Construction of human kinase knock-out library by using CRISPR/Cas9 technique / 实用医学杂志

Objective CRISPR/Cas9 genome-editing technique provides an novel method for whole genome editing in eukaryotic cells.Recently,we found that gene subtype library with smaller size and focused pur-pose is more economical and practical. In this study,we aimed to target kinases,a group of pivotal cell signal transducers,to construct a kinase knock-out library using CRISPR/Cas9 technique.The construction strategy wll al-so be discussed. Methods 10 sgRNA was designed for each kinase target.After oligo pool synthesis by semicon-ductor chip,the oligos were eluted from the chip. The oligo templates were amplified and cloned into Cas9 vector and transformed into Stble3 competent cells.Monoclonal colonies were selected for DNA sequencing. Results(1) GO analysis of 507 cell kinases showed that the cell kinases took part in a wide range of cell signaling.(2)The sgRNA pool with about 140 bp in length was successfully amplified by using oligo pool as the template and univer-sal PCR primers.(3)In 40 identified library clones,34 clones were sequenced successfully. Among them,the DNA sequencing results of 25 samples were completely consistent with the designed target sequences.But there are some mutations in the primers of 9 samples.Failure in bacteria shaking,DNA sequencing and other factors were ex-isted in the other clones. Conclusion The CRISPR/Cas9 kinase knock-out library can be widely used for screen-ing the important kinases which may mediate cell proliferation,metastasis,drug resistance and autophagy.This li-brary will play an important role in clarifying the development of disease associated with kinases.

Development of portable vital signs monitoring system for field severe patient / 医疗卫生装备

Objective To design a portable vital signs monitoring system to monitor and analyze severe patient vital signs in field conditions.Methods The communication protocols of all medical devices were analyzed.The information on the vital signs were acquired periodically from medical devices with the signal transducer based on embedded technique,and then sent to the computer for analysis.The abnormalities were displayed on the portable terminal.Results The system met the desired requirements,and facilitated the medical personnel dedicated to treatment.Conclusion The system contributes to information sharing for the changes of vital signs in field conditions,so that the doctor can take measures in time to enhance the efficiency and reliability of the treatment.

Changes of microRNA expression profiles in Vero cells induced by HSV-2 LAT overexpression / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the changes in the microRNA expression profile of Vero cells induced by HSV-2 LAT overexpression.</p><p><b>METHODS</b>The full-length open reading frame of HSV-2 LAT was synthesized and cloned into pRetroQ- AcGFP1-C1 vector, and the recombinant retrovirus expressing HSV-2 LAT was packaged. Using a microRNA microarray, the microRNA expression profile changes in Vero cells were analyzed after infection with the recombinant retrovirus.</p><p><b>RESULTS</b>In Vero cells infected with the recombinant retrovirus for stable HSV-2 LAT overexpression, 5 microRNAs (hsa-miR-23a*, kshv-miR-K12-3, hsa-miR-943, hsa-miR-634, and hsa-miR-1270) were up-regulated and 5 (hsa-miR-181a-2*, hsa-miR-450b-5p, hsa-miR-31, hsa-miR-24, and kshv-miR-K12-12*) were down-regulated.</p><p><b>CONCLUSION</b>The expression of HSV-2 LAT can induce changes in microRNA expression profile in Vero cells.</p>

Preparation of a monoclonal antibody against fusion protein preM/EIII of WNV / 南方医科大学学报

<p><b>OBJECTIVE</b>To prepare a monoclonal antibody (mAb) against the fusion protein preM/EIII of West Nile virus (WNV) for clinical detection of WNV.</p><p><b>METHODS</b>Sp2/0 cells were fused with the spleen cells of BALB/c mice immunized with the recombinant fusion protein preM/EIII expressed in E. coil to obtain the hybridoma cell line that secreted preM/EIII mAb. The hybridoma cells were injected into the peritoneal cavity of BALB/c mice and the ascites was collected and purified. The specificity and titer of the obtained mAb were determined using ELISA and Western blotting.</p><p><b>RESULTS</b>One hybridoma cell line secreting preM/EIII mAb, named ME1, was obtained. The titer of the purified mAb was 10(-6). Identified as a mAb of the Ig subclass IgG1, ME1 was capable of specific reactions with preM/EIII protein and WNV without cross-reactions with other viruses such as JEV, SLEV, YFV and DENV. The accuracy of clinical testing of MNV with ME1 was 97.78%.</p><p><b>CONCLUSION</b>The mAb against preM/EIII obtained have a high specificity and accuracy in clinical testing of MNV and can be used in clinical diagnosis of MNV infection.</p>

Effect of ferric nanopar ticles on the mechanical properties of methyl vinyl silicone rubber / 国际生物医学工程杂志

Objective To explore mechanical property changes of methyl vinyl silicone rubber modified by ferric nanoparticles and its dispersed phase.Methods Mechanical properties such as Shore A hardness,tensile strength,elongation at break,tearing rate of permanent deformation and tearing strength of pre-prepared ironic nanoparticle enhanced silicone rubber and carbon-coated ferric particle reinforced silicone rubber were tested according to national standards.A thermal field emission scanning electron microscope (TFE-SEM) was used to investigate the morphology of both surface and fracture of the composite materials and to observe the dispersion of ferric nanoparticles in them.Results Mean values of Shore A hardness,tensile strength,elongation at break,tearing permanent deformation rate and tear strength of modified composites increased with the increasing amounts of ferric nanoparticles,however,when the quota of ironic nanoparticles in the composite formula were greater than 17 phr,carbon-coated ferric nanoparticles more than 19 phr,the mean values of tensile strength of two composites stopped increasing and presented the declining trend.When the quota of ferric nanoparticles in the formula exceeding 15 phr,the mean values of elongation at break and tear strength began to decrease in the formula ratio of silicone rubber/ferric nanoparticles up to 85:15,while the Shore A hardness of samples increased all the way.Ferric nanoparticles dispersed evenly on the surface of composites.Nanopowder aggregation in the fracture surface of both composites could be observed at the formula ratio of 85:15 of silicone rubber/iron nanoparticle and 87:13 of silicone rubber/carbon-coated iron specimen.Conclusion Effect of iron nanapareticles and carbon-coated ferric nanoparticles on the mechanical properties of the reinforced methyl vinyl silicone rubber depends on the nanoparticle size,additive amount and agglomeration.

Diagnostic role of Golgi protein 73 and its gene determination in primary hepatic carcinoma / 中华检验医学杂志

Objective To explore the diagnostic value of GP-/3 protein in gene detection in the patient of primary hepatic carcinoma, to discuss the joint roles of serum GP73 and AFP, and provide a novel method for the diagnosis for PHC and screening for high-risk population. Methods ELISA was used to detect the serum level of GP73 and AFP in 73 cases of PHC, 13 cases of hepatic cirrhosis, 32 cases of hepatitis and 62 cases of health people. SYBR Green real time fluorescence quantitative PCR was used to detect the relative value of GP73 mRNA in the peripheral blood cells of each group. Comparative Ct method was used to evaluate the relative expression levels. Eight cases of normal liver tissues and 8 cases of PHC tissues were detected at the same time to compare the relative expression levels. Results Kruskal-Wallis test showed that the serum levels of GP73 and AFP had significant differences between four groups(H value were 89. 6 and 52.0, P ＜ 0. 01) and the whole blood GP73 mRNA had no significant differences(H =4. 33, P ＞ 0. 05). Mann-Whitney test showed that the serum levels of GP73 had significant differences among PHC groups[166. 7 (162. 7-231.8) μg/L] and liver cirrhosis[57. 3 (46. 6-113. 6) μg/L], hepatitis[29. 6(26. 2-54. 5) μg/L], health group[25.1 (20. 8-29. 4) μg/L] (U value were 246, 297, 349, P ＜ 0. 01).The A FP levels of the four groups were 380. 9 (258.5-503.2) μg/L, 3.8 (1.3-14. 5) μg/L, 5. 1 (2. 4-7. 8)μg/L and 2. 8(2. 2-5.7) μg/L. It also showed significant differences (U value were 246,419 and 790,P ＜0. 01). The GP73 mRNA expression of PHC liver tissues(12. 64) was significant higher than normal liver tissues (1.00). The critical values for GP73 and AFP was determined to be 123. 2 μg/L and 10. 6 μg/L through the 8OC curves. Under the critical value the sensitivity of GP73 and AFP were 65.8% and 53.4% ,and the specificity of CP73 and AFP were 95.3% and 92. 5% respectively. Joint detection could increase the sensitivity up to 79. 5%, and achieve the high specificity of 90. 7%. Conclusions As a new diagnositic marker of primary hepatic carcinoma, GP73 protein has the very good sensitivity and specificity. The GP73 mRNA in the whole blood sample could not be used for the diagnosis of PHC. But it woule be a good molecular marker for diagnosis of PHC in the liver tissue sample. The joint detection of GP73 and AFP could improve PHC diagnostic performance, and provide an effective approcach to the PHC high-risk screening.

Experimental study of kansui root and LMWH combined therapy for severe acute pancreatitis / 中国普通外科杂志

Objective To investigate if there is a synergism of kansui root and low molecular weight heparin(LMWH) in combined treatment of SAP in rats.Methods SD rats were randomly divided into 5 groups:Sham group(A group),severe acute pancreatitis group(B group),kansui root treatment group(C group),LMWH treatment group(D group),combined treatment group(E group).The serum TNF-?,IL-6 level and the ratio of TXB2/6-keto-PGF1? in pancreatic tissue,and the histological examination were evaluated.Results The values of TNF-??IL-6 and the ratio of TXB2/6-keto-PGF1? in C,D,E group were significantly lower than those in B group at the time point of 6,12,24h.But there were no significant differences in C,D,E group at all the time points.The histological examination showed that the pancreas was normal in group A;the pathological changes were milder in C,D,E group than that in B group.Conclusions Combined treatment of kansui root and LMWH was effective in SAP,but there was no synergism,and also no side effects.