Objective To explore the proper dosage of establishment of stable hepatic oval cells （HOC） prolif-eration model by using 2-acetaminofluorene （2-AAF） combined with two-third partial hepatectomy （2/3 PH） surgery， and to explore isolated and cultured method of HOC in vitro. Methods The 174 Wistar rats were randomly divided into 4 experimental groups （each group enrolled 30 rats）， saline group （n=30）， and untreated group （n=24）. Rats of 4 experi-mental groups were underwent gavage of 5， 10， 15， and 20 mg/（kg ? d） 2-AAF， corresponding to the groups from No.1 to No.4 group. Rats of saline group received saline gavage and rats of untreated group didn’t received any treatment. A standard 2/3 PH surgery was performed on the 5th day after gavage， then the same gavage method was still administrated as preoperation untill rats were sacrificed. The liver tissues of 6 selected rats were adopted and identified by HE staining and immunohistochemical staining on 4， 8， 12， and 16 days after PH for observation of the proliferation of HOC in every group， on 4 days， levels of alanine aminotransferase （ALT） and aspartate aminotransferase （AST） were tested in addition. HOC were isolated and purified by collagenase perfusion method and percoll gradient centrifugation. Results The surv-ival rates of untreated group，saline group，No.1 group，No.2 group，No.3 group，and No.4 group were 100% （24/24），93% （28/30），93% （28/30），90% （27/30），90% （27/30），and 80% （24/30） respectively. Compared with the saline group and untreated group， the levels of serum ALT and AST increased significantly in No.2， No.3， and No.4 group on the 4th day after PH （P＜0.05）. The results of HE staining showed that No.2， No.3， and No.4 group were observed visibly different level of damage at liver tissue， and the proliferation level of HOC were most obviously in No.3 and No.4 group. The results of immunohistochemical staining revealed that proliferation cells were positively expressed oval cell marker-6 （OV-6）. The number of OV-6 positive cells were increased significantly with the increase of dosage of 2-AAF between 4 days and 12 days after operation， and proliferation levels were related with dosages of 2-AAF （P＜0.05）. In all cultured cells， 80% of cells were OV-6 positive cells after isolation and culture by using collagenase perfusion method and percoll gradient centrifugation. Conclusions The methods of gavage of 2-AAF at 15 mg/（kg ? d） combined with 2/3 PH surgery can establish the HOC proliferation model on the 12th day， as well as the rats have lower mortality and better tolerance， especially. The collagenase perfusion method and percoll gradient centrifugation can be used to isolate HOC effectively.
Objective To investigate the effect of adiponectin on proliferation of airway smooth muscle cells( ASMCs) , and explore its possible mechanism. Methods ASMCs were derived fromrat airway tissue and were cultured in vitro. RT-PCR was used to verify the expression of adiponectin receptors on ASMCs. Then ASMCs were treated with adiponectin at different concentrations( 5, 10, 20, 40, 80 μg/mL) for different periods of time( 1, 12, 24, 48, 72 hours) , respectively. The absorbsence ratios of adiponectin at different concentrations were determined by MTT assay. The adenosine monophosphate-activated protein kinase( AMPK) and phosphorylated AMPK( pho-AMPK) in ASMCs were quantified by Western blot after being treated with adiponectin at different concentrations ( 5, 10, 20, 40 μg/mL) for 48 hours. ResultsThe inhibition of adiponectin on ASMCs was showed in dose-dependent manner( r = 0. 324, P lt; 0. 01) and time-dependent manner( r = 0. 607, P lt; 0. 05) . Western blot indicated that the expression of pho-AMPK increased with the increased concentrations of adiponectin( r =0. 607, P lt; 0. 01) . The ratio of pho-AMPK/AMPK were ( 27. 66 ±1. 03) % , ( 31. 91 ±0. 86 ) %, ( 75. 52 ±2. 67) % , and ( 84. 50 ±1. 05) % ,respectively, with significant differences between each concentrations of adiponectin( P lt; 0. 05) . There was no expression of pho-AMPK in the control group. Conclusion Adiponectin can significantly inhibit ASMCs’proliferation by activating AMPK.
Objective To summarize the role of matrix metalloproteinases (MMPs) in occurrence and development of gastric cancer. Methods Domestic and international publications online involving MMPs of gastric cancer in recent years were collected and reviewed. Results The occurrence and development of gastric cancer was a multi-step and multi-factorial complicated progress, whose etiology and pathogenesis were still unclarified. MMPs were a class of proteolytic enzymes, which played an important role in the proliferation, metastasis, angiogenesis of gastric cancer and apoptosis of tumor cells and their surrounding normal cells by regulating the microenvironment of the growth of tumor. Conclusion MMPs promote the evolution of gastric cancer in variable ways, the mechanisms of which should be comprehended to provide a theoretical basis for the future treatment of gastric cancer.
ObjectiveTo study the effect of titanium particles on the proliferation, differentiation, and cytomorphology of osteoblasts, and to explore the possible internal relations and mechanism. MethodsCalvarial osteoblasts were separated from 10 newborn Sprague Dawley rats by repeated enzyme digestion, and were cultured in vitro. The cells were identified by alkaline phosphatase (ALP) staining and alizarin red staining. The cells at passage 3 were cultured with titanium particles culture medium at concentrations of 0.01, 0.05, 0.1, 0.5, and 1 mg/mL (0.01, 0.05, 0.1, 0.5, and 1 mg/mL groups). The absorbance (A) values were detected by cell counting kit 8 at 7 days after cultured to compare the effect of titanium particles at different concentrations on proliferation, and median lethal concentration was screened out. The expression of collagen type I was detected by ELISA to observe the effect of titanium particles on differentiation. The osteoblasts co-cultured with titanium particles of median lethal concentration (experimental group) for 7 days, and double fluorescence staining with FITC-phalloidine and propidium iodide was performed. The cytomorphology variation of osteoblasts after swallowing titanium particles was observed under laser scanning confocal microscope. The osteoblasts at passage 3 cultured with culture medium without titanium particles served as control group. ResultsThe cultured cells were identified as osteoblasts by ALP staining and alizarin red staining. Different concentrations of titanium particles could inhibit osteoblasts proliferation and differentiation in varying degrees, showing significant difference when compared with the control group at 7 days after culture (P<0.05). The cell proliferation and differentiation were decreased with increased titanium particles concentration; significant differences were found between the other groups (P<0.05) except 0.01 and 0.05 mg/mL groups (P>0.05). The median lethal concentration of titanium particles was 0.5 mg/mL. Laser scanning confocal microscope showed cellular shrinking, microfilaments distortion, pseudopodia contraction of osteoblasts that swallowed titanium particles in the experimental group. ConclusionTitanium particles can inhibit proliferation and differentiation of osteoblasts. The effect may be related to variation of cytomorphology after swallowing titanium particles.
Objective To investigate the effect of the serum from severe burn patients on the biology characteristics of human umbilical cord mesenchymal stem cells (hUCMSCs) in vitro, so as to explore the feasibility of hUCMSCs transplantation for treating severe burn. Methods The 3rd passage of hUCMSCs were randomly divided into 3 groups: 10% fetal bovine serum group (group A), 10% normal serum group (group B), and 10% burn serum group (group C). At 24 hours, 72 hours, and 6 days after culture, the cell morphology and density were observed by inverted microscope; the cell proliferation was assessed by MTT; after 6 days of culture, the cell cycle by propidium iodide staining and flow cytometry, the apoptosis by acridine orange/ethidium bromide staining, and the cell senescence by β-galactosidase staining; the levels of tumor necrosis factor α (TNF-α), interleukin 1 (IL-1), platelet-derived growth factor (PDGF), and insulin-like growth factor 1 (IGF-1) in serum were detected by a double-antibody sandwich ELISA kit. Results hUCMSCs were long spindle/polygon in 3 groups. The cell fusion of group C was obviously faster than that in group A and group B. The cell proliferation curves showed that the velocity and number of cell proliferation in group C were significantly higher than those in group A and group B at 2-6 days after culture (P lt; 0.05). The rates of proliferation period (S) of hUCMSCs were 9.21% ± 1.02%, 11.79% ± 1.87%, and 20.54% ± 2.03%, respectively in groups A, B, and C at 6 days, and group C was significantly higher than that of group A and group B (P lt; 0.05). The hUCMSCs showed normal morphology and structure in 3 groups, and no apoptosis cells was observed. The positive cells percentage of group C (2.6% ± 0.1%) was significantly lower than that of group A (4.8% ± 0.2%) and group B (3.8% ± 0.4%) (P lt; 0.05). The levels of TNF-α, IL-1, PDGF, and IGF-1 in group C were significantly higher than those in group B (P lt; 0.05). Conclusion The higher levels of cytokines in serum from the severe burn patients can significantly stimulate hUCMSCs proliferation, prevent cells apoptosis, and reduce cells senescence. Therefore, it is feasible to use hUCMSCs transplantation for treating severe burn patients.
ObjectiveTo study the expression levels of miR-339-3p and miR-339-5p in the gastric carcinoma cell lines(SGC-7901, BGC-823, and MKN-45) and gastric surface epithelium(GES-1);detect the relationship between miR-339-3p and miR-339-5p and the gastric carcinoma cell lines in vetrio experiment through the gain of function, and further significance is suggested. MethodsSYBR greenⅠreal time PCR was performed to access the expression of miR-339-3p and miR-339-5p in different cell lines(SGC-7901, BGC-823, MKN-45, and GES-1). The expression levels of miR-339-3p and miR-339-5p were verified by real time PCR experiment again after transfecting miR-339-3p mimics and miR-339-5p mimics. After that, the changes of MKN-45 cells apoptosis and proliferation at 72 h after transfection were detected by flow cytometry and CCK-8 method. ResultsThe expression levels of miR-339-3p and miR-339-5p in gastric carcinoma cell lines(SGC-7901, BGC-823, and MKN-45) were down regulated. Compared with the control group, the apoptosis of MKN-45 cell line was significantly higher(P < 0.05), the ability of proliferation of MKN-45 cell line decreased after transfecting miR-339-3p mimics and miR-339-5p mimics within 72 hours(P < 0.01). ConclusionThe expression levels of miR-339-3p and miR-339-5p significantly decreased in the gastric carcinoma cell lines(SGC-7901, BGC-823, and MKN-45) in contrast with gastric surface epithelium. MiR-339-3p and miR-339-5p may be involved in the apoptosis and proferation of the gastric carcinoma.
Objective To investigate the role of DNA methylation on regulation of cell apoptosis and proliferation in ischemia-reperfusion of small intestine. Methods Thirty-five male Wistar rats were randomly divided into normal group， sham operation group， and ischemia-reperfusion group. The apoptotic cell was assessed by TUNEL and electron microscopy and the expression of Ki-67 was examined by immunohistochemistry in the small intestinal parts （villi epithe-lium， crypt epithelium， and lamina propria mucosa of small intestine）. The DNA methylation was detected by DNA histo-endonuclease-linked detection of methylated DNA sites. Results ①The apoptotic positive cells increased at 3 h， 6 h，and 12 h after ischemia-reperfusion in the villi epithelium， crypt epithelium， and lamina propria mucosa of small intestine as compared with the normal group and sham operation group （P＜0.01）；Moreover， the apoptotic cells in the lamina propria mucosa of small intestine were identified as T cells by electron microscopy. ②The expressions of Ki-67 markedly increased at 3 h， 6 h， 12 h， and 24 h after ischemia-reperfusion in the villi epithelium cells as compared with the normal group and sham operation group （P＜0.01）. ③The weak expression of DNA methylation was found in the villi epith-elium and crypt epithelium in the normal group and sham operation group， the b expression was examined in the crypt epithelium cells nearby stem cell site in the ischemia-reperfusion of small intestine， the change of expression was gradually weak from crypt epithelium to villi epithelium. Conclusion This initial results indicate that the DNA methyl-ation in the ischemia-reperfusion of small intestine might regulate cell apoptosis and proliferation.
Objective To observe the effect of bone forming protein 4 (BMP4) on the proliferation and migration of human retinal pigment epithelium (RPE) cells under oxidative stress, and to preliminarily explore its effect on epithelial-mesenchymal transition (EMT) of RPE cells. MethodsHuman RPE cells cultured in vitro were divided into normal group, pure 4-hydroxynonenal (HNE) group (4-HNE group), 4-HNE+NC group and 4-HNE+ small interfering BMP (siBMP4) group. The effect of 4-HNE on the proliferation of RPE cells was detected by thiazole blue colorimetry. The effects of 4-HNE and BMP4 on cell migration were determined by cell scratch test. The expression of BMP4 was detected by immunofluorescence staining, Western blot and real-time quantitative polymerase chain reaction. The transfection efficiency of siBMP4 was observed by fluorescence microscopy. Mitochondrial reactive oxygen species (MitoSOX) were detected by flow cytometry. The expression of EMT markers E-cadherin and Fibronection were detected by immunofluorescence assay. t-test was used for comparison between the two groups, and one-way analysis of variance was used for comparison between the three groups. ResultsCompared with normal group, cell proliferation and migration ability of 4-HNE group were significantly enhanced, with statistical significance (t=21.619, 24.469; P＜0.05). The expression of BMP4 in cells was significantly increased, and the difference was statistically significant (t=19.441, P＜0.05). The relative expression levels of BMP4 mRNA and protein were also significantly increased, with statistical significance (t=26.163, 37.163; P＜0.05). After transfection with siBMP4 for 24 h, the transfection efficiency of BMP4 in RPE cells was＞90%. Compared with 4-HNE group and 4-HNE+NC group, the relative expression levels of BMP4 protein (F=27.241), mRNA (F=36.943), cell mobility (F=46.723) and MitoSOX expression levels (F=39.721) in normal group and 4-HNE+siBMP4 group were significantly decreased. The differences were statistically significant (P＜0.05). The epithelial marker E-cadherin increased significantly, while the mesenchymal marker Fibronection decreased significantly, with statistical significance (F= 51.722, 45.153; P＜0.05). ConclusionsBMP4 inhibits RPE proliferation and migration under oxidative stress. BMP4 is involved in inducing EMT in RPE cells.
Objective To investigate the inhibitory effect of proliferation cell nuclear antigen (PCNA) antisense oligonucleotides mediated by liposome transfection on hepatocellular carcinoma cell proliferation. MethodsThe antisense oligonucleotides were complementary to 18mer sequences next to the start codon of PCNA mRNA sequences. The human hepatocellular carcinoma cell line Bel7404 was treated with antisense oligonucleotides. The inhibition of proliferation was estimated by MTT method. We compared the deference between the liposome mediated transfection technique and direct transfection technique. ResultsThe cell proliferation was inhibited effectively by antisense oligonucleotides. A sense sequence oligomer showed no effect.Liposome mediated transfection could enhance the inhibitory effect. Conclusion Liposome mediated transfection could enhance the inhibitory effect of PCNA antisense oligonucleotides on hepatocellular carcinoma cell proliferation.
ObjectiveTo explore the effects of several immunosuppressants on the proliferation of pheochromocytoma 12 (PC12) and L929 cells. 〖WTHZ〗Methods Different concentrations of methylprednisolone(10-3,10-4, 10-6and 10-8 mol/L), cyclosporin A(CsA,10-5 ,10-6 , 10 -7and 10-8 mol/L) and FK506 (10-6 ,10-7 , 10-8and 10-9mol/L)were administrated to the PC12 and L929 cells, while control group was given no drugs. At 24, 48 and 72 hours after administration, the cell proliferationwasmeasured with MTT methods respectively. The results were compared and analyzed statistically. Results High concentration methylprednisolone (10-3 mol/L) and low concentration CsA (10-8-10-7mol/L) could promote the proliferation of PC12 cells within 48 hours after administration, after that, the proliferation effects were no longer significant. There were no promotion effects for different concentrations of FK506. Under high concentrations, both CsA (10-6 -1×10-5 mol/L) and methylprednisolone (10-3 mol/L) could significantly inhibit the proliferationof L929 cells after 24 hours of administration. And high concentration (10-6mol/L) FK506 could promote the proliferation of L929 cells transitorily (only for 48 hours after administration). Conclusion 10-3 mol/L methylprednisolone and 10-8 -10-7mol/L CsA can promote the proliferation of PC12 cells for a short period of time. Both 10-3 mol/L methylprednisolone and 10-6-10-5mol/L CsA can significantly inhibit proliferation of L929.