【 Abstract 】 Objective Overexpressions of epidermal growth factor (EGF) and EGF receptor have been associated with progression and invasive phenotype of pancreatic cancer. However, the underlying molecular mechanism by which EGF worked in pancreatic cancer cells has not been completely understood. In this study, effect of EGF on the invasion and metastasis of pancreatic cancer cells and its regulatory mechanism were investigated. Methods The effects of EGF on the proliferation, adhesion and invasion of pancreatic cancer cells were detected by WST-1 proliferation assay, adhesion assay and invasive assay, respectively. The activity and expression of MMP-2 and MMP-9 were examined by zymography, Western blot and RT-PCR, respectively. The activity of NF- κ B was examined by EMSA. Results EGF could significantly promote the invasiveness of pancreatic cancer cells but did not affect cell proliferation or adhesion. The expressions of NF- κ B and MMP-9 were significantly increased by EGF, but EGF did not affect the activity and expression of MMP-2. Furthermore, EGF stimulated the NF- κ B binding activity. Pretreatment with NF- κ B inhibitors, pyrrolidine dithiocarbamate (PDTC), could significantly inhibit the activity of NF- κ B induced by EGF. Meanwhile, the EGF-induced expression and activity of MMP-9, as well as cell invasiveness were also inhibited by NF- κ B inhibitor. Conclusion EGF could increase the expression and promote the invasiveness of MMP-9 via the activation of NF- κ B in pancreatic cancer cells, which implies that NF- κ B inhibitant, such as PDTC, may diminish the invasiveness of pancreatic cancer cells.
Objective To construct lentiviral vector carrying the human hepatocyte growth factor (hHGF) gene, and then to get hHGF gene/modified bone marrow mesenchymal stem cells (BMSCs) by infecting the BMSCs. Methods The hHGF gene was obtained with PCR from pcDNA-hHGF plasmid. The recombination lentiviral vector plasmid hHGF was constructed with Age I digestion and gene recombinant, then was identified with PCR and sequencing. Mediated by Lipofectamine2000, the three plasmids system of lentiviral vector including pGC-E1-hHGF, pHelper 1.0, and pHelper 2.0 was co-transfected to 293T cells to produce hHGF gene. The supernatant was collected and concentrated by ultracentrifugation and the titer of lentivirus was measured by real-time quantitative PCR. The BMSCs were infected by the constructed lentivirus and the multipl icities of infection (MOI) was identified with fluorescent microscope, the efficiency of infection with flow cytometry (FCM) analysis, the hHGF level with ELISA analysis, and the expression of hHGF gene with RT-PCR. Results Lentiviral vector carrying hHGF gene was constructed successfully. The titer of lentivirus was 1 × 108 TU/mL. The infection efficiency of BMSCs by hHGF lentiviral was high and reached 98% by FCM, and the best MOI was 10. A great mount of green fluorescence was observed with the fluorescent microscope at 28 days after infection. Peak concentration of hHGF secreted by BMSCs/hHGF reached 40.5 ng/mL at 5 days. The concentration could maintain a high level until 28 days after infection. RT-PCR showed that BMSCs/hHGF could express hHGF gene. Conclusion By lentiviral vector, hHGF gene was integrated into BMSCs genome, and it can express stably.
ObjectiveTo explore the effect and mechanisms of bone marrow mesenchymal stem cells (BMSCs) on healing quality of acetic acid-induced gastric ulcer.
MethodsForty-eight clean grade male Wistar rats were used to establish the model of gastric ulcer with acetic acid and were randomly divided into 3 groups after 3 days of modeling, 16 rats each group. After the abdominal cavity was open and stomach was pulled out, no treatment was given in group A, 150 μL phosphate buffered saline (PBS) and 150 μL BMSCs at passage 4+PBS (1×108 cells/100 μL) were injected into the gastric wall surrounding the ulcer at 5 different points in groups B and C respectively. After 10 days, the ulcer area was measured, the mucosal thickness and the number of dilated glands were tested in the regenerative mucosa by histological method. And the expression of vascular endothelial growth factor (VEGF) was detected at ulcerative margin by immunohistochemical method.
ResultsThe ulcer area in group C was significantly smaller than that of groups A and B (P<0.01), but no significant difference was found between groups A and B (P>0.05). HE staining showed that group C had thicker regenerative gastric mucosa, less dilated glands, and more regular mucosal structure than groups A and B, showing significant differences in regenerative gastric mucosa thickness and dilated glands number (P<0.01), but no significant difference between groups A and B (P>0.05). Immunohistochemical staining showed that the positive expression of VEGF in the ulcer margin mucosa of group C was significantly higher than that of groups A and B. The integral absorbance (IA) value of VEGF expression in group C was significantly higher than that in groups A and B (P<0.01), but no significant difference between groups A and B (P>0.05).
ConclusionBMSCs can accelerate ulcer healing by the secretion of VEGF, and improve the quality of ulcer healing.
ObjectiveTo explore the relationship between Beclin 1 level and lymph node metastasis in patients with non-small cell lung cancer.MethodA total of 204 surgical specimens of patients with non-small cell lung cancer from September 2011 to September 2016 were collected in our hospital. There were 116 males and 88 females . Beclin 1 levels were detected by Western blotting. There were 116 males and 88 females at average age of 55.3±11.2 years. The patients were divided into three groups including a group N0 (no lymph node metastasis), a group N1(intralobar and interlobar lymph node metastases, and no mediastinal lymph node metastasis), and a group N2 (mediastinal lymph node metastasis). The differences of Beclin 1 levels in tumor tissues and lymph nodes of patients with N0, N1 and N2 were statistically analyzed.ResultsAmong 204 patients of lung cancer, 36 patients were squamous cell carcinoma and 168 patients were adenocarcinoma. The levels of Beclin 1 in tumor tissues of N0, N1 and N2 groups decreased gradually with a statistical difference (P<0.05). In the three groups, the levels of Beclin 1 in the lung hilum and intrapulmonary lymph nodes (N1 Beclin 1) of N1 and N2 groups were less than that of N0 group with a statistical difference (P<0.01). In the three groups, the level of Beclin 1 in the mediastinal lymph nodes (N2 Beclin 1) of N2 group was less than that of the N0 and N1 groups with a statistical difference (P<0.01). In the N1 group, the level of N1 Beclin 1 was less than that of N2 group (P<0.01). In the N2 group, though the level of N1 Beclin 1 was less than N2 Beclin 1, there was no statistical difference (P>0.05). ConclusionBeclin 1 level can be used as a reference index to judge the benign and malignant lung masses, and lymph node Beclin 1 level can be used as an important reference index to help determine whether there is lymph node metastasis in lung cancer.
Objective To investigate the expression of ATP-binding cassette superfamily G member 2 （ABCG2） in liver cancer cell lines and the relationship between ABCG2 expression and liver cancer drug resistance，and observe the difference of function between the ABCG2 positive cells and negative cells．Methods The expressions of ABCG2 in four liver cancer cell lines （PLC/PRF/5，7402，7701，and 7721） were detected by flow cytometry．IC50 of 5-fluorouracil （5-FU） and adriamycin were calculated．The expression of the ABCG2 in the 7721 cell lines was observed by immunofluorescence staining．The ABCG2 positive and negative cells were selected by the axenic flow sorting method，the difference of function between the ABCG2 positive cells and negative cells was compared．Results The positive expression rate of ABCG2 in the cell line 7721 was highest among four liver cancer cell lines（P＜0.05），and the ABCG2 positive and negative cells had clear bimodal．IC50 of 5-FU and adriamycin to the cell line 7721 were higher than those of the other three cell lines （P＜0.05） except for 5-FU to the cell line 7701．There was no difference in cell proliferation between ABCG2 positive cells and negative cells．Cell cycle analysis showed that the ABCG2 positive cells had more quiescent cells as compared with the negative cells（P＜0.05）．Conclusions ABCG2 expresses in a variety human liver cancer cell lines，ABCG2 positive cells have some stem cell features like drug resistance and more quiescent cells comparing with the negative cells．
ObjectiveTo investigate the regulatory role of DAB2IP in proliferation effect of colon cancer cells by salinomycin. MethodsCell counting kit 8 (CCK8) assay was used to detect median inhibitory concentration (IC50) of salinomycin on HT29 and SW480 cells. Colon cancer cells with stable knock-down of DAB2IP (HT29-shDAB2IP) and control cells (HT29-shcon) were constructed by lentivirus plasmid. And colon cancer cells with stable over-expression of DAB2IP (SW480-DAB2IP) and control cells (SW480-con) were constructed using pCI plasmid. The proliferation effect of salinomycin on stable knock-down or over-expression of DAB2IP by CCK8 assay in the colon cancer cell was identified. The colon cancer stem cells makers CD44, CD24, and CD133 were investigated using real-time PCR. ResultsThe salinomycin had obvious inhibitory effects on the proliferations of HT29 and SW480 cells, the IC50 value was 20.0 μmol/L and 10.0 μmol/L, respectively. The stable knock-down of DAB2IP could significantly enhance the inhibitory effect of salinomycin on the proliferation in HT29 cells (P<0.05) and stable over-expression of DAB2IP could significantly decrease the inhibitory effect of salinomycin on the proliferation in SW480 cells (P<0.05). Further the result of real-time PCR detection showed that the expressions of cancer stem cells markers CD44, D24, and CD133 were significantly increased after stable knock-down of DAB2IP in the HT29 cells (P<0.05), while the expressions were significantly decreased after stable over-expression of DAB2IP in the SW480 cells (P<0.05). ConclusionsFrom initial results of this study, salinomycin could suppress proliferation of colon cancer cells. DAB2IP might weaken proliferative inhibitory effect of salinomycin by inhibiting expressions of cancer cells stem in colon cancer cells.
In recent years, with the improvement of the sensitivity of examination equipment and the change of people's living environment and diet, the rate of thyroid cancer has risen rapidly, which has increased nearly five folds in 10 years. The pathogenesis, clinical manifestation, biological behavior, treatment and prognosis of thyroid carcinoma of different pathological types are obviously different. Papillary thyroid carcinoma (PTC) can develop at any age, which accounts for about 90% of thyroid cancer. It progresses slowly and has favourable prognosis, but lymph node metastasis appears easily. Whether PTC is accompanied by lymph node metastasis has an important impact on its prognosis and outcome. The Raf murine sarcoma viral oncogene homolog B(BRAF)gene mutation plays a crucial role in PTC lymph node metastasis. Having an in-depth understanding of the specific role and mechanism of BRAF gene mutation in PTC is expected to provide new ideas for diagnosis and treatment of PTC.
ObjectiveTo investigate the effect of echinococcus granulosus protoscolices on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into fibroblasts.MethodsFemur bone marrow of 4-week-old C57BL/6 mice was taken and BMSCs were isolated and cultured by adherent culture. Echinococcus granulosus protoscolices was extracted from the liver of sheep infected with echinococcus granulosus. The experiment was divided into two groups. The experimental group was co-cultured with the 3rd generation BMSCs and the echinococcus granulosus protoscolices, and the control group was the 3rd generation BMSCs. Before and after co-culture, the morphology of BMSCs and the activity of echinococcus granulosus protoscolices were observed by inverted microscope. After cultured for 1, 3, 5, and 7 days, the mRNA expressions of transforming growth factor β1 (TGF-β1), collagen type Ⅰ, and collagen type Ⅲ were detected by real-time fluorescent quantitative PCR, the protein expressions of TGF-β1, collagen type Ⅰ, collagen type Ⅲ, Smad7, and phosphorylated Smad2/3 were detected by Western blot, and the contents of collagen type Ⅰ and collagen type Ⅲ in the supernatant of the two groups were detected by ELISA.ResultsAfter 7 days of co-culture, the morphology of BMSCs changed into fusiform and irregular triangle, which was closer to the mouse fibroblasts. The relative mRNA expressions of TGF-β1, collagen type Ⅰ, and collagen type Ⅲ in the experimental group were significantly higher than those in the control group; the relative protein expressions of TGF-β1, collagen type Ⅰ, collagen type Ⅲ, and phosphorylated Smad2/3 in the experimental group were significantly higher than those in the control group, and the relative protein expression of Smad7 in the experimental group was significantly lower than that in the control group; the contents of collagen type Ⅰ and collagen type Ⅲ in the supernatant of the experimental group were significantly higher than those in the control group. The differences between the two groups were significant (P<0.05).ConclusionEchinococcus granulosus protoscolices may promote the secretion of collagen type Ⅰ, collagen type Ⅲ, and TGF-β1 by TGF-β1/Smad signal pathway, which can promote the fibrosis of BMSCs that related to the formation of fibrocystic wall by echinococcosis.
Objective
To explore the paracrine effect of bone marrow mesenchymal stem cells (BMSCs) on dexamethasone-induced inhibition of osteoblast function in vitro.
Methods
The serum free conditioned medium of mouse BMSCs cultured for 24 hours was prepared for spare use. The 3rd passage of MC3T3-E1 cells were divided into 4 groups: the control group (group A), dexamethasone group (group B), dexamethasone+BMSCs conditioned medium (1:1) group (group C), and BMSCs conditioned medium group (group D). After 24 hours of culture, the alkaline phosphatase (ALP) content was determined; the protein expressions of RUNX2 and Osteocalcin were detected by Western blot; and the gene expressions of collagen type I-α 1 (COL1A1), RUNX2, ALP, and Osteocalcin were detected by real-time fluorescence quantitative PCR (RT-qPCR); alizarin red staining was used to observe calcium nodules formation at 21 days.
Results
After cultured for 24 hours, ALP content was significantly lower in groups B, C, and D than group A, and in group B than groups C and D (P < 0.05), but no significant difference was found between groups C and D (P > 0.05). The relative protein expression of RUNX2 of group B was significantly lower than that of groups A, C, and D (P < 0.05), but difference was not significant between groups A, C, and D (P > 0.05). The relative protein expression of Osteocalcin was significantly lower in group B than groups A, C, and D, in groups A and C than group D (P < 0.05), but difference had no significance between groups A and C (P > 0.05). The relative gene expressions of RUNX2, Osteocalcin, COL1A1, and ALP of groups B, C, and D were significantly lower than those of group A (P < 0.05); the relative gene expressions of RUNX2, Osteocalcin, and ALP were significantly higher in group D than groups B and C, in group C than group B (P < 0.05). The gene expression of COL1A1 was significantly higher in group D than group B (P < 0.05), but difference was not significant between groups B and C, and between groups C and D (P > 0.05). The cells of group A all died at 6 days after culture; at 21 days, the calcium no dule staining was positive by alizarin red in groups B, C and D, and the degree of the staining gradually increased from groups B to D.
Conclusion
BMSCs conditioned medium can alleviate the inhibitory effect of dexamethasone on osteoblasts function.
Regarding to the channel selection problem during the classification of electroencephalogram (EEG) signals, we proposed a novel method, Relief-SBS, in this paper. Firstly, the proposed method performed EEG channel selection by combining the principles of Relief and sequential backward selection (SBS) algorithms. And then correlation coefficient was used for classification of EEG signals. The selected channels that achieved optimal classification accuracy were considered as optimal channels. The data recorded from motor imagery task experiments were analyzed, and the results showed that the channels selected with our proposed method achieved excellent classification accuracy, and also outperformed other feature selection methods. In addition, the distribution of the optimal channels was proved to be consistent with the neurophysiological knowledge. This demonstrates the effectiveness of our method. It can be well concluded that our proposed method, Relief-SBS, provides a new way for channel selection.
