[The number of regulatory T cells in spleen and serum-related cytokines increase in mouse model of psoriasis induced by imiquimod].

Objective To explore the role of regulatory T cells (Tregs) and its related cytokines in mouse model of psoriasis induced by imiquimod. Methods Female BALB/c mice were divided into control group and model group of psoriasis induced by imiquimod, with ten mice in each group. The psoriasis model was established by smearing imiquimod cream on the back of mice. HE staining was performed to observe the pathological changes of skin tissues. Flow cytometry was used to detect the number of Tregs in spleen and the levels of serum IL-10 and TGF-ß1 was detected by ELISA. Results The mouse model of psoriasis was successfully established. Compared with the control group, the percentage of Tregs in spleen of the mouse model group significantly increased, and the levels of serum IL-10 and TGF-ß1 also increased. Conclusion The number of Tregs and related cytokines increase in mouse model of psoriasis.

[IL-6 promotes the activation of rat astrocytes and down-regulation of the expression of Kir4.1 channel].

Objective To identify the effects of interleukin-6 (IL-6) on astrocytes activation, and the regulation of the expression of inwardly rectifying potassium 4.1 (Kir4.1) channels in astrocytes. Methods Astrocytes were separated from the cerebral cortex of newborn SD rats, and cultured in the presence of IL-6 or combined with interleukin-6 receptor antagonist (IL-6Ra). CCK-8 assay was performed to measure cell viability. The expression level of Kir4.1 channels in astrocytes was measured using quantitative real-time PCR and Western blot analysis. Results IL-6 promoted the proliferation of astrocytes in a dose- (0-30 ng/mL) and time- (0-24 hours) dependent manner. After astrocytes were treated with IL-6 (30 ng/mL) for 24 hours, the levels of Kir4.1 mRNA and protein decreased significantly, and this down-regulation could be attenuated by IL-6Ra. Conclusion IL-6 promotes the activation of astrocyte and down-regulation of the expression of Kir4.1 channel.

siRNA-induced CD44 knockdown suppresses the proliferation and invasion of colorectal cancer stem cells through inhibiting epithelial-mesenchymal transition.

CD44 has shown prognostic values and promising therapeutic potential in multiple human cancers; however, the effects of CD44 silencing on biological behaviors of cancer stem cells (CSCs) have not been fully understood in colorectal cancer. To examine the contribution of siRNA-induced knockdown of CD44 to the biological features of colorectal CSCs, colorectal CSCs HCT116-CSCs were generated, and CD44 was knocked down in HCT116-CSCs using siRNA. The proliferation, migration and invasion of HCT116-CSCs were measured, and apoptosis and cell-cycle analyses were performed. The sensitivity of HCT116-CSCs to oxaliplatin was tested, and xenograft tumor growth assay was performed to examine the role of CD44 in HCT116-CSCs tumorigenesis in vivo. In addition, the expression of epithelial-mesenchymal transition (EMT) markers E-cadherin, N-cadherin and vimentin was quantified. siRNA-induced knockdown of CD44 was found to inhibit the proliferation, migration and invasion, induce apoptosis, promote cell-cycle arrest at the G1/G0 phase and increase the sensitivity of HCT116-CSCs to oxaliplatin in HCT116-CSCs, and knockdown of CD44 suppressed in vivo tumorigenesis and intrapulmonary metastasis of HCT116-CSCs. Moreover, silencing CD44 resulted in EMT inhibition. Our findings demonstrate that siRNA-induced CD44 knockdown suppresses the proliferation, invasion and in vivo tumorigenesis and metastasis of colorectal CSCs by inhibiting EMT.

LncRNA LINC01116 Contributes to Cisplatin Resistance in Lung Adenocarcinoma.

BACKGROUND: Long non-coding RNAs (lncRNAs) have been found to contribute to cisplatin resistance in several cancers; however, the role of lncRNA LINC01116 in cisplatin resistance remains unknown in non-small-cell lung cancer. This study aimed to examine the contribution of LINC01116 to cisplatin resistance in lung adenocarcinoma (LAD). MATERIALS AND METHODS: Cisplatin-resistant A549/DDP cells were generated by treatment with cisplatin by dose escalation. LINC01116 expression was compared between A549 and A549/DDP cells, and between cisplatin-resistant and non-resistant LAD specimens. The cell viability, colony formation, proliferation, migration and invasion were measured using MTT and Transwell assays, and cell apoptosis and cell cycle were detected using flow cytometry. The expression of E-cadherin and Vimentin was quantified. LAD xenografts were modeled in nude mice to investigate the role of LINC01116 on the resistance of LAD to cisplatin. RESULTS: MTT assay measured the IC50 values of 13.49 ± 1.62 and 3.52 ± 1.33 µg/mL for A549/DDP and A549 cells, respectively. LINC01116 was overexpressed in cisplatin-resistant LAD specimens and A549/DDP cells (P < 0.05). Knockdown of LINC01116 inhibited cell viability, proliferation, migration and invasion, promoted apoptosis and enhanced the sensitivity to cisplatin in A549/DDP cells, while LINC01116 overexpression promoted cell viability, proliferation, migration and invasion, inhibited apoptosis and reduced the sensitivity to cisplatin in A549 cells. LINC01116 knockdown resulted in a 2.1-fold increase in E-cadherin expression and a 56% reduction in Vimentin expression in A549/DDP cells, and LINC01116 overexpression resulted in a 45% reduction in E-cadherin expression and a 1.82-fold increase in Vimentin expression in A549 cells. CONCLUSION: Dysregulation of lncRNA LINC01116 expression results in resistance of LAD to cisplatin via the EMT process. Our findings support the oncogenic role of LINC01116 to promote the development of cisplatin resistance in LAD, and LINC01116 may be a novel predictor of poor response to cisplatin.

Rat Bone Marrow-Derived Mesenchymal Stem Cells Promote the Migration and Invasion of Colorectal Cancer Stem Cells.

BACKGROUND: Colorectal cancer is one of the most common cancers and the second leading cause of cancer-related deaths worldwide. Targeting cancer stem cells (CSCs) may be a novel strategy for the treatment of colorectal cancer. Previous studies have shown that bone marrow-derived MSCs (BM-MSCs) promote tumor growth and metastasis. However, the role of rat BM-MSCs in the biological behaviors of colorectal CSCs remains unclear until now. MATERIALS AND METHODS: BM-MSCs were isolated from rats and characterized. CSCs were enriched from HCT116 cells using the microsphere culture method, and the microspheres incubated for at least 10 passages were termed HCT116-CSCs that were characterized. The effects of rat BM-MSCs on migration and invasion of HCT116-CSCs were examined using transwell migration and invasion assays and xenograft tumor growth assay. RESULTS: Rat BM-MSCs appeared typical stem cell morphology. Flow cytometry revealed positive CD29 and CD44 expression in rat BM-MSCs at passage 3, and rat BM-MSCs were found to differentiate into osteocytes following incubation in osteogenic induction medium. Microscopy, flow cytometric detection of stem cell surface markers, colony-formation assay and transwell migration and invasion assays characterized the successful preparation of HCT116-CSCs, and subcutaneous injection of HCT116-CSCs produced xenograft tumors in nude mice, while HE staining of the xenograft tumors displayed cancer specimen shapes. Transwell migration and invasion assays showed that rat BM-MSCs promoted the migration and invasion of HCT116-CSCs, and injection of rat BM-MSCs was found to promote the growth of the mouse xenograft tumor derived from HCT116-CSCs. CONCLUSION: Rat BM-MSCs promote the migration and invasion of colorectal CSCs, and colorectal CSCs may be a potential target for the therapy against colorectal cancer.

Association of CD44 and CD24 phenotype with lymph node metastasis and survival in triple-negative breast cancer.

BACKGROUND: CD44+CD24-/low phenotypes are associated with poor outcome of triple-negative breast cancer (TNBC); however, the role of the CD44+CD24-/low phenotype in lymph node metastasis and survival has not been fully understood in TNBC. METHODS: A total of 51 TNBC patients were included. CD44 and CD24 expression was determined using immunohistochemistry by which CD44 and CD24 were double-immunostained. Overall survival (OS) and disease-free survival (DFS) were estimated using the Kaplan-Meier method. RESULTS: The proportion of the CD44+CD24-/low phenotype was 33.3% in TNBC specimens without lymph node metastases and 69.0% in those with lymph node metastases. In addition, the CD44+CD24-/low phenotype correlated significantly with tumor size, histologic classification, TNM stage, and lymph node metastasis (P < 0.05). The CD44+CD24-/low phenotype was detected in 69.0% of TNBC patients with lymph node metastases, and 51.7% of TNBC patients without lymph node metastases. In TNBC patients without lymph node metastases, the median DFS and OS were 18.2 and 28 months in cases with a CD44+CD24-/low phenotype and 26.5 and 42.5 months in those without a CD44+CD24-/low phenotype (P < 0.05), and in TNBC patients with lymph node metastases, the median DFS and OS were 17.2 and 25.7 months in cases with a CD44+CD24-/low phenotype and 24.5 and 39.3 months in those without a CD44+CD24-/low phenotype, respectively (P < 0.05). CONCLUSIONS: CD44 and CD24 are independent prognostic markers for patients with TNBC. The CD44+CD24-/low phenotype correlates with more aggressive clinicopathologic features and is strongly associated with poor prognosis in patients with TNBC.

[Construction and optimization of an in vitro induction system for bone marrow-derived mesenchymal stem cell differentiation into hematopoietic cells].

Objective To construct the hematopoietic microenvironment system simulating different hematopoietic sites during the embryonic stages for in vitro inducing the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) into hematopoietic cells, and to initially evaluate the efficiency of differentiation. Methods Stereomicroscopy and HE staining were used to observe the anatomical parts and morphology of yolk sac (YS), placenta (PL) and fetal liver (FL) of E11.5 mice. The embryonic YS, PL and FL tissues from the stages of E7.5-E9.5, E10.5-E12.5 and E13.5-E15.5 were collected for stromal cell culture, and conditioned culture media were prepared, namely, yolk sac stromal cell conditioned medium (YSSC-CM), placental SC-CM (PLSC-CM) and fetal liver SC-CM (FLSC-CM), and co-cultured with in vitro expanded SD rat BMSCs. The experimental cells were divided into control group, interleukin 6(IL-6) combined with stem cell factor (SCF) treatment group, YSSC-CM treatment group, PLSC-CM treatment group and FLSC-CM treatment group. After co-culture for 7-9 days, the floating cells in culture medium were collected. Giemsa staining was used to exam cell morphology. Direct immunofluorescence was conducted to detect CD34 and CD45 expression. Colony formation assay was performed to detect granulocyte/macrophage colony formation unit (GM-CFU) to identify differentiated cells. Results Under a stereomicroscope, PL and FL of mouse embryos had the same position as human embryos, while YS was wrapped on the outer side of embryo body and amniotic membrane. HE staining showed that PL vessel labyrinth, FL sinusoids and YS blood islands were rich in blood cells. The inverted phase-contrast microscope and cell counting results indicated that the floating cells in the culture medium of the YSSC-CM group, the PLSC-CM group and the FLSC-CM group significantly increased compared with the control group and the IL-6 combined with SCF group, especially the FLSC-CM group had the most. The floating cells in YSSC-CM group, PLSC-CM group and FLSC-CM group were similar in morphology to lymphoid or mononucleoid cells as showed by Giemsa staining, and expressed hematopoietic cell-specific surface markers CD34 and CD45. The number of hematopoietic cell colonies formed was the most in the FLSC-CM group, followed by the PLSC-CM group, and the least in the YSSC-CM group. The control group and IL-6 combined with SCF treatment group had no changes. Conclusion By collecting YS, PL, and FL according to the stage and time sequence, YSSC-CM, PLSC-CM, and FLSC-CM prepared can induce the differentiation of SD rat's BMSCs into hematopoietic cells, and FLSC-CM-treated cells have better differentiation efficiency.

[Comparison of biological characteristics of bone marrow mesenchymal stem cells cultured in vitro from SD rats and BALB/c mice].

Objective To assess the biological characteristics of bone marrow mesenchymal stem cells (BMMSCs) cultured in vitro from SD rats and BALB/c mice. Methods BMMSCs derived from the bone marrow of SD rats or BALB/c mice were separated and purified by whole bone marrow culture and differential adherence methods, and then the cells were expanded by subculture successively. The growth and morphology of the primary and third-passage BMMSCs were observed by inverted phase-contrast microscopy and HE staining, the growth curve of the third-passage cells was drawn by cell counting. The cell surface antigen makers CD29, CD44, CD34, and CD45 on the third-passage BMMSCs were detected by flow cytometry. The third-passage BMMSCs were induced to differentiate into osteoblasts after exposed to bone-inducing media for 3 weeks, and then were identified by alizarin red staining. Results The primary bone marrow cells from SD rats formed the colony earlier than those from BALB/c mice. The primary and third-passage SD-BMMSCs were spindle-shaped, uniform in size and more in flocks, while the morphology of BALB/c-BMMSCs was more complex, being round, triangular or spindle-shaped, smaller, fewer and frost-like. HE staining showed the colony of the third-passage SD-BMMSCs was more typical than that of BALB/c-BMMSCs, and the cell morphology was more homogeneous. The growth curves of the third-passage BMMSCs of the 2 species presented a similar "S" shape, however, SD-BMMSCs had better growth vigor. Their differentiation into osteocytes was observed and both of the third-passage BMMSCs could differentiate into osteoblasts, but the mineralized nodules formed by SD-BMMSCs were more obvious. Flow cytometry revealed that the CD29 and CD44 expression levels in both of the third-passage BMMSCs were high and the CD34 level was low. The CD45 expression level in the SD-BMMSCs was low, while that was high in the BALB/c-BMMSCs. Conclusion The bone marrow of SD rats and BALB/c mice are suitable sources for BMMSCs, and the BMMSCs isolated and purified by whole bone marrow culture and differential adherence methods have typical morphology, immunophenotype and differentiation capacities of MSCs. However, the BMMSCs from SD rats can be separated and cultured more easily, have a higher purity and grow faster as compared with the ones from BALB/c mice.

Efficacy and Safety of Vinorelbine Plus Cisplatin vs. Gemcitabine Plus Cisplatin for Treatment of Metastatic Triple-Negative Breast Cancer After Failure with Anthracyclines and Taxanes.

BACKGROUND This study aimed to compare the efficacy and safety of vinorelbine plus cisplatin (NP regimen) vs. gemcitabine plus cisplatin (GP regimen) for treatment of metastatic TNBC after failure with anthracyclines and taxanes. MATERIAL AND METHODS A total of 48 patients with metastatic TNBC that failed in anthracyclines and taxanes treatment were enrolled and randomly grouped. Patients in the NP group (n=22) were given 25 mg/m² vinorelbine on days 1 and 8 and 25 mg/m² cisplatin on days 2-4 of each 21-day cycle, while subjects in the GP group (n=26) were administered 1000 mg/m² gemcitabine on days 1 and 8 and 25 mg/m² cisplatin on days 2-4 of each 21-day cycle. The treatment response and adverse events were compared between the 2 groups every 2 cycles. RESULTS The ORR, DCR, and median TTP were 45.5%, 77.3%, and 5 months in the NP group, and 46.2%, 80.8%, and 5.2 months in the GP group, and no significant differences were observed in ORR, DCR, and median TTP between the 2 groups (P>0.05). The major adverse events included grade I-II bone marrow inhibition, gastrointestinal reactions, and phlebitis, and a lower incidence of thrombocytopenia and rash and a higher incidence of phlebitis was found in the NP group than in the GP group (P<0.05). CONCLUSIONS Either NP or GP regimen is active and tolerated in treatment of metastatic TNBC with anthracyclines and/or taxanes resistance, which may be used as a salvage treatment for metastatic TNBC.

[IL-1ß promotes the proliferation of astrocytes and downregulates the expression of Kir4.1].

Objective To explore the impact of IL-1ß on the proliferation of astrocytes and the expression of the inwardly rectifying potassium channel 4.1 (Kir4.1) in astrocytes. Methods Astrocytes were isolated from cerebral cortex of newborn SD rats and cultured in the presence of IL-1ß or IL-1ß combined with interleukin-1 receptor antagonist (IL-1Ra). The effect of IL-1ß on the cell cycle of astrocytes was measured with flow cytometry; the level of Kir4.1 mRNA was determined by quantitative real-time PCR. The level of Kir4.1 protein was detected by Western blotting. Results IL-1ß promoted astrocyte proliferation in a time- and dose-dependent manner. After astrocytes were treated with IL-1ß (10 ng/mL) for 24 hours, the levels of Kir4.1 mRNA and protein significantly decreased, but IL-1Ra significantly inhibited IL-1ß-induced decrease of Kir4.1. The downregulation of Kir4.1 mRNA and protein expressions could be partially reverted when IL-1ß was removed. Conclusion IL-1ß could promote the proliferation of cultured astrocytes, and IL-1ß may be a key factor influencing the expression of Kir4.1 in astrocytes.

[Glycyrrhizinic acid modulates the immunity of MRL/lpr mice and related mechanism].

Objective To investigate the immunological effect of glycyrrhizic acid (GA) on MRL/lpr mice and its underlying mechanism. Methods The research included 10 wild type C57BL/6 mice and 40 MRL/lpr mice. MRL/lpr mice were randomly assigned to four groups: MRL/lpr control, MRL/lpr treated with dexamethasone (1.5 mg/kg), MRL/lpr treated with GA (20 mg/kg) and GA (40 mg/kg), 10 mice in each group. The serum levels of interleukin 4 (IL-4) and interferon-γ (IFN-γ) were tested by ELISA. The ratio of Th1/Th2 cell subsets in spleen was analyzed by flow cytometry. The protein levels of GATA-binding protein 3 (GATA3), T-bet, p-JAK3, JAK3, phosphorylated signal transducer and activator of transcription 3 (p-STAT3), STAT3, p-NF-κBp65, NF-κBp65, p-IκBα, IκBα in spleen were detected by Western blot analysis. Results GA decreased serum levels of IL-4 and increased IFN-γ, modulated the balance of Th1/Th2 cell subsets in spleen markedly. GA inhibited the protein levels of GATA3, p-JAK3, p-STAT3, p-NF-κB, p-IκBα and increased T-bet protein in MRL/lpr mice. Conclusion Administration of GA can ameliorate the immune function in MRL/lpr mice.

[Distribution of postsynaptic density protein 95 (PSD95) and synaptophysin during neuronal maturation].

Objective To investigate the expressions of postsynaptic density protein 95 (PSD95) and synaptophysin (SYP) in primary culture of neurons at different times. Methods The expressions of PSD95 and SYP were detected using immunofluorescence technique in primary culture of cerebral cortical neurons at 3 days in vitro (3DIV), 7DIV and 14DIV. Results PSD95 was mainly distributed in the cell body at 3DIV, in the cell body, protuberance end and branch of neurons at 7DIV, and in the cell body as well as protuberance with a stippled pattern at 14DIV. The expression of SYP was not obvious at 3DIV, and it was seen mainly in nucleus at 7DIV, and in nucleus and protuberance showing a stippled pattern at 14DIV. Conclusion PSD95 and SYP are mainly located in the soma and nucleus of neurons in the early stage of neuron maturation, and finally mostly move to the protuberance with a stippled pattern, which indicates that PSD95 and SYP may originate from the different sites, but they are closely related to the formation and maturation of synapse.

[Lipopolysaccharide induces astrocyte activation and downregulates the expression of Kir4.1 channel].

OBJECTIVE: To explore the influence of lipopolysaccharide (LPS) on the activation of astrocytes and the expression of the inwardly rectifying K(+) channel Kir4.1. METHODS: Astrocytes were separated from the cerebral cortex of newborn SD rats and cultured in the presence of LPS or LPS combined with interleukin-1 receptor antagonist (IL-1ra). The cell vitality was detected by MTT assay; the expression of glial fibrillary acidic protein (GFAP) was analyzed by immunocytochemistry; the production of IL-1ß was tested by ELISA; the expression of Kir4.1 and IL-1ß mRNAs were determined by quantitative real-time PCR. RESULTS: LPS promoted the activation of astrocytes in a time- and dose-dependent manner. LPS significantly increased the production of IL-1ß and the expression of IL-1ß mRNA, while decreased the expression of Kir4.1 mRNA in cultured astrocytes. Compared with the LPS group, IL-1ra could effectively reversed the above two results. CONCLUSION: The cultured astrocytes could be activated by LPS; LPS-induced downregulation of Kir4.1 mRNA in cultured astrocytes might be related with the inflammatory cytokine IL-1ß.