To construct a library of mouse single chain variable fragment (scFv) antibody against ofloxacin using phage display and recombinant antibody technique, specific anti-ofloxacin scFv was screened and 3D structure was homology modeling. Total RNA was extracted from hybridoma cell of ofloxacin mAb, and was used to amplify VH and VL gene by RT-PCR using random primer. Then they were linked by a DNA linker encoding (G1y4 Ser) 3 as VH-linker-VL sequence forming scFv by SOE(splicing by overlap extension) PCR. These fragments were inserted into phage T7 after double digestion and transformed with host bacteria BLT5403. 3 ×105 pfu / ml single chain antibody phage libraries were successfully constructed. Four positive phage scFv clones were screened by direct competitive ELISA after four times of enriched procedure in the order of adsorption-elution-amplificatio, 3D structure of specific scFv was homology modeling finally. This research lays a foundation for further massive expression of anti-ofloxacin scFv.

Objective To investigate the diagnostic value of acridine orange fluorescene(AO-F) in bladder cancers. Methods One thousand and sixteen bladder cancer patients were reviewed retro-spectively. The positive-rates of AO-F in different stages, grades, size, quantity, position of tumors, hematuria and treatment ways were evaluated. Results The total positive rate of AO-F was 78.05 % (793/1016). The positive-rate was 74.69% (611/818) in superficial stage and 91.92% (182/198) in invasive bladder cancer, 67.24% (351/522) in grade Ⅰ and Ⅱ , 90. 37% (413/457) in grade Ⅲ. The percentage of positive AO-F was 80.30% (750/934) in patients with hematuria, 52.44% (43/82) in patients without hematuria. The percentage was 79.87% (710/889) when the tumor size was more than 2 cm, 65.35% (83/127) when size less than 2 cm. 83.07% (363/437) sample was positive in multiple tumors, 74.27% (430/579) in single tumor. The percentage was 77.21% (105/136) in tumors involving trigone or neck of bladder, 78.07% (687/880) in tumors without involving these re-gions. There was 69.68% (393/564) in treatment with TURBt, 87.87% (268/305) in partial resec-tion, 91.74% (100/109) in total resection. A good association was observed between stage, grade, hematuria appearance, tumor size, quantity of carcinoma, treatment way and AO-F positive-rate, and a linear correlation was present between grade, stage and positive cytology. There was no significant association between position of the tumor and AO-F positive-rate. Conclusions The function of AO-F is significant in diagnosis of bladder cancer.

Objective Anti-RNase virus-like particles containing HCV RNA 5'-UTR were used as positive samples in national external quality assessment ( EQA ) to evaluate the competency of clinical Laboratories for the quantification of HCV RNA and analyze the possible problems of domestic kits. Methods The quality control samples with target values in EQA panels were distributed nationally twice by National Center of Clinical Laboratory (NCCL) to participating laboratories for the quantification of HCV RNA in 2008 and 2009. Each panel consisted of 5 samples. All participants were required to carry out the detection and to return results in expected time. Positive samples were virus-like particles which had been calibrated against the WHO HCV International Standard (NIBSC96/798)and the results of positive samples from participants should be in the range of target value of logarithm ± 0. 5. The 2nd panel in 2008 contained the common HCV genotypes and the 2nd panel in 2009 contained serial diluted samples of genotype 1b. The results of positive samples detected with 3 different lots reagent (21001,21078 and 21097) from the 2nd EQA in 2009 were statistically analyzed using the analysis of variance, then Dunnett'S T3 and Tamhane'S T2 were used if heterogeneity of variance was found. Results There was 390 participating laboratories in 2008 and 428/426 in 2009. The percentages of laboratories within the range of target value of logarithm ± 0. 5 for varied genotypes were different. The percentages of laboratories for 1b were more than 91%, for 2a were 93.7% and 74. 2% ,for6 were 83.3% and 80. 3%. The CVfor the low-level sample was higher than that for the high-level sample in the same year. The numbers of laboratories reporting false-negative samples in 2008and the 2nd in 2009 were 5, 1 and 10 respectively. Statistical differences were found among the results of four quality control serum samples using 3 different reagents( F = 288.23, 324. 79, 291.98 and 261.16,P ＜0. 01 ). Conclusion The competency for detecting low concentration samples and samples with genotype 2a or 6 needs to be improved.

Objective To study the effect of PIM-1 gene silence by RNA interference (RNAi) on the growth of human prostate cancer xenograft tumor in nude mice. Methods The xenograft tumor model of human prostate cancer was established by injecting PC-3 cells in armpits of 12 nude mice. After modeling, the nude mice were randomly divided into three groups: interference plasmid group (injecting with RNAi recombinant plasmid), empty plasmid group and negative control group (liposome every), 4 mice in each group. Mice were injected every 2 days for 5 times. The tumor volumes of xenografts were measured during experiment, and the curve of tumor growth was drawn accordingly. The quality of tumor was measured, and the inhibitory rate of tumor was calculated at the end of the experiments. The expression levels of PIM-1, c-MYC mRNA and protein in xenograft tumors were detected by real-time PCR and Western blot assay, respectively. Furthermore, immunohistochemistry staining was used to verify the expression of PIM-1. Results The xenograft tumor model of human prostate cancer was established successfully. The volume of tumor was significantly decreased 6 days after the injection treatment in interference plasmid group than that of empty plasmid group and negative control group. The effect of suppressing tumor growth was remarkable. The expression levels of PIM-1 mRNA and protein were down-regulated significantly in interference plasmid group than those of other two groups. The immunohistochemical staining of PIM-1 showed the same changes. There was no significant difference in c-MYC protein level between the three groups. But interestingly, the c-MYC mRNA level was significantly decreased in interference plasmid group than that of other two groups. Conclusion The silence of PIM-1 gene by RNAi recombinant plasmid can result a significant growth suppression of the human prostate cancer xenograft tumors in nude mice. The expression of c-MYC gene is down-regulated at translation level in the therapeutic group concomitantly. PIM-1 may be a promising target of gene therapy for prostate cancer.

Objective:To study the expression of miRNA-7 in B lymphocytes of primary Sj?gren's syndrome (pSS) and its relationship with phosphatase andtensin homolog deleted (PETN) and disease activity.Methods:Twenty newly diagnosed outpatient and inpatient pSS patients were used as case group collected from January 2017 to December 2019 of Qinghai Provincial People's Hospital. Twentyhealthy persons were used as the control group. Disease-related indicators of the case group were collected. Quantitative polymerase chain reaction (RT-qPCR)was used to detect miRNA-7 and PETN mRNA expression in B lymphocytes of the two groups and the consistency between miRNA-7 expression in the plasma and B lymphocytes of the case group was analyzed. Western Blotting method was used to detect the PETN protein in B lymphocytes of the two groups. Correlation analysis was used to analyze the relationship between miRNA-7 expression in B lymphocytes and disease activity in the case group. Linear regression analysis was performed between miRNA-7 and PETN mRNA.Results:The expression of miRNA-7 (0.53±0.17) in the B cells increased and the expression of PTEN mRNA (0.88± 0.24) and protein (0.51±0.12) in the case group were reduced compared with that of miRNA-7(0.39±0.11), PTEN mRNA(2.32±0.30) and protein(1.03±0.21) of the control group. The above differences were statistically significant ( t=2.990, P<0.05; t=16.98, P<0.05; t=8.41, P<0.05). Linear regression showedthat PTEN miRNAwas negatively correlated with miRNA-7 ( b=-0.78, P<0.01), the expression of miRNA-7 in the case group was positively related with EULAR Sj?gren′s syndrome Disease Activity Index (ESSDAI), IgG, IgA, anti-SSB and was negatively correlated with C4 and WBC. Conclusion:There is a certain relationship between miRNA-7 and disease activity. MiRNA-7 may participate in the pathogenesis of pSS byregulating PETN in B cells of pSS. MiRNA-7 has certain clinical value for disease activity evaluation.

ObjectiveTo evaluate the performance of antinuclear antibody (ANA) detection in clinical laboratories.Methods There were 2 external quality assessments (EQA) scheme for nuclear antibody detection.The panel consisting of 5 samples was distributed.Each participant laborotory of the EQA program was required to report the ANA qualitative results,patterns,titers and anti-double strain DNA (dsDNA) antibody,anti-extractable nuclear antigen(ENA) antibody,the percent agreements of which were calculated respectively.ResultsThe number of laboratories performing ANA test with IIF increased from 77.6％ ( 149/192 )in 2006 to 82.2％ (342/416) in 2011,while the number of laboratories performing ANA test with ELISA was in the range of 14.5％ ( 53/365 ) and 16.0％ ( 52/326 ).The positive percent agreements of IIF was over 98％.The positive percent agreement of ELISA were all over 90％.IIF showed more satisfying positive percent agreements than ELISA every year.Over 90％ of the laboratories reported correct results for samples with granular ANA pattern except 0613 and 0624.Over 95％ of the laboratories reported correct results for samples with homogeneous ANA pattern.Two samples with centromere pattern were correctly detected by 88.5％ ( 161/182 ),79.0％ ( 147/186 ) of the laboratories in 2007,while the sample with centromere pattern was correctly detected by 98.4％ (299/304), which indicated an improvement in the detection of centromere pattern.In ANA positive results,the lowest percentage of the laboratories reporting the median result was 36％ (94/261),while the highest percentage was only 85.5％(224/262).The satisfied results of anti-ENA antibody were over 90％.And those of anti-dsDNA antibody was over 85％.ConclusionsIIF is the most common method for ANA screening in clinical laboratories.ELISA is also used in some laboratories.The two methods reported satisfying results in ANA test.The detection of anticentromere antibodies is improved.But the results of ANA titer reported are unsatisfactory. ANA detection in routine practice needs to be improved by standardization.

Objective To observe the effect of acupuncturing Lianquan point combined with rehabilitation on severe pseudobulbar palsy.Methods Sixty patients with severe pseudobulbar palsy were divided into the control group and therapy group with 30 cases in each group. The patients of the control group were treated with medication, nasal feeding and vein nutrition. The patients of the therapy group were added with acupuncturing Lianquan point combined with rehabilitation besides methods the control group used. After two periods of treatment, the effects of two groups were compared.Results The overall efficient rate was 96.7% in the therapy group, and 73.3% in the control group, the former was obviously superior to the latter ( P<0.05).Conclusion Acupuncturing Lianquan point combined with rehabilitation can improve the therapeutic effect of severe pseudobulbar palsy.

ObjectiveTo establish rat model of traumatic brain injury combined with hemorrhagic shock.Methods Rat models of traumatic brain injury (produced by free fall impact method) combined with hemorrhagic shock (produced by venous injury method) were established and the related physiological parameters were recorded.The neurological impairment score,cerebral edema degree and blood brain barrier (BBB) were determined by using neurofunction scales,dry-wet method and Evans blue (EB) respectively.HE staining and immunohistochemical staining were applied to evaluate the pathological changes in brain sections.ResultsBlood pressure dropped from 95 mm Hg to 25 mm Hg within three minutes after modeling and maintained around 60 mm Hg one hour later.Neurological impairment score was increased dramatically.The ratio of water content in the brain tissue was elevated nearly from 77％ to 81％.The concentration of EB residual in the brain tissue was increased more than one fold.Neuronal pathological abnormalities,including neuron shrinking,dark eosinophilic staining,perineuronal vacuole in HE staining,and positive staining of β-amyloid precursor protein (β-APP) in immunohistochemical staining were also observed. ConclusionsRat models of traumatic brain injury combined with hemorrhagic shock are successfully established.In addition,the main pathological changes,such as cerebral edema,disruption of BBB,neuron damage,and expression of β-APP are replicated.

Objective Through the design of comprehensive experiments, the precise medical philo-sophy was put into the medical molecular biology experimental teaching, and to explore its effect. Methods Eight-year medical students of Grade 2012 and Grade 2013 in the Fourth Military Medical University were chosen as the teaching subjects. Experimental group consisted of 36 students of Grade 2013, while control group consisted of 30 students of Grade 2012. Precision medicine-based learning was applied in experimental group while traditional learning method was adopted by the control group. At the end of the course, students of two groups were implemented theoretical and experimental skills assessment; through questionnaire students were required to evaluate the effect of teaching methods and the number of two groups of students who asked questions after class greater than or equal to 1 times was counted to evaluate the students' learning enthusiasm. SPSS 15.0 software was used to make t test and Chi-square analysis for the data of the students. Results The assessment results showed that the experimental group was better than control group, especially in the section of comprehensive experimental design [(16.7 ± 2.04) vs. (13.9 ± 2.87), P=0.000]. The results from questionnaire showed that the satisfaction degree of experimental group was also higher than that of control group in many respects, including learning interests, innovation capability, knowledge mastery, cognition of precision medicine and clinical research, satisfaction with the teaching method (P<0.05). And students' learning enthusiasm and the proportion of the number of students asking questions in the experimental group were higher than the control group (P=0.000). Conclusions Precision medicine-based learning not only changes the situation of slavish imitation and passive acceptance in traditional learning, but also arouses students' interest in study and helps students to cultivate clinical thinking, which is com-mensurate to the characteristics of precision-medicine era.

Objective To evaluate the agreement of antinuclear antibody (ANA) titer reported in clinical laboratories and analyze possible problems in clinical laboratories.Methods Experiment survey.The panel consisting of 5 samples was distributed to 533 laboratories.Each panel contains one negative sample and 4 positive samples,which were from individuals of autoimmune disease.ANA titer system was divided into traditional titer system with two-fold dilution and titer system with 3.2 times dilution.Clinical laboratories were required to report the ANA titers and initial screening dilution according to the standard used in routine work.Results were expressed as median titers and range for acceptable performance.As to titer system with two-fold dilution,acceptable performance on proficiency testing was defined by a result equal to median titer ± two two-fold dilutions.While as to titer system with 3.2 times dilution,acceptable performance on proficiency testing was defined by a result equal to median titer ± 3.2 times dilutions.The laboratories percentages with acceptable performance were calculated to evaluate their agreements.Results 412 laboratories reported ANA titer results,of which 11.9％ (49/412)reported results with two-fold dilution titer system,88.1％ (363/412)reported results with 3.2 times dilution titer system.The median titers of sample 1211,1212,1213 and 1215 reported with two-fold titer system were 1 ∶ 640,1∶ 320,＞ 1 ∶ 1280 and1∶ 160,respectively.The agreement within the median ANA titer reported with two-fold dilution titer system ranged from 24.5％ (12/49) to 57.1％ (28/49) and the lowest percentage of the results within the acceptable limits was 87.8％ (43/49).The median titers of sample 1211,1212,1213 and 1215 reported with 3.2 times dilution titer system were 1 ∶ 1000,1∶ 1000,＞ 1 ∶ 3200 and 1 ∶ 320,respectively.The agreement within the median ANA titer reported with 3.2 times dilution titer system ranged from 63.3％ (31/49)to 83.7％ (41/49).The lowest percentage of the results within the acceptable limits was 98.0％ (48/49).Conclusions The results of ANA titer reported are unsatisfactory.Standardization for reagent,microscopy,procedure and result interpretation is necessary to improve the agreement of ANA titer report in different laboratories.