ObjectiveTo observe the protective effect of dl-3-n-Butylphthalide (NBP) on apoptosis of retinal Müller cells induced by hydrogen peroxide (H2O2).MethodsHuman retinal Müller cells cultured in vitro were divided into normal control group, model group (H2O2 group) and experimental group (H2O2+NBP group). The cells in the H2O2 group and H2O2+NBP group were cultured with 200 μmol/L H2O2 for 2 h. Then the culture solution of the H2O2 group replace with complete medium and the H2O2+NBP group replace with complete medium containing 1 μmol/L NBP. The normal control group was a conventional cultured cells. Müller cells were identified by immunofluorescence staining. Hematoxylin-eosin (HE) staining was used to observe the apoptosis morphological changes. MTT assay was used to detect the activity of of retinal Müller cells after after 24 h and 48 h of NBP intervention. Hoechst33258 staining was used to observe the apoptosis. LIVE/DEAD ® cell activity/cytotoxicity kit was used to detect cell viability. Dichlorofluorescein diacetate (DCFH-DA) + endoplasmic reticulum (ER) red fluorescent probe (ER-Tracker Red) double staining was used to observe the expression level of reactive oxygen species (ROS) in ER of cells. One-way ANOVA combined with Dunnett statistical method were used for data analysis.ResultsHE staining showed that the number of cells in H2O2+NBP group was higher than that in H2O2 group. MTT assay showed that after 24 h and 48 h of NBP intervention, the differences in cell viability between the normal control group and the H2O2 group, the H2O2 group and the H2O2+NBP group were statistically significant (t=28.96, 3.658, 47.58, 20.33; P＜0.001, 0.022). The results of Hoechst33258 showed that the nuclear nucleus of a few cells in the H2O2+NBP group was crescent-shaped and the nuclear fragmentation was reduced, and the blue fluorescence of the remaining cells was uniform. The LIVE/DEAD ® cell activity/cytotoxicity kit showed that the number of dead cells with red fluorescence in the H2O2 group increased significantly, and the number of viable cells with green fluorescence decreased significantly. In the H2O2+NBP group, the number of viable cells with green fluorescence increased, and the number of dead cells with red fluorescence decreased. The double staining results of DCFH-DA+ER-Tracker Red showed that the green fluorescence intensity of H2O2 group was significantly enhanced; the green fluorescence intensity of H2O2+NBP group was lower than that of H2O2 group.ConclusionNBP alleviates H2O2-induced apoptosis of human retinal Müller cells by inhibiting ROS production.
ObjectiveTo observe the effect of polypyramidine tract binding protein-associated splicing factor (PSF) towards advanced glycation end products (AGEs) induced the apoptosis of Müller cells in vitro.MethodsExperimental study. Müller cells were cultured and divided into groups according to the project design, plasmid enhanced green fluorescent protein-PSF were transfected into the cells to achieve the overexpression of PSF Müller cells in vitro, then cells were exposed to AGEs and the Morphological changes were observed by HE staining and Hoechst 33258 staining while the survival rate of cells were detected by MTT assay. The effects of PSF on AGEs-induced Müller apoptosis was measured by Cell Death Detection ELISA kit. Meanwhile, 2′,7′-dichlorofluorescin diacetate staining was performed to monitor the protective effects of PSF on AGEs-induced Müller cells ROS.ResultsThe morphology of cells in normal group was full and the cytoplasm staining was uniform. In N+AGEs group and Vec+AGEs group, cell volume decreased, cytoplasm was dense and concentrated, and eosinophilic staining was enhanced. The cell morphology of PSF+AGEs group was still full, with uniform cytoplasm staining and uniform nucleus staining. The viability of N+AGEs group, Vec+AGEs group and PSF+AGEs group were 0.42±0.11, 0.35±0.12 and 0.68±0.12. The apoptosis values were 1.08±0.16, 0.96±0.20 and 0.44±0.08. The intracellular ROS levels were 28 833.67±3 550.06, 28 356.67±4 854.81, 186 163.00±382.54. Compared with N+AGEs group and Vec+AGEs group, the cell viability of PSF+AGEs group was significantly improved (F=20.65, P=0.000), cell apoptosis value (F=43.43, P=0.000) and intracellular ROS level (F=18.86, P=0.000).ConclusionPSF overexpression play a protective role in AGEs-induced apoptosis by inhibiting the production of ROS in Müller cells.
ObjectiveTo investigate the influence of programmed cell death protein-1 (PD-1) monoclonal antibody on the anti-lung cancer effect of cytokine-induced killer cells (CIK) which were programmed in vitro. MethodsPeripheral blood mononuclear cells from 20 patients (8 males and 12 females with an average age of 56.45±5.89 years ranging from 42 to 65 years) diagnosed with advanced lung cancer from January to May 2019 at the Department of Oncology of Dalian Central Hospital were collected and induced to amplify into CIK cells in vitro. PD-1 monoclonal antibody combined with CIK cell culture group, individual cell culture group and PD-1 monoclonal antibody group were set up to detect the cell killing activity of CIK cells against lung cancer under different effective target ratio conditions, and the ratio of perforin and granzyme positive expression in PD-1 monoclonal antibody combined CIK cell culture group and individual CIK cell culture group was detected by flow cytometry. ELISA method was used to detect the interleukin-2 (IL-2), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) cytokine secretion levels in the two groups.ResultsThe killing effect of CIK cells on A549 lung cancer cells increased with the increase of effective target ratio by CCK8, and PD-1 monoclonal antibody increased the killing effect of CIK cells on A549 lung cancer cells under different effective target ratio, E∶T=5∶1 (28.5%±1.9% vs. 20.3%±1.8%), 10∶1 (40.6%±2.4% vs. 31.7%±2.1%), 20∶1 (57.4%±3.5% vs. 44.7%±3.8%), 40∶1 (74.1%±8.3% vs. 60.8%±5.3%). The killing effect of PD-1 monoclonal antibody combined with CIK cells and CIK cells alone on A549 lung cancer cells was statistically different (P<0.05). The killing effect of cells in both groups on lung cancer A549 cells was stronger than that of the PD-1 monoclonal antibody group (P<0.01). The results of flow cytometry showed that PD-1 monoclonal antibody increased the positive ratio of perforin and granzyme release in CIK cells, and the positive ratios of perforin release (46.7%±3.5%% vs. 35.1%±2.2%) and granzyme release (34.6%±3.8% vs. 25.7%±3.3%) in PD-1 monoclonal antibody combination with CIK cells group and CIK cells group were statistically different (P<0.05). Similarly, the secretion levels of IL-2, TNF-α, and IFN-γ cytokines were also increased in the PD-1 monoclonal antibody combined with CIK cells group compared with the CIK group (5 409.0±168.8 pg/mL vs. 4 300.0±132.3 pg/mL, 252.7±16.7 pg/mL vs. 172.5±8.6 pg/mL, 327.2±23.5 pg/mL vs. 209.7±16.0 pg/mL, P<0.05).ConclusionPD-1 monoclonal antibody can promote the release of tumoricidal substances in CIK cells and improve the killing effect of CIK cells on lung cancer A549 cells. It is speculated that the infusion of PD-1 monoclonal antibody before CIK cell adoption in lung cancer patients may be more beneficial to the treatment of disease. PD-1 monoclonal antibody combined with CIK cell therapy is promising as a new type of lung cancer immunotherapy.
ObjectiveTo systematically review the efficacy and safety of autologous natural killer cells (NK) cells for the treatment of malignant tumors. MethodsThe PubMed, Web of Science, and Embase databases were electronically searched to collect clinical studies on autologous NK cells for the treatment of malignant tumors from inception to July 1, 2023. Two reviewers independently screened literature, extracted data and assessed the risk of bias of the included studies. Descriptive analysis of the results were conducted. ResultsA total of 15 studies were included. The most common tumor type was non-small cell lung cancer. The dose of NK cell injections usually ranged from 7.0×107 to 7.0×109 cells, with a treatment interval of 14-21 days and a frequency of 3-6 injections. The overall response rate for NK cell therapy ranged from 0% to 77.78%. The main adverse effects were fever (3.98%), fatigue (1.99%), rash (0.4%), and dizziness (1.20%). ConclusionCurrent evidence shows that autologous NK cell therapy is safe for treating malignant tumors, and some studies have shown that NK cell therapy has a relieving effect. Due to the limited quality and quantity of the included studies, more high-quality studies are needed to verify above conclusion.
Objective To investigate the expression of induced heat shock protein (HSP) 70 in ratprime;s retinal neurons (RNs) and Muuml;ller cells, and evaluate the protective effect of HSP 70 on RNs injured with glucose deprivation and glutamate. Methods Ratprime;s RNs and Muuml;ller cells cultured in vitro were treated with heat shock (42℃ for 1 hour), and duration of the expression of HSP70 was detected by immunocytochemical techniques. Viability of the cells was measured by methyl thiazolyl tetrazolium (MTT) chromatometry after incitant toxic injury with glucose deprivation (0.56 mmol/L glucose for 6 hours) and glutamate (100 mu;mol/L for 6 hours). Simultaneously, the expression was interdicted by HSP70. Results Hypereffective expression of HSP70 was found in cultured RNs and Muuml;ller cells after heat shock. The viability of RNs pretreated by heat shock after injured with glucose deprivation and glutamate significantly increased which could be interdicted by HSP70 antibody. Conclusion Hypereffective expression of HSP 70 may be induced by heat shock, which enhances the ability of tolerance of RNs to the incitant toxic injury by glucose deprivation and exitotoxicity. (Chin J Ocul Fundus Dis, 2005,21:110-113)
31 case of advanced primary liver cancer were treated by using IL-2 and LAK cells in which 15 cases were combined with surgery (group A) and 16 cases were combined with chemotherpy (Group B). 7～14 months follow-up showed: In group A there was no recurence and metastasis, and the cell-mediated immunity was obviously improved. In group B, the average life time was more than 5.84 months, the tumor average diameter dicreased in 10 cases ,and the cee-mediated immunity was also improved. The role of immunotherapy combined with surgery or chemotherapy was discussed.
Neural stem cell is a kind of stem cells that can differentiate into neural and glial cells. While Müller cells, the main endogenous neural stem cell in retina,have the features to reentry into the cell cycle and differentiate into neural cells after retinal damage. Although it is highly effective for retinal Müller cell differentiation spontaneously after retinal injury in vertebrates, this feature is rigorous restricted in mammals. Recently, some transcription factors,such as Ascl1, Sox2, Lin28, Atoh7, are sufficient to drive quiescent Müller cells back in proliferation to generate new retinal neurons. Moreover, combining Ascl1 expression with a histone deacetylase inhibitor can bypass the limitation and increase the generation of new neurons in the adult retina. These regenerated neurons integrate the existing neuronal network and are able to respond to light, indicating that they can likely be used to restore vision. While these results are extremely promising, the regenerative response is still limited, likely because the proliferative capacity of mammalian Müller cells is low compared to their zebrafish counterparts. It is indeed necessary to identify new factors increasing the efficiency of the regenerative response.
Objective To investigate the effect of long-term intraocular retention of domestic perfluorocarbon liquid (PFCL) on morphology and histology of ocular tissues. Methods A total of 18 New-Zealand rabbits were randomly divided into 3 experimental groups, whose left eyes underwent intraocular injection with 0.3, 0.6, and 1.0 ml PFCL, respectively. All of the right eyes of the rabbits were in the control group. The morphological, electrophysiological and histological changes of the ocular tissue were observed 4, 8, and 12 weeks after the injection. Results No clinically significant retinopathy but only mild morphological changes were found in group 1 and 2, while obvious morphological and histological changes were found in group 3. Mild morphological and histological changes were found in all of the rabbits 4-8 weeks after the injection while significant ones were found 8-12 weeks after the injection. The results of electroretinography indicated a statistically significant decline of amplitude of b wave in group 3. Conclusions Long-term intraocular retention of few PFCL may cause mild histological changes but not affect the clinical function. Plentiful PFCL remains in eyes may lead to toxic reaction to the ocular tissue. (Chin J Ocul Fundus Dis, 2006, 22: 128-130)
Obiective lt;brgt;To investigate the change of the activity of proliferation in cultivated Muuml;ller cells treated by advanced glycation endoproducts (AGEs), and the effect of these changes on expression of occludin in bovine retinal vascular endothelial cells (BREC). lt;brgt;Methods lt;brgt;The cultivated Muuml;ller cells were devided into normal growth group and cultured with AGEs group. The cultured BREC were devided into 4 groups:group 1, without any medium; group 2, with normal growth Muuml;ller cell medium (MCM); group 3,MCM treated by AGEs; group 4, without cell as the control. Enzyme-linked immuno sorbent assay was used to detect the content of occludin in the medium in the 4 groups. lt;brgt;Results lt;brgt;The content of expression of occludin was the most in group 2, less in group 1, and the least in group 3. lt;brgt;Conclusion lt;brgt;AGEs may promote the abnormal proliferation of Muuml;ller cells and inhibit the expression of occludin in BREC. lt;brgt;(Chin J Ocul Fundus Dis, 2006, 22: 28-30)
Objective To observe the effects of bevacizumab on aquaporin 4 (AQP4) expression in human retinal Muuml;ller cells in vitro under hypoxia. To explored the mechanism of treating retinal edema with bevacizumab. Methods Human Muuml;ller cells were cultured using the enzymatic digestion method. Transmission electron microscopic analysis and immunofluorescence staining identified Muuml;ller cells. With semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), the expression of AQP4 mRNA and vascular endothelial growth factor （VEGF） mRNA in Muuml;ller cells cultured under different concentration of COCl2 for different hours were observed. The expression of AQP4 mRNA in Muuml;ller cells cultured using CoCl2 precultured with 200 mu;g/ml bevacizumab was measured. Results More than 95% of primary cells showed positive reaction to glial fibrillary acidic protein, glutamine synthetase, vimentin and alpha;-smooth muscle actin with immunofluorescence staining. Characteristic 8-10 nm intracellular filaments could be seen in the cytoplasm viewed with transmission electron microscopy. The results using RT-PCR showed that CoCl2 increased the AQP4 and VEGF mRNA expression in Muuml;ller cells in a dose and time dependent manner (r=0.952, 0.954;P＜0.05). The expression of AQP4 mRNA in Muuml;ller cells was increased by VEGF (F=12.43,P＜0.05). The expression of AQP4 mRNA was significantly decreased by bevacizumab (F=2 370.37，P＜0.05). Conclusion Bevacizumab can down-regulate the expression of AQP4 mRNA in human Muuml;ller cells under hypoxic conditions partially by VEGF path, which may be a mechanism for treating retinal edema with bevacizumab.