RESUMO
Objective@#To set up a chyle (n-hexane) testing method based on n-hexane extraction system.@*Methods@#The hydrothorax specimen which was positive in chyle testing was selected and the feasibility was evaluated by replace Diethyl ether with n-hexane. The optimal extraction volume ratio for the new chyle testing was confirmed and sultan Ⅲ dye process was improved. Then, the new chyle testing method and its standard operation process was established.A total of 120 specimens with triglyceride(TG)in different levels were collected and divided into three groups with 40 specimens for each group (group A. TG≤1.7 mmol/L; B. 5.6<TG<12.8 mmol/L; C. TG≥12.8 mmol/L). Then, 50 body fluid specimens were collected and used to confirm the above test results. A total of 134 real body fluid specimens were used to assessment the clinical using by review and calculating the clinical coincidence rate.@*Results@#N-hexane can replace Diethyl ether in the Chyle test. The extraction volume ratio of 1 ml sample plus 400 μl N-hexane is the best one and the sultan Ⅲ tube dying method is better than former direct smear. Compared with the old Chyle test using Diethyl ether, the new Chyle test using N-hexane is in good consistency with the Kappa value 0.95. The clinical coincidence rate of the above Chyle test reaches 94.8%(127/134) after the verification of 134 specimens.@*Conclusions@#A new N-hexane test is established successfully. It was convenient to operate with high security and little contamination and thus has a wide clinical application value.(Chin J Lab Med, 2018, 41: 527-530)
RESUMO
Objective To knock out the GP73 gene in H22 cells originating in mice using CRISPR/Cas9 gene editing system and construct H22 GP73 gene knockout stable strain for identification of its functions .Methods Two pairs of sgRNAs that could specifically identify the upstream and downstream of GP 73 gene first promoter were designed before a recombinant eukaryotic expressional plasmid was constructed using pX 459 .After enzyme digestion and sequencing , two pairs of recombinant plasmids were co-transfected into H22 cells before puromycin was used to screen positive cells and monoclonal cells which stably knocked out GP 73 gene were developed .The knockout effect was measured by Western blotting.Cell Titer 96? AQueous One Solution Assay was used to detect the effect on cell reproductive capacity when the GP73 was knocked out .The transferability was detected through wound healing test .Results The result of Western blotting suggested that GP73 protein was undetected in the construction of H22 GP73 knockout gene stable strain after transfection.The transfer and reproduction slowed down .Conclusion H22 GP73 gene knockout stable strain can be successfully built using CRISPR/Cas9 gene editing system ,thus facilitating studies on the function of GP 73 in hepatocarcinogenesis .