In recent years,more and more of the enzyme proteins have been carried out using recombinant microorganism bioreactor for large scale production.For reasons of improved the catalytic capability and environmental suitability,or enhanced expression level of the protein,a variety of genetic engineering technology according to protein molecule modification have been applied extensively.Major strategies and achievements of molecular modification for microbial enzyme,such as site-directed mutagenesis,error-prone PCR,DNA shuffling and optimum codon design were reviewed.

Adenosine deaminase (ADA)plays an important role in the regulation of normal immune system.The altered activ-ity of serum ADA could associate with some autoimmune diseases(AID).Various studies abroad have found the elevated ac-tivity of serum ADA compared with healthy controls in many kinds of AID such as systemic lupus erythematosus (SLE), rheumatoid arthritis(RA),et al (P<0.05).Many studies foused on the correlation of altered activity of serum ADA with the disease severity,but there is still controversy.To explore the diagnostic value of serum ADA in AID,make a review a-bout the application research progress of ADA in several AID.

Objective: To investigate the effect of enolase 1 (ENO1) expression on proliferation, apoptosis and migration of lung cancer PC14 cells. Methods: ENO1 over-expression vector-pcDNA3.1/ENO1 was constructed and transfected into PC14 cells at logarithmic growth phase with liposome LipofectamineTM 2000. G418 was used to screen PC14 cells that stably expressing ENO1. The effects of ENO1 over-expression on proliferation, migration and apoptosis of PC14 cells were detected by CCK-8 method, scratch-healing assay and flow cytometry, respectively. Results: The ENO1 over-expression cell model was successfully constructed. Compared with PC14-vehicle and wild-type PC14 cells, the mRNA and protein expression levels of ENO1 in PC14-ENO1 cells were significantly elevated (all P<0.05), and the proliferation of PC14-ENO1 cells was significantly increased (all P<0.05). The relative mobility of PC14ENO1 cells was significantly higher than that of pcDNA3.1-vehicle cells and wild-type PC14 cells ([13.26±1.13]% vs [8.46±1.11]%, [7.86±1.00]%, both P<0.05). There was no significant difference in apoptotic rate among PC14-ENO1, PC14-vehicle and PC14 cells (all P> 0.05) Conclusion: Over-expression of ENO1 promotes proliferation and migration of lung cancer PC14 cells.

Objective To construct anti-astrocyte elevated gene-1(AEG-1)single-chain variable antibody (V23)prokaryotic ex-pression vector,and to conduct the protein purification and immunological activity detection.Methods The Primer5 software was applied to design the primers aiming at the gene sequence of the antibody anti-AEG-1 single-chain variable region for constructing the prokaryotic expression plasmid of PRsetC/V23.After the enzyme digestion by the restriction enzyme Pst1 and correct DNA se-quencing,the prokaryotic expression plasmid was led to E.coli BL21 ,the prokaryotic expression engineering strain containing the V23 gene was constructed.After the induction with IPTG,the interest protein was purified by the magnetic beads with the HIS tag,and the content of interest protein was determined by the SDS-PAGE electrophoresis.Western blot and ELISA were adopted to detect the immune activity of the nti-AEG-1 single-chain variable region antibody.Results For the constructed prokaryotic expres-sion plasmid PRsetC/V23,the single enzyme digestion and sequencing analysis displayed that the constructed V23 gene was com-pletely consistent to the designing sequences.After IPTG induction,SDS-PAGE electrophoresis showed an apparent protein band at 31×103 ,the Western blot detection showed a specific AEG-1 response band at 80 ×103 ,the ELISA test showed the positive re-sults.Conclusion The PRsetC/V23 prokaryotic expression plasmid and the V23 prokaryotic expression engineering strain are suc-cessfully constructed,this engineering strain can express anti-AEG-1 single-chain variable region antibody protein,and the protein has good immune activity.

BACKGROUND:With the development of tissue engineering, autologous chondrocyte implantation is often used to repair cartilage defects. And poor integration is one of the common reasons that lead to failure repairing. Many models in vitro are used for related studies.
OBJECTIVE:To develop an interface integrated model of tissue engineered cartilage repair in vitro and to evaluate the effect.
METHODS:Cartilage integration model in vitro was established in pigs. Total y 21 cartilaginous rings were obtained and divided into agarose gel group (n=18) and control group (n=3). In agarose gel group, cartilage rings were covered with agarose gel. Chondrocytes were separated and implanted into the ring. The leakage of cells around the cartilage rings was observed. The sections were stained for histological observation at 1, 2, 4 weeks. The average area of neochondrocytes was measured and compared.
RESULTS AND CONCLUSION:The results from the control group were not processed, because there was no chondrocyte aggregate formation in the center of the explant ring due to earlier chondrocyte leakage outside the explant. While no chondrocytes were found outside the explant ring in the agarose gel group. Tissue sections of the agarose gel group were stained by hematoxylin and eosin, alcian blue, Safranin-O and col agen type II immunohistochemistry at 1, 2, 4 weeks. Neochondrocytes proliferated within cartilage ring, and produced extracellular matrix. After 2 weeks of incubation, these inserted chondrocytes were significantly increased. There was no statistical y significant increment between 2 weeks and 4 weeks (P>0.05), although the area was further increased by 4 weeks. This model provides a convenient simulation of the cartilage integration process in vitro and has a potential application in studies of cartilage integration and cartilage tissue engineering.

Objective To explore the regularity and potential mechanisms of medicinal and edible herbs(MEHs)in the treatment of myelosuppression through the retrieval,summary,sorting and visual analysis of relevant literature.Methods Literature about MEHs treatment for myelosuppression was reviewed in document databases,such as Web of Science,CNKI,Wanfang Data Knowledge Service Platform,China Science and Technology Journal Database,and China Biology Medicine Disc.Multivariate statistical analysis was performed using the SPSS and CiteSpace software to explore the frequency,efficacy and correlation of MEHs,as well as the potential mechanisms of MEHs in treating myelosuppression.Results A total of 123 recipes involving 170 traditional Chinese medicines(including 38 MEHs)were screened out.Five pairs of MEHs core combinations in the treatment of myelosuppression were obtained by cluster analysis.Their main functions included benefiting qi and nourishing blood,invigo-rating spleen and dispelling dampness,replenishing qi and solidifying kidney.The potential mechanisms were associated with many related signal pathways,such as Janus kinase 2-signal transducer and activator of transcription 5 andβ-catenini.Conclusion MEHs such as radix astragali combined with angelica sinensis,poria cocos and codonopsis pilosula are mainly used clinically to treat myelosuppression induced by chemotherapy.They play their therapeutic effects by promoting proliferation and delaying senescence of hematopoietic stem cells.

Objective To study the effect of vibration training with adjusted frequency on changes in explosive force of lower limbs, balance and muscle function around ankle joints of patients with functional ankle instability (FAI), so as to provide an empirical basis for rehabilitation training of FAI patients in clinic. Methods Twenty-six FAI patients were randomly divided into the experimental group (n=14) and the control group (n=12). The experimental group received 8-week rehabilitation training with vibration intervention, while the control group only received 8-week rehabilitation training. Changes in maximum power, average power, maximum speed and average speed of the injured limb during vertical jump with single leg, changes in distances during long jump, changes in time during one-leg standing with eyes open and closed，changes in contract time (tc), relax time (tr) and displacement (Dm) of medial gastrocnemius (GM), lateral gastrocnemius (GL) and tibialis anterior (TA) muscles before and after training were measured and compared. Results In the experimental group, the maximum power and maximum speed of the injured limb during vertical jump with single leg, the distance during long jump with single leg and the time during one-leg standing with open and closed eyes were significantly improved, and the increase was higher than that of the control group. The increase of tc of all muscles in the experimental group was smaller than that of the control group, but tr and Dm did not show any regularity. Conclusions Vibration training with adjusted frequency can effectively improve the explosive force and balance ability of lower limbs of FAI patients, and promote the tc shortening of GL, GM and TA muscles, but whether vibration training with adjusted frequency can reduce muscle tension and promote muscle relaxation is still not clear.

OBJECTIVE: To study the clinical value of detecting carcinoembryonic antigen levels in pleural effusion (PCEA) and serum (SCEA) and their ratio (P/S) in the differential diagnosis of pleural effusions resulting from tuberculosis and lung cancer. METHODS: This retrospectively study was conducted among 82 patients with pleural effusion caused by pulmonary tuberculous (TB; control group) and 120 patients with pleural effusion resulting from lung cancer in our hospital between April, 2016 and March, 2018. PCEA, SCEA and P/S were compared between the two groups and among the subgroups of lung cancer patients with squamous cell carcinoma (SqCa), adenocarcinoma (ACA), small cell carcinoma (SCLC). The receiveroperating characteristic curve (ROC) analysis was used to confirm the optimal critical value to evaluate the diagnostic efficiency of different combinations of PCEA, SCEA and P/S. RESULTS: PCEA, SCEA and P/S were significantly higher in the overall cancer patients and in all the 3 subgroups of cancer patients than in the patients with TB ( < 0.05). The areas under the ROC curve of PCEA, SCEA and P/S were 0.925, 0.866 and 0.796, respectively; PCEA had the highest diagnostic value, whose diagnostic sensitivity, specificity, accurate rate, and diagnostic threshold were 83.33%, 96.34, 88.61%, and 3.26 ng/ml, respectively; SCEA had the lowest diagnostic performance; the diagnostic performance of P/S was between that of SCEA and PCEA, but its combination with SCEA greatly improved the diagnostic performance and reduced the rates of misdiagnosis and missed diagnosis. Parallel tests showed that the 3 indexes combined had significantly higher diagnostic sensitivity than each or any two of the single indexes ( < 0.05), but the diagnostic specificity did not differ significantly. The area under the ROC curve of combined detections of the 3 indexes was 0.941 for diagnosis of lung cancer-related pleural effusion, higher than those of any other combinations of the indexes. CONCLUSIONS The combined detection of PCEA, SCEA and P/S has a high sensitivity for diagnosis of lung cancer-related pleural effusion and provides important information for rapid and accurate diagnosis of suspected cases.
Carcinoembryonic Antigen ; analysis ; blood ; Case-Control Studies ; Diagnosis, Differential ; Humans ; Lung Neoplasms ; blood ; complications ; Pleural Effusion ; blood ; diagnosis ; immunology ; Pleural Effusion, Malignant ; blood ; chemistry ; diagnosis ; ROC Curve ; Retrospective Studies ; Sensitivity and Specificity ; Tuberculosis, Pulmonary ; complications

[摘 要] 目的：探讨干扰B7-H4表达对乳腺癌细胞增殖、凋亡、周期以及相关下游分子表达的影响。方法：利用脂质体转染技术分别将特异性靶向B7-H4的siRNA（siB7-H4）及其阴性对照（siNC）转染至对数生长期的乳腺癌T47D和MCF-7细胞，分别命名为T47D-siB7-H4、T47D-siNC、MCF-7-siB7-H4和MCF-7-siNC组。用qPCR法和WB法验证siRNA干扰效果及其对细胞周期分子cyclin D1表达的影响，CCK-8法和FCM分别检测干扰B7-H4表达对T47D和MCF-7细胞增殖、周期和凋亡的影响，qPCR法检测B7-H4干扰对E2F家族相关转录因子表达的影响。结果：成功构建干扰B7-H4表达的乳腺癌T47D和MCF-7细胞。与T47D-siNC和MCF-7-siNC组相比，T47D-siB7-H4和MCF-7-siB7-H4组细胞中B7-H4 mRNA和蛋白表达水平均显著降低、细胞增殖能力显著降低（均P<0.01），并伴有G1/S期细胞周期阻滞以及cyclin D1表达下调（均P<0.01），但细胞凋亡率差异无统计学意义（均P>0.05）。与T47D-siNC相比，干扰B7-H4后T47D细胞中E2F1、E2F2、E2F7和E2F8 mRNA水平有不同程度的降低（均P<0.01）；与MCF-7-siNC相比，干扰B7-H4后MCF-7细胞中E2F1、E2F2、E2F3、E2F7和E2F8 mRNA水平均有不同程度的降低（P<0.05或P<0.01）。结论：干扰乳腺癌细胞B7-H4表达可下调cyclin D1和E2F家族相关转录因子的表达，导致细胞周期阻滞并抑制细胞增殖。

Objective:To compare the efficacy of the combination of abidol, lopinavir/ritonavir plus recombinant interferon α-2b (rIFNα-2b) and the combination of lopinavir/ritonavir plus rIFNα-2b for patients with COVID-19 in Zhejiang province.Methods:A multicenter prospective study was carried out to compare the efficacy of triple combination antiviral therapy and dual combination antiviral therapy in 15 medical institutions of Zhejiang province during January 22 to February 16, 2020. All patients were treated with rIFNα-2b (5 million U, 2 times/d) aerosol inhalation, in addition 196 patients were treated with abidol (200 mg, 3 times/d) + lopinavir/ritonavir (2 tablets, 1 time/12 h) (triple combination group) and 41 patients were treated with lopinavir/ritonavir (2 tablets, 1 time/12 h) (dual combination group). The patients who received triple combination antiviral therapy were further divided into three subgroups: <48 h, 3-5 d and >5 d according the time from the symptom onset to medication starting. The therapeutic efficacy was compared between triple combination group and dual combination group, and compared among 3 subgroups of patients receiving triple combination antiviral therapy. SPSS 17.0 software was used to analyze the data.Results:The virus nucleic acid-negative conversion time in respiratory tract specimens was (12.2±4.7) d in the triple combination group, which was shorter than that in the dual combination group [(15.0±5.0) d] ( t=6.159, P<0.01). The length of hospital stay in the triple combination group [12.0 (9.0, 17.0) d] was also shorter than that in the dual combination group [15.0 (10.0, 18.0) d] ( H=2.073, P<0.05). Compared with the antiviral treatment which was started within after the symptom onset of in the triple combination group, the time from the symptom onset to the viral negative conversion was 13.0 (10.0, 17.0), 17.0 (13.0, 22.0) and 21.0 (18.0, 24.0) d in subgroups of 48 h, 3-5 d and >5 d, respectively ( Z=32.983, P<0.01), while the time from antiviral therapy to viral negative conversion was (11.8±3.9), (13.5±5.1) and (11.2±4.3) d, respectively( Z=6.722, P<0.05). Conclusions:The triple combination antiviral therapy of abidol, lopinavir/litonavir and rIFNα-2b shows shorter viral shedding time and shorter hospitalization time, compared with the dual combination antiviral therapy; and the earlier starting triple combination antiviral therapy will result in better antiviral efficacy.