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In mammal, the myocardium loss cannot be recovered spontaneously due to the negligible proliferation ability of mature mammalian cardiomyocyte. However, accumulated evidence has shown that terminally differentiated mammalian cardiomyocyte also has proliferation potency, which can be mediated by several mechanisms. Here, we reported that circNCX1, the most abundant circular RNA in mammalian hearts, can affect the proliferation of murine cardiomyocytes. The level of circNCX1 is significantly elevated during heart development. Forced expression of circNCX1 inhibits cardiomyocyte proliferation, while silencing of endogenous circNCX1 in cardiomyocyte shows reversed effect in vitro. Mechanistically, circNCX1 functions via negatively regulating transcription activator BRG1. It bridges BRG1 and FBXW7 to enhance the ubiquitination and degradation of BRG1, decreasing the expression of BMP10 to lead cell cycle arrest. In summary, our study first revealed that circNCX1 is a modulator of cardiomyocyte proliferation.
Subject(s)
Cell Proliferation , DNA Helicases , Myocytes, Cardiac , Nuclear Proteins , RNA, Circular , Transcription Factors , Ubiquitination , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/cytology , Animals , Transcription Factors/metabolism , DNA Helicases/metabolism , Nuclear Proteins/metabolism , Nuclear Proteins/genetics , Mice , RNA, Circular/metabolism , RNA, Circular/genetics , HumansABSTRACT
Objective To construct Raji-Luc lymphoma cells with CD19 knockout using CRISPR/Cas9 technology and preliminarily validate their immune escape ability.Methods PB-CRISPR-CD19 small guide RNA(sgRNA)plasmids was constructed,the optimal sgRNA sequence was screened,and Raji-Luc cells with pCAG-PBase,PB-CD19 sgRNA,and PB-CRISPR-Cas9 were co-transfected.Stable knockout monoclonal cell lines were screened by flow sorting and limit dilution method and the knockout effect was verified through gene sequence testing.The expression of luciferase on the surface of the cell line was detected by microplate reader,CD19 CAR-T and CD38 CAR-T previously constructed in the laboratory were used as effector cells,and the immune escape ability of Raji-Luc CD19 KO cell line was verified by universal luciferase chemiluminescence method.Results The transfection efficiency of Raji-Luc CD19 KO cells prepared by electro transfection was high,and the knockout efficiency of the two monoclonal cells was more than 99%.There was no significant difference in luciferase expression compared to the original Raji-Luc cells,and CD19 CAR-T cells could not be activated to the kill them.Conclusion Successfully constructed Raji-Luc CD19 KO lymphoma cell line.
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In the online teaching of Biochemistry course, a variety of network resource platforms (such as Zhihuishu learning network, teleconference, WeChat, QQ, etc) were used to establish a learning community. The teaching content and teaching plan were carefully designed and implemented, enriching the knowledge system of the learning community. And then blending teaching was performed through the combination of live broadcasting and online interaction. In addition to teaching students the basic knowledge of biochemistry, it is also combined with clinical cases and life examples to interact and discuss with students in various forms, giving full play to the advantages of learning community and improving the quality and effect of online learning.
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Objective To investigate the intervention effect of Xuebijing Injection on the differentiation of bone marrow hematopoietic stem cells (HSC) in septic mice. Methods Fifty-four male C57BL/6N mice were randomly divided into three groups: normal control group, model group and Xuebijing group, each group with 18 mice. The mouse models of sepsis were duplicated by intra-peritoneal injection of 10 mg/kg E. coli lipopolysaccharide (LPS) method. Starting from the day of modeling, Xuebijing Injection 20 mL/kg was intravenously injected into the tail vein in Xuebijing group, once a day for consecutive 4 days; the normal control and model groups were intravenously injected with normal saline at the same dose and site for 4 days. The bone marrow cells of the femur and tibia of the mice were isolated after 4 days of various treatments in the three groups, and the proportions of bone marrow HSC Lin-Sca-1+c-Kit+ (LSK) and hematopoietic progenitor cells Lin-Sca-1-c-Kit+ (LS-K) of each group were detected by flow cytometry. Results Finally, 14 mice were included in the normal control group, 17 in the model group, and 12 in the Xuebijing group. With the prolongation of time, the body weight of the normal control group gradually increased, the body masses of the model group and the Xuebijing group were decreased first and then increased, reaching a peak at 96 hours after the model was established, but they were still significantly lower than the body mass of normal control group (g: 19.81±0.27, 19.58±0.39 vs. 22.23±0.30, both P < 0.05). Compared with the normal control group, the proportions of LSK, LS-K, long-term HSC (LT-HSC), and megakaryocyte-erythroid progenitor cells (MEP) were all significantly increased in the model group [LSK: (16.62±1.28)% vs. (12.89±0.83)%, LS-K: (44.77±1.77)% vs. (30.34±0.90)%, LT-HSC: (6.88±0.48)% vs. (1.83±0.24)%, MEP: (13.89±1.26)% vs. (9.38±0.66)%, all P < 0.05], the proportion of multipotential progenitor cells (MPP) was significantly decreased [(2.41±0.34)% vs. (5.99±0.59)%, P < 0.05]. Compared with the model group, the LSK and myeloid progenitor (CMP) of the Xuebijing group were significantly reduced [LSK: (12.25±0.69)% vs. (16.62±1.28)%, CMP :(0.31±0.05)% vs. (0.55±0.13)%, both P < 0.05], and LS-K, LT-HSC, MEP showed a decreasing trend [LS-K: (42.75±2.48)% vs. (44.77±1.77)%, LT-HSC:(5.98±0.70)% vs. (6.88±0.48)%, MEP: (10.94±1.36)% vs. (13.89±1.26) %], but the differences were not statistically significant (all P > 0.05). There were no significant differences in the proportions of short-term HSC (ST-HSC) and granulocyte-macrophage progenitor cells (GMP) among the three septic groups (all P > 0.05). Conclusion Xuebijing Injection can improve the differentiation function of bone marrow cells in septic mice, which may be possibly related to the inhibition of pathological proliferation of bone marrow hematopoietic stem cells and progenitor cells in septic mice by Xuebijing Injection.
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Objective To explore psychological resilience, anxiety, depression, coping styles and social supports status among parents of preterm infants of ophthalmic clinic, and analyze their relationship. Methods A total of 217 parents of preterm infants at ophthalmic clinic of hospital were selected by convenience sampling method and investigated by self- designed general information questionnaire, Connor-Davidson Resilience Scale, Self-rating Anxiety Scale, Self-rating Depression Scale, Social Support Rating Scale, and Simplified Coping Style Questionnaire. Results The total score of psychological resilience was (67.48 ± 14.20) points. The average score of positive coping styles dimension was (1.98±0.50) points, and negative coping style dimension score was (1.19±0.55) points. The social support score was moderate with a total score of (42.75 ± 6.17) points, the anxiety and depression got a total score of (36.77 ± 8.17) points and (39.67 ± 9.02) points respectively. Psychological resilience was negatively correlated with anxiety and depression (r=-0.363--0.242, P<0.01), and was positively correlated with coping styles and the social support (r=0.141-0.312, P<0.05 or 0.01). Multi-factor linear regression analysis showed that depression and social support were the influence factors of psychological resilience(t=-4.376, 2.516, P<0.01 or 0.05). Conclusions The parents of preterm infants are at the poor psychological states. Coping styles and depression are the important factors which affect psychological resilience among parents of preterm infants. Nurses should assess the psychological status of the parents, provide targeted interventions to relieve stress, guide the parents use social support effectively, and improve their psychological state.
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Objective To observe the influence of N-myc downstream-regulated gene 2 (NDRG2) on the growth and invasive ability of human colon cancer cell line SW620,and to explore its mechanism.Methods pcDNA3.1-NDRG2 and siRNA-NDRG2 were transfected transiently respectively into SW620 by Lipofectamine TM 2000,untreated cells as the control group.Western blotting was used to investigate the expression of NDRG2 and matrix metalloproteinase-2 (MMP-2).Matrigel invasion assay was used to study the invasive abilities of SW620 cells in all groups.The growth curve was determined through 3-(4,5-dimethyl-2-thiazoly)-2,5-diphenyl-2H-tetrazolium bromide method.Result After transfecting pcDNA3.1-NDRG2 into the SW620 cells,the protein level of NDRG2 increased and the expression of MMP-2 declined markedly.After transfecting siRNA-NDRG2 into the SW620 cells,the protein level of NDRG2 declined and the expression of MMP-2 increased markedly.In addition,compared with the control group (75.80 ± 4.82),the numbers of transmembrane cells in pcDNA3.1 group (56.20 ± 7.40) and in siRNA group (94.20 ± 9.23) were significantly different (t =13.102,P =0.000;t =11.820,P =0.000).The growth curve showed that:compared with the control group (0.67 ±0.01),the absorbance of the fifth day after transfection in pcDNA3.1 group (0.46 ±0.01) and in siRNA group (0.91 ± 0.02) were different significantly (t =9.561,P =0.000;t =10.922,P =0.000).Conclusion NDRG2 can reduce the invasion and proliferation ability of colon cancer cell SW620,and its mechanism may be related to the down-regulation of MMP-2 expression.
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<p><b>OBJECTIVE</b>To synthesize artificial antigen of the chlorogenic acid (CGA), a constituent in Traditional Chinese Medicine, then optimize the reaction conditions, so that to supply the basis in material and technique for study on Chinese medicine injection of adverse reactions in-depth.</p><p><b>METHOD</b>The complete antigen of chlorogenic acid (CGA-BSA) was synthesized by CGA coupled to carrier protein bovine serum albumi (BSA) using carbodiimide method. We used the orthogonal design test to optimize reaction conditions and used the MALDI-TOF-MS and UV motheds to determine the coupling rate of the CGA-BSA.</p><p><b>RESULT</b>The coupling rate were 20 and 18.8, counting by MALDI-TOF-MS and UV, respectively; while the best reaction conditions were NHS: CGA, EDC: CGA, CGA: BSA were 4: 1, 1: 1, 200: 1, respectively, with the pH 9.</p><p><b>CONCLUSION</b>The complete antigen is preparated successfully. Which supplied the basis to advanced study of correlation.</p>
Subject(s)
Antigens, Plant , Chemistry , Chlorogenic Acid , Chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Spectrophotometry, UltravioletABSTRACT
<p><b>OBJECTIVE</b>To detect DNA and chromosomes instabilities during the progression of tumors and screen new molecular markers coupled to putative or unknown oncogenes and/or tumor suppressor genes.</p><p><b>METHODS</b>Five kinds of tumors, in a total of 128 specimens, were analyzed by random amplified polymorphic DNA (RAPD) PCR. Bands representing instabilities were recovered, purified, and cloned, labeled as probes for Southern and Northern blot analysis and DNA sequencing.</p><p><b>RESULTS</b>Sample 5 and 3 of the gastric cancer tissues showed the highest genomic changes and the average detectability in five cancers was up to at least 40% (42.2% - 49.4%). There were significant differences in the ability of each primer to detect genomic instability, which ranged from 27% (primer 8) to 68% (primer 2). Band B is a single copy fragment, and was found to be an allelic loss in gastric and colon cancers. It is a novel sequence and was registered in GenBank with Accession Number AF151005. Further analysis revealed that it might be part of a cis-regulatory element of a new tumor suppressor gene, containing a promoter of cis-action "CACA" box, an enhancer of "GATA" family and a start codon.</p><p><b>CONCLUSIONS</b>It was impossible or difficult to get great achievements for cancer treatments with the procedure of gene therapy only to one oncogene or one tumor suppressor gene because the extensive DNA variations occurred during the progression of tumor. RAPD assay connected with other techniques was a good tool for the detection of genomic instabilities and direct screening of some new molecular markers related to tumor suppressor genes or oncogenes.</p>
Subject(s)
Humans , Base Sequence , Blotting, Southern , Colonic Neoplasms , Genetics , Pathology , Liver Neoplasms , Genetics , Pathology , Lung Neoplasms , Genetics , Pathology , Molecular Sequence Data , Neoplasms , Genetics , Pathology , Polymorphism, Restriction Fragment Length , Random Amplified Polymorphic DNA Technique , Sequence Analysis, DNA , Stomach Neoplasms , Genetics , PathologyABSTRACT
ObjectiveTo detect the instabilities of DNA and chromosome in gastric carcinoma. Methods A total of 33 gastric cancer specimens were analyzed by RAPD(random amplified polymorphic DNA) PCR with nine 10-base arbitrary primers for detecting instabilities of DNA and chromosome. ResultsSample 5 and 3 showed the highest genomic changes and that there were significant differences in the ability of each primer to detect genomic instability ranging from 21% to 85%.ConclusionsThe genetic instabilities often concentrated on some special loci of chromosome e.g. repetitive sequences. It is difficult to influence the result of cancer treatment in gene therapy targeting at only one oncogene or tumor suppressor gene because of the extensive DNA variations occurred during the progression of tumor.