Objective:This study aims to analyze the differences in the alternative splicing pattern of ADAR2 among glioma cell lines U87, U251, A172, and normal human astrocyte HA1800. Methods:A-to-I editing level at the Q/R-Site of GluR-2 was analyzed by RT-PCR and sequencing. Real-time PCR was performed to detect the expression level of each alternatively splicing variant using a specific primer that was confirmed to amplify only the targeted template and not other alternatively spliced variant fragments. Results:We verified that the Q/R-Site of GluR-2 is under-edited in glioma cell lines. Real-time PCR revealed that the ADAR2 pre-mRNA splic-ing pattern has no significant difference at exons 1a and 2 between glioma cell lines and normal human astrocyte. We also detected that the amount of alternative splicing variants, including exon 5a, was higher than that of alternative splicing variants not including exon 5a in human glioma cell lines. However, the expression of alternative splicing variants, including exon 5a, was lower than that of alterna-tive splicing variants not including exon 5a in human astrocyte. Conclusion:Evident differences in splicing were observed at the site of exon 5a between glioma cell lines and normal human astrocytes. The difference in the alternatively splicing pattern at exon 5a may be attributed to the decreased activity of ADAR2.

Objective: To observe the clinical efficacy of grain-sized moxibustion in treating chemotherapy-induced myelosuppression for non-small cell lung cancer (NSCLC) and its effect on quality of life (QOL). Methods: Eighty NSCLC patients admitted to the Inpatient Department of Zhejiang Cancer Hospital between September 2016 and March 2018 were recruited and divided into an observation group and a control group by random number method, with 40 cases in each group. The two groups both received chemotherapy with paclitaxel plus cisplatin (TP regimen). The control group received oral administration of leucogen tablets starting from the first day of chemotherapy, 20 mg each time, three times a day, for consecutive 14 d; the observation group was additionally given grain-sized moxibustion, once a day, five days per week at a two-day interval, until the fourteenth day. The myelosuppression severity was observed and compared between the two groups prior to chemotherapy, at the 3rd, 7th and 14th days of chemotherapy; the QOL in the two groups was evaluated before chemotherapy, at the 14th and 21st days of chemotherapy. Results: Regarding myelosuppression, the peripheral blood indicators increased significantly at the 3rd day of chemotherapy in both groups (P<0.05 or P<0.01); at the 7th and 14th days of chemotherapy, the peripheral blood indicators presented a decreasing tendency in the two groups, but the level in the observation group was still significantly higher than that before chemotherapy (P<0.01); at the 3rd, 7th and 14th days of chemotherapy, the peripheral blood indicators in the observation group were higher than those in the control group (P<0.05 or P<0.01); the occurrence rate of myelosuppression in the observation group was significantly lower than that in the control group (P<0.01). The QOL score in the observation group was markedly higher than that in the control group at the 14th and 21st days of chemotherapy (both P<0.05). Conclusion: Grain-sized moxibustion can effectively improve myelosuppression after chemotherapy for NSCLC, reducing its occurrence and enhancing the patient's QOL.

Objective:To investigate the diagnostic value of conventional endoscopy (CE) and endoscopic ultrasonography (EUS) for invasion depth prediction of superficial gastric cancer.Methods:A total of 84 patients with superficial gastric cancer underwent both CE and EUS before treatment at Beijing Shijitan Hospital from January 2011 to December 2019. The patients were divided into CE affirmation group (47 cases) and CE non-affirmation group (37 cases) according to the endoscopist′s affirmation in the results of CE. Diagnostic accuracy of each method was compared with the histology of the resected specimen. And influential factors for the diagnosis were analyzed.Results:The overall accuracy in determining the invasion depth of superficial gastric cancer was 73.8% (62/84) for CE and 81.0% (68/84) for EUS respectively ( P=0.092). In CE affirmation group, the diagnostic accuracy of CE was significantly higher than that in the CE non-affirmation group [93.6% (44/47) VS 48.7% (18/37), χ2=21.656, P<0.001]. Twenty (23.8%) of 84 lesions were over-staged by CE, dignosed as surgical candidates, and 8 (40.0%) of the over-staged diagnosis were modified by additional EUS assessment. Multivariate logistic analysis showed that influential factors associated with observer affirmation included uneven surface of lesion ( OR=5.076, 95% CI: 1.628-15.821, P=0.005), margin elevation ( OR=3.831, 95% CI: 1.238-11.857, P=0.020) and undifferentiated carcinoma ( OR=6.887, 95% CI: 1.882-25.204, P=0.004). Conclusion:For patients of CE affirmation in the invasion depth, the diagnostic accuracy is high. For those of non-affirmation, additional EUS can improve the diagnostic accuracy and help to develop a more appropriate regime.

This study was purposed to explore the effect of specific small interfering RNA targeting at bcr-abl fusion gene and its combination with p27 gene clone on the proliferation, cell cycle and apoptosis of chronic myeloid leukemia (CML) cell line K562. CML cell line K562 was used as the study object. A 21nt siRNA targeting at the fusion site of b3:a2 mRNA in bcr-abl fusion gene was designed, synthesized and transfected into the K562 cells as RNA interference group. Northern blot was used to detect the bcr-abl fusion gene, Western blot was used to detect the expression of P210 protein and apoptosis-related protein BCL-xL after the transfection. Meanwhile, p27 gene was amplified from peripheral blood mononuclear cells by RT-PCR, and was confirmed to be correct by sequencing, then p27-pcDNA3.1 vector was constructed and transfected into K562 cell line by Lipofectin. After being selected with G418, p27-pcDNA3.1-K562 cell clone stably expressing p27 was isolated. P27 protein was identified by Western blot. Finally, siRNA and p27 gene clone were together applied to K562 cells, the cell survival rate was tested by MTT. The cell cycle and the apoptosis were tested by flow cytometry. The result showed that in contrast with the control group, the expression level of bcr-abl fusion gene was much lower in siRNA group, about 18.4% of K562 cells in siRNA group were apoptotic at 24 hours after siRNA transfection, and the expression of apoptosis-associated protein BCL-xL was greatly down-regulated. The expression of P27 protein could be detected by Western blot in p27-pcDNA3.1-K562 cells. The strong inhibition of cell proliferation was observed in p27-pcDNA3.1-K562 cells, as compared with control K562 cells. The count of p27-pcDNA3.1-K562 cells in G(0)/G(1) phase increased apparently, but that in S phase declined greatly. Cell cycle was arrested in G(0)/G(1) phase. After the combination of p27-pcDNA3.1-K562 cells with specific siRNA, the percentage of apoptosis obviously increased and cell survival rate significantly declined. It is concluded that the specific siRNA distinctly inhibits the expression of bcr-abl fusion gene, and can induce K562 cell apoptosis. The combination of specific siRNA with P27 gene clone displays a synergy of inhibition and pro-apoptosis effects to K562 cells.
Apoptosis ; Cell Cycle ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p27 ; genetics ; Fusion Proteins, bcr-abl ; genetics ; Humans ; K562 Cells ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection

Graft Rejection ; Humans ; Liver Transplantation

The aim of the manuscript was to optimize formulations and preparation technologies of cataplasm of white mustard seed varnish, and to evaluate its anti-asthma effect on rats. The single factor experiments included spreading thickness, types of crosslinking agents, dihydroxyaluminum aminoacetate amount, sodium polyacrylate amount, types of adhesive agents with human sense as the evaluation index. Blank cataplasm matrix was optimized by the orthogonal experiment with the amount of glycerine, citric acid, and sodium carboxymethylcellulose as the major influential factors. Initial adhesive force, peeling strength and human sense were as the evaluation index. The optimized formulation of blank cataplasm were as followings: glycerine-water-ethanol-PEG400-dihydroxyaluminum aminoacetate-citric acid-sodium carboxymethylcellulose-sodium carboxymethylcellulose 2 : 8 : 0.8 : 0.4 : 0.07: 0.15 : 0.1 : 0.5. The active ingredients of white mustard seed, corydalis, and gansui root were extracted by alcohol extraction method. Asiasarum volatile oil was extracted by oil extractor. The optimized drug loading amount was 11% with initial adhesive force, peeling strength and human sense as the evaluation index. Asthma rats model were established by sensitized with ovalbumin and nose-scratching time as the evaluation index. High dose (17%) group of drug-loaded cataplasm had the obvious inhibition effect on nose-scratching time of rats (P = 0.037 < 0.05). In comparison, middle dose (11%), low dose (4%) and positive-control groups had no obvious inhibitive effect on rats. White mustard seed cataplasm supplied a novel choice for anti-asthma therapy. And the overall pharmacodynamics assessment will be carried out on molecular level in near future.
Animals ; Anti-Asthmatic Agents ; administration & dosage ; chemistry ; Asthma ; drug therapy ; Chemistry, Pharmaceutical ; Drugs, Chinese Herbal ; administration & dosage ; chemistry ; Female ; Humans ; Male ; Mustard Plant ; chemistry ; Rats ; Rats, Sprague-Dawley ; Seeds ; chemistry

In recent years, layer-by-layer self-assembly (LbL) has developed rapidly. It has been widely used in various industries such as medicine and metallurgy because of its simplicity, flexibility and controllability. In the study of drug delivery system, hollow microcapsules constructed by LbL method as drug carrier have great advantages in drug release, circulation in vivo and bioavailability, providing a technical platform for targeted drug release. In this paper, we summarize the types of film-forming materials and the driving force used in LbL technology, the way of loading drug into hollow micro capsule, and the variety of loaded drugs. We focus on the release mechanism, its evaluation and safety evaluation of self-assembled film as drug carrier in vivo and in vitro. The review shows the great application prospect of LbL technology in the field of drug delivery.

OBJECTIVEIn order to fully understand hepatitis c virus (HCV) genotype 3b, 1a, 2b and 6a infection in China, We built HCV 5'-noncoding region (5'-NCR) of different genotypes and subtypes.

METHODSThe classification HCV into variable genotypes (subtypes) was carried on by programs A, B and C A. Using a combination of three restriction endonuclease BHH' (BsrB I, Hae II, Hinf I) digestions at the same time. The distinct genotypes were classified into 5 groups: genotype 1 (1a, 1b), 6a, 2 (2a, 2b), genotype 3 (3a, 3b), genotype4 (4a). B. With regard to genotype 1, we could distinguish subtype 1a from 1b using BstU I digestion. C. Using restriction endonuclease Hae III, genotype 2a, 2b, 3b, 4a, 6a are differentiated respectively.

RESULTS(1) HCV genotype 1a, 1b, 2a, 2b, 3a, 3b, 4a, 6a are fully discriminated by comparison with the genotypes regular samples. (2) Of the 93 patients, HCV genotype distribution in China was 66.67% for 1b, 18.28% for 2a, 3.23% for 1b/2b, 3b, 2b respectively. 2.15% for 2a/2b, 1b/2a respectively. 1.08% for 1a.

CONCLUSIONThis research indicated that adoption of HCV 5'-NCR A B C restriction endonuclease digestions techniques, might be sensitive and efficient to detect HCV and discriminate HCV genotype (subtypes) 1a to 6a.

5' Untranslated Regions ; chemistry ; DNA Restriction Enzymes ; Genotype ; Hepacivirus ; classification ; genetics ; RNA, Viral ; analysis

OBJECTIVETo explore whether coal tar pitch smoke extract (CTP) induced pyroptosis in human bronchial epithelial cells (BEAS-2B).

METHODSBEAS-2B cells were treated with different concentrations of CTP (1, 3 µg/ml) for 8h and 24 h, respectively. Lactic dehydrogenase (LDH) activity and interleukin-1 beta (IL-1β) levels in the supernatants of cell culture media were measured with LDH activity or human IL-1β ELISA kit, respectively. The activity of Caspase-1 was measured with Caspase-1 colorimetric assay kit.

RESULTSThe activity of caspase-1 in 1 and 3 µg/ml CTP groups were (9.29 ± 0.30) and (8.67 ± 0.59) µmol/ml respectively which were both significantly increased compared to that (7.42 ± 0.59) µmol/ml in the control group (P < 0.05) after 8 h exposure, but there was no significant difference in the activity of LDH and levels of IL-1β in the cell culture media among the CTP and control groups. 24 h after exposure, the activity of LDH in the CTP (1, 3 µg/ml) groups were (1323.03 ± 28.53) and (1148.45 ± 16.42) U/dl respectively which were significantly higher than that (1091.93 ± 26.64) U/dl in the control group (P < 0.05), and the levels of IL-1β in the CTP (1 and 3 µg/ml) groups were (125.37 ± 25.00) pg/ml and (92.04 ± 19.09) pg/ml respectively which were significantly higher than that (46.20 ± 14.43) pg/ml in the control group (P < 0.05), but there was no significant difference in the activity of Caspase-1 among CTP and control groups (P < 0.05).

CONCLUSIONCTP treatment induced early increase in caspase-1 activity followed by the increase in LDH activity and IL-1 levels, indicative of pyroptosis in human bronchial epithelial cells.

Apoptosis ; Bronchi ; cytology ; Caspase 1 ; metabolism ; Cell Line ; Coal Tar ; adverse effects ; Epithelial Cells ; cytology ; Humans ; Interleukin-1beta ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Smoke ; adverse effects

AIM:To explore the effect of microRNA-221 (miR-221) on the proliferation of lung cancer A549 cells, and to investigate its mechanism .METHODS: The A549 cells were transfected with miR-221 mimics by Lipo-fectamine 2000.The expression of miR-221 was detected by RT-qPCR.The expression of PTEN at mRNA and protein le-vels was detected by RT-qPCR and Western blot , respectively .The cell proliferation was examined by CCK-8 assay and colony formation assay.The 3'-UTR of PTEN was cloned into luciferase reporter vector and its enzymatic activity was detec-ted to verify whether miR-221 targeted to PTEN.RESULTS:The expression level of miR-221 in the A549 cells was sig-nificantly increased after transfection with miR-221 mimics as compared with negative control group and blank group ( P<0.01).The mRNA and protein levels of PTEN were significantly down-regulated compared with control group and blank group ( P <0.05 ) .In addition , miR-221 over-expression significantly promoted the proliferation of A 549 cells ( P <0.05).Moreover, miR-221 inhibited the enzymatic activity of luciferase reporter vector of PTEN.CONCLUSION:Over-expression of miR-221 significantly promotes the proliferation ability of human lung cancer A 549 cells by down-regulation of PTEN.