As main recipient cells for porcine reproductive and respiratory syndrome virus (PRRSV), porcine alveolar macrophage (PAM) are involved in the progress of several highly pathogenic virus infections. However, due to the fact that the PAM cells can only be obtained from primary tissues, research on PAM-based virus-host interactions remains challenging. The improvement of induced pluripotent stem cells (iPSCs) technology provides a new strategy to develop IPSCs-derived PAM cells. Since the CD163 is a macrophage-specific marker and a validated receptor essential for PRRSV infection, generation of stable porcine induced pluripotent stem cells lines containing CD163 reporter system play important roles in the investigation of IPSCs-PAM transition and PAM-based virus-host interaction. Based on the CRISPR/Cas9- mediated gene editing system, we designed a sgRNA targeting CD163 locus and constructed the corresponding donor vectors. To test whether this reporter system has the expected function, the reporter system was introduced into primary PAM cells to detect the expression of RFP. To validate the low effect on stem cell pluripotency, we generated porcine iPSC lines containing CD163 reporter and assessed the pluripotency through multiple assays such as alkaline phosphatase staining, immunofluorescent staining, and EdU staining. The red-fluorescent protein (RFP) expression was detected in CD163-edited PAM cells, suggesting that our reporter system indeed has the ability to reflect the expression of gene CD163. Compared with wild-type (WT) iPSCs, the CD163 reporter-iPSCs display similar pluripotency-associated transcription factors expression. Besides, cells with the reporter system showed consistent cell morphology and proliferation ability as compared to WT iPSCs, indicating that the edited-cells have no effect on stem cell pluripotency. In conclusion, we generated porcine iPSCs that contain a CD163 reporter system. Our results demonstrated that this reporter system was functional and safe. This study provides a platform to investigate the iPS-PAM development and virus-host interaction in PAM cells.
Swine ; Animals ; Induced Pluripotent Stem Cells/metabolism* ; Receptors, Cell Surface/genetics* ; Antigens, CD/metabolism* ; Porcine respiratory and reproductive syndrome virus/genetics*

OBJECTIVE: To assess the effect of neutralizing CD96 on natural killer (NK) cell functions in mice with pulmonary infection and explore the possible mechanism. METHODS: Male BALB/c mice were randomly divided into infection group (Cm group), anti-CD96 treatment group (anti-CD96 group) and control group (=5). In the former two groups, was inoculated intranasal administration to establish mouse models of pulmonary infection, and the mice in the control group received intranasal administration of the inhalation buffer. In anti-CD96 group, the mice were injected with anti-CD96 antibody intraperitoneally at the dose of 250 μg every 3 days after the infection; the mice in Cm group received intraperitoneal injections of saline. The body weight of the mice was recorded daily. The mice were sacrificed 5 days after infection, and CD96 expression was detected by quantitative real-time PCR and Western blotting. HE staining and pathological scores were used to evaluate pneumonia of the mice. The inclusion body forming units (IFUs) were detected in the lung tissue homogenates to assess lung tissue chlamydia load. Flow cytometry and ELISA were used to assess the capacity of the lung NK cells to produce interferon-γ (IFN-γ) and regulate macrophages and Th1 cells. RESULTS: infection inhibited CD96 expression in NK cells of the mice. Compared with those in Cm group, the mice in antiCD96 mice showed significantly milder lung inflammation ( < 0.05) and reduced chlamydia load in the lung tissue ( < 0.05). Neutralizing CD96 with anti-CD96 significantly enhanced IFN-γ secretion by the NK cells ( < 0.05) and augmented the immunoregulatory effect of the NK cells shown by enhanced responses of the lung macrophages ( < 0.05) and Th1 cells ( < 0.05). CONCLUSIONS Inhibition of CD96 alleviates pneumonia in -infected mice possibly by enhancing IFN-γ secretion by NK cells and augmenting the immunoregulatory effect of the NK cells on innate and adaptive immunity.
Animals ; Antigens, CD ; metabolism ; Chlamydia Infections ; complications ; immunology ; physiopathology ; Chlamydia muridarum ; Interferon-gamma ; genetics ; metabolism ; Killer Cells, Natural ; metabolism ; Lung Injury ; etiology ; genetics ; prevention & control ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL

OBJECTIVE: To investigate the role of MTBP in regulating the migration and invasion of human prostate cancer cells. METHODS: The baseline expressions of MTBP in 3 different human prostate cancer cells lines (22RV1, DU145 and Lncap) were detected using Western blotting. The cells were transfected with a small interfering RNA (siRNA) for MTBP knockdown or MTBP plasmid for MTBP overexpression, and 48 h later, the cells were examined for MTBP expression with Western blotting; the changes in the migration abilities of the cells were evaluated using wound healing assay and Transwell assay, and the cell invasiveness was assessed using Matrigel Transwell assay. The expression of E-cadherin protein, a marker of epithelial mesenchymal transition (EMT), was detected using Western blotting. RESULTS: MTBP expression was the highest in DU145 cells followed by Lncap cells, and was the lowest in 22RV1 cells, indicating a positive correlation of MTBP expression with the level of malignancy of human prostate cancer cells. Transfection of the cells with siRNA or MTBP plasmids efficiently lowered or enhanced the expressions of MTBP in human prostate cancer cells. Wound healing assay showed that inhibition of MTBP expression decreased the migration ability of the prostate cancer cells, and MTBP overexpression significantly promoted the migration of the cells ( < 0.01). Transwell assay showed that MTBP knockdown significantly lowered the migration and invasion ability of the cells, while MTBP overexpression markedly increased the number of migrating and invading cells ( < 0.01); Western blotting results showed that MTBP knockdown increased the expression of E-cadherin protein, and MTBP overexpression decreased E-cadherin expression in the prostate cancer cells. CONCLUSIONS MTBP overexpression promotes the migration and invasion of human prostate cancer cells possibly relation to the induction of EMT.
Antigens, CD ; metabolism ; Cadherins ; metabolism ; Carrier Proteins ; genetics ; metabolism ; Cell Line, Tumor ; Cell Movement ; Epithelial-Mesenchymal Transition ; Gene Expression Regulation, Neoplastic ; Gene Knockdown Techniques ; Humans ; Male ; Neoplasm Invasiveness ; Prostatic Neoplasms ; metabolism ; pathology ; RNA, Small Interfering ; Transfection

To investigate the expressions of mucosal barrier proteins in colon cell line DLD-1 under hypoxic environment and its mechanism. Methods After DLD-1 cells were treated separately with hypoxia(l% O),vitamin D(100 nmol/L),or vitamin D plus hypoxia for 48 hours,the expressions of vitamin D receptor(VDR),tight junction proteins zonula occludens-1(ZO-1),occludin,Claudin-1,and adherent junction protein(E-cadherin)were determined by Western blot.Stable VDR knock-down(Sh-VDR)DLD-1 cell line and control DLD-1 cell line were established by lentivirus package technology and the protein expressions after hypoxia treatment were detected. Results Compared with control group,the expressions of occludin,Claudin-1,and VDR increased significantly after hypoxia treatment(all <0.001).In addition to the protein expressions of occludin,Claudin-1 and VDR,the expressions of ZO-1 and E-cadherin were also obviously higher in vitamin D plus hypoxia group than in single vitamin D treatment group(all <0.001).After hypoxia treatment,Sh-VDR cell line showed significantly decreased expressions of ZO-1(<0.001),occludin(<0.05),Claudin-1(<0.01)and E-cadherin(<0.001)when compared with untreated Sh-VDR cell line. Conclusion VDR acts as a regulator for the expressions of intestinal mucosal barrier proteins under hypoxia environment in DLD-1 colon cell line,indicating that VDR pathway may be another important protective mechanism for gut barrier in low-oxygen environment.
Antigens, CD ; metabolism ; Cadherins ; metabolism ; Cell Hypoxia ; Cell Line ; Claudin-1 ; metabolism ; Colon ; cytology ; Humans ; Occludin ; metabolism ; Receptors, Calcitriol ; metabolism ; Tight Junctions ; Vitamin D ; pharmacology ; Zonula Occludens-1 Protein ; metabolism

OBJECTIVETo investigate the effect of steadily down-regulating the expression of VE-cadherin on the chemotheraputic sensitivity of K562 cells, and explore its possible mechanism.

METHODSSpecifically targeting interference sequences carrying human VE-cadherin were designed, the recombinant lentiviral vector containing the IRES-GFP and NEO segment was constructed; recombinant lentivirus was generated by three-plasmids packing system, and transfected into K562 cells, then the cells steadily down-regulated were sorted. CCK-8 assay was performed to evaluate the VE-cadherin of chemotherapeutic (Imatinib) sensitivity of K562 cells. The apoptosis was analyzed by flow cytometry with Annexin V/7-AAD double labeling. The expressions of CD133 and ALDH1 mRNA were determined by real time PCR. The protein expressions of VE-cadherin, BCR-ABL and β-catenin were analyzed by Western blot.

RESULTSThe recombinant lentiviral vector pLB-shVEC-NEO-IRES-GFP was successfully constructed, packed into the lentivirus, then the K562 cells steadily down-regulating VE-cadherin expression was obtained. When VE-cadherin was down-rengulated in K562 cells, the proliferation rate was reduced while the the apoptosis rate was increased; the mRNA levels of CD133 and ALDH1 also were reduced; BCR-ABL fusion protein was not obviously changed; the total β-catenin protein, as well as the nuclear β-catenin protein were decreased in the K562/shVEC cells. Conclution: K562 cells are more susceptible to chemotherapy when VE-cadherin is down-regulated, that may be realized via reducing the stability and the nuclear transfer of β-catenin protein.

Antigens, CD ; metabolism ; Apoptosis ; Cadherins ; metabolism ; Cell Proliferation ; Fusion Proteins, bcr-abl ; Humans ; K562 Cells

OBJECTIVE: To explore the role of SIRT1 in the occurrence of epithelial-mesenchymal transition (EMT) in EC-9706 and Eca-109 cells and the possible mechanism. METHODS: Three chemically synthesized siRNA targeting SIRT1 were transfected into EC-9706 and Eca-109 cells with the non-transfected cells and cells transfected with the negative siRNAs as controls. Real-time PCR and Western blotting were used to detect the expressions of SIRT1, E-cadherin, vimentin, Snail, Twist1 and ZEB in the cells. Transwell invasion assay and wounding healing assay were used to examine the changes in the invasion and metastasis abilities of the cells after transfection. RESULTS: EC-9706 and Eca-109 cells transfected with SIRT1 siRNA1 and SIRT1 siRNA3 showed significantly decreased mRNA and protein expressions of SIRT1 ( < 0.05). Transwell invasion assay and wounding healing assay showed that transfection with SIRT1 siRNA1 and SIRT1 siRNA3 caused significantly lowered invasion and metastasis abilities in EC-9706 and Eca-109 cells ( < 0.05). In EC-9706 and Eca-109 cells transfected with SIRT1 siRNA1 and SIRT1 siRNA3, the expression level of E-cadherin was significantly increased while the expressions of vimentin, Snail and Twist were significantly lowered ( < 0.05). CONCLUSIONS SIRT1 participates in the invasion and metastasis of EC-9706 and Eca- 109 cells probably by inducing EMT via regulating the expression of Snail.
Antigens, CD ; metabolism ; Cadherins ; metabolism ; Cell Line, Tumor ; Cell Movement ; Epithelial-Mesenchymal Transition ; physiology ; Humans ; Neoplasm Invasiveness ; Nuclear Proteins ; metabolism ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; metabolism ; Sirtuin 1 ; genetics ; metabolism ; Snail Family Transcription Factors ; metabolism ; Transfection ; Twist-Related Protein 1 ; metabolism ; Vimentin ; metabolism ; Zinc Finger E-box-Binding Homeobox 1 ; metabolism

OBJECTIVE: To investigate the regulation mechanism of RhoA signaling pathway during the enamel formation by using the EGFP-RhoA^{Dominant Negative} (EGFP-RhoA^DN) transgenic mice model, from the aspect of adherens junctions, and to provide a theory basis for mechanism of enamel development defects. METHODS: The enamel thickness of mandibular first molars of EGFP-RhoA^DN transgenic mice and wild type (WT) mice were observed by scanning electronic microscopy at 20 kV, and the enamel thickness of the distal face of the central cusp was measured at 10 locations via analysis by ImageJ (Rasband, 1997-2009). The enamel organs from mandibular first molars from postnatal-4-day (P4) EGFP-RhoA^DN mice and wild type mice were isolated, and the total RNA and protein were extracted from the epithelium of the enamel organs. The expression level of the adherens junctions components in ameloblasts layer of the postnatal-4-day EGFP-RhoA^DN transgenic mice and wild type mice mandibular first molars were detected by real-time PCR and Western blot assay. RESULTS: The EGFP-RhoA^DN transgenic mice had decreased enamel thickness in their bilateral mandibular first molars versus those of control group (n=20), and enamel thickness was (84.60±0.20) μm vs. (106.24±0.24) μm, P<0.05. The protein expressions of E-cadherin, α-E-catenin and pan-cadherin in ameloblasts layer of postnatal-4-day EGFP-RhoA^DN transgenic mice molars were down-regulated, and the protein level of β-catenin in ameloblasts layer of P4 EGFP-RhoA^DN transgenic mice molars was up-regulated. The mRNA level of E-cadherin in ameloblasts layer of P4 EGFP-RhoA^DN transgenic mice molars was down-regulated versus that of WT mice, and the gene expression of E-cadherin was 0.93±0.01 vs. 1.00±0.02, P<0.05. The mRNA level of β-catenin in ameloblasts layer of P4 EGFP-RhoA^DN transgenic mice molars was up-regulated versus that of WT mice, and the gene expression of β-catenin was 1.23±0.03 vs. 1.00±0.05, P<0.05. CONCLUSION In the mandibular first molars of EGFP-RhoA^DN transgenic mice, the enamel formation was disrupted and the adherens junctions of EGFP-RhoA^DN transgenic mice ameloblasts were implicated during amelogenesis. RhoA signaling pathway may play a critical role in enamel development by altering the adherens junctions in ameloblasts.
Adherens Junctions ; Ameloblasts ; Amelogenesis ; Animals ; Antigens, CD ; Cadherins/metabolism* ; Dental Enamel/metabolism* ; Enamel Organ ; Humans ; Mice ; Mice, Transgenic ; Molar ; Signal Transduction ; alpha Catenin ; beta Catenin ; rhoA GTP-Binding Protein/physiology*

Asthma is a chronic disease of airway inflammation due to excessive T helper cell type 2 (Th2) response. Present treatment based on inhalation of synthetic glucocorticoids can only control Th2-driven chronic eosinophilic inflammation, but cannot change the immune tolerance of the body to external allergens. Regulatory T cells (Tregs) are the main negative regulatory cells of the immune response. Tregs play a great role in regulating allergic, autoimmune, graft-versus-host responses, and other immune responses. In this review, we will discuss the classification and biological characteristics, the established immunomodulatory mechanisms, and the characteristics of induced differentiation of Tregs. We will also discuss the progress of Tregs in the field of asthma. We believe that further studies on the regulatory mechanisms of Tregs will provide better treatments and control strategies for asthma.
Antigens, CD/analysis* ; Apyrase/analysis* ; Asthma/immunology* ; Cell Differentiation ; Cytokines/metabolism* ; Humans ; Lymphocyte Transfusion ; T-Lymphocytes, Regulatory/immunology*

OBJECTIVETo observe the effects of Compound Zhebei Granule (, CZBG) combined with chemotherapy on surface markers of leukemia stem cell (LSC) in the bone marrow of patients with acute myeloid leukemia (AML).

METHODSSeventy-eight patients with AML received bone marrow aspiration and the percentages of CD34(+) CD123(+) and CD33(+) CD123(+) cells were tested using flow cytometry method. A total of 24 refractory or relapsed AML patients were enrolled and treated with one cycle of standard chemotherapy combined with CZBG. Bone marrow samples were obtained before and after treatment, and the percentages of CD34(+) CD123(+) and CD33(+) CD123(+) cells were examined by flflow cytometry.

RESULTSCompared with refractory or relapsed AML patients, patients achieved remission had a significant lower percentage of CD34(+) CD123(+) cells(P<0.01) and CD33(+) CD123(+) cells (P<0.01), indicating that controlling the LSC percentage may be important for patients with AML to achieve sustainable remission. Compared with those before treatment, the expression levels of CD34(+) CD123(+) were significantly decreased after CZBG combined with chemotherapy treatment (P<0.01). The percentages of CD34(+) CD123(+) cells and CD33(+) CD123(+) in patients achieving complete remission after CZBG combined with chemotherapy treatment were both significantly lower than those in patients with nonremission (P<0.01).

CONCLUSIONCZBG combining chemotherapy could reduce the percentages of CD34(+) CD123(+) and CD33(+) CD123(+) LSC, which might improve the clinical efficacy of refractory or relapsed AML.

Antigens, CD ; metabolism ; Antineoplastic Agents ; therapeutic use ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Biomarkers, Tumor ; metabolism ; Bone Marrow Cells ; drug effects ; metabolism ; pathology ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Humans ; Leukemia, Myeloid, Acute ; drug therapy ; pathology ; Male ; Middle Aged ; Neoplastic Stem Cells ; metabolism ; pathology ; Remission Induction

Artemisinin is an anti-malarial drug with poor water solubility and oral absorption; so a variety of derivatives based on the parent nucleus have been developed. Compared with artemisinin, dihydroartemisinin (DHA) has a stronger anti-malaria activity, and has the advantages of high metabolic rate and better water solubility. Recent studies have discovered that DHA has a good inhibitory effect on tumor cells, which is closely related to the peroxide bridge in its molecular structure. Since tumor cells need more Fethan normal cells, there are a large number of transferrin receptors on the tumor cell membrane. DHA can break the peroxide bridge in the presence of Fe, and the free radicals generated can play its lethal effect on tumor cells. In addition, DHA can promote endocytosis of transferrin receptor, and thus prevent cancer cells from taking Fefrom microenvironment. This article reviews the anti-tumor molecular mechanism of DHA, including accelerating oxidative damage, inducing apoptosis, inhibiting the growth, proliferation and invasion of tumor cells, reversing tumor multidrug resistance.
Antigens, CD ; drug effects ; metabolism ; Antineoplastic Agents ; pharmacokinetics ; pharmacology ; Apoptosis ; drug effects ; Artemisinins ; metabolism ; pharmacokinetics ; pharmacology ; Endocytosis ; drug effects ; Free Radicals ; chemical synthesis ; pharmacology ; Humans ; Iron ; metabolism ; Neoplasms ; drug therapy ; physiopathology ; Oxidative Stress ; drug effects ; Receptors, Transferrin ; drug effects ; metabolism