Objective To explore the effects of Zhaoke defibrase and anti alpha;vbeta;3mAb (23C6) on the adhesion and immigration of bovine retinal vascular endothelial cells. Methods The culture dishes coated with vitronectin (Vn) and collagen，assays of adhesion and immigration were performed 60 minutes after different concentration of Zhaoke defibrase and anti-alpha;vbeta;3 mAb was added to the bovine retinal vascular endothelial cells. The apoptosis of bovine retinal vascular endothelial cells induced by Zhaoke defibrase and anti-alpha;vbeta;3 mAb was detected by electron microscopy. Results Both Zhaoke defibrase and anti-alpha;vbeta;3 mAb inhibited the adhesion and immigration of bovine retinal vascular endothelial cells in a dose-dependent manner. The inhibited concentration (IC50) of Zhaoke defibrase was less than 0.05 mu;mol/L, while (IC50) of anti-alpha;vbeta;3 mAb was more than 2.5 mu;mol/L. 81.8% endothelial cells adhering to Vn were inhibited by 0.1 mu;mol/L Zhaoke defibrase, while 76.3% by endothelial cells adhering to Vn were inhibited by 10 mu;mol/L anti-alpha;vbeta;3 mAb. Typical apoptosis cells were found in bovine retinal vascular endothelial cells after affected by Zhaoke defibrase and anti-alpha;vbeta;3 mAb. Conclusion Both Zhaoke defibrase and anti- alpha;vbeta;3mAb can significantly inhibit the adhesion and immigration of bovine retinal vascular endothelial cells to extracellular matrix， and the mechanism may lie in inducing the apoptosis of endothelial cells. (Chin J Ocul Fundus Dis, 2005,21:118-121)
ObjectiveTo investigate the effects of overexpression of alpha/beta hydrolase domain-containing protein 5 (ABHD5) on the invasion and migration of human colon cancer cell line HCT116 and the pathway of adenosine monophosphate-activated protein kinase (AMPK)/mechanistic target of rapamycin (mTOR).MethodsThe expression of ABHD5 in colon cancer tissues and its relationship with clinicopathological features was analyzed by UALCAN database. HCT116 cells were cultured in vitro and transfected with ABHD5 recombinant plasmid, then they were divided into control group, negative transfection group and ABHD5 transfection group. Real time quantitative PCR (qRT-PCR) was used to detect the expression of ABHD5 mRNA in HCT116 cells. The proliferation of HCT116 cells was detected by CCK-8 method. Transwell assay was used to detect the invasion and migration of HCT116 cells. The expression of matrix metalloprotein 9 (MMP-9), E-cadherin, Snail, and AMPK/mTOR pathway proteins p-AMPK, AMPK, p-mTOR and mTOR were detected by Western blot.ResultsThe results of the UALCAN showed that compared with normal colon tissues, the expression of ABHD5 mRNA in colon cancer tissues was decreased (P<0.05), and which in the adenocarcinoma and the N1 stage was lower than that of the mucinous adenocarcinoma (P<0.05) and N0 stage (P<0.05), respectively. Compared with the control group and the negative transfection group, the expression of ABHD5 mRNA in the ABHD5 transfection group was increased (P<0.05), the proliferation inhibition rate of HCT116 cells in the ABHD5 transfection group was increased (P<0.05), the numbers of migration and invasion cells in the ABHD5 transfection group were decreased (P<0.05), the expressions of MMP-9, Snail, p-mTOR and mTOR were reduced, and the expressions of E-cadherin, p-AMPK and AMPK were increased (P<0.05).ConclusionsThe overexpression of ABHD5 can inhibit the invasion and migration of colon cancer HCT116 cells, activate AMPK, and inhibit the expression of mTOR. It suggests that ABHD5 may play a role in inhibiting colon cancer by affecting AMPK/mTOR pathway.
ObjectiveTo study the effects of ATP citrate lyase (ACLY) gene on proliferation, apoptosis, invasion, and lipid metabolism of colon cancer cells.MethodsColon cancer cells HCT116 were transfected with lentiviral knockdown ACLY gene in vitro and divided into three groups according to cell treatment: HCT116 cells with ACLY gene knockdown as knockdown group, empty vector transfected cells as negative control group, and untreated colon cancer HCT116 cells as blank control group. After the stable new cell line was screened with puromycin, the expression of ACLY protein was detected by Western blot method, the lipid production of cells was detected by triglyceride test kit, the proliferation ability of cells was detected by CCK-8 method, the apoptosis rate was detected by flow cytometry, and the migration ability of cells was detected by cell scratch test.ResultsThe cell survival rate of the knockdown group was lower than those of the blank control group and the negative control group at 120 h, but there was no significant difference among the three groups at 24 h and 48 h. Compared with the negative control group and the blank control group, the apoptosis rate in the knockdown group increased, the 24 h migration ability and the level of intracellular triglyceride decreased.ConclusionACLY gene knockdown can inhibit the proliferation, apoptosis, and migration of colon cancer cells, and its mechanism may be related to the decrease of lipid synthesis ability of colon cancer cells.
ObjectiveTo interpret the mechanisms of vascular repair disorders in steroid-induced avascular necrosis of the femoral head (SANFH) via detection of the changes of proliferation, migration, and macrophage migration inhibitory factor (MIF)/vascular endothelial growth factor (VEGF) expressions of endothelial cells (ECs) under hypoxia/glucocorticoid. MethodsAccording to culture conditions, human umbilical vein ECs (HUVECs) at passage 3 were divided into group A (normal), group B (1.0×10-6 mol/L dexamethasone), group C (hypoxia), and group D (hypoxia+1.0×10-6 mol/L dexamethasone). The cell activity was detected by AlamarBlue; the number of viable cells was detected in live/dead cell staining; the cell morphology was observed after cytoskeleton staining; cell migration ability was compared by scratch test; and the levels of MIF and VEGF expressions were detected by ELISA. ResultsAt 24 hours after culture, the cell activity and the number of living cells in group C were significantly higher than those in the other 3 groups, showing significant difference between groups (P < 0.05), and group D had the worst cell activity and least living cells. Cytoskeleton staining showed that cells had normal morphology in groups A and B; cells had rich cytoskeleton and secretion granules in group C; cytoskeleton form disorder and nucleus pyknosis were observed in group D. Scratch test showed that the cell migration ability of group C was strongest while cell migration ability of group D was weakest. Accumulated concentration of MIF and VEGF in 4 groups significantly increased with time extending. Accumulated concentration of MIF in group C were significantly higher than that in other 3 groups at each time point (P < 0.05). Within 24 hours after intervention, stage concentration of MIF during 1-8 hours was significantly lower than that during 0-1 hour and 8-24 hours in every group (P < 0.05). Stage concentration of MIF in group C was significantly higher than other groups during 0-1 hour and 8-24 hours (P < 0.05). Within 2 hours after intervention, stage concentration of MIF in 4 groups during 0.5-1 hour was significantly higher than that during other stages (P < 0.05). Accumulated concentration of VEGF in group C was significantly higher than that in other groups at 8 and 24 hours (P < 0.05). The stage concentration of VEGF in groups C and D during 8-24 hours was significantly higher than that during 0-1 hour and 1-8 hours (P < 0.05). There was no significant difference in the stage concentration of VEGF within and among group A, B, C, and D at every stage within 2 hours after intervention (P > 0.05). ConclusionIn hypoxia environment, the proliferation and migration of ECs is enhanced, and the secretion of VEGF and MIF is increased. High concentration of dexamethasone will suppress the process above, which induces vascular repair disorders and aggravating SANFH.
Objective Tissue engineered bone implanted with sensory nerve can effectively promote angiogenesis and repair of bone defects. To investigate the effects of calcitonin gene-related peptide (CGRP) on proliferation and migration of human umbilical vein endothelial cells (HUVECs) for further revealing the mechanism of tissue engineered bone implanted with sensory nerve promoting angiogenesis. Methods HUVECs were collected from human umbilical core, and identified through von Willebrand factor (vWF) and CD31 immunofluorescence. The HUVECs were treated with CGRP and were ivided into 6 groups according to CGRP concentration: group A (0 mol/L), group B (1 × 10—12 mol/L), group C (1 × 10—11 mol/L), group D (1 × 10—10 mol/L), group E (1 × 10—9 mol/L), and group F (1 × 10—8 mol/L). The expression of the CGRP1 receptor (CGRP1R) was observed in HUVECs by cell immunofluorescence. The growth rate of HUVECs was detected through AlarmarBlue at 1, 2, 3, 4, and 5 days. Transwell chamber was used to detect the abil ity of cell migration. ELISA assay was used to detect the vascular endothel ial growth factor (VEGF) secretion and the protein expression of focal adhesion kinase (FAK) was examined using Western blot. Results HUVECs were identified through morphology, vWF and CD31 immunofluorescence. HUVECs expressed CGRP1R. CGRP could stimulate HUVECs prol iferation in a time- and concentration-dependent manners; the cell growth rates of groups B-F were significantly higher than that of group A at all time (P lt; 0.05); group F had highest cell growth rate. The number of cell migration of group B-F was significantly higher than that of group A (P lt; 0.05), which increased more than 3 times. Groups B-F had higher amount of VEGF than group A (P lt; 0.05), and groups C and D had highest amount of VEGF. FAK expression of groups B-F was significantly increased at 3, 7, and 10 days after CGRP treatment when compared with group A (P lt; 0.05). Conclusion CGRP may enhance the proliferation and migration of HUVECs by increasing the secretion of VEGF and expression of FAK.
ObjectiveTo study the expression and role of homeobox transcription antisense intergenic ribose nucleic acid (HOTAIR) in CD133-positive gastric cancer cells, which was classified to long non-coding RNA (LncRNA). MethodsImmune magnetic cell sorting (MACS) was performed to sort CD133-positive and CD133-negative cells of KATO-Ⅲgastric cancer cells, then reverse transcription polymerase chain reaction (RT-PCR) was performed to detect the expressions of HOTAIR mRNA and CD133 mRNA. After the intervention of small interfering RNA (siRNA) for CD133-positive KATO-Ⅲcells, RT-PCR method was performed to detect the expression of HOTAIR mRNA to select siRNA who had the best silent effect. The selected-siHOTAIR was used to silent the expression of HOTAIR, then the expressions of CD133 mRNA, E-cadherin mRNA, and N-cadherin mRNA were detected by RT-PCR. At last, Transwell experiments were performed to detect the migration ability and invasion ability. Results①?RT-PCR test results showed that, the expression levels of CD133 mRNA and HOTAIR mRNA in CD133-positive group were significantly higher than those of CD133-negative group and no separation group (P < 0.05).②?After interference of siHOTAIR, the expression levels of HOTAIR mRNA in siHOTAIR1 group, siHOTAIR2 group, and siHOTAIR3 group were all significantly lower than those of blank control group and negative control group (P < 0.05), and the expression levels of HOTAIR mRNA in siHOTAIR2 group was lower than those of siHOTAIR1 group and siHOTAIR3 group (P < 0.05). The results indicated that siHOTAIR2 had the best interference efficiency.③?The expression levels of CD133 mRNA and N-cadherin mRNA in siHOTAIR2 group were lower than those corresponding indicators of blank control group and negative control group (P < 0.05), but the expression level of E-cadherin mRNA was higher than those of blank control group and negative control group (P < 0.05). Transwell experiment results showed that, number of cells which through the cell membrane in siHOTAIR2 group was lower than those of blank control group and negative control group (P < 0.05). ConclusionThe expression of HOTAIR mRNA in CD133-positive KATO-Ⅲgastric cancer cells was higher than that of CD133-negative cells, interfering the expression of HOTAIR mRNA can reduce the expression of CD133 mRNA in CD133-positive KATO-Ⅲgastric cancer cells, and can inhibit cell migration and invasion.
Objective To confirm the association between tail femur distance (TFD) and lag screw migration or cutting-out in the treatment of intertrochanteric fracture with the third generation of Gamma nail (TGN). Methods The clinical data of 124 cases of intertrochanteric fracture treated with TGN internal fixation and followed up more than 18 months between January 2012 and December 2015 were reviewed and analyzed. There were 52 males and 72 females, with an age of 46-93 years (mean, 78.5 years). According to AO/Association for the Study of Internal Fixation (AO/ASIF) classification, 43 cases were type 31-A1, 69 cases were type 31-A2, and 12 cases were type 31-A3. The time from injury to operation was 1-10 days (mean, 2.9 days). According to the fracture healing of the patients, the patients were divided into the healing group and failure group. The age, gender, height, bone mineral density (BMD), fracture AO/ASIF classification, the time from injury to operation, and the TFD value at 1 day after operation were recorded and compared. The risk factors for the migration or cutting-out of lag screw were analyzed by logistic regression. Results There were 111 cases in healing group, the healing time was 80-110 days (mean, 95.5 days). There were 13 cases in failure group, including 2 cases of lag screw cutting-out and 11 cases of significant migration. Except for the TFD value at 1 day after operation in failure group was significantly higher than that in the healing group(t=5.14, P=0.00), there was no significant difference in gender, age, height, BMD, fracture of AO/ASIF classification, and the time from injury to operation (P>0.05) between 2 groups. logistic regression analysis showed that TFD value was a risk factor for the migration or cutting-out of lag screw (B=1.22, standardized coefficient=0.32, Wald χ2=14.66, P=0.00, OR=3.37). Conclusion The patients with higher TFD value had higher risk of postoperative lag screw migration or cutting-out. This result indicates that the appropriate length of the lag screw is helpful to reduce TFD value and prevent postoperative lag screw migration or cutting-out.
ObjectiveTo investigate the regulatory mechanism of thioredoxin binding protein (TXNIP)/nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) pathway in the occurrence and development of breast cancer.MethodsThe resected 15 cases of breast cancer tissues and their adjacent tissues in our hospital from September 2019 to June 2020 were selected, and the immunohistochemistry was used to detect the expression levels of TXNIP and NLRP3 in breast cancer and its adjacent tissues. Three kinds of breast cancer cell lines (MDA-MB231, MCF-7 and SKBR3) and normal breast epithelial cell line (HMEC) were collected. Western blot was used to detect the relative expression levels of TXNIP and NLRP3 in three kinds of breast cancer cell lines and HMEC cell line. MDA-MB231 cancer cells were divided into blank control group (normal culture without any treatment), TXNIP overexpression group (Ad-TXNIP group, transfected with adenovirus vector carrying TXNIP overexpression sequence), Ad-TXNIP negative control group (Ad-eGFP1 group, transfected of empty adenovirus vector without TXNIP overexpression sequence), NLRP3 overexpression group (Ad-NLRP3 group, transfected with adenovirus vector containing NLRP3 overexpression sequence), TXNIP and NLRP3 overexpression co-transfection group (Ad-TXNIP+Ad-NLRP3 group, co-transfection of adenovirus vector carrying TXNIP and NLRP3 overexpression sequence), TXNIP overexpression and Ad-NLRP3 negative control (Ad-eGFP2) co-transfection group (Ad-TXNIP+Ad-eGFP2 group,co-transfection of adenovirus vector carrying TXNIP overexpression sequence and empty adenovirus without NLRP3 overexpression sequence). After 24 hours of transfection and culture, CCK-8 method was used to detect the MDA-MB231 cells proliferation. Transwell chamber method was used to detect MDA-MB231 cells migration and invasion. Nude mice tumorigenicity test was used to detect the tumorigenicity of the MDA-MB231 cells in vivo. Western blot was used to detect the expressions of TXNIP, NLRP3, proliferation marker protein (Ki-67), caspase-1, vascular endothelial growth factor (VEGF), interleukin (IL)-1β, IL-18 and caspase-1 precursor protein (pro-caspase-1) in the MDA-MB231 cells.ResultsCompared with the adjacent tissues, the relative expression level of TXNIP decreased (P<0.05) and the relative expression level of NLRP3 increased (P<0.05) in breast cancer tissues. Compared with normal breast epithelial cell line (HMEC cell line), the relative expression levels of TXNIP in MDA-MB231, MCF-7 and SKBR3 breast cancer cell lines were decreased (P<0.05), and the relative expression levels of NLRP3 were increased (P<0.05). Compared with the blank control group, the relative expression levels of TXNIP, NLRP3, IL-1β, IL-18, pro-caspase-1 and caspase-1 were increased (P<0.05), the relative expression levels of Ki-67 and VEGF, the proliferation activity, invasion and migration ability of MDA-MB231 cells and tumor weight were decreased (P<0.05) in the Ad-TXNIP group and the Ad-NLRP3 group. Compared with the Ad-TXNIP group and the Ad-NLRP3 group, the relative expression levels of TXNIP, NLRP3, IL-1β, IL-18, pro-caspase-1 and caspase-1 were further increased (P<0.05), the relative expression levels of Ki-67 and VEGF, the proliferation activity, invasion and migration ability of MDA-MB231 cells and tumor weight were further decreased (P<0.05) in the Ad-TXNIP+Ad-NLRP3 group.ConclusionsIn breast cancer tissues and breast cancer cell lines, TXNIP is low expression and NLRP3 is high expression. They can interact with each other to promote pyroptosis and inhibit the proliferation, invasion and migration of breast cancer cells.
ObjectiveTo investigate the effects of thrombospondin-1 active fragment (TSP-1) synthetical peptide VR-10 on proliferation and migration of rhesus choroidal-retinal endothelial (RF/6A) cell and the expressions of apoptosis relative genes in RF/6A cell. MethodsThe survival rate of RF/6A cell were detected by methyl thiazolyl tetrazolium, and migration ability was measured by transwell chamber after exposure to 1.0 μg/ml TSP-1 and synthetic peptide VR-10 (0.1, 1.0, 10.0 μg/ml) for different times (6, 12, 24, 48 hours). Caspase-3 and factor associated suicide (FAS) protein levels were measured by Western blot. The mRNA level of bcl-2 and FAS ligand (FASL) were measured by reverse transcription-polymerase chain reaction (RT-PCR). ResultsThe survival rate of RF/6A cells was determined by the treatment time and concentration of TSP-1(1.0 μg/ml) and the synthetic peptide VR-10 (0.1, 1.0, 10.0 μg/ml). The lowest survival ratio of RF/6A was 78% (P < 0.001) when cells were treated by 10 μg/ml synthetic peptide VR-10 after 48 hours. TSP-1 and synthetic peptide VR-10 could inhibit migration of RF/6A cells in transwell chamber (P < 0.001). 10.0 μg/ml synthetic peptide VR-10 had the strongest effect, 1.0 μg/ml TSP-1 was the next. Migration inhibition rate was increase with the increase of the concentration of VR-10 (P < 0.001). There was no significant differences between 0.1 μg/ml and 1.0 μg/ml VR-10 (P=0.114). Western bolt showed that RF/6A cell in control group mainly expressed the 32×103 procaspase-3 forms. To 10.0 μg/ml VR-10 treated group, it showed decreased expression of procaspase-3 (32×103) and concomitant increased expression of its shorter proapoptotic forms (20×103). Compared with control group, expression of FAS peptides were significantly increased in 10.0 μg/ml VR-10 treated group. Compared with control group, expression of FasL mRNA was significantly increased in 10.0 μg/ml VR-10 treated group(t=39.365, P=0.001), but the expression of bcl-2 mRNA was decreased(t=-67.419, P=0.000). ConclusionTSP-1 and synthetic peptide VR-10 had the ability to inhibit proliferation and migration of endothelial cell, and also induce apoptosis by increasing FAS/FASL expression and repressing bcl-2 expression.
ObjectiveTo investigate the effects of pipecolic acid oxidase (PIPOX) on the proliferation, apoptosis, migration and invasion of primary liver cancer cells. MethodsImmunohistochemical staining and analysis of The Cancer Genome Atlas (TCGA) database were used to examine the PIPOX expression levels in liver cancer tissues and paired adjacent normal tissues, and studied their relationship with patient prognosis. Liver cancer cell lines stably overexpressing or knocking out PIPOX were constructed to explore PIPOX’s impact on liver cancer cell proliferation, apoptosis, migration and invasion by conducting in vitro functional experiments such as CCK-8, EdU, apoptosis detection, and Transwell assays. In vivo, nude mice subcutaneous tumor models and lung metastasis models were used to verify PIPOX’s effect on liver cancer growth and metastasis. Real-time quantitative polymerase chain reaction (RT-qPCR) and western blot were both employed to detect the expression of epithelial-mesenchymal transition (EMT) markers in liver cancer cells. ResultsImmunohistochemical staining and TCGA database analysis revealed that PIPOX expression was significantly lower in liver cancer tissues compared to paired adjacent normal tissues (P<0.05). Prognostic analysis indicated shorter overall survival and disease-free survival in PIPOX low expression group (P<0.05). In vitro gain- and loss-of-function experiments showed that PIPOX significantly inhibited liver cancer cell migration and invasion (P<0.05), while having no significant effects on their proliferation and apoptosis (P>0.05). Animal experiments also confirmed that PIPOX significantly inhibited liver cancer lung metastasis (P<0.05), but had no significant effects on tumor growth (P>0.05). Finally, RT-qPCR and western blot results revealed that PIPOX promoted the expression of the epithelial marker E-cadherin (P<0.05) and inhibited the expression of mesenchymal markers (N-cadherin, vimentin, Snail) (P<0.05). ConclusionsPIPOX significantly inhibits liver cancer cell migration and invasion, potentially via suppressing the EMT process. However, PIPOX does not significantly affect liver cancer cell proliferation and apoptosis.