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Purpose@#Drug resistance is one of the main causes of chemotherapy failure in patients with small cell lung cancer (SCLC), and extensive biological studies into chemotherapy drug resistance are required. @*Materials and Methods@#In this study, we performed lncRNA microarray, in vitro functional assays, in vivo models and cDNA microarray to evaluate the impact of lncRNA in SCLC chemoresistance. @*Results@#The results showed that KCNQ1OT1 expression was upregulated in SCLC tissues and was a poor prognostic factor for patients with SCLC. Knockdown of KCNQ1OT1 inhibited cell proliferation, migration, chemoresistance and promoted apoptosis of SCLC cells. Mechanistic investigation showed that KCNQ1OT1 can activate transforming growth factor-β1 mediated epithelial-to-mesenchymal transition in SCLC cells. @*Conclusion@#Taken together, our study revealed the role of KCNQ1OT1 in the progression and chemoresistance of SCLC, and suggested KCNQ1OT1 as a potential diagnostic and prognostic biomarker in SCLC clinical management.
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Objective@#To investigate the relationship between miRNA-196b-5p and miRNA-99a-5p expression and autophagy and apoptosis in multiple myeloma cells.@*Methods@#Human myeloma cell line U266 and normal CD138+ plasma cells were selected as the research objects. The subjects were divided into 45 cases of multiple myeloma patients and 40 healthy controls. The expression of miRNA-196b-5p and miRNA-99a-5p was measured by real-time quantitative PCR, and Western blot was used to determine the expression of autophagy related protein LC3-Ⅱ, LC3-Ⅰ, P62, Beclin-1 expression, apoptosis related protein CL caspase3, CL caspase7, Bcl-2, Bax, and TGF-β/Smad pathway associated proteins TGF-β1, Smad2/3, p-Smad3 and Smad7. The cell apoptosis rate was determined by flow cytometry. The correlation between miRNA expression level and clinical characteristics of multiple myeloma patients was analyzed.@*Results@#Compared with normal plasma cells, the expression of miRNA-196b-5p in myeloma cells increased significantly (0.43±0.15 vs 2.44±0.63 or 2.02±0.85, all P<0.001), the expression of miRNA-99a-5p was significantly decreased (1.87±0.61 vs 0.62±0.15 or 0.80±0.33, P<0.001), LC3-Ⅱ/LC3-Ⅰ increased significantly (P<0.05), Beclin-1 expression increased significantly (P<0.05), P62 expression decreased significantly (P<0.05). The expression of Bax, CL caspase3 and CL caspase7 decreased significantly (P<0.05), and the expression of Bcl-2 increased significantly (P<0.05) and apoptosis rate significantly decreased (P<0.05). After transfected with miRNA-196b-5p mimic or miRNA-99a-5p inhibitor, the LC3-Ⅱ/LC3-Ⅰ of CD138+ plasma cells increased significantly (P<0.05), the expression of Beclin-1 increased significantly (P<0.05), P62 expression decreased significantly (P<0.05), and the apoptosis rate significantly decreased (P<0.05). However, after autophagy inhibitor of 3-MA was administered, the apoptotic rate of the above reaction system did not change significantly (P>0.05). The expression of miRNA-196b-5p and miRNA-99a-5p was significantly correlated with DS and ISS stage in multiple myeloma patients (P<0.05).@*Conclusion@#miRNA-196b-5p and miRNA-99a-5p are closely related to the clinical characteristics of patients with multiple myeloma. The overexpression of miRNA-196b-5p and down regulation of miRNA-99a-5p could inhibit the apoptosis of myeloma cells by up regulation of autophagy, and the mechanism is related to the activation of the TGF-β/Smad signaling pathway.
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Objective The present study aimed to explore the expression and purification of a fusion protein of human TIM-4 and EGFP in Escherichiacoli(E.coli)and evaluate its bioactivity.Methods The cDNA fragments of human TIM-4 and EG-FP genes were respectively amplified by RT-PCR and cloned into prokaryotic expression vector pET-28a. The constructed re-combinant plasmid pET-28a-TIM-4-EGFP was transformed to E. coli BL21 (DE3)for the expression under the induction of IPTG.The expressed protein was purified by Ni-NTA resin.Recombinant protein was analyzed by SDS-PAGE and Western blotting ,and its binding activity to the apoptotic cells was detected under the fluorescence microscope.Results The TIM-4-EG-FP vector was constructed and expressed in E. coli. The TIM-4-EGFP fusion protein was identified and verified by SDS-PAGE and Western blotting.Our results demonstrated that all the TIM-4-EGFP fusion proteins recognize and bind directly to apoptot-ic cells ,but not to viable cells.We further verified that the interactions of TIM-4-EGFP with apoptotic cells were blocked by TIM-4-Ig fusion proteins.Conclusion We successfully constructed a fusion protein encoding human TIM-4 and EGFP ,and ex-pressed it in E.coli. The fusion protein shows a readily obtainable source of biologically active TIM-4 ,which has considerable potential for further studies on human TIM-4 and its receptor.
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Objective To study the influence of residual methylene blue after plasma viral inactivation on the human immune cell function by using the peripheral blood mononuclear cell(PBMC) .Methods PBMC were isolated by adopting the Ficoll‐Hypaque density gradient centrifugation method and co‐cultured for 72 h in presence of specific T cell stimulating factors(Anti‐CD3/28 and Anti‐CD28) ,with or without different concentration of methylene blue .The culture supernatant was collected and detected the cyto‐kines secretion situation by ELISA .After 66 h culture ,CCK‐8 dye was added and continueously cultured for 4-6 h ,the prolifera‐tion was determined at A450 .Results The high‐concentration doses of methylene blue (1 .25 ,2 .5 ,5 μmol/L groups) had signifi‐cantly inhibiting effect on the proliferation of PBMC stimulated by Anti‐CD3/28(P< 0 .01) ,its OD value was decreased from 0 .897 ± 0 .385 to 0 .632 ± 0 .334 ,0 .524 ± 0 .254 and 0 .445 ± 0 .287 respectively ,showing certain dose dependent effect .The high concentrations of methylene blue (1 .25 ,2 .5 ,5 μmol/L groups) could down‐regulate interleukin(IL)‐17a ,IL‐10 and interferon (IFN)‐γ secreted by anti‐CD28 induced PBMC ,moreover showing a dose dependent effect .1 .25 ,2 .5 ,5 μmol/L methylene blue af‐fected the IL‐17a level secreted by PBMC from (406 ± 57)pg/mL descending to (276 ± 38) ,(192 ± 31) ,(134 ± 24)pg/mL respec‐tively ;affected PBMC to secrete IL‐10 ,its level was reduced from (184 ± 15) pg/mL to (132 ± 13) ,(110 ± 12) ,(42 ± 8)pg/mL ;af‐fected PBMC to secrete IFN‐γ,its level was deduced from (4 512 ± 187)pg/mL to (2 876 ± 143) ,(2 234 ± 153) ,(1 988 ± 112)pg/mL respectively .Conclusion High concentrations of methylene blue (≥1 .25 μmol/L ) has the significant inhibiting effect on the proliferation and cytokine secretion functions of PBMC .In other words ,the residual methylene blue concentration in viral inactiva‐tion plasma (≤0 .33 μmol/L) has no obvious effect on the immune function of PBMC ,but whether this concentration of methylene blue having the effect on human pure T cell immune function needs to be further evaluated and studied .
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<p><b>OBJECTIVE</b>To investigate the hematological and molecular characteristics of hemoglobin Q-Thailand in Guangxi, so as to provide reference data for hemoglobinopathy screening.</p><p><b>METHODS</b>A total of 51088 samples were screened by capillary electrophoresis. Samples suspected with Hb Q-Thailand were processed with blood cell count and DNA sequencing. Gap-PCR and PCR-reverse dot blotting were used for the detection of common mutations of alpha and beta thalassemia.</p><p><b>RESULTS</b>The carrier rate of Hb Q-Thailand in Guangxi was 0.06%. The hematological phenotype index(HGB, MCV, MCH, Hb Q-Thailand, Hb A2, Hb QA2) of 28 Hb Q-Thailand heterozygous samples were (125.60±22.30) g/L, (78.22±4.81) fl, (25.79±2.14) pg, (27.37±2.72)%, (1.89±0.22)%, (0.69±0.16)%, respectively, and of 2 Hb Q-Thailand heterozygous combined with beta-thalassemia samples were (125.00±18.39) g/L, (69.65±5.02) fl, (22.00±0.0) pg, (14.80±0.71)%, (4.45±0.07)%, (0.95±0.71)%, respectively. A statistical difference was found in hematological phenotype index between the two groups except HGB (P<0.05).</p><p><b>CONCLUSION</b>In Guangxi, the detected Hb Q-Thailands were mainly heterozygous. Part of Hb Q-Thailand heterozygotes had normal red blood cell parameters, but can still be detected by hemoglobin electrophoresis. When combined with other types of thalassemia, these heterozygotes may still exhibit reduced MCV and MCH or various degrees of anemia.</p>
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Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Female , Humans , Infant , Male , Middle Aged , Young Adult , Base Sequence , China , Electrophoresis, Capillary , Genotype , Hemoglobins, Abnormal , Genetics , Metabolism , Heterozygote , Molecular Sequence Data , Mutation , Phenotype , Thalassemia , Blood , GeneticsABSTRACT
Purpose To investigate the expression and clinical significance of p63 in nodular lymphocyte predominant Hodgkin lym-phoma (NLPHL) and classical Hodgkin lymphoma (CHL). Methods 15 cases of NLPHL and 54 cases of CHL were stained for CD45, CD30, p63, PAX5, CD20, CD15, Oct-2, BOB1, MUM1, EMA, EBV-LMP1 and Ki-67 by immunohistochemical methods of EliVision. EBER were detected by in situ hybridization method in 12 cases of CHL. Results The expression of p63 in NLPHL (53. 3%, 8/15) was significantly higher than that in CHL (0, 0/54) (P<0. 05). Conclusions p63 protein is frequently expressed in NLPHL and helpful in the differential diagnosis between NLPHL and CHL.
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<p><b>OBJECTIVE</b>To raise awareness of the pathogenesis and diagnosis of thalassemia by reporting one case of α thalassemia patient with a large deletion fragment and analyzing the pedigree.</p><p><b>METHODS</b>Firstly, blood cells and hemoglobin electrophoresis analysis were used for screening of thalassemia, and then three common kinds of deletional α thalassemia in Chinese was detected by Gap-PCR, three common kinds of non- deletional α thalassemia and seventeen common mutations of β thalassemia in Chinese were analyzed by using PCR- RDB. The unknown mutation of samples was identified with Multiplex Ligation-dependent Probe Amplification (MLPA) and DNA sequencing.</p><p><b>RESULTS</b>The proband female presented with microcytic hypochromic anemia(hemoglobin 71 g/L, mean corpuscular volume 52.4 fl, mean corpuscular hemoglobin 16.1 pg), and hemoglobin A2 1.4%. The identified large deletion fragment length was 21 925 bp, so far which had not been reported in the world and was named -α²¹·⁹. It was registered in USA DNA database and GenBank accession number as KF360979. The genotype of her mother and father and brother were αα/-α²¹·⁹, --(SEA)/-α³·⁷, αα/-α³·⁷ respectively, and the genotype of her and her sister were the same of --(SEA)/-α²¹·⁹. Her husband gene of thalassemia had no mutation, so prenatal diagnosis of thalassemia was not carried out in the pregnant woman.</p><p><b>CONCLUSION</b>The discovery of -α(21.9) deletion mutation was enriched the DNA mutation gene database of thalassemia, and had important significance for genetic counseling and thalassemia prenatal diagnosis.</p>
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Female , Humans , Male , Young Adult , Pedigree , Sequence Deletion , alpha-Thalassemia , GeneticsABSTRACT
T cell immunoglobulin mucin (TIM) family plays a key role in regulating immune responses. In this study, the interactions of human TIM family with apoptotic cells were evaluated in order to provide a foundation for further study on the roles of human TIM genes in apoptosis. Nine kinds of pEGFP-N1 eukaryotic expression vectors containing different lengths of the three members of human TIM genes for the expression of TIM-EGFP and the vectors for the expression of TIM-Fc fusion proteins were constructed. It was found that human TIM proteins could recognize and bind to apoptotic cells directly, but not to viable cells. The interactions of sTIM-1-EGFP, sTIM-3-EGFP and sTIM-4-EGFP with apoptotic cells were blocked by TIM-1-Ig, TIM-3-Ig and TIM-4-Ig fusion proteins respectively. In addition, human TIM proteins mediated the recognition of apoptotic cells and bound to apoptotic cells directly via the IgV domains. In conclusion, the TIM family may play a key role in the regulation of apoptosis. Our data also suggest that human TIM proteins probably serve as novel proteins for the detection of the early cellular apoptosis.
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BACKGROUND: Foreign studies have demonstrated that the blue light at 470 nm inhibits melatonin secretion and displays the most obvious biorhythm regulation. To date, light-emitting diode (LED) applied in regulating biorhythm remains poorly explored. OBJECTIVE: To explore whether a certain intensity of LED (blue) light could induce retinal injury in rats. DESIGN, TIME AND SETTING: Randomized, controlled animal experiment was performed at the Animal Laboratory of Peking University Third Hospital between May 2007 and April 2008. MATERIALS: A total of 32 SD rats and 16 BN rats were provided by Animal Department of Peking University Third Hospital. METHODS: A total of 16 SD and 16 BN rats were respectively randomly divided into test and control groups. Test group rats were placed in light boxes which were controlled by blue LED (wavelength 470 nm) at a intensity of 300-350μW/cm2, 4 hours everyday for 3 days. The remaining SD rats were placed in light box which was controlled by blue LED (wavelength 470 nm) at a intensity of 120-150μW/cm2, 4 hours everyday for 3 days. The control rats were not treated. MAIN OUTCOME MEASURES: At the second day after light irradiation, the rats of all groups were sacrificed and both eyeballs were harvested. The frozen sections were subjected to hematoxylin-eosin staining to observe changes of rat retina. RESULTS: A total of 48 rats were included in final analysis. The retina of SD rats became thinning and disorderly arranged following blue LED irradiation at density of 300-350μW/cm2, but the retina of BN rat remained unchanged similar to control group. After blue LED irradiation at density of 120-150μW/cm2, the retina of SD rat remained unchanged similar to control group. CONCLUSION: Blue LED light source irradiation at a intensity of 300-350μW/cm2 is safe to pigment-protected retina, and at a intensity of 120-150μW/cm2 does not injury retina of different races of rats.