Construction of recombinant lentivirus vector containing retinoic acid-induced gene G and its effect on human lung cancer A549 cell line / 中国癌症杂志

Background and purpose:Retinoic acid-induced gene G (RIG-G) is a tumor suppressor gene which is cloned by NB4 cell line from a acute promyelocytic leukemia cell. This study aimed to investigate the effect ofRIG-G in lung cancer cells A549 by constructing a lentiviral vector expressing RIG-G under doxycycline (DOX) regulation.Methods:RIG-G gene ampliifcation was performed by quantitative real-time PCR (qRT-PCR). pLenti6/TO/V5-GIM-RIG-G lentiviral vector withGFP was built by LR recombination system. The concentration of pLenti6/TO/V5-GIM-RIG-G lentiviral vector andTet-on lentiviral vector were measured by virus titer method. After infecting A549 cells, stably transfected lines were selected via limiting dilution analysis.RIG-G gene expression was examined by immunolfuorescence staining and Western blot assay. Cellular proliferation was determined by CCK-8 assay.Results:The concentrations of pLenti6/TO/V5-GIM-RIG-G lentiviral vector andTet-on lentiviral vector were 1.0×108TU/mL and 4×109 VP/mL, respectively. RIG-G was expressed in lentivirus infected A549 cells after adding DOX, and the amount of cells withGFP could be observed by lfuorescence microscopy.After the expression of RIG-G protein, the prolif-eration activity of A594 cell was signiifcantly inhibited compared to the control group (1.168±0.107vs 2.099±0.162, P<0.05).Conclusion:The regulated expression ofRIG-G gene was established in A549 lung cancer cell line. The RIG-G protein has potential abilities to inhibit the proliferation of lung cancer cell A549.

Mechanisms of myeloid cell RelA/p65 in cigarette smoking-induced lung cancer growth in mice / 中华肿瘤杂志

<p><b>OBJECTIVE</b>The aim of this study was to investigate the mechanism of cigarette smoking (CS)-induced lung cancer growth in mice.</p><p><b>METHODS</b>RelA/p65⁻/⁻ mice and WT mice were used to establish mouse models of lung cancer. Both mice were divided into two groups: air group and CS group, respectively. Tumor number on the lung surface was counted and maximal tumor size was evaluated using HE staining. Kaplan Meier (K-M) survival curve was used to analyze the survival rate of the mice. Expression of Ki-67, TNF-α and CD68 in the tumor tissue was determined by immunohistochemical analysis, and cyclin D1 and c-myc proteins were examined by Western blot. Apoptosis of tumor cells was analyzed using TUNEL staining. The concentrations of inflammatory cytokines TNF-α, IL-6 and KC in the mouse lung tissues were evaluated by ELISA.</p><p><b>RESULTS</b>Compared with the WT air group, the lung weight, lung tumor multiplicity, as well as maximum tumor size in the WT mice exposed to CS were (1.5 ± 0.1)g, (64.8 ± 4.1) and (7.6 ± 0.2) mm, respectively, significantly increased than those in the WT mice not exposed to CS (P < 0.05 for all). However, there were no statistically significant differences between RelA/p65⁻/⁻ mice before and after CS exposure (P > 0.05 for all). Kaplan-Meier survival analysis showed that CS exposure significantly shortened the life time of WT mice (P < 0.05), and deletion of RelA/p65 in myeloid cells resulted in an increased survival compared with that of the WT mice (P < 0.05 for all). The ratios of Ki-67 positive tumor cells were (43.4 ± 2.9)%, (60.6 ± 5.4)%, (12.8 ± 3.6)% and (15.0 ± 4.2)% in the WT air group, WT CS groups, RelA/p65⁻/⁻ air groups and RelA/p65⁻/⁻ CS groups, respectively. After smoking, the number of Ki-67-positive cells was significantly increased in the WT mice (P < 0.05). However, there was no significant difference between the RelA/p65⁻/⁻ groups before and after smoking (P > 0.05). The apoptosis rate of WT air, WT CS, RelA/p65⁻/⁻ air and RelA/p65⁻/⁻ CS groups were (11.6 ± 1.7)%, (13.0 ± 2.0)%, (13.2 ± 2.0)% and (11.0 ± 1.4)%, respectively, with no significant difference among them (P > 0.05). Expression of cyclin D1 and c-myc was induced in response to CS exposure in lung tumor cells of WT mice. In contrast, their expressions were not significantly changed in the RelA/p65⁻/⁻ mice after smoke exposure. CS exposure was associated with an increased number of macrophages infiltrating in the tumor tissue, in both WT and RelA/p65⁻/⁻ mice (P < 0.05). The concentrations of IL-6, KC and TNF-α were significantly increased after CS exposure in the lungs of WT mice (P < 0.05).</p><p><b>CONCLUSIONS</b>Cigarette smoking promotes the lung cancer growth in mice. Myeloid cell RelA/p65 mediates CS-induced tumor growth. TNFα regulated by RelA/p65 may be involved in the lung cancer development.</p>

Using informationalized management to achieve real-time monitoring of turnaround time / 中华检验医学杂志

With the extensive application of informationalized management systems for barcode specimens , the degree of informatization is becoming higher and higher in medical laboratory.Informationalized management combines modern information technology and advanced management concepts , transforms or reengineers the laboratory operation and business process.Test specimen turnaround time ( TAT) is an important factor affecting the quality of the inspection.By analyzing the test process of each time node , establish the suitable specimens monitoring program for clinical requirements and real -time monitor the key nodes in test processes ,which will effectively shorten TAT , improve reporting timeliness rate and avoid clinical complaints.

Study on effect of NFκB-p65 pathway inhibition in macrophages on cigarette smoke induced non-small cell lung cancer cell NCI-H520 cells proliferation / 肿瘤研究与临床

Objective To investigate the mechanism of cigarette smoke promotes non-small cell lung cancer (NSCLC) NCI-H520 cell line proliferation mediated by macrophages with method of blocking NFκB-p65 pathway by RNAi.Methods To co-culture NCI-H520 cells with primary macrophages or U937 cell line,the Transwell Inserts system was used in cell co-culture model.NFκB activation was confirmed by electrophoretic mobility shift assay (EMSA) and Western blot analysis.U937 cells were transfected with NFκB-p65 shRNA plasmid to abrogate the NFκB activation,by BrdU ELISA,the effect of cigarette smoke extract (CSE) promoted NCI-H520 cells proliferation were assessed,inflammatory factors TNF and IL-6 expressions were analysed by ELISA.Results Exposure of CSE enhanced NFκB-p65 nuclcus translocation and activated the NFκB pathway.CSE did not promote NCI-H520 cells proliferation alone (P ＞ 0.05),but after 4 days coincubation with macrophages,the proliferation of NCI-H520 cells was significantly increased (P ＜0.01),addition of CSE to the co-culture much more enhanced this effect (P ＜ 0.01).After NFκB-p65 was blocked by RNAi,it significantly reduced NFκB-p65 protein expression and inhibited NFκB activation in U937 p65-cells,and markedly inhibited U937 cells induced proliferation of NCI-H520 cells and IL-6,TNF secretion (P ＜ 0.01).Conclusion Cigarette smoke promotes NCI-H520 cells proliferation mediated by macrophages.Blockade of NFκB pathway with RNAi in macrophages can reduced cigarette smoke induced inflammatory factors secretion in macrophages,and significantly inhibit cigarette smoke promoted tumor proliferation.

Screening of infection of malaria parasite with, atypical eosinophil distributions in the WBC scattergram and alarm in Sysmex XE-2100 hematology analyzer / 中华检验医学杂志

Objective To explore a quick and feasible method of screening malaria parasite by using a Sysmex XE-2100 hematology analyzer though alarm information on high eosinophil count and atypical eosinophil distributions in the WBC scattergram. Methods Sysmex XE-2100 hematology analyzer was used for complete blood cell analysis. Microscopic review was used when high eosinophil count and atypicaleosinophil distributions in the WBC scattergram were found. If the review showed normal eosinophil cells, wecontinued to focus on red cell for searching malaria parasite. Results Among 1 501 specimens showing higheosinophil counts and atypical eosinophil distributions, nine cases with normal eosinophil cells were indisaccordance with the hematology analyzer, six of them showed high eosinophil count in the Sysmex XE-2100 hematology analyzer, whose distribution was located close to neutrophil clusters in scattergram. The otherthree had an abnormal WBC scattergram. There was no gap between eosinophil clusters and neutrophilclusters, which brought no classified results. But all the nine specimens had been found the trophozoite,schizont and merozoite in blood smears. Conclusions There were great possibility of the existence of themalaria parasite in specimens when hematology analysis showed high eosinophil count and atypical eosinophildistributions in the WBC scattergram in a Sysmex XE-2100 hematology analyzer, although these alarm wasnot comfirmed by microscopic review. This method provides not only a simple and reliable way for quickscreening malaria parasite but also has a great value in preventing the undetected ratio on malaria parasite.

Study on the Polymorphism of Intron 22 (CA)n Repeat within FVIII Gene in Dai, Yi and Han Populations of Yunnan Province / 中国实验血液学杂志

This study was designed to investigate the polymorphism of intron 22 (CA)n repeat within FVIII gene in Dai, Yi and Han populations of Yunnan province. PCR, DNA sequencing technique and PAGE were used in this study. The results showed that three different alleles corresponding to 24, 25 and 26 dinucleotides were found at this locus and the allele frequency ranged from 1.20% to 57.7% in Dai and from 16.43% to 63.00% in Yi populations. In Han population, five different alleles with 24, 25, 26, 27 and 28 (CA)s were found, the allele frequency ranged from 8.97% to 32.00%. Heterozygotes of intron 22 (CA) repeat within FVIII gene were not found in the three populations. In conclusion, it was obvious that the locus cannot be acted as DNA genetic marker of FVIII gene in the three populations in Yunnan.