Va by RNAi led to the inhibition of PG cells motility and migration, indicating that there might exist correlation between the expression of myosin Va and cancer progression.

Objective To set up the quality standard of Wanbile Wan. Method s Fructus Psoraleae, Radix Puerariae, Radix paeoniae Alba and Radix Glycyrrhiza e Preparatae in Wanbile Wan were identified by TLC .Isopsoralen was determined b y TLC scanning. The chromatographic conditions were: high performance silica gel G plate as lamellated plate, n- hexane - ethyl acetate (8 ∶ 2)being the exp ander, serrated scanning with single- wavelength reflection, beam stricture of 0.4? 0.4 mm, linear parameter SX=3 and scanning wavelength at 300 nm. Results The TLC spots were highly specific, clear and concentrated without interferen ce of the presence of negative controls. Isopsoralen showed a good linearity wit hin the range of 0.064～ 1.536 ? g, r=0.9980, average recovery rate was 100.7 % , and RSD was 2.0% .Conclusion This method can be used for the quality co ntrol of Wanbile Wan.

ObjectiveReport an operation of off-pump occlusion of trans-thoracic minimal invasive surgery (OPOTTMIS) adopted on the therapy of 92 patients suffered with simple congenital heart diseases (CHD) of atrial septal defect ( ASD),ventricular septal defect ( VSD ) and patent ductus arteriosus (PDA) and summarize the experiences.Methods The clinical data of 92 CHD patients administrated with OPOTTMIS in our hospital during the time of July 2008 and July 2011 were analyzed retrospectively.Moreover,the relevant literatures were reviewed and the different kinds of treatments and their outcomes of CHD were compared.These patients were 3-56 years old and weighed 8.0-54.5 kg,with male and female 38 and 54 cases,including ASD 52 cases,VSD 26 cases and PDA 14 cases,respectively.Within the initial 3 months of postoperation,all the patients were followed up and managed to take the re-examination with electrocardiogram and echocardiogram one time for each month,and 3 months later,at least one time for every 3-6 months.Results91 cases were performed the OPTTMIS procedure successfully,1 child with sieve-like ASD transferred to open-heart surgery and 1 adult( 1.1％ ) PDA died on the second day after operation for the pulmonary hypertension crisis.4 cases appeared mild residual shunt including 2 of ASD,1 of VSD and 1 of PDA.2 cases showed Ⅱ stage atrial ventricular block ( AVB ) within ASD and VSD,respectively.Also,post-operation hemothorax happened to 1 case ASD and 1 case VSD for the active bleeding of cardiac puncture site and the exploration and hemostatic operation were performed.91 (98.9％) patients were performed the OPOTTMIS operation successfully,the total complications within 72 hours was 6.5％ (6/92),respectively.There were no heavy complications happened during peri-operation such as cardiac rupture,infective endocarditis,stroke,hemolytic and thrombus formation.90 patients outcomes of 3 months to 4 years follow-up with the ECG and UCG examination showed that there were no Ⅲ stage atrioventricular block,no obvious occluder shift and broken and no moderate cardiac valve regurgitation,no evident blood flow restriction of left ventricular outflow tract and descending aorta,only found mild residual shunt within 2 cases ( VSD and PDA each) and heart expansion compared to pre-operation within 2 PDA patients.However,all the patients'heart functions were in grade Ⅰ to grade Ⅱ according to NYHA standard.ConclusionOPOTTMIS is a safe,feasible and effective option on simple congenital heart diseases.

Objective To construct miRNA-29b1 gene knockout mice based on CRISPR/Cas9 technology. Methods To design and synthesize sgRNA according to the miRNA-29b1 sequence in Genbank .sgRNA and Cas9 were transcribed to RNA in vitro, these RNA were then microinjected into zygotes of C 57BL/6 mice.After mouse birth, the genome DNA was extracted and sequenced to identify its genotype; meanwhile , real-time PCR was used to assay the expression of miRNA-29b1 in the heart, liver, spleen, lung and kidney of mutated mice .Result A 20 bp sgRNA targeted on miRNA-29b1 was synthesized and transcribed to RNA with Cas 9.After microinjection, miRNA-29b1 gene-mutated mice were obtained.The sequencing results showed that there were two types of genotype for the mutated mice , one was 10 bp deletion, and another was 23 bp deletion accompanied with a 3 bp insertion.Compared with the wild-type mice, the expression of miRNA-29b1 in the heart, liver, spleen, lung and kidney was reduced significantly .Conclusions miRNA-29b1 gene knockout mice are constructed successfully by using CRISPR /Cas9 technology.

Objective To compare the clinical effects of vesselplasty and percutaneous kyphoplasty in the treatment of osteoporotic thoracolumbar vertebral fracture in the elderly.Methods A retrospective case control study was conducted to analyze 52 cases of OVCFs treated from August 2013 to May 2015.According to treatment method,the patients were assigned to vesselplasty (Group A) and percutaneous kyphoplasty (Group B).Group A (25 cases,38 vertebrae) included 11 males and 14 females,aged (63.6 ±8.3)years (range,60-89 years).In Group A,there were nine cases of T11,10 T12,13 L1,and six L2.Group B (27 cases,41 vertebrae) included 10 males and 17 females,aged (64.1 ±9.6)years (range,63-87 years).In Group B,there were seven cases of T11,13 T12,12 L1,and nine L2.The bone cement leakage before operation,after operation,and at the last follow-up were recorded.The operation time,average fluoroscopy frequency,visual analog score (VAS),vertebral reduction height,Cobb angle,and Oswestry disability index (ODI) changes were compared between two groups.Results All patients were followed up for 6-18 months (mean,12 months).The operation time was (29.3 ± 4.1)minutes in Group A and (35.7 ± 5.2) minutes in Group B (P ＜ 0.05).The number of fluoroscopy was (9.3 ± 1.5) times in Group A and (13.1 ±3.7)times in Group B (P ＜0.05).The VAS at the last follow up was (1.3 ±0.3) points in Group A and (1.4 ± 0.3) points in Group B;the ODI at the last follow up was 32.5 ± 6.7in Group A and 30.5 ± 5.3 in Group B;the injured vertebral height at the last follow up was (85.3 ±9.7)％ in Group A and (82.7 ±10.4)％ in Group B;the Cobb angle at the last follow up was (11.3 ±5.3) ° in Group A and (12.7 ± 6.1) ° in Group B.VAS,vertebral reduction height,and Cobb angle were all improved significantly compared with those before operation (P ＜ 0.05),but there was no significant difference between two groups (P ＞0.05).The leakage rate was 3％ in Group A and 26％ in Group B (P ＜ O.05).Conclusion Both vesselplasty and percutaneous kyphoplasty can quickly relieve the pain and effectively restore the height of injured vertebra.But vesselplasty can reduce bone cement leakage more effectively,thus being a better treatment for osteoporotic thoracolumbar vertebral fracture in the elderly.

Objective To analyze the correlations of D-dimer and the Global Registry of Acute Coronary Events (GRACE) score with long-term heart failure (HF) in patients with acute myocardial infarction (AMI). Methods A total of 398 patients with AMI were selected as research objects and divided into normal D-dimer group (n=309) and elevated D-dimer group (n=89) based on the D-dimer level. Cox proportional hazard model was used to analyze the risk factors for long-term HF in both groups. Time-dependent receiver operating characteristic (ROC) curve was used to analyze the values of D-dimer, GRACE score and their combination in predicting long-term HF. According to the GRACE score and D-dimer level, 398 patients were divided into low-value group (181 patients with normal D-dimerand low GRACE score), high-value group (70 patients with elevated D-dimer and high GRACE score), and middle-value group (147 patients did not meet the conditions of the low-value and high-value groups). Kaplan-Meier method was used to analyze the occurrence of long-term HF in the three groups. Point-biserial analysis was used to analyze the correlation between elevated D-dimer and the occurrence of long-term HF. Results The number of patients with long-term HF in the elevated D-dimer group was 2.3 times of the normal group. D-dimer and GRACE score were independent risk factors for long-term HF in patients with AMI (P < 0.05). Both D-dimer and GRACE score had certain predictive values for the occurrence of HF at 5 years after AMI, but the predictive efficiency of GRACE score was better. The incidence of long-term HF in the high-value group was significantly higher than that in the low-value group and the middle-value group (P < 0.01). D-dimer was significantly positively correlated with the occurrence of long-term HF after AMI (P < 0.001). Conclusion Both D-dimer and GRACE score are independent risk factors for long-term HF in patients with AMI, and the two indexes have certain predictive value for the occurrence of long-term HF. Patients with both elevated indicators are high-risk groups for long-term HF.

To develop a rapid differential detection method for porcine epidemic diarrhea virus(PEDV)and transmissible gastroenteritis virus(PEDV),M gene sequences of PEDV and TGEV were compared,the inner and outer primer pairs and probes were designed according to the highly conserved region.PEDV-Probe was labeled with FAM at5'end and BHQ1 at 3'end.TGEV-Probe was labeled with CY5.5 at the 5'end and BHQ2 at the 3'end.After optimizing the reaction condi-tions and system,a dual fluorescent RT-LAMP assay for PEDV and TGEV rapid identification was established.The amplification could be completed within 60 min in a 63 ℃ water bath and then stopped at 85 ℃ for 10 min.Then the tubes were placed in a multicolor imaging system,and the re-sults could be observed under 520 nm and 690 nm dual channels.There was no cross-reaction when other common swine viral pathogens were detected by this method.The sensitivity of the assay was evaluated with a 10-fold diluted recombinant plasmid as templates.The lowest detection limit was 102 copies/μL recombinant plasmid,which was 10 times more sensitive than the conventional RT-PCR method.Seventy-two PEDV-positive samples,49 TGEV-positive samples,and 40 PEDV and TGEV co-infected samples were detected from 175 anal swab samples of diarrheic piglets by the established method,which were all higher than the detection rates of the conventional RT-PCR method.The dual fluorescent RT-LAMP method established in this study can be used to amplify the target gene in an ordinary water bath without gel electrophoresis,which provides technical sup-port for rapid and convenient differential diagnosis of PED and TGE and simultaneous detection of PEDV and TGEV co-infection.

Objective: To understand the prevalence of school bullying and depressive symptoms among the Han, Yi, and Tibetan adolescents in western China, and to explore the relationships between bullying roles, bullying frequency and depression symptoms. Methods: A combination method of typical sampling and cluster sampling was used to recruit 3 115 participants from six primary and middle schools in Cangxi County and Ganluo County in Sichuan Province, and Lhasa City in Tibet Autonomous Region from April to November 2020. All participants completed self filled questionnaires. Multiple Logistic regression was used to analyze the association between school bullying and depression symptoms. Results: A total of 881 participants (28.3%) had experienced school bullying in the past 6 months, and 892 participants(28.6%) were detected with depressive symptoms. The rates of school bullying in the Han, Yi, and Tibetan adolescents were 20.5%, 46.4% and 23.8%, respectively. Among them, the rates of victims were 15.3%, 25.0% and 11.8%, respectively; the rates of bully were 2.3%, 5.7% and 6.7%, respectively; the rates of bully victims were 2.9%, 5.7% and 5.4%, respectively. The prevalence of depressive symptoms in the Han, Yi, and Tibetan adolescents were 20.4%, 37.9% and 36.5%, respectively. After adjusting for age, grade, ethnicity, smoking, and drinking, the odds for depressive symptoms among victims ( AOR=1.98, 95%CI =1.61- 2.45 ), bully ( AOR=2.63, 95%CI =1.81-3.82), and bully victims ( AOR=3.33, 95%CI =2.44-4.54) were significantly higher than those without school bullying experience( P <0.01). And the risks increased with the increasing bullying frequency. Conclusion School bullying and depressive symptoms of the Yi and the Tibetan adolescents in western China were higher than those of the Han adolescents. Meanwhile, participation in school bullying and the high frequency of bullying were closely related to depressive symptoms. Future interventions should pay attention to various bullying roles and minority adolescents.

Objective:To investigate the methylation status of SDC2, PPP2R5C and ADHFE1 genes in stool and their values in the screening of colorectal cancer and precancerous lesions.Methods:From August 2020 to March 2021, 64 patients with colorectal cancer, 72 patients with adenoma, 33 patients with hyperplastic polyps and 59 healthy people were recruited from Qingdao Central Hospital Affiliated to Qingdao University, and the morning stool samples were collected from the research subjects. The genomic DNA was extracted and modified with sulfite. The methylation status of SDC2, PPP2R5C and ADHFE1 genes were detected by methylation specific polymerase chain reaction (MSP), and the fecal occult blood test (FOBT) was performed. Taking the pathological results as the gold standard, receiver operating characteristic (ROC) curve and area under the curve (AUC) were used to compare the effect of combined detection of methylation of three genes and FOBT in predicting colorectal cancer and precancerous lesions. R-Studio software was used to construct a nomogram for the prediction of colorectal cancer with combined detection of gene methylation in stool and other clinical features, and the calibration and validation were performed.Results:The positive rates of combined detection of methylation of SDC2, PPP2R5C and ADHFE1 genes in stool were higher than those of FOBT in colorectal cancer+adenoma [74.3% (101/136) vs. 47.1% (64/136), χ2 = 23.20, P = 0.001], colorectal cancer [90.6% (58/64) vs. 70.3% (45/64), χ2 = 8.91, P = 0.003] and adenoma [59.7% (43/72) vs. 26.4% (19/72), χ2 = 14.43, P = 0.002]. There was no significant difference in the positive rates in hyperplastic polyps [21.2% (7/33) vs. 6.1% (2/33), χ2 = 0.12, P = 0.125] and healthy controls [10.2% (6/59) vs. 8.5% (5/59), χ2 = 4.01, P = 1.000]. The combined detection of gene methylation was better than FOBT in the prediction of colorectal cancer + adenoma [AUC: 0.85 (95% CI 0.80-0.91) vs. 0.71 (95% CI 0.64-0.78), P < 0.05], especially in the prediction of adenoma [AUC: 0.82 (95% CI 0.74-0.89) vs 0.64 (95% CI 0.57-0.69), P < 0.001]. The sensitivity and specificity of ADHFE1 gene methylation status in predicting colorectal cancer were high (90.6% and 96.6%). In colorectal cancer patients over 50 years old, the positive rate of combined detection of gene methylation was higher than that of FOBT [90.2% (55/61) vs. 68.9% (42/61), P < 0.05]. The nomogram calibration curve for predicting colorectal cancer constructed based on the combined detection of gene methylation and each clinical feature showed a high degree of concordance between the predicted and observed diagnostic performance of colorectal cancer. Conclusions:The methylation levels of SDC2, PPP2R5C AND ADHFE1 genes in stool are increased in patients with colorectal cancer or adenoma. The combined detection of gene methylation is expected to be a non-invasive method for the screening of colorectal cancer and precancerous lesions.