[An interlaboratory comparison study on the detection of RUNX1-RUNX1T1 fusion transcript levels and WT1 transcript levels].

Objective: To investigate the current status and real performance of the detection of RUNX1-RUNX1T1 fusion transcript levels and WT1 transcript levels in China through interlaboratory comparison. Methods: Peking University People's Hospital (PKUPH) prepared the samples for comparison. That is, the fresh RUNX1-RUNX1T1 positive (+) bone morrow nucleated cells were serially diluted with RUNX1-RUNX1T1 negative (-) nucleated cells from different patients. Totally 23 sets with 14 different samples per set were prepared. TRIzol reagent was added in each tube and thoroughly mixed with cells for homogenization. Each laboratory simultaneously tested RUNX1-RUNX1T1 and WT1 transcript levels of one set of samples by real-time quantitative PCR method. All transcript levels were reported as the percentage of RUNX1-RUNX1T1 or WT1 transcript copies/ABL copies. Spearman correlation coefficient between the reported transcript levels of each participated laboratory and those of PKUPH was calculated. Results: ①RUNX1-RUNX1T1 comparison: 9 samples were (+) and 5 were (-) , the false negative and positive rates of the 20 participated laboratories were 0 (0/180) and 5% (5/100) , respectively. The reported transcript levels of all 9 positive samples were different among laboratories. The median reported transcript levels of 9 positive samples were from 0.060% to 176.7%, which covered 3.5-log. The ratios of each sample's highest to the lowest reported transcript levels were from 5.5 to 12.3 (one result which obviously deviated from other laboratories' results was not included) , 85% (17/20) of the laboratories had correlation coefficient ≥0.98. ②WT1 comparison: The median reported transcript levels of all 14 samples were from 0.17% to 67.6%, which covered 2.6-log. The ratios of each sample's highest to the lowest reported transcript levels were from 5.3-13.7, 62% (13/21) of the laboratories had correlation coefficient ≥0.98. ③ The relative relationship of the reported RUNX1-RUNX1T1 transcript levels between the participants and PKUPH was not always consistent with that of WT1 transcript levels. Both RUNX1-RUNX1T1 and WT1 transcript levels from 2 and 7 laboratories were individually lower than and higher than those of PKUPH, whereas for the rest 11 laboratories, one transcript level was higher than and the other was lower than that of PKUPH. Conclusion: The reported RUNX1-RUNX1T1 and WT1 transcript levels were different among laboratories for the same sample. Most of the participated laboratories reported highly consistent result with that of PKUPH. The relationship between laboratories of the different transcript levels may not be the same.

MiR-34b-5p knockdown attenuates bleomycin-induced pulmonary fibrosis by targeting tissue inhibitor of metalloproteinase 3 (TIMP3).

OBJECTIVE: To examine the effects of microRNA-34b-5p (miR-34b-5p) on bleomycin-induced lung fibrosis in mice and the underlying mechanism. MATERIALS AND METHODS: TIMP3-deficient (Timp3-/-) and wild-type mice were administered with bleomycin before to detect the miR-34b-5p expression using quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Luciferase reporter assay was used to identify the target of miR-34b-5p in human lung fibroblast MRC-5. MiR-34b-5p was then silenced in vivo before lung histologic analysis and evaluation of extracellular matrix (ECM) genes as well as myofibroblast marker. The protein and mRNA expression levels were detected by Western blot and qRT-PCR, respectively. RESULTS: We found that miR-34b-5p was significantly increased in lung tissues from bleomycin-stimulated mice. TIMP3 was identified as a direct target of miR-34b-5p by using dual-luciferase reporter assay, and enhanced expression of miR-34b-5p led to a decrease in TIMP3 whereas miR-34b-5p knockdown was responsible for TIMP3 elevation in MRC-5 cells. MiR-34b-5p knockdown in vivo attenuated the bleomycin-induced pulmonary fibrosis in wild-type mice, displayed by a reduced expression of Col1A1, fibronectin (Fn), and α-SMA. Furthermore, histological examination of lung sections also verified a diminishing fibrotic phenotype caused by the miR-34b-5p knockdown. But in Timp3-/- mice, down-regulation of miR-34b-5p did not exert an effect on the severe fibrotic lung injury after bleomycin exposure. CONCLUSIONS: MiR-34b-5p knockdown appears to enhance the resistance to bleomycin by regulating its target gene TIMP3 during the pathogenesis of lung fibrosis.

[A Study on the establishment of immune thrombocytopenia model induced by anti-platelet GPâ bα antibodies].

Objective: To establish primary immune thrombocytopenia (ITP) animal model induced by anti-platelet membrane glycoprotein GPⅠbα antibodies AN51 and R300. Methods: Twenty guinea pigs (6-8 week) were divided into 4 groups. Five guinea pigs in each group were intravenously injected with different doses of AN51 (0.05, 0.1, 0.2 µg/g) and 0.2 µg/g IgG as control. The whole blood was collected from inner angular venous plexus. Platelets number was determined by an automated cell counter and Swiss-Jim method. Then, the similar protocol was used to establish ITP nude mice model by intraperitoneal injection of different concentrations of anti-platelet GPⅠbα antibody R300, respectively. Results: ①Five minutes after intravenous injection of AN51 at 0.05, 0.1 and 0.2 µg/g, the platelet counts of guinea pigs reduced about 0-5%, 50%-60% and 70%-80% compared to the control group, respectively. The difference was statistically significant (P<0.01) . ②Six hours after intraperitoneal injection of R300 at 0.05, 0.1, 0.2 µg/g, the platelet counts of nude mice decreased about 20%-30%, 60%-70% and 80%-90% compared to the control group, respectively. The difference was statistically significant (P<0.01) . The nude mice, injected 0.2 µg/g R300 once a day for 2 weeks, showed typical ITP clinical manifestations including large number of petechiaes or ecchymoses on limbs, head and abdomen. Conclusion: AN51 at 0.2 µg/g and R300 at 0.2 µg/g could establish stable ITP model in guinea pigs and nude mice respectively.