ObjectiveTo establish the system of isolation, cultivation, and identification of the neural stem cells (NSCs) from subventricular zone (SVZ) of neonatal mice so as to seek for the appropriate seed cells for potential therapeutic interventions of neurological disorders. MethodsNSCs were isolated enzymatically and mechanically from SVZ of neonatal mice and cultured. The cellular morphology was observed by inverted microscopy. Immunocytochemical stainings of anti-Nestin and anti-SOX-2 were used to identify NSCs of passage 3. To study the differentiation of NSCs, NSCs were plated into 24-wells in the medium supplemented without epidermal growth factor (EGF) and basic fibroblastic growth factor (bFGF) for 3 or 7 days. To compare the differentiation and proliferation potential of NSCs with different cultivation time, the BrdU pulse-labeling method and MTT test were used. To identify neurons and astrocytes, the anti-β-tubulin Ⅲ (Tuj-1) and anti-glial fibrillary acidic protein (GFAP) staining were used. ResultsThe cells of the SVZ can be isolated and cultured in vitro, and these cells began to form neurospheres after cultured for 3 days at primary passage. While cultured for 7 days, these cells formed more neurospheres, and the volume of the neurospheres became bigger than neurospheres cultured for 3 days. In addition, after cultured for 7 days, the phenomena of fusion of neurospheres and adherent differentiation of neurospheres were observed under inverted microscope. These cells were provided with the typical phenotype of NSCs. The immunofluorescence staining results revealed that these cells showed positive immunoreactivity to Nestin and SOX-2. During the 4 hours BrdU pulse, the number of proliferated NSCs cultured for 3 days (75.817±2.961) was significantly higher than that of NSCs cultured for 7 days (56.600±4.881) (t=3.366, P=0.028). The results of MTT assay revealed that the absorbance (A) value of NSCs cultured for 3 days (0.478±0.025) was significantly higher than that of NSCs which were cultured for 7 days (0.366±0.032)(t=2.752, P=0.011). After cultivated without EGF and bFGF, the percentage of Tuj-1 and GFAP positive cells in NSCs was 23.1%±3.7% and 23.7%±3.8% for 3 days and was 40.1%±3.6% and 37.1%±4.5% for 7 days, respectively, all showing significant differences (t=3.285, P=0.030; t=3.930, P=0.017). ConclusionThe NSCs from SVZ of neonatal mice have potentials of self-renewal and multipotential differentiation in vitro. With different cultivation time, the potentials of proliferation and differentiation of NSCs are different.
Objective To observe the effects of SARS-CoV-2 infection on the morphology, proliferation, apoptosis, cell cycle, and immune response function of mouse retinal photoreceptor cells (661w cells). MethodsA cell experiment. Logarithmic growth phase 661w cells were cultured in vitro and transfected with angiotensin-converting enzyme 2 (ACE2) overexpressing lentivirus to construct ACE2 overexpressing 661w cells that could be infected with SARS-CoV-2 pseudovirus (hereafter referred to as ‘pseudovirus’). The 661w cells were divided into three groups: the normal group (untreated), the siACE2 group (overexpressing ACE2 and not infected with the pseudovirus) and the infected group (overexpressing ACE2 and infected with the pseudovirus), in which the infected group was 5 TU/ml pseudovirus group, 15 TU/ml pseudovirus group, 30 TU/ml pseudovirus group and 50 TU/ml pseudovirus group, and the cells were infected with the pseudovirus for 12, 24, 48 and 72 h, respectively. The infected group was infected with 5 TU/ml pseudovirus group, 15 TU/ml pseudovirus group, 30 TU/ml pseudovirus group and 50 TU/ml pseudovirus group, respectively, for 12, 24, 48 and 72 h. Fluorescence microscopy was used to observe the transfection efficiency of ACE2; protein immunoblotting (Western blot) was used to detect the relative expression level of ACE2 in the cells; light microscope was used to observe the morphology of the cells in the normal and the infected groups; cell proliferation was detected by Cell Counting Kit-8 (CCK8) assay; flow cytometry was used to detect the cell cycle; Western blot and real-time quantitative polymerase chain reaction (qPCR) were used to detect the relative expression of interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α), B lymphocytoma-2 (Bcl-2), Bcl-2-associated X-protein (Bax) proteins and mRNA in the cells of siACE2 group, infected group (30 TU/ml pseudovirus group); qPCR was used to detect the relative expression of nuclear factor (NF)- κB1 and NF-κB2, as well as NF- kB enhancer (P65) and precursor protein (P100) in cells of the siACE2 group and the infected group (30 TU/ml pseudovirus group). One-way ANOVA was used for comparison between multiple groups; t-test was used for comparison between two groups. Results Compared with the siACE2 group, the cells in the infected group showed different degrees of crumpling, and with the increase of the concentration and time of pseudovirus induction, the crumpling of the cells worsened, and the number of cells decreased. Compared with the normal group, the cells in the infected group showed a gradual decrease in cell viability with the prolongation of pseudovirus induction time, and the difference was no statistically significant (F=0.840, 0.412, 1.498, 1.138; P＞0.05), and the apoptotic index of the cells induced in the 30 and 50 TU/ml pseudovirus group was significantly elevated, and the difference was statistically significant (F=2.523, 6.716, 3.477, 3.421; P＜0.05). At 72 h of pseudovirus induction, compared with the siACE2 group, the G1 phase cells in the 30 TU/ml pseudovirus group were significantly increased, and the difference was statistically significant (t=3.812, P＜0.05); the relative expression of IL-6, TNF-α, Bax protein and mRNA in the cells was up-regulated (t=7.601, 6.039, 3.088, 5.193, 6.427, 7.667; P＜0.05), the relative expression of Bcl-2 protein and mRNA was down-regulated (t=3.614, 6.777; P＜0.05), and the relative expression of NF-κB1, NF-κB2, P65, and P100 mRNA was significantly up-regulated with statistically significant differences (t=3.550, 3.074, 3.307, 4.218; P＜0.05). ConclusionSARS-CoV-2 infection may inhibit photoreceptor cell proliferation, promote apoptosis and cycle blockade by activating the NF-κB signalling pathway.
ObjectiveTo analyze the expression of Hsa-miR-29c in gastric cancer and its mechanism of action, and to explore its relationship with clinicopathological characteristics and prognosis of gastric cancer patients.MethodsTheoverexpression of Hsa-miR-29c in gastric cancer cell lines of MKN28 and MKN45 were established by lentivirus transfection (transfection group), and the control group of empty lentivirus (negative control group) was established. The expressions of Hsa-miR-29c in cells of the two groups after transfection were detected by real time polymerase chain reaction (qRT-PCR), and the proliferation and clonogenesis of cells in the two groups were detected by CCK-8 and plate cloning. The expression of extracellular matrix protein 1 (ECM1), type Ⅰ collagen (Col Ⅰ), smooth muscle actin(α-SMA), matrix metalloproteinase-2 (MMP-2), and tissue inhibitor of metalloproteinase-1 (TIMP-1) in the two groups were detected by Western blot. qRT-PCR and immunohistochemistry were used to detect the expression of Hsa-miR-29c in 70 gastric cancer tissues and adjacent tissues respectively, and then analyzed its relationship with the clinicopathological features and prognosis of gastric cancer.ResultsThe stable expression of Hsa-miR-29c gastric cancer cell line was successfully constructed in this research, the expression of Hsa-miR-29c in the transfection group was significantly higher than that in the negative control group (P<0.05). The proliferation and clone forming ability of MKN28 and MKN45 cells in the transfection group were significantly lower than those in the negative control group (P<0.05). Compared with the negative control group, the expression of Col Ⅰ and TIMP-1 in MKN28 and MKN45 cells were increased after transfection, while the expression levels of ECM1, α-SMA, and MMP-2 were significantly decreased, with significant differences between the two groups (P<0.05). The expression level of Hsa-miR-29c in gastric cancer tissues was significantly lower than that of adjacent tissues (P<0.05), and the positive expression rate was not related to age, sex, and pathological type (P>0.05), but related to tumor size, TNM stage, tumor differentiation, and lymph node metastasis (P<0.05). The mean survival time (MST) of patients with negative expression of Hsa-miR-29c was significantly shorter than that of patients with positive expression (P=0.029).ConclusionsHsa-miR-29c is down expressed in gastric cancer, and is related to the clinical characteristics and prognosis of it. The overexpression of Hsa-miR-29c can inhibit the proliferation of gastric cancer cells, and the mechanism may be related to the inhibition of extracellular matrix (ECM) signaling pathway.
Objective To observe the proliferation and migration of endothelial cells after 30% total burn surface area (TBSA) of deep partial thickness scald, and the effect of basic fibroblast growth factor (bFGF) on angiogenesis during wound healing.Methods A total of 133 male Wistar ratswere divided randomly into normal control (n=7), injured control group (n=42), bFGF group (n=42) andanti-c-fos group (n=42). The apoptosis expression of fibroblasts was determinedwith in situ hybridization and the changes of proliferation cell nuclear antigen(PCNA), focal adhesion rinase(FAK), c-fos and extracellular signalregulated kinase(ERK) proteins expression were detected with immunohistochemistry staining technique after 3 hours, 6 hours, 1 day, 3 days, 7 days, 14 days and 21 days of scald.Results In injured control group and bFGF group, theproliferation rate of the vascular endothelial had evident changes 7 days and14 days after scald； the expression of FAK was increased 14 days after scald. ERK proteins expression was different between injury control group and bFGF group at initial stage after scald. Stimulation of ERKs by bFGF led to up-regulation of c-fos and b expression of FAK. Conclusion Exogenous bFGF extended the influence on wound healing process by ERK signaling pathway, affecting migration cascade of vascular endothelial cell. The oncogene proteins play an important role on accelerating angiogenesis duringwound healing.
Objective To investigate the impacts of neoadjuvant chemotherapy on the expression of insulin-like growth factor-1 receptor (IGF-1R) and on operation procedure and the significance of prognosis. Methods The expression of IGF-1R in 40 patients with breast cancer before and after neoadjuvant chemotherapy was measured by immunohistochemistry. The diagnosis was proved by core biopsy. All the patients took the TAC chemotherapy regimen. Modified radical operation was performed after two chemotherapy cycles and the IGF-1R expression was measured again. The clinical effect of neoadjuvant chemotherapy was assessed according to WHO criterion by measuring the size of tumor by physical examination and B type ultrasound. Results After neoadjuvant chemotherapy the tumor size shrank in 29 patients, there was no CR (complete response) or PD (progressed disease) to be documented. IGF-1R expression could be downregulated in 25 patients. Conclusion Neoadjuvant chemotherapy can inhibit the tumor growth by downregulation of the expression of IGF-1R.
An experimental model of proliferative vitretinopathy(PVR) induced by macrophages was used for the evaluation of drug efficacy of daunomycin encapsulated in liposomes in the treatment of PVR.Five mu;g daunomycin(n=40),10mu;g daunomycin-liposome(DL,n=30)and 0.1 ml saline or empty liposomes(n=40,as controls)were injected into the rabbit vitreous after macrophage injection.Retinal detachment developed in 77.5% of the control eyes on day 28,compared to 33.3% of the eyes treated with DL(P＜0.01)and 50% of the daunomycin-treated eyes(P＜0.05).The results suggest that encapsulation in liposomes of cytotoxic agents can enhance drug efficacy.The phasic course of development of PVR is important in the selection of particular drugs. （Chin J Ocul Fundus Dis,1993,9:77-80）
Objective To explore whether blood exosome carrying miR-140-3p can regulate the malignant progression of small cell lung cancer (SCLC) through targeting ubiquitin-conjugating enzyme E2C (UBE2C). MethodsThis study was consisted of bioinformatics analysis, clinical research, cell analysis, and animal experiments. We searched GEO database for data of SCLC related microRNA (miRNA) dataset GSE19945, mRNA dataset GSE40275, and GSE60052. T-test was used to detect the differential expression of miR-140-3p in normal tissues and SCLC tissues in the dataset, and the expression of miR-140-3p in different tissues and extracellular vesicles was analyzed through a database. SCLC tissue and paired cancerous tissues excised at Yongzhou Central Hospital were collected between December 2021 and December 2022, and healthy volunteers 7 days before the start of the study was selected. Quantitative real-time polymerase chain reaction was used to detect the expression level distribution of miR-140-3p and UBE2C in tissue samples of SCLC patients and healthy volunteers. SCLC patients were divided into low expression and high expression groups based on the median expression level, and the correlation between the expression levels of miR-140-3p and UBE2C and patient pathological parameters was analyzed. 20 male nude mice was selected. The nude mice were randomly divided into 4 groups: miR-140-3p, UBE2C analog negative control group, and analog control group, with 5 mice in each group. Immunohistochemical detection system was used to detect tumor tissue sections in nude mice. Results A total of 45 patients and 30 healthy volunteers were included. SCLC malignant progression was significantly associated with the expression of miR-140-3p and UBE2C. The expression of miR-140-3p was low in blood-derived exosomes from SCLC patients. Overexpression of miR-140-3p inhibited the proliferation (47.33±2.52 vs. 107.67±10.69, P<0.05), migration [(11.63±2.62)% vs. (31.77±4.30)%, P<0.05] and invasion (44.33±3.06 vs. 102.67±8.50, P <0.05) and promoted their apoptosis [(14.48±1.20)% vs. (10.14±1.21)%, P<0.05]. Bioinformatics analysis yielded the target gene UBE2C of miR-140-3p. In vitro experiments further demonstrated that miR-140-3p directly targetd UBE2C to inhibit SCLC cell proliferation, migration, invasion, epithelial mesenchymal transition, and promote apoptosis. Mouse xenotransplantation experiments showed that miR-140-3p mimic significantly inhibited tumor growth. ConclusionTherefore, the miR-140-3p extracellular vesicle and the oncogenic gene UBE2C may be potential targets for inhibiting the malignant progression of SCLC.
Objective To study the effects of adenosine 2A receptor activation on activation, proliferation, and toxicity of T lymphocytes stimulated by phytohemagglutinin (PHA) in vitro. Methods A model of activated T cells was established by stimulating the cells with PHA. Those T cells were treated with different concentrations of adenosine 2A receptors agonist (0.01 μmol/L, 0.1 μmol/L, 1 μmol/L, and 10 μmol/L CGS21680). The expressions of CD69, CD25 and proliferation of T cells were measured by fluorescent antibody stain and flow cytometry. ELISA method was used to detect IL-2 and INF-γ levels. Results All concentrations of CGS21680 significantly inhibited the expressions of CD25 and CD69 on PHA-stimulated T cells surface and proliferation of T cells （Plt;0.05， Plt;0.01）. IL-2 and INF-γ secreted by T cells were significantly suppressed, too （Plt;0.01）. Conclusion Activation of adenosine 2A receptor can effectively inhibit the activation, proliferation, and toxicity of T cells in vitro.
Objective To investigate the spatial and temporal regulation effect of VEGF on human fetal retinal vascularization and angiogenesis. Methods The posterior segmental retinas from 54 human fetuses of the 9th week to the 40th week were studied by immunohistodhemistry standing for the expressions of VEGF and PCNA. Results 1. The distribution of VEGF espression was spiking and the peaks were during the 9th-13th and around the 26th week. 2. PCNA immunoreactivity was localized in spindle cells and vascular endothelial cells. The expression level was fluctuated during the developmental process. The peaks were during the 9th-13th and around the 21st week. In these periods, the spindle cells kept proliferating and differentiating, and remodelled subsequently to form the inner side retinal vessels. From the 26th or 34th week, the PCNA immununoreactivity is fully expressed in the vascular endothelial cells of the inner and outer margin of inner nuclear layer(INL) and kept to full terms. 3. Significant positive correlation were shown between the content of VEGF in the retina and that of PCNA in spindle cells and vascular endothelial cells(r=0.736,p＜0.01). Conclusion VEGF was positively involved in modulating human fetal retinal vascularization and angiogenesis. (Chin J Ocul Fundus Dis,1999,15:12-15)
ObjectiveTo establish the co-culture model of mice's early embryo and tumor cells in Vitro to observe the embryonic development and biologic behavior of tumor cells in the same microenvironment and discuss their interaction. MethodsWe acquired 2-cell embryos from mice, and then co-cultured them with tumor cell lines of mice in Vitro. We observed the development of embryos in Vitro and the rates of 4-cell embryos, morula and blastocyst formation. The transwell chamber was used for culture. Methylthiazolyldiphenyl-tetrazolium method was used to test the proliferative activity of tumor cells, while the flow cytometry was used to test its apoptosis. The interaction of co-cultured embryos and tumor cells was analyzed by propidium iodide staining and immunohistochemical technique. ResultsThe co-cultured 2-cell embryos could continue surviving and developing. The rates of 4-cell embryos, morula and blastocyst formation increased significantly in the co-cultured group (P<0.05). There was no significant difference in the proliferative activity and apoptosis of tumor cells between the co-cultured group and the control group (P>0.05). Tumor-free ring formed between the trophoblast and tumor cells. We could observe tumor cells stacked around the tumor-free ring. However, no difference in expression of proliferating cell nuclear antigen and B-cell lymphoma leukemia-2 was observed in tumor cells stacking around the tumor-free ring compared with those elsewhere. ConclusionThe development of 2-cell embryos is enhanced in the co-culture model. The proliferative activity and apoptosis of tumor cells are not affected in this model. A tumor-free ring can form between trophoblast and tumor cells. However, the proliferative activity of tumor cells is not affected by this ring.