Objective To explore the effects of bile from patients with cholecystolithiasis on the growth of human gallbladder carcinoma cells GBC-SD and the potential correlation between cholecystolithiasis and gallbladder carcinoma. Methods Cholecystolithiasis bile (CB) and normal bile (NB) specimens were used for this study. The proliferative effects of bile were measured by methabenzthiazuron (MTT) assay and cell cycle and apoptosis were analyzed by flow cytometry. Results CB can significantly promote the proliferation of GBC-SD cells, GBC-SD proliferative index increased significantly after treated with 1% CB for 48 h （P＜0.05）.The Sphase fraction of CB 〔（49.26±8.07）%〕 increased remarkably (P＜0.05) compared with that of NB 〔（25.54±6.57）%〕, and the CB percentage of G0/G1 phase 〔（40.59±9.12）%〕 decreased remarkably (P＜0.05) compared with NB 〔(60.64±13.42）〕%. Conclusion CB can promote the proliferation of human gallbladder carcinoma GBC-SD cells.
Abstract： Objective To construct a nesprin-siRNA lentiviral vector（LV-siNesprin）, transfect it into bone marrow mesenchymal stem cells （MSCs）, and observe morphology changes of MSCs. Methods According to the target gene sequence of nesprin, we designed and synthesized four pairs of miRNA oligo, which were then annealed into double-strand DNA and identified by sequencing. MiRNA interference with the four kinds of plasmids （SR-1,SR-2,SR-3, andSR-4） were transfected into rat vascular smooth muscle cells, and reverse transcriptase chain reaction(RT-PCR) and Western blotting were performed to detect the interference effects and filter out the most effective interference sequence. We used the best interference sequence carriers and pDONR221 to react together to get the entry vectors with interference sequence. Then the objective carrier pLenti6/V5-DEST expressing both entry vectors and lentiviral vectors was restructured to get lentiviral expression vector containing interference sequence （LV-siNesprin＋green fluoresent protein （GFP））, which was packaged and the virus titer was determined. LV-siNesprin＋GFP was transfected to MSCs, and the expression of nesprin protein（LV-siNesprin＋GFP group，GFP control group and normal cell group）was detected by Western blotting. The morphology of MSCs nuclear was observed by 4’,6-diamidino-2-phenylindole （DAPI） stain. The proliferation of MSCs （LV-siNesprin＋GFP group，GFP control group and normal group） was detected by 3-(4,5-dimethylthia- zol-2-yl)-2,5-diphenyltetrazolium bromide（MTT） after lentivirus transfected to MSCs at 24, 48, 72, and 96 hours. Results The four pairs of miRNA oligo were confirmed by sequencing. Successful construction of LV-siNesprin was confirmed by sequencing. The best interference with miRNA plasmid selected by RT-PCR and Western blotting was SR-3. Lentiviral was packaged, and the activity of the virus titer of the concentrated suspension was 1×106 ifu/ml. After MSCs were transfected with LV-siNesprin, nesprin protein expression significantly decreased, and the nuclear morphology also changed including fusion and fragmentation. The proliferation rate of MSCs in the LV-siNesprin＋GFP group was significantly slower than that of the GFP control and normal cell groups by MTT. Conclusion Nesprin protein plays an important role in stabilizing MSCs nuclear membrane, maintaining spatial structure of MSCs nuclear membrane，and facilitating MSCs proliferation.
Objective To investigate the effect of carboxymethylated chitosan (CMCS) on the proliferation, cell cycle, and secretion of neurotrophic factors in cultured Schwann cells (SCs). Methods SCs were obtained from sciatic nerves of 20 Sprague Dawley rats (3-5 days old; male or female; weighing, 25-30 g) and cultured in vitro, SCs were identified and purified by immunofluorescence against S-100. The cell counting kit 8 (CCK-8) assay was used to determine the proliferation of SCs. The SCs were divided into 4 groups: 50 μg/mL CMCS (group B), 100 μg/mL CMCS (group C), 200 μg/mL CMCS (group D), and the same amount of PBS (group A) were added. The flow cytometry was used to analyze the cell cycle of SCs; the real-time quantitative PCR and Western blot analysis were used to detect the levels of never growth factor (NGF) and ciliary neurotrophic factor (CNTF) in cultured SCs induced by CMCS. Results The purity of cultured SCs was more than 90% by immunofluorescence against S-100; the CCK-8 results indicated that CMCS in concentrations of 10-1 000 μg/mL could promote the proliferation of SCs, especially in concentrations of 200 and 500 μg/mL (P lt; 0.01), but no significant difference was found between 200 and 500 μg/mL (P gt; 0.05). CMCS at a concentration of 200 μg/mL for 24 hours induced the highest proliferation, showing significant difference when compared with that at 0 hour (P lt; 0.01). The percentage of cells in phase S and the proliferation index were significantly higher in groups B, C, and D than in group A (P lt; 0.05), in groups C and D than in group B (P lt; 0.05); and there was no significant difference between group C and group D (P gt; 0.05). Real-time quantitative PCR and Western blot results showed that the levels of NGF and CNTF in groups B, C, and D were significantly higher than those in group A (P lt; 0.05), especially in group D. Conclusion CMCS can stimulate the proliferation, and induce the synthesis of neurotrophic factors in cultured SCs.
Objective To evaluate the effect of Schwann cell (SC) on the proliferation of marrow mesenchymal stem cells (MSCs) and provide evidence for application of SC in construction of the tissue engineered vessels.Methods SC and MSCs were harvested from SD rats(weight 40 g). SC were verified immunohstochemically by the S-100 staining, and MSCs were verified by CD 44, CD 105, CD 34 and CD 45. The 3rd passages of both the cells were cocultured in the Transwell system and were amounted by the 3H-TDR integration technique at 1, 3, 5 and 7 days,respectively. The results were expressed by the CPM(counts per minute, CPM) values. However, MSCs on both the layers were served as the controls. The Westernblot was performed to assess the expression of the vascular endothelial growth factor (VEGF), its receptor Flk-1, and the associated receptor neuropilin 1(NRP-1) in SC, the trial cells, and the controls. Results SC had a spindle shape in the flasks, and more than 90% of SC had a positive reaction for the S-100 staining.MSCs expressed CD44 and CD105, and had a negativesignal in CD 34 and CD 45. The CPM values of MSCs in the trial groups were 2 411.00±270.84，3 016.17±241.57，6 570.83±2 848.27 and 6 375.8±1 431.28at 1, 3, 5 and 7 days, respectively. They were significantly higher in their values than the control group (2 142.17±531.63，2 603.33±389.64，2 707.50±328.55，2 389.00±908.01), especially at 5 days （P＜0.05）. The Western blot indicated that VEGF was expressedobviously in both the SC group and the cocultured MSCs grou,p and was less visible in the control cells. The expressions of Flk-1 and NRP-1 inthe cocultured MSCs were much ber than in the controls. Conclusion SC can significantly promote the proliferation of MSCs when they are cocultured. The peak time of the proliferation effect appeared at 5 days. This effect may be triggered by the up-regulation of VEGF in MSCs, which also leads to the upregulation of Flk-1 and NRP-1 .
Objective To observe the effects of keratinocytes on proliferation and collagen secretion of fibroblasts. Methods The conditioned medium,collected from cultured keratinocytes, was added to the cultured fibroblasts as the tested groups(12.5％, 25% and 50％ groups) and DMEM as control group. The MTT, hydroxyproline coloricmetric method and flow cytometer were employed to measure the fibroblast proliferation, the collagen secretion andthe change of the cell cycle.Results In fibroblast proliferation, the absorbency(A) value of tested groups was significantly different from that of the control group (P＜0.01). A value increased as increasing concentration, there was statistically significant difference betweetheconcentrations of 25%,50% and the concentration of 12.5%(P＜0.01), but no statistically significant difference between the concentrations of 25% and 50%(P＞0.01). In collagen secretion, there was no statistically significant difference between the tested groups and the control group(P＞0.01), and between the tested groups(P＞0.01). In cell cycle, 50% of conditioned medium could make the fibroblast pass the limit of G1/S and S/G2 period, the cell rates of S,G2-M period increased. Conclusion The conditioned medium from keratinocytes can increase fibroblasts proliferation, have little effect on general collagen secretion.
OBJECTIVE: To investigate the effects of Ginsenoside Rb1 on the proliferation of Schwann cell cultured. METHODS: The sciatic nerve from SD rats was cultured in vitro; 10 micrograms/ml, 20 micrograms/ml, 200 micrograms/ml and 1 mg/ml Ginsenoside Rb1 was applied on the fifth day of culture. The proliferation of Schwann cells of sciatic nerves was determined in different time by MTT assay and thymidine incorporation assay. RESULTS: 10 micrograms/ml of Ginsenoside Rb1 significantly induced Schwann cell proliferation better than DMEM cell culture medium, but higher concentrations of Ginsenoside Rb1 at 1 mg/ml significantly inhibited the proliferation of Schwann cells, whereas 200 micrograms/ml of Ginsenoside Rb1 had similar effects to DMEM culture medium. CONCLUSION: Ginsenoside Rb1 at the optimal concentration is effective on inducing the proliferation of Schwann cells, but at higher concentration is cytotoxic for Schwann cells.
It was reported in this article that a preparation of acid/heat-stable peptides (AHSP) from pig serum with a molecular-weight less than 18 ku a without antigenity and toxicity could exert enhancement effect on wound healing. Two pieces of polyvinyl alcohol (PVA) sponge implanted in rat dorsal subcutaneous pouchs of 20 mice were selected as the wound model. The subcutaneous pouch having one piece of sponge was taken as the experimental group and the other as the control. Injection of 50 microliters of such peptide preparation into the test sponge was performed once a day from the time of injury on for 5 consecutive times, while 50 microliters of BSA (5 mg/ml) into the control sponge in the same way. The levels of total DNA, protein and hydroproline in AHSP-treated sponge were observed significantly higher than those in the control sponge on the 7th and 10th days after wounding (P lt; 0.05). No significant difference was seen on the 14th postinjury day (P gt; 0.05). The effect of AHSP on proliferation of wound fibroblast cultured in vitro was also detected. In conclusion, such peptides derived from pig serum had the activity to accelerate wound healing without resultant excessive healing and its direct stimulation of the proliferation of wound fibroblast was probably one of the way which AHSP exerted its action.
Objective To investigate the effect of berbamine (BBM) on the proliferation and apoptosis of retinoblastoma (RB) HXO-RB44 cells and its possible mechanism in vitro.Methods RB cells in logarithmic growth phase were divided into BBM treated group and control group. RB cells in BBM treated group were cultured with different concentrations of BBM (2,4,8,16 and 32 mg/L) for 24,48 and 72 hours, respectively. The proliferation was assayed by methyl Thiazolyl tetrazolium (MTT). RB cells were cultured with different concentrations of BBM (4,8 and 16 mg/L) for 24 hours. The early apoptotic rates were detected by flow cytometry; the expression of bcl-2 and Bax were measured by enzyme-linked immunosorbent assay (ELISA) and the activity of Caspase-3 was detected by colorimetric assay.Results BBM could obviously inhibit the proliferation of RB cells in a time and dose dependent manner (24 hours: F=70.547,P＜0.01; 48 hours: F=603.438,P＜0.01; 72 hours: F=577.521,P＜0.01). The IC50 value at 24,48 and 72 hours were 25.26, 10.94 and 6.25 mg/L, respectively. Necrosis rates of control group and BBM treated group were (1.25plusmn;0.45)%, (4.10plusmn;2.95)%, (4.39plusmn;0.21)% and (10.54plusmn;4.38)% respectively; the difference between two groups was statistically significant (F=6.527,P＜0.05). Apoptotic and necrosis rates in advanced stage of control group and BBM treated group were (2.13plusmn;0.71)%, (5.45plusmn;2.31)%, (9.86plusmn;3.18)% and (11.10plusmn;1.70)%, respectively. The difference between two groups was statistically significant (F=10.845,P＜0.05). Early apoptotic rates of control group and BBM treated group were (0.51plusmn;0.26)%, (2.68plusmn;0.35)%, (5.97plusmn;0.50)% and (11.22plusmn;1.17)%, respectively. The difference between two groups was statistically significant (F=144.976,P＜0.01). In addition, BBM dose-dependently reduced bcl-2 level and increased Bax expression, causing the reduction of the bcl-2/Bax protein ratio as well as increased the Caspase-3 activity in RB cells remarkably (bcl-2: F=835.726,P＜0.01; bax: F=111.963, P＜0.01;Caspase-3:F=298.058,P＜0.01).Conclusions BBM can inhibit the proliferation and induce apoptosis or necrosis of RB cells in vitro, down regulating the expression of bcl-2, up regulating the expression of Bax. Along with increased Caspase-3 activity these may be the apoptotic mechanisms.
Objective To observe the influence of the indomethacin on the proliferative and invasive activity of OCM-1 human choroidal melanoma cells. Methods OCM-1 cells were cultured with different concentrations of indomethacin (25, 50, 100, 200, 400 mu;mol/L ), and their proliferation were assessed by methyl thiazolyl tetrazolium（MTT）, invasive behaviors were examined by cell invasion assays, expression of survivin and VEGF were evaluated by reverse transcriptase polymerase chain reaction（RT-PCR）, immunofluorescence staining, ELISA and western blot analysis. Result All concentrations of indomethacin in this study can inhibit the proliferation and invasion of OCM-1 cells in a time and dosage-dependant manner（MTT/24 h：F=19.642，P＜0.01；MTT/48 h：F=136.597，P＜0.01；MTT/72 h：F=582.543，P＜0.01；invasion assays：F=54.225，P＜0.01）. Immunofluorescence staining indicated that survivin and VEGF mainly expressed in the cytoplasm of OCM-1 cells. Survivin mRNA in OCM-1 cells was inhibited by 100, 200, 400 mu;mol/L indomethacin（F=16.679，P＜0.01）. The concentrations of survivin were (787.3plusmn;47.37), (257.0plusmn;26.21), (123.3plusmn;8.02) pg/ml in control group and 100, 400 mu;mol/L indomethacin groups, respectively. Survivin expression was also significantly down-regulated in indomethacin-treated cells by Western blot analysis.Indomethacin had no effects on VEGF expression in OCM-1 cells.Conclusions Indomethacin can inhibit proliferation and invasion of OCM-1 cells in vitro,down-regulated expression of survivin may be the mechanism.
Objective To investigate the influence of vascular endothelial growth factor (VEGF) antagonist bevacizumab on the growth of human choroidal melanoma (CM) OCM-1 cell xenografts in nude mice, and to explore the probable mechanism.Methods OCM-1 cells were subcutaneously implanted on 18 nude mice to establish ectopic model of human CM. The nude mice with the tumor of 5 mm in diameter were randomly divided into three groups: untreated group (group A), normal saline （NS） group (group B), drug treated group (group C). Bevacizumab was intraperitoneally injected for 14 consecutive days in group C, and the same volume of NS was used at a same way in group B. The volume and weight of implanted tumor as well as inhibitory rates of drug on tumor were calculated, ki67 and survivin proteins were measured with immunohistochemistry, and the mRNA expression of VEGF and survivin were assessed by RT-PCR.Results The volume and weight of tumor was (598.86plusmn;321.81) mm3, (0.66plusmn;0.15) g; (1 715.15plusmn;278.16) mm3, (1.54plusmn;0.39) g and (1 750.23plusmn;206.36) mm3, (1.54plusmn;0.31) g in groups C, A and B, respectively. There were significant differences between group C and A (F=34.53, P=0.00) and group C and group B (F=8.69, P=0.01). The inhibitory rate of these three groups were 57.14%, 5.31%, 6.25%, respectively, and the proliferation index (PI) of ki67 in these three groups were (51.85plusmn;1.32)%, (46.30plusmn;1.39)%, (27.90plusmn;0.90)%, respectively, there were significant differences in ki67 PI between C group and A or B group (H=15.17, P=0.00). The expression of survivin mRNA was (0.49plusmn;0.02), (0.82plusmn;0.05) and (0.61plusmn;0.05) in groupss C, A and B, respectively, there were significant differences between C group and A or B group (F=15.17, P＜0.05) . The expression of VEGF mRNA was (0.32plusmn;0.08), (0.73plusmn;0.07), (0.80plusmn;0.04) in groups C, A and B, significant difference was found between group C and A or B group (F=12.05，P＜0.05). Conclusion Bevacizumab can inhibit the growth of human CM in nude mice probably by inhibiting the activity of VEGF and downregulating survivin expression of the tumor as well as inhibiting the growth of the tumor.