Ratio Of Positive Cells Research Articles

Rapamycin Promote the Inhibition Rate of Proliferation on SHI-1 Cells and Enhance the Efficacy of Chemotherapy in Npg Leukemia Mice

Acute myeloid leukemia (AML) is a malignant hematologic disease. The remission rate of traditional chemotherapy is about 70-80%. In order to improve the efficacy of chemotherapy, some new drug which could enhance the efficacy of chemotherapy should be explored. Rapamycin is an immunosuppressive agent which can regulate cell proliferation, autophagy and other activities by inhibiting mTOR signal pathway. The studies of rapamycin as an anti-leukemia agent were limited. We had successfully establish a leukemia model in NPG mice with systemic leukemia infiltration using the human acute monocytic leukemia cell line SHI-1 cells. So in this study, the rapamycin and/or chemotherapy were used to treat the SHI-1 cells in vitro and NPG leukemia mice to investigate the efficacy of rapamycin in the treatment of AML. Rapamycin alone could effectively inhibit the proliferation of SHI-1 cells in vitro, and the inhibition rate were 35.4% which lower than the inhibition rate chemotherapy group (49.9%) using cytarabine and adriamycin. The combination with rapamycin and chemotherapy could significantly enhanced the inhibition rate to 60.3% (P<0.05) (Fig A). At day 19 after SHI-1 were inoculated to the NPG mice from tail vein, nearly 1% CD45 and CD33 positive cells were found in the peripheral blood. Then the NPG mice were divided to 4 groups to receive treatment. Control group without any treatment, rapamycin group were intraperitoneally injected with rapamycin (10mg/kg) every day for 5 days, chemotherapy group were intraperitoneally injected with cytarabine (100mg/kg for 5 days) and adriamycin (1mg/kg every other day for 3 times), combined group were treated with rapamycin, cytarabine and adriamycin. At the first day after treatment, one NPG mice were sacrificed randomly in four groups. The weight of the spleen in control group, rapamycin group, chemotherapy group and combination group were 78.6 mg, 48.9 mg, 16.1 mg, 11.7 mg respectively (Fig B). The ratio of human CD33 and CD45 positive cells in peripheral blood, bone marrow and spleen of NPG mice were 20.2%, 23.2% and 16.8% in control group, 2.02%, 2.68% and 3.31% in rapamycin group, 13.6%, 14.2% and 5.50% in chemotherapy group, 0.31%, 2.49% and 1.22% in combined group respectively (Fig C). Histopathological results showed that the degree of leukemia infiltration in the organs of NPG in the rapamycin group, chemotherapy group, and the combined group was significantly less than that of the control group. The median survival time of NPG mice in control group was 30.5 days. The treatment of rapamycin or chemotherapy could significantly prolong the media survival time of NPG mice to 35.0 and 34.0 days (P<0.05). When combined rapamycin with chemotherapy, the life time of NPG mice were significantly extended to 39 days (P<0.05) (Fig D). When NPG mice were died, more neoplasms were grow in the organs such as kidney, liver, stomach, mesenteric, mediastinum, bladder, spine, skin and neck in the NPG mice of control group, the treatment of rapamycin, chemotherapy and combined treatment would significantly decrease the counts of infiltrative organ with leukemia cells (Fig E). Altogether, our studies verified that rapamycin could inhibit the growth of SHI-1 cells and enhance the efficacy of chemotherapy in vitro. In NPG leukemia mice, the treatment of rapamycin alone could gain the efficacy as in chemotherapy group, when combined with chemotherapy, rapamycin had synergistic effect to significantly enhance the efficacy of chemotherapy to prolong the median survival time and decrease the degree of leukemia infiltration in NPG mice. DisclosuresNo relevant conflicts of interest to declare.

Effect of aspirin on breast cancer stem cells and stemness of breast cancer

Objective: To explore the effect of aspirin on the stemness of breast cancer cells and apoptosis induction of breast cancer stem cells. Methods: The 4T1 cells cultured with stem cell culture medium were screened, and immunofluorescence technique, flow cytometry and tumor-forming experiment in vivo were applied to test stem cell characteristics of the tumor spheres. After dealt with aspirin, the apoptosis rate of 4T1 stem cells was analyzed by flow cytometry. The 4T1 cells were cultured in vitro and treated with aspirin, then flow cytometry analysis was used to detect the expression of aldehydedehy drogenase1 (ALDH1), and the expression of stemness genes was tested by Western blot . Then, after culturing the cells with medium containing basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), B27 and N2, the ability of sphere-forming was observed and recorded by microscopy. In vivo BALB/c mice inoculated with 4T1 stem cells were randomly divided into the control group, 10 mg/kg, 30 mg/kg and 100 mg/kg aspirin groups. After 10 days, the mice were dealt with aspirin or NS for 15 days, then the tumor growth was observed and recorded. Results: The ratio of ALDH1 positive cells was up to 78.55%, and 4T1 tumor sphere had a postive expression of ALDH1 and sex determining region Y-box 2 (SOX2). In vivo tumorigenesis abilities of tumor sphere with 1×10(2) 4T1 stem cells could be 75%, while the ratio of normal cells was zero. The ratio of Aspirin-induced apoptosis of 4T1 stem cells at early stage and and late stage increased from 0.36% to 21.61%, and from 4.21% to 21.38%, respectively. Flow cytometry and Western blot assay results indicated that aspirin could reduce the expression of ALDH1, SOX2, octamer-binding transcription factor 4 (OCT4) and NANOG in 4T1 cells. Sphere-forming experiments results showed that aspirin could inhibit sphere forming ability of breast cancer cells. In vivo, aspirin inhibited the growth of tumors with a dose-dependent manner. Conclusion: Aspirin could induce apoptosis of cancer stem cells and reduce stemness of breast cancer, and thus play a growth-inhibiting action on breast cancer.

Micro-osteoperforations accelerate orthodontic tooth movement by stimulating periodontal ligament cell cycles.

The aim of this study was to investigate the mechanism of how micro-osteoperforations (MOPs) accelerate tooth movement. We focused on inflammation, cell proliferation, and apoptosis ofperiodontal ligament cells and performed immunostaining of MOPs exposed to tumor necrosis factor-alpha (TNF-α), proliferating cell nuclear antigen (PCNA), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) during experimental tooth movement. Eleven-week-old male Wistar rats were divided into 2 groups: (1) 10g of orthodontic force applied to the maxillary first molar (TM) and (2) force application plus 3 small perforations of the cortical plate (TM+MOPs). On days 1, 4, 7, 10, and 14 after force application, we investigated tooth movement and alveolar bone microstructure using microcomputed tomography (n=5). We also determined the expression of TNF-α and PCNA in the pressure sides of periodontal ligaments via an immunohistochemical analysis. The expression of apoptotic cells was also determined by the TUNEL method. The tooth movement in the TM+MOPs group was significantly greater on days 4 to 14 than in the TM group. The TM+MOPs group showed statistically significant decreases in bone volume/tissue volume ratio and bone mineral density compared with the TM group. The ratios of TNF-α positive cells in the TM+MOPs group were increased on days 1, 4. 7, and 10 compared with the TM group. The ratios of PCNA positive cells in the TM+MOPs group were increased on days 1, 4, and 7 compared with the TM group, and the ratios of TUNEL positive cells in the TM+MOPs group were increased on days 1 and 7 compared with the TM group. These results suggest that MOPs may accelerate tooth movement through activation of cell proliferation and apoptosis of periodontal ligament cells.

Clinical analysis of IgG4-related lung disease

Objective: To improve the understanding and treatment of IgG4-related lung disease (IgG4-RLD) by analyzing the clinical characteristics of patients. Methods: A total of 13 patients with IgG4-related lung disease (IgG4-RLD) diagnosed by pathology at Zhongshan Hospital affiliated to Fudan University during December 2007 to December 2017 were included. The clinical characteristics, chest CT, pathological features, serum IgG4 levels, therapy and prognosis of these 13 patients were analyzed. Results: The 13 patients with IgG4-RLD included 8 men and 5 women, with an average age of (51.1±14.8) years. The clinical manifestations were varied, 10 patients showed respiratory symptoms, mainly including cough (n=9), expectoration (n=6), hemoptysis (n=6), chest tightness (n=2) and breathless (n=2). Multiple organs were involved in 12 cases. Chest CT showed solid lung nodules (n=9), ground glass shadows (n=1), alveolar-interstitial infiltration (n=3), bronchovascular lesions (n=3), and the solid lung nodules was predominant. Enlargement of lymph nodes in the mediastinal and bilateral hilum of the lung were present in 10 cases. Serum IgG4 levels were elevated in all the 13 cases, with an average concentration of (7.92 ±13.98)g/L. Quite amount of lymphocytes, plasma cell infiltration and fibrosis were common pathologic findings. Immunohistochemical staining showed a large number of IgG4 positive plasma cells infiltration with IgG4 positive plasma cells count 10-320/HP in 13 cases. The ratio of IgG4 positive plasma cells to IgG positive plasma cells was higher than 40%. Eight patients received glucocorticoid therapy alone, five received glucocorticoids with immunosuppressant therapy. After treatment, 10 patients were with remission, and disease progressed in 3 patients, but no death. Conclusions: The clinical manifestations and imaging features of IgG4-RLD are diverse, and lesions can often involve multiple organs. The diagnosis could be made based on pathological features and IgG4 serum levels. Glucocorticoid can be used as the first choice for the treatment of IgG4-RLD.

NEAT1 contributes to the CSC-like traits of A549/CDDP cells via activating Wnt signaling pathway

Long non-coding RNAs (lncRNAs) have been identified to exert crucial roles in tumorigenesis and can serve as novel biomarkers for cancer therapy including lung cancer. Cisplatin is a first-line chemotherapeutic agent in non-small cell lung cancer (NSCLC), but the therapeutic effect is unsatisfactory, partly due to drug resistance. Emerging evidence showed that chemo-resistance is associated with acquisition of cancer stem cell (CSC)-like properties. Cisplatin resistance remains a major obstacle in the treatment of lung cancer, and its mechanism is still not fully elucidated. Meanwhile, CSCs have been involved in tumor metastasis, tumor recurrence and chemotherapy resistance. So far, the mechanism of nuclear enriched abundant transcript 1 (NEAT1) in modulating CSCs in lung cancer remains barely known. Therefore, we aimed to explore the correlation between NEAT1 and cancer stem cells in lung cancer. In our current study, we observed that CSC-like traits were much more enriched in cisplatin-resistant A549/CDDP cells. In addition, NEAT1 was obviously up-regulated in A549/CDDP cells compared with parental A549 cells. Knockdown of NEAT1 decreased the CSC-like properties of A549/CDDP cells through inhibiting tumor cell sphere volume, repressing CSC-like biomarkers levels and restraining CD44 positive cell ratios. Oppositely, overexpression of NEAT1 enhanced the stemness respectively. Moreover, it has been reported that Wnt pathway is implicated in many vital cellular functions including cancer stem cells. Here, it was exhibited that Wnt signal pathway was inactivated by knockdown of NEAT1 whereas activated by NEAT1 overexpression in A549/CDDP cells. Taken these together, it was indicated that NEAT1 could exert a novel biological role in NSCLC chemo-resistance.

Regenerative effect of microcurrent according to intensity on calf muscle atrophy in immobilised rabbit

The intensity of microcurrent therapy is important in the treatment of muscle damage. In one study, when an intensity of 100–500 μA was applied after muscle damage, the healing process, including amino acid transport, triphosphate generation, and protein synthesis, increased by 30–40% above the control level. On the contrary, when the intensity exceeded 1000 μA, these biostimulatory effects were reversed. Therefore, we aims to investigate the regenerative effect of microcurrent electric stimulation according to intensity on gastrocnemius (GCM) muscle atrophy in rabbits induced by cast immobilization Fifteen male New Zealand white rabbits were randomly allocated into 3 groups of 5 rabbits. Right GCM muscle was used for immobilization by cast for 2 weeks (IC). IC (group 1), IC and Microcurrent stimulation (MS: 25 μA) for 2 weeks after CR (group 2), and IC and Microcurrent stimulation (MS: 5000 μA) for 2 weeks after CR (group 3). Atrophic change of Rt. calf circumference, Compound muscle action potential (CMAP) of Rt. tibial nerve, thickness of Rt. GCM by ultrasound was calculated. Muscle composition of GCM muscle and cross sectional area (CSA) of muscle fibers was measured. Proliferating cell nuclear antigen (PCNA) and bromodeoxyuridine (BrdU) positive cell ratio was calculated as the number of BrdU positive cells per muscle fiber. Mean atrophic changes of Rt. calf circumference, amplitude of CMAP on Rt. tibial nerve, and Rt. medial and lateral GCM muscle thickness in group 2 was significantly lesser than those in group 1 and 3 ( P < .05). Mean CSA of medial GCM type 1 muscle fibers and PCNA and BrdU ratio in group 2 were significantly greater than those in group 1 and group 3, respectively ( P < .05). The result shows that a low-intensity microcurrent promotes more effectively than high-intensity in regeneration of GCM type 1 muscle atrophy.

Nicotine enhances mesangial cell proliferation and fibronectin production in high glucose milieu via activation of Wnt/β-catenin pathway.

Diabetic nephropathy (DN) is a major complication of diabetes mellitus. Clinic reports indicate cigarette smoking is an independent risk factor for chronic kidney disease including DN; however, the underlying molecular mechanisms are not clear. Recent studies have demonstrated that nicotine, one of the active compounds in cigarette smoke, contributes to the pathogenesis of the cigarette smoking-accelerated chronic kidney disease. One of the characteristics of DN is the expansion of mesangium, a precursor of glomerular sclerosis. In the present study, we examined the involvement of Wnt/β-catenin pathway in nicotine-mediated mesangial cell growth in high glucose milieu. Primary human renal mesangial cells were treated with nicotine in the presence of normal (5 mM) or high glucose (30 mM) followed by evaluation for cell growth. In the presence of normal glucose, nicotine increased both the total cell numbers and Ki-67 positive cell ratio, indicating that nicotine stimulated mesangial cell proliferation. Although high glucose itself also stimulated mesangial cell proliferation, nicotine further enhanced the mitogenic effect of high glucose. Similarly, nicotine increased the expression of Wnts, β-catenin, and fibronectin in normal glucose medium, but further increased mesangial cell expression of these proteins in high glucose milieu. Pharmacological inhibition or genetic knockdown of β-catenin activity or expression with specific inhibitor FH535 or siRNA significantly impaired the nicotine/glucose-stimulated cell proliferation and fibronectin production. We conclude that nicotine may enhance renal mesangial cell proliferation and fibronectin production under high glucose milieus partly through activating Wnt/β-catenin pathway. Our study provides insight into molecular mechanisms involved in DN.

Vitamin D3 regulates mononuclear phagocyte polarization induced by serum from patients with ankylosing spondylitis

To investigate the effect of vitamin D3 on polarization of monocyte macrophages induced by serum from patients with ankylosing spondylitis (AS). Twenty AS naïve patients and 20 healthy controls from Wenzhou People's Hospital during January 2016 and December 2017 were enrolled. The macrophages were differentiated from THP1 cells induced by phorbol 12-myristate 13-acetate (PMA), and then co-cultured with the serum from healthy subjects (control group) or AS patients. Vitamin D3 was added in the medium mixed with serum from AS patients. Flow cytometry was used to analyze the ratio of CD68 and CD206 positive cells, and RT-PCR was performed to detect the mRNA expression of inducible nitric oxide synthase(iNOS) and arginase-1(Arg-1). THP1 cells could be polarized into mononuclear-macrophages with the induction of PMA. The proportion of CD206 positive cells in AS-serum group was lower than that in the control group (t=9.434, P<0.05), while the proportion of CD68 positive cells was higher than that in the control group (t=43.920, P<0.05). The proportion of CD206 positive cells in vitamin D3 group was higher than that in AS-serum group (t=8.895, P<0.05), while the proportion of CD68 positive cells was lower than that in AS-serum group (t=9.089, P<0.05). mRNA expression of Arg-1 in AS-serum group was lower than that in the control group (t=8.899, P<0.05), while mRNA expression of iNOS was higher than that in the control group (t=3.656, P<0.05). mRNA expression of Arg-1 in vitamin D3 group was higher than that in AS-serum group (t=6.219, P<0.05), while mRNA expression of iNOS was lower than that in AS-serum group (t=5.876, P<0.05). Vitamin D3 can regulate the polarization of mononuclear macrophages for immunoregulation in patients with AS.

The experimental study on endoplasmic reticulum stress-participated outer hair cell apoptosis in cadherin 23 gene mutant mice

Objective: To test the mechanism and upstream pathway of outer hair cell apoptosis in Cadherin 23 (Cdh23) gene mutant mice. Method: The mutant Cdh23(erl/erl)(erl) mice were collected as the study group, while the C57BL/6J (B6) mice were chosen as the control group. A total of 70 mice per group were used in this study. The study group and control group underwent auditory-evoked brainstem response (ABR) tests at the same age. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to detect outer hair cell(OHC) apoptosis. The qRT-PCR was conducted to test the expression of ER stress markers immunoglobulin-binding protein (BiP) and C/EBP homologous protein (CHOP) mRNA. The expression and location of BiP and CHOP protein in OHC were detected by immunostaining. The expression of BiP protein in cochleae was identified by Western blot. The expression and location of CDH23 protein in OHC were discovered by immunostaining. Results: The ABR thresholds in erl mice were significantly higher than those in B6 mice at the age of 1 and 3 months (both P<0.05). The surface preparation with TUNEL staining confirmed OHC apoptosis in erl mouse cochleae which showed a higher TUNEL positive cell ratio than B6 mouse(t=11.291, P<0.01). The ER stress marker Bip and Chop mRNA were upregulated in the erl mouse inner ear, when compared with those in the B6 mouse(both P<0.05). The BiP protein extracted from the erl mouse cochleae was significantly higher than that of B6 mouse measured by Western blot (t=3.66, P=0.02). Immunostaining showed that BiP and CHOP were highly detected in the OHC in erl mouse cochleae, and was mainly detected in the perinuclear region of OHC. However, a bare BiP and CHOP signal were shown in B6 mouse cochleae. The CDH23 protein was specifically localized at the top of the OHC in B6 mice, indicating the localization of the tip links in hair bundle stereocilia. On the contrary, the CDH23(erl) protein was found to be localized from the top to the nuclei of the OHC in erl mice. Portions of the CDH23(erl) proteins failed to reach the top of the hair bundles and remained in the OHC cytoplasm. Conclusion: As the downstream response of the Cdh23 gene mutation, portions of the mutant CDH23(erl) protein was accumulated in ER lumen resulting in the increase of ER loading and ultimately triggered ER stress and hair cell apoptosis in erl mouse cochleae.

Prolyl isomerase Pin1 is required sperm production by promoting mitosis progression of spermatogonial stem cells

A prolyl isomerase Pin1 deficient (Pin1−/−) male mice had severe testicular atrophy. We investigated the function of Pin1 in spermatogenesis by analyzing the Pin1−/− mice at reproductive age. Pin1−/− mice had lessαPLZF positive spermatogonia (undifferentiated spermatogonia) than wild type (WT). Nevertheless, the Pin1−/− testis contained approximately the same number of GFRα1 positive spermatogonia (SSCs in steady state) as the WT testis. Furthermore, degeneration of the spermatogenia appeared in seminiferous tubules of 10 months old Pin1−/− mouse testis, and abnormal shape GFRα1 positive spermatogonia were observed. In Pin1−/− spermatogonia, the ratio of the phospho-histone H3 positive cells (mitotic cells) in GFRα1-positive spermatogonia was higher than that of WT. These results suggest that Pin1 promotes the progression of the mitotic cell cycle of SSC in steady-state, which is required for the sperm production from SSCs.

MiR-143 suppresses the osteogenic differentiation of dental pulp stem cells by inactivation of NF-κB signaling pathway via targeting TNF-α

BackgroundDental pulp stem cells (DPSCs) are multipotent and play an important role in repairing damaged and/or defective dentinogenesis/osteogenesis. Recent studies have documented the implication of miR-143 in osteogenic differentiation of DPSCs. Nevertheless, the detailed mechanisms of miR-143 involved in the osteogenic differentiation of DPSCs remain to be further elaborated. MethodsIsolated DPSCs were incubated with osteogenic differentiation medium to induce osteogenic differentiation. qRT-PCR and western blot were performed to determine the expressions of miR-143 and tumor necrosis factor α (TNF-α). Luciferase reporter assay was used to confirm whether TNF-α was a target of miR-143. Osteogenic differentiation of DPSCs was evaluated by alkaline phosphatase (ALP) activity assay, ALP staining, and western blot analyses of osteogenic-markers including bone morphogenetic protein 2 (BMP2), ALP, runt-related transcription factor 2 (RUNX2) and collagen type I (COLI). ResultsmiR-143 was downregulated and TNF-α was upregulated during osteogenic differentiation of DPSCs. miR-143 posttranscriptionally regulated TNF-α expression in DPSCs by binding to its 3′UTR. miR-143 overexpression suppressed osteogenic differentiation of DPSCs, as demonstrated by the decrease of ALP activity, ALP positive cell ratio, as well as BMP2, ALP, RUNX2, and COLI expressions. Moreover, miR-143 reversed TNF-α-induced osteogenic differentiation of DPSCs. Finally, the osteogenic differentiation of DPSCs induced by miR-143 inhibitor was attenuated following inactivation of nuclear factor kappa B (NF-κB) signaling pathway. ConclusionmiR-143 suppressed the osteogenic differentiation of DPSCs by blockade of NF-κB signaling pathway via targeting TNF-α.

Expression of CGRP neurotransmitter and vascular genesis marker mRNA is age-dependent in superior cervical ganglia of senescence-accelerated prone mice.

Calcitonin gene-related peptide (CGRP) is a neurotransmitter that is released from the superior cervical ganglion (SCG) and causes head and neck pain. The morphological properties of human SCG neurons, including neurotransmitter content, are altered during aging. However, morphological changes in CGRP in the SCG during aging are not known. Therefore, we investigated CGRP and other markers in the SCG during aging in an aging model of senescence-accelerated prone mouse (SAMP8) and senescence-accelerated resistant mice (SAMR1) using real-time RT-PCR mRNA analyses and in situ hybridization. The abundance of neurotransmitter (CGRP, NPY, TRPV1), vascular genesis marker (CD31, LYVE-1), and cytochrome C mRNA differed between 12-week-old and 24-week-old SAMP8 and SAMR1. Abundance of TRPV1, CD31 and cytochrome C mRNAs of SAMP8 decreased between 12- and 24-week-old. The ratio of CGRP mRNA positive cells and CGRP mRNA abundance levels of the SCG of aging mouse such as SAMP8 have already been also higher than that of SAMR1 at 12-week-old. The CGRP positive shrunken ganglion cells was increased from 12- to 24-weeks-old mouse in SAMR1 and SAMP8. The SCG primarily affected the internal and external carotid arteries, larynx thyroid gland, and pharyngeal muscle during aging.

Effects of cyclin D1 gene silencing on cell proliferation, cell cycle, and apoptosis of hepatocellular carcinoma cells.

This study aims to investigate the effects of Cyclin D1 silencing on cell cycle, cell proliferation, and apoptosis of hepatocellular carcinoma cells (HCC). Cells were divided into the blank group, negative control group (HCC cells transfected with control shRNA), Cyclin D1 shRNA group (HCC cells transfected with Cyclin D1 shRNA), and the normal group (human normal liver L-02 cells). Expressions of Cyclin D1, Caspase-3, Bcl-2, and C-myc were detected by RT-qPCR and Western blotting. Cell proliferation was detected by Cell Counting Kit-8. Cell cycle and apoptosis were detected by flow cytometry. Tumor xenograft in nude mice was performed to detect in vivo tumorigenesis. HCC tissues and HCC cells exhibited elevated expression levels of Cyclin D1. Cyclin D1 expression levels was found to be correlated with tumor size and tumor staging. Compared with the normal group, the blank group showed enhanced cell proliferation, a reduction in the amount of cells in G0/G1 phase, increased number cells in S and G2/M phase, reduced apoptosis, elevated expressions of Cyclin D1, Bcl-2, and C-myc, decreased Caspase-3 activity and significant tumorigenicity. In comparison with the blank group, the Cyclin D1 shRNA group revealed weakened cell proliferation, reduced cells in S and G2/M phase, increased cells in G0/G1 phase, increased Annexin V positive cell ratio, decreased expression of Cyclin D1, Bcl-2, and C-myc, elevated Caspase-3 activity and inhibited tumorigenicity. In conclusion, Cyclin D1 gene silencing suppresses cell proliferation and inhibits cell apoptosis, which may be a new target approach in the treatment and management for HCC.

Relevance of sample preparation for flow cytometry.

Flow cytometry is a useful tool for diagnosis and minimal residual disease (MRD) study of hematological diseases. Standard sample preparation protocols are characterized by stain-lyse-wash (SLW). To prevent nonspecific bindings and achieve high sensitivity in MRD studies, lyse-wash-stain-wash (LWSW) is required. To our knowledge, no comparison between the two methods has been performed. We compared mean fluorescence intensity (MFI), stain index, signal-to-noise ratio, and percentage of positive cells of 104 antibodies and of 13 selected antibodies tested in 10 samples simultaneously prepared with the two methods. MFI and percentages of positive cells obtained by the two methods did not show significant differences and showed a very high correlation. Stain index and signal-to-noise ratio presented higher values for kappa and lambda surface chains in LWSW samples and a trend of higher values for the other antibodies in SLW samples. We suggest to use LWSW method also at diagnosis to obtain more comparable antibody intensity expressions when samples from the same patient are processed for MRD evaluation after bulk lysis. Moreover, LWSW can prevent nonspecific bindings, shows no differences in the identification and quantitation of the populations of interest, and reduces acquisition of cell debris.

JNK inhibitor alleviates apoptosis of fetal neural stem cells induced by emulsified isoflurane.

Isoflurane can provide both neuroprotection and neurotoxicity in various culture models and in rodent developing brains. Emulsified Isoflurane (EI) is an emulsion formulation of isoflurane, while its underlying molecular mechanism of developemental nerve toxicity largely remains unclear. We hypothesized that EI induced fetal neural stem cells (FNSCs) apoptosis, endoplasmic reticulum (ER) stress and c-Jun N-terminal kinase (JNK) activation.FNSCs were isolated from the cortex of SD rats during 14 days of gestation. The cell viability, cell apoptotic rates and the expression of apoptosis-related protein Caspase3, inositol requiring enzyme 1 (IRE1), poly (adenosine diphosphate-ribose) polymerase (PARP), Bax, Bcl-2, JNK, p-JNK and XBP1 were determined. Specific inhibition was performed by siRNA-targeting of JNK in FNSCs. EI could increase the p-JNK, JNK and caspase3 protein expression, the JNK pathway was activated by EI, and EI-induced apoptosis was blocked by inhibiting JNK pathway with SP600125 or JNK-small interfering RNA (siRNA), EI enhanced the level of IRE1, PARP, Bax/Bcl-2 and XBP1, which led FNSCs to apoptosis and ER stress. Meanwhile, dilatation of the ER lumens in FNSCs treated by EI for 24 h was significant. Green fluorescent protein (GFP) positive cell ratios were significantly decreased by FNSCs transfecting with JNK gene silencing. JNK was efficiently silenced in siRNA-JNK1 group.The results provided in-vitro evidence which supports that the underlying mechanisms of EI-induced apoptosis are the induction of ER stress and sequent JNK activation. Together, these data suggest that JNK inhibiting might be applied for improving therapeutic outcomes in anesthestics-induced neurotoxicity.Highlights:1. Prolonged treatment with high-dose EI decreased the survival level of FNSCs by inducing apoptosis and inhibiting proliferation via the JNK signaling pathway.2. EI induced ER stress and sequent JNK activation.3. JNK inhibiting might be applied for improving therapeutic outcomes in anesthestics-induced neurotoxicity

Low-dose DHA-induced astrocyte proliferation can be attenuated by insufficient expression of BLBP in vitro

Docosahexaenoic acid (DHA) is an n-3 long chain polyunsaturated fatty acid (PUFA) that is involved in a wide range of cellular processes in human cells. Brain lipid binding protein (BLBP) exhibits a high affinity for n-3 PUFAs, especially DHA, but the precise functional contributions of DHA and BLBP in astrocytes are not clear. We analyzed cell viability and the ratio of Ki67 positive cells after manipulating DHA and/or BLBP levels in cultured astrocytes, and found that low-dose DHA stimulated proliferation of astrocytes, whereas this proliferative effect could be attenuated by downregulation of BLBP. Moreover, we found that astrocyte proliferation was directly regulated by BLBP independently of DHA. Taken together, low-dose DHA-induced astrocyte proliferation was disturbed by insufficient BLBP; and besides acting as a fatty acid transporter, BLBP was also involved in the proliferation of astrocytes directly.

Re-expression of pro-fibrotic, embryonic preserved mediators in irradiated arterial vessels of the head and neck region.

Surgical treatment of head and neck malignancies frequently includes microvascular free tissue transfer. Preoperative radiotherapy increases postoperative fibrosis-related complications up to transplant loss. Fibrogenesis is associated with re-expression of embryonic preserved tissue developmental mediators: osteopontin (OPN), regulated by sex-determining region Y‑box9 (Sox9), and homeoboxA9 (HoxA9) play important roles in pathologic tissue remodeling and are upregulated in atherosclerotic vascular lesions; dickkopf-1 (DKK1) inhibits pro-fibrotic and atherogenic Wnt signaling. We evaluated the influence of irradiation on expression of these mediators in arteries of the head and neck region. DKK1, HoxA9, OPN, and Sox9 expression was examined immunohistochemically in 24irradiated and 24nonirradiated arteries of the lower head and neck region. The ratio of positive cells to total cell number (labeling index) in the investigated vessel walls was assessed semiquantitatively. DKK1 expression was significantly decreased, whereas HoxA9, OPN, and Sox9 expression were significantly increased in irradiated compared to nonirradiated arterial vessels. Preoperative radiotherapy induces re-expression of embryonic preserved mediators in arterial vessels and may thus contribute to enhanced activation of pro-fibrotic downstream signaling leading to media hypertrophy and intima degeneration comparable to fibrotic development steps in atherosclerosis. These histopathological changes may be promoted by HoxA9-, OPN-, and Sox9-related inflammation and vascular remodeling, supported by downregulation of anti-fibrotic DKK1. Future pharmaceutical strategies targeting these vessel alterations, e. g., bisphosphonates, might reduce postoperative complications in free tissue transfer.

Abstract 51: Effects of Human Immunodeficiency Virus-1 gp120 Viral Proteins on Endothelial Senescence and microRNA Expression

Human immunodeficiency virus (HIV)-1 is associated with profound endothelial cell damage and dysfunction. HIV-1 envelope glycoprotein 120 (gp120) is a toxic viral protein released from infected cells and promotes endothelial cell dysfunction. Endothelial cell senescence is a key factor underlying endothelial dysfunction and, in turn, vascular disease risk. It is now clear that microRNAs (miRs) play a pivotal role in regulating cellular senescence. The aim of this study was to determine whether gp120 induces endothelial cell senescence; and if so, whether the cellular expression of senescence-related miRs (mir-34a, -146a and -217) are adversely affected by gp120. Cultured HAECs (3 rd passage) were plated at a density of 5.0x10 5 cells/condition. Cells were treated with media alone or media containing gp120: either lav gp120 (X4; 100ng/mL) or Bal gp120 (R5; 100ng/mL) for 24 h. Thereafter, cells were stained for senescence-associated β-galactosidase (β-gal) for 14 h. Cells from 5-7 random microscope fields were counted and (%) senescence was calculated as the ratio of β-gal positive cells to total cells. To determine miR expression, RNA was extracted from 1.0x10 5 cells and was quantified using RT-PCR. Cellular expression was normalized to RNU6 and calculated as fold change in ΔΔCt from control (N=4, experimental units). Both X4 (32.0±0.7% vs 18.3±1.7%) and R5 (30.2±3.8% vs 18.3±1.7%) significantly increased cellular senescence vs control. Moreover, the magnitude of increase (~65%) in senescent cells was similar between the glycoproteins. Cellular expression of pro-senescence miR-34a (1.60±0.04 fold) was increased (~60%; P&lt;0.05) in response to X4 treatment compared with control. However, expression of miR-146a (1.34±0.36 fold) and miR-217 (1.38±0.36 fold) was not significantly affected by X4. In response to R5, cellular expression of miR-34a (1.23±0.07 fold) was significantly increased (~23%) and miR-146a (1.23±0.07 fold) was significantly decreased (~80%); miR-217 was not influenced by R5 (1.05±0.13 fold). These data indicate that HIV-1 viral protein gp120 induces endothelial cell senescence and adversely affects cellular expression of senescence-associated miRs. miR dysregulation may underlie the pro-senescent effects of gp120.

Expressions of annexins and epidermal growth factor receptor in pediatric middle ear cholesteatoma

Objective To explore the expressions of annexin and epidermal growth factor receptor(EGFR) in pediatric middle ear cholesteatoma. Methods Twenty-three children with middle ear cholesteatoma and 26 children with normal skin of external auditory canal(control group) were selected from the children enrolled in the First Affiliated Hospital of Zhengzhou University from August 2014 to March 2016.The expressions of annexin A1 (AnxA1), AnxA2 and EGFR mRNA in cholesteatoma and normal tissues were analyzed by quantitative real-time PCR (qPCR). Protein expressions of AnxA1, AnxA2 and EGFR were evaluated by using Western blot and immunohistochemistry methods. Results The expressions of AnxA1, AnxA2 and EGFR mRNA were significantly increased in cholesteatoma compared with the control group(AnxA1: 4.68±1.77 vs.2.65±0.96, U=111.5, P<0.001; AnxA2: 3.89±1.00 vs.2.47±0.81, U=84.5, P<0.001; EGFR: 4.97±1.85 vs.3.50±0.95, U=153.5, P=0.004). AnxA1 and AnxA2 mRNA expressions were positively correlated with EGFR mRNA (AnxA1 and EGFR: r2=0.283 2, P=0.009; AnxA2 and EGFR: r2=0.213 5, P=0.027). Compared with the control group, protein expressions of AnxA1, AnxA2 and EGFR were markedly enhanced (AnxA1: 0.450±0.031 vs.0.320±0.026, U=102.4, P<0.001; AnxA2: 0.568±0.024 vs.0.365±0.028, U=94.6, P<0.001; EGFR: 0.397±0.021 vs.0.228±0.017, U=128.4, P<0.001). The results of immunohistochemistry analysis showed that the ratio of AnxA1, AnxA2 and EGFR positive cells were higher than those in the control group(AnxA1: 65.22% vs.38.46%, χ2=9.296, P=0.026; AnxA2: 69.57% vs.46.15%, χ2=8.378, P=0.039; EGFR: 69.57% vs.50.00%, χ2=10.574, P=0.014). Conclusions The expressions of AnxA1, AnxA2 and EGFR are upregulated in pediatric cholesteatoma, with AnxA1 and AnxA2 expressions positively correlated with EGFR. Key words: Annexin A1; Annexin A2; Epidermal growth factor receptor; Cholesteatoma; Child

Expression and Asymmetric Division of Numb in Leukemia Cell K562 Line

To investigate the role of asymmetric division in leukemia cells through detection of expression and asymmetric division of Numb in differentiated and undifferentiated K562 cells. Firstly, Hemin was used to induce K562 cell differentiation, and the expression of Numb was detected by the real-time quantitative RT-PCR and flow cytometry. After K562 cells were synchronized by nocodazole, the Numb protein was labeled by immunohistochemical staining, followed by the determination of the terminally differentiated cells through confocal microscopy. The fluorescence intensity was calculated by Image J software, and the cell division pattern was analyzed on the basis of the fluorescence intensities of Numb in 2 divided daughter cells. Compared with the undifferentiated K562 cells, the level of Numb mRNA expression increased 2.3 times (P<0.001). The ratio of Numb positive cells was(67.37±5.01)% in differentiated K562 cells, while that was (43.97±5.72)% in undifferentiated K562 cells (P<0.01). Compared with undifferentiated K562 cells, the ratio of cells with asymmetric division in differentiated K562 cells increased 18.3%, the percentage of cells with symmetry self-renewal reduced 49.7%(P<0.001) and that with symmetry differentiation increased 32%(P<0.001). In differentiated K562 cells, expression of Numb and proportion of cells with asymmetric division were higher than that in undifferentiated cells. With the differentiation of leukemia cells, the proportion of cells with asymmetrical division increases, and the proportion of cells with symmetrical self-renewal decreases. The stemness of leukemia cells is maintained mainly through the symmetrical self-renewal.