1.A study of the functions of autografted hemispleen
Shiyan REN ; Minggui XU ; Aochuan WANG
Journal of Third Military Medical University 1984;0(01):-
One hundred and forty-five mice were randomized into 4 groups:Group Ⅰ consisted of 25 mice with sham operation,group Ⅰ of 40 mice with hemisplenectomy,group Ⅲ of 55 mice with hemisplenic autotransplantation,and group Ⅳ of 25 mice with splenectomy.Twelve weeks after operation,it was found under optical microscopy,transmission electron microscopy and scanning electron microscopy that the autografted hemispleen was viable.The contents of alpha-naphthol acetylate esterase (ANAE) and acid phosphatase (ACP) in the splenic tissue were measured with microspectrophotometer.ACP level decreased continuously in group Ⅰ (0.462?0.013),group Ⅱ (0.304?0.009),and group Ⅲ(0.180?0.014) (P
2.The establishment of the uPA inducible expression system
Lixiang CHEN ; Xiaojing ZHOU ; Wenwen LIU ; Wenjiang ZHOU ; Xiaonan REN ; Shiyan YU ; Xiaohui ZHOU
Chinese Journal of Comparative Medicine 2015;(1):1-8
Objective To establish uPA inducible expression system using recombinant retroviral system for the further construction of inducible uPA-SCID animal model .Methods The Inducible expression system need to construct two plasmids:pLNHXO1O2-Alb-GLUC-FMN2A -rtTA and pLNHXO5O6-TRE2-uPA-IRES-ZsGreen respectively. Both plasmids were based on retroviral vector pLNHX , Albumin promoter gene ( Alb) and rtTA gene or uPA gene and ZsGreen were obtained by PCR reaction and inserted into pLNHX .The Gaussia enzyme fluorescent element ( GLUC) was used to monitor rtTA expression in pLNHXO1O2-Alb-GLUC-FMN2A-rtTA, and the ZsGreen for uPA expression monitoring in pLNHXO5O6-TRE2-uPA-IRES-ZsGreen.The correct constructed plasmids were transfected into packaging cell line GP 2-293 to gain recombinant viral particles .NIH/3T3 cells were infected with these viral particles and selected with G 418.Gene expression in the surviving cells was confirmed by the PCR method .Results The recombinant retroviral vectors harbouring target genes were successfully cloned .The rtTA gene in pLNHXO1O2-Alb-GLUC-FMN2A-rtTA was expressed, and uPA can be induced to express in pLNHXO5O6-TRE2-uPA-IRES-ZsGreen by doxycycline (Dox) when the plasmid transfected into the HepG-Tet-on cell.The constructed recombinant two retroviral vectors were transfected into GP 2-293 packaging cells respectively to gain infectious viral particles .Then,NIH/3T3 cells were infected with these viral particles and single-cell clones which stably expressed the transgenes were successfully established .Conclusion This study primarily established uPA inducible expression system , it laid a foundation for the murine model of inducible liver damage , and provided a novel technical platform for further building the liver humanised murine models for viral hepatitis studying .
3.Role of Preoperative Peripheral Blood Inflammatory Parameters and Postoperative Lymph-node Ratio in Prognosis of Patients with Gastric Cancer Undergoing Chemotherapy
Wenzhen REN ; Honghao WANG ; Tian XIANG ; Gao LIU
Cancer Research on Prevention and Treatment 2023;50(5):490-497
Objective To analyze the role of preoperative peripheral blood inflammatory parameters and postoperative lymph-node ratio (LNR) in the prognosis of gastric cancer patients treated with chemotherapy. Methods The neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), systemic immune inflammatory response index (SII), lymphocyte-to-monocyte ratio (LMR), Onodera prognostic nutritional index (OPNI), and LNR of 108 patients with gastric cancer were classified into high and low groups to analyze their prognostic value on the overall survival (OS) of gastric cancer patients treated with chemotherapy. The independent prognostic indicators were plotted in columns to predict the survival rate of gastric cancer patients. Results NLR was statistically significant in the prognostic assessment of gastric cancer patients treated with chemotherapy (
4.Mechanism of Polyphyllin Ⅰ Extract in Activating Hippo Signal to Induce Apoptosis and Autophagy of Colorectal Cancer Cells
Yuliang REN ; Hongling OU ; Hui WU ; Fang WAN ; Ying LIU ; Yuan SI
Chinese Journal of Experimental Traditional Medical Formulae 2023;29(19):126-135
ObjectiveTo study the inhibitory effect of polyphyllin Ⅰ (PPI) on the growth of colorectal cancer cells and its molecular mechanism. MethodRKO cells were cultured and divided into a blank group and PPI treatment groups with concentrations of 0.6, 0.8, 1.0 μmol·L-1, respectively. HRT18 cells were cultured and divided into a blank group and PPI treatment groups with concentrations of 1.2, 1.4, 1.6 μmol·L-1, respectively. The effects of PPI on the proliferation and morphology of colorectal cancer were detected by cell proliferation toxicity assay, trypan blue exclusion assay, plate clone formation assay, and confocal high-intension cell imaging analysis system. Flow cytometry was used to detect the apoptosis rate of colorectal cancer cells. The pQCXIP-GFP-LC3 plasmid transfection assay was used to detect the formation of autophagosomes in colorectal cancer cells after PPI treatment. Western blot was used to detect the expression of apoptosis-related proteins Caspase-3, Caspase-8, and poly ADP ribose polymerase (PARP), the expression of autophagy related protein LC3Ⅱ, and the expression and phosphorylation of Hippo signaling pathway proteins LATS1 and YAP. In the plvx-Flag-YAP plasmid transfection assay, YAP was overexpressed and treated with PPI, and the proliferation of colorectal cancer cells was detected by cytotoxicity assay. The expression of LC3Ⅱ and PARP in colorectal cancer cells was detected by Western blot. SwissADME predicted pharmacokinetic parameters of PPI. ResultAs compared with the blank group, the survival rate and clone formation ability of colorectal cancer cells in the PPI group were significantly decreased (P<0.01), the cell area of colorectal cancer cells in the PPI group was significantly decreased, and the roundness of colorectal cancer cells was significantly increased (P<0.01). As compared with the blank group, the apoptosis rate of colorectal cancer cells in PPI treatment groupw was significantly increased (P<0.01), the expression of apoptotic proteins Caspase-3 and Caspase-8 protein precursor in PPI treatment groups was decreased, and the cleavage of PARP was increased (P<0.01). As compared with the blank group, the expression level of autophagy-related protein LC3Ⅱ in colorectal cancer cells in PPI treatment groups was significantly increased, and the formation of autophagosomes was promoted (P<0.01). As compared with the blank group, the expression of YAP protein in colorectal cancer cells in PPI treatment groups was significantly decreased, and the expressions of phosphorylated LATS1 and YAP were significantly increased (P<0.01). As compared with the blank group, overexpression of YAP could significantly antagonize the effect of PPI on apoptosis, autophagy activation, and proliferation inhibition of colorectal cancer cells. SwissADME simulation results showed that PPI had good drug like activity. ConclusionPPI can induce apoptosis and autophagy of colorectal cancer cells through targeted activation of Hippo signaling pathway, thereby inhibiting their proliferation.
5.Mechanism of polyphyllin A in inhibition of proliferation and induction of apoptosis of gastric cancer cells by directly targeting ETS-1.
Liang ZHANG ; Yi-Lian XIONG ; Yu-Liang REN ; Xue-Wen LIU ; Yuan SI ; Ying LIU
China Journal of Chinese Materia Medica 2022;47(6):1650-1657
The present study investigated the mechanism of polyphyllin A(PPA) in inhibiting gastric cancer(GC) cells. GC cells(SGC7901 and MGC803 cell lines) were treated with PPA at different concentrations. The effect of PPA on the proliferation of GC cells was detected by MTT assay, real-time cell analysis(RTCA) assay, and clone-forming assay, respectively. Reactive oxygen species(ROS) of GC cells was detected by flow cytometry. The change of mitochondrial membrane potential was detected by JC-1 assay. The expression and phosphorylation levels of apoptosis-related proteins(caspase-9, caspase-3, and PARP) and proteins related to the signaling pathway(ETS-1, CIP2 A, and Akt) were detected by Western blot. The binding sites of PPA to ETS-1 were analyzed by molecular docking. The affinity of PPA and ETS-1 was detected by drug affinity responsive target stability(DARTS) assay. PPA had a significant inhibitory effect on the proliferation and colony formation of GC cells at a low concentration. The PPA groups showed increased ROS and decreased mitochondrial membrane potential. PPA down-regulated the precursor expression of caspase-9 and caspase-3 and promoted the cleavage of PARP, suggesting that PPA induced the apoptosis of GC cells through the mitochondrial pathway. PPA significantly reduced expression levels of CIP2 A and the phosphorylation of downstream Akt. Molecular docking showed that PPA bound to the ETS domain of ETS-1, the transcription factor of CIP2 A, and formed hydrogen bonds with Pro319 and Asp317. DARTS assay further confirmed that PPA significantly prevented the hydrolysis of ETS-1 by pronase, which was inductive of the direct binding effect of PPA and ETS-1. PPA inhibits the proliferation and induces the apoptosis of GC cells by directly targeting ETS-1 to down-regulate the ETS-1/CIP2 A/Akt signaling pathway.
Apoptosis
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Cell Line, Tumor
;
Cell Proliferation
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Humans
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Molecular Docking Simulation
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Stomach Neoplasms/metabolism*
6.Hydnocarpin inhibits malignant progression of triple negative breast cancer via CNOT4-mediated ubiquitination and degradation of YAP.
Hong-Ling OU ; Hui WU ; Yu-Liang REN ; Yuan SI ; Zhong-Qi DUAN ; Xue-Wen LIU
China Journal of Chinese Materia Medica 2023;48(16):4483-4492
This study aims to investigate the effect and mechanism of hydnocarpin(HC) in treating triple negative breast cancer(TNBC). Cell counting kit-8(CCK-8), xCELLigence real-time cellular analysis(RTCA), and colony formation assay were employed to determine the effects of HC on the proliferation of two TNBC cell lines: MDA-MB-231 and MDA-MB-436. The effects of HC on the migration and invasion of TNBC cells were detected by high-content analysis, wound-healing assay, and Transwell assay. The changes in the epithelial-mesenchymal transition(EMT) and the expression of invasion-and migration-associated proteins [E-cadherin, vimentin, Snail, matrix metalloproteinase-2(MMP-2), and MMP-9] were detected by Western blot. Western blot and RT-qPCR were employed to determine the protein and mRNA levels of Yes-associated protein(YAP) and downstream targets(CTGF and Cyr61). TNBC cells were transfected with Flag-YAP for the overexpression of YAP, and the role of YAP as a key target for HC to inhibit TNBC malignant progression was examined by CCK-8 assay, Transwell assay, and wound-healing assay. The pathway of HC-induced YAP degradation was detected by the co-treatment of proteasome inhibitor with HC and ubiquitination assay. The binding of HC to YAP and the E3 ubiquitin ligase Ccr4-not transcription complex subunit 4(CNOT4) was detected by microscale thermophoresis(MST) assay and drug affinity responsive target stability(DARTS) assay. The results showed that HC significantly inhibited the proliferation, colony formation, invasion, and EMT of TNBC cells. HC down-regulated the protein and mRNA levels of CTGF and Cyr61. HC down-regulated the total protein level of YAP, while it had no effect on the mRNA level of YAP. The overexpression of YAP antagonized the inhibitory effects of HC on the proliferation, migration, and invasion of TNBC cells. HC promoted the degradation of YAP through the proteasome pathway and up-regulated the ubiquitination level of YAP. The results of MST and DARTS demonstrated direct binding between HC, YAP, and CNOT4. The above results indicated that HC inhibited the malignant progression of TNBC via CNOT4-mediated degradation and ubiquitination of YAP.
Humans
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Triple Negative Breast Neoplasms/metabolism*
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Matrix Metalloproteinase 2/metabolism*
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Cell Line, Tumor
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Cell Proliferation
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Cell Movement
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Ubiquitination
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RNA, Messenger/metabolism*
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Epithelial-Mesenchymal Transition
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Transcription Factors/metabolism*
7.N-acetylcysteine reduces artesunate-induced pancreatic carcinoma cell death by activating protective autophagy via the AMPK/mTOR pathway
Zijing REN ; Hongxia XU ; Xingyue LI ; Yue WANG ; Jiajia MA ; Peiyang ZHOU
Journal of Xi'an Jiaotong University(Medical Sciences) 2022;43(3):354-360
【Objective】 In this study, reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC) was used to explore the inhibitory effect and mechanism of artesunate (ART) on pancreatic carcinoma (PC) cells. 【Methods】 Different concentrations of ART interfered with 3 PC cell lines CFPAC-1, Capan-2 and BxPC3. Cell viability was measured by CCK8; cell migration ability was measured by Transwell method, and the expressions of migration-related proteins E-cadherin, N-cadherin and Vimentin were measured by Western blotting. ROS probe DCFH-DA was used to measure intracellular ROS; LC3 cell immunofluorescence (IF) was used to detect the formation of intracellular autophagosomes. After adding NAC or autophagy inhibitor 3-MA, the cell viability was tested again by CCK8, and the expressions of p-AMPK/ AMPK, p-mTOR/mTOR, p62 and LC3Ⅱ/Ⅰ were detected by Western blotting. 【Results】 ART inhibited the growth of CFPAC-1 and Capan-2 in a time- and dose-dependent manner. After treatment of CFPAC-1 and Capan-2 cells with 200 μmol/L of ART for 48 h, the expression of E-cadherin was upregulated, while N-cadherin and Vimentin were downregulated, and the cell migration ability was significantly reduced. ART significantly upregulated intracellular ROS level and promoted the formation of autophagosomes. NAC could reduce the inhibitory effect of ART on CFPAC-1 and Capan-2 cells, upregulate p-AMPK/AMPK, P62 and LC3Ⅱ/Ⅰ, downregulate the expression of p-mTOR/mTOR, and intensify autophagy. 3-MA could not reverse the inhibitory effect of ART on PC cells. 【Conclusion】 ART is dependent on ROS, but not on autophagy, in exerting an anti-pancreatic carcinoma effect. NAC attenuates the inhibitory effect of ART on PC cells by activating protective autophagy through AMPK/mTOR signaling pathway.
8.Extraction of exosome by gel electrophoresis microfluidic chip and determination of miRNA-21 in exosome of human plasma.
Dan LUO ; Fengying RAN ; Lun WU ; Juan ZHANG ; Fangling REN ; Jingjian LIU ; Binqiang ZHANG ; Qinhua CHEN
Chinese Journal of Biotechnology 2021;37(2):663-672
We developed a high-efficiency microfluidic chip for extracting exosomes from human plasma. We collected peripheral blood from normal human, designed and fabricated a microfluidic chip based on nanoporous membrane and agarose gel electrophoresis to isolate exosomes. The extracted exosomes were characterized by transmission electron microscopy, nanosight and Western blotting, the morphology, concentration and particle size of exosomes were identified and analyzed. Meanwhile, we used ultracentrifugation and microfluidic chip to isolate exosomes separately. The particle size and concentration of the exosomes extracted by two methods were compared and analyzed, and their respective extraction efficiency was discussed. Finally, the expression level of miRNA-21 in exosomes was analyzed by RT-PCR. The microfluidic chip isolated (in 1 hour) high-purity exosomes with size ranging from 30-200 nm directly from human plasma, allowing downstream exosomal miRNA analysis. By comparing with ultracentrifugation, the isolation yield of microfluidic chip was 3.80 times higher than ultracentrifugation when the volume of plasma sample less than 100 μL. The optimized parameters for exosome isolation by gel electrophoresis microfluidic chip were: voltage: 100 V; concentration of agarose gel: 1.0%; flow rate of injection pump: 0.1 mL/h. The gel electrophoresis microfluidic chips could rapidly and efficiently isolate the exosomes, showing great potential in the research of exosomes and cancer biomarkers.
Exosomes
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Humans
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MicroRNAs/genetics*
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Microfluidics
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Plasma
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Ultracentrifugation
9.Effects of HGF on GVHD and Th1/Th2-related cytokines in ALL mice after allo-BMT.
Yun-Jin XIA ; Qing-Ping GAO ; Chu-Cheng WAN ; Fan-Jun CHENG ; Wei-Min WANG ; Ren-Ci GUO
Journal of Experimental Hematology 2005;13(1):35-38
To observe the effects of hepatocyte growth factor (HGF) on graft-versus-host disease (GVHD) and Th1/Th2 related cytokines in mice with acute lymphoblastic leukemia (ALL) after allogenic bone marrow transplantation (allo-BMT), BALB/c mice were conditioned by total body irradiation with 11 Gy and then were transplanted with allogeneic bone marrow after establishing ALL model. BALB/c mice were divided into groups A and B. The mice of group A were injected subcutaneously with HGF from day 0 to 7 after allo-BMT, and the mice of group B were injected subcutaneously with PBS from day 0 to 7 after allo-BMT. The symptoms of GVHD and the GVHD pathological changes of liver and small intestine and skin were observed. The serum levels of both IFN-gamma and IL-4 were determined by ELISA. The results showed that the score of GVHD in group A was lower than that in group B (P < 0.05). The levels of IFN-gamma in both groups A and B were all higher than that in normal group (P < 0.05 and P < 0.001, respectively), However, the level of IFN-gamma in group A was lower than that in group B (P < 0.01). The levels of IL-4 in both group A and B were all lower than that in normal group (P < 0.05), but the level of IL-4 in group A was higher than that in group B (P < 0.05). It is concluded that HGF can alleviates the severity of GVHD, because of its balancing the Th1/Th2-related cytokines after allo-BMT.
Animals
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Bone Marrow Transplantation
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adverse effects
;
methods
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Cytokines
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blood
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Enzyme-Linked Immunosorbent Assay
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Female
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Graft vs Host Disease
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immunology
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prevention & control
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Hepatocyte Growth Factor
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pharmacology
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Interferon-gamma
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blood
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Interleukin-4
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blood
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Male
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Mice
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Mice, Inbred BALB C
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Precursor Cell Lymphoblastic Leukemia-Lymphoma
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blood
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immunology
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surgery
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Th1 Cells
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immunology
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Th2 Cells
;
immunology
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Transplantation, Homologous
10.Effect of vascular endothelial growth factor on bone marrow-derived mesenchymal stem cell proliferation and the signaling mechanism.
Jun ZHANG ; Shan-shan XIE ; Xiao-xia HAN ; Jin-tao REN ; Fu-ran LV ; Jun-ming TANG ; Fei ZHENG ; Ling-yun GUO ; Jian-ye YANG ; Xia KONG ; Lei ZHANG ; Yong-zhang HUANG ; Jia-ning WAN
Journal of Southern Medical University 2011;31(10):1697-1700
OBJECTIVETo observe the effect of vascular endothelial growth factor (VEGF) on bone marrow-derived mesenchymal stem cell (MSC) proliferation and explore the signaling mechanism involved.
METHODSMSC culture was performed following the classical whole bone marrow adhering method. The characteristics of MSC were identified by induction of multi-lineage differentiation and flow cytometry for surface marker analysis (CD34, CD45, CD29, and CD90). Following the addition of 50 nmol/L wortmannin, 50 µmol/L PD98059, 30 µmol/L SB203580, 10 µmol/L H89, 20 µmol/L Y27632, 1 µmol/L rapamycin, 10 µmol/L straurosporine, 6 nmol/L Go6976, or 50 µmol/L Pseudo Z inhibitors in the cell culture, the MSC were treated with 20 ng/ml VEGF and the changes of the cell proliferation rate was measured with MTT assay.
RESULTSCultured MSC were capable of multi-linage differentiation and did not express VEGF-R, CD29 or CD90. Treatment with 20 ng/ml VEGF obviously promoted MSC proliferation, and this effect was inhibited partially by p38 mitogen-activated protein kinase (MAPK) inhibitor rapamycin, PD98059, SB203580, Go6976, and straurosporine.
CONCLUSIONSVEGF promotes MSC proliferation in close relation to the AKT-PKC pathway, in which PKC signal pathway may play the central role.
Animals ; Bone Marrow Cells ; cytology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Female ; Male ; Mesenchymal Stromal Cells ; cytology ; Protein Kinase C ; metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Vascular Endothelial Growth Factor A ; pharmacology