ObjectiveTo explore the effect of fetal bovine serum (FBS) of different concentrations in the culture medium on osteogenic growth peptide (OGP) promoting bone marrow mesenchymal stem cells (BMSCs) proliferation and differentiation. MethodsBMSCs were separated from limb bones of 8 Sprague Dawley rats (5 weeks old) and purified by adherence method, and BMSCs at passage 3 were divided into 4 groups according to OGP concentration: OGP 1×10-10 mol/L group, OGP 1×10-9mol/L group, OGP 1×10-8 mol/L group, and control group without OGP; and 0, 2%, 5%, 8%, and 10%FBS concentration gradient was used in each group. The cell proliferation rate was detected by MTT method at 1, 3, 5, 7, 9, and 12 days after culture, and the activity of intracellular alkaline phosphatase (ALP) was determined by the method of p-nitrophenyl phosphate disodium at 9 days after culture. ResultsBMSCs showed adherent growth, rapid proliferation, long fiber vortex, and typical morphology. MTT analysis showed that cells could not sustain proliferation when FBS concentration was less than 5% in each group; when FBS concentration was above 8%, cells proliferated continually. Proliferation promoting effect of OGP 1×10-8 mol/L and 1×10-9 mol/L groups was significantly higher than that of the control group in all serum concentrations (P<0.05); when FBS concentration was lower than 10%, the proliferation promoting effect of OGP 1×10-8 mol/L group was significantly higher than that of the other 2 OGP groups (P<0.05), but when FBS concentration was 10%, OGP 1×10-8 mol/L group had no advantage of promoting proliferation. ALP test results showed that as the FBS concentration increased, ALP activity of all groups also significantly increased (P<0.05). Under the condition of 5%FBS and 8%FBS, the ALP activity of each OGP group was significantly greater than that of the control group, and it was the highest in OGP 1×10-8 mol/L group (P<0.05). Under the condition of 10%FBS, the ALP activity of each OGP group was still greater than that of the control group (P<0.05), but no significant difference was found between the OGP 1×10-8 mol/L group and OGP 1×10-9 mol/L group (P>0.05). ConclusionThe concentration of 8%FBS is the best concentration of serum for OGP promoting the proliferation and differentiation of BMSCs, and the most suitable concentration of promoting the proliferation and differentiation of BMSCs is OGP 1×10-8 mol/L.
Objective To observe the effects of extracellular-signal regulated kinase (ERK) 1/2 inhibitor U0126 on hepatoma carcinoma cell proliferation and apoptosis. Methods Hepatoma SMMC-7721 cell strain was divided into blank control group and different concentrations of U0126 groups. The proliferation inhibition was measured by MTT assay. FCM was used to analyze the cell cycle distribution and apoptosis. Results U0126 obviously inhibited cell proliferation, induced cell apoptosis and G0/G1 phase cell cycle arrest. There were significant differences between control group and different concentrations of U0126 groups on cell proliferation and apoptosis （P＜0.05， P＜0.01）. Conclusion Blocking ERK1/2 pathway may be an important treatment strategy for liver cancer.
【Abstract】Objective To explore the clinical significance of β-catenin expression in pancreatic carcinoma.Methods The immunohistochemical staining was performed to detect the expression of β-catenin in the specimens of 46 patients with pancreatic carcinoma and the results were statistically analyzed.Results The abnormal expression rate on the membrane was 54.3%, the poorer the differentiation, the higher the abnormal expression rate. The levels of the cases in whom metastasis occurred were much higher than those without metastasis. The abnormal cytoplasm expression rate was 21.7%,which had not significant correlation with the clinical indexes, such as staging, tumor size, grading and metastasis. In 23 patients who accepted intervention chemotherapy before operation, the cytoplasm expression rate in those with tumor mass smaller was 0, which was evidently lower than that of those without tumor mass change (33.3%). Moreover, the abnormal membrane and cytoplasm expression rates had remarkable concordance (63.0%).Conclusion The abnormal membrane expression of β-catenin may accelerate metastasis, and the abnormal expression of β-catenin in cytoplasm may result in cell proliferation.
ObjectiveTo observe the effects of endovascular radiation (ER) on the proliferation and apoptosis of medial smooth muscle cells (SMC) and to discuss the possible mechanisms of radiation in the prevention of vascular restenosis (RS) in rabbits after carotid endarterectomy (CEA).MethodsForty rabbits undergoing CEA were randomly divided into four groups (each group=10) and given a radiation dose of 0, 10, 20 and 40 Gy 32P respectively. Rabbits were killed on the 3rd, 7th, 14th, 28th and 56th day after operation. The specimens were collected and histopathologic examinations were done.ResultsProliferation apparently occurred in the intima and media of carotid the lumen became narrow in the control group on the 14 th, 28 th and 56 th day after operation. While in the radiation groups, proliferation was apparently suppressed and the lumen was much less narrowed (P＜0.05). The apoptosis rate of SMCs and PCNA positive cells increased on the 3rd day after operation and reached the peak on the 7th day. There was statistical difference between the ER groups and control group (P＜0.01). The effects were much more evident in 20 Gy and 40 Gy groups compared with 10 Gy group (P＜0.01).ConclusionER may prevent RS by suppressing SMC proliferation and migration as well as inducing SMC apoptosis. The effects are positively correlated with radiation doses. SMC proliferation and apoptosis occur in the early period after balloon injury, while hyperplasia of intima and medial happens later.
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 assess the effects of Radix Salviae Miltiorrhizae (RSM) on patency and proliferation lesion of autologous vein to artery grafts in the earlymiddle stage.MethodsAutologous jugular vein was grafted into abdominal artery in the rats. The rats were divided into two groups: RSM group and control group. The rats in RSM group were fed with RSM ［24 g/(kg·d )］,which began 1 day before operation and continued until harvesting. Vein grafts were harvested at 1,3 days, 1, 2, 4 and 8 weeks after surgery for examining the patency, thickness of intimamedia and expression of proliferating cell nuclear antigen (PCNA). ResultsNo significant differences existed in patency of vein grafts between the two groups (Pgt;0.05). The intimamedia thickness of the vein grafts in RSM group decreased 1/3 compared with control group at 2, 4 and 8 weeks (P＜0.01). The PCNA positive cells in RSM group reduced significantly as compared to the control group (P＜0.01). ConclusionRSM can inhibit proliferation lesion of vein grafts but has no influence on patency of vein grafts in the earlymiddle stage.
Objective To study the relationship between early gastric cancer and MiB-1 expression. Methods Resected early gastric cancer from 99 cases were studied by immunohistochemical method. Results MiB-1 proliferation index (MiB-1 PI) was related to patient ’s age, tumor size, location and depth of invasion. MiB-1 PI was higher in differentiated adenocarcinomas than in undifferentiated adenocarcinomas(P＜0.01). Cancers with lymphatic vessel invasion or lymph node metastasis had higher MiB-1 PI (P＜0.05). The postoperative survival was related to MiB-1 proliferation grade (MiB-1 PG), being higher in MiB-1 PG 1,2 grade than that in MiB-1 PG 3,4 grade. Conclusion Even in the early stage of gastric cancer, tumor proliferation activity was related to patients’ prognosis.
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 extract and identify primary culture rat pulmonary arterial smooth cells ( PASMCs) , and investigate the effects of hypoxia on the proliferation of PASMCs. Methods Rat PASMCs were separated by the method of tissue block anchorage, and the cellular morphology was observed under light microscope. The cells were identified by projection electron microscopy, and α-smooth muscle actin ( α-SMactin)in the cells was identified by immunohistochemistry and immunofluorescence. The primary cultured PASMCs were exposed to normoxic and/ or hypoxia condition for 2, 6, 12, 24, 48 hours respectively, thenMTT assay and PCNA ( proliferating cell nuclear antigen) immunohistochemistry were used to detect the proliferation of PASMCs. Results The cells tended to be long spindle and grew in the “peak-valley”mode under light microscope. Immunology results showed that endochylema was stained in brownish yellow, and the positive rate was beyond 96% . There were dense patch, dense body and many filaments in endochylema under projection electron microscopy. MTT assay demonstrated that the A values of PASMCs expose to hypoxia were higher than that of nomoxia. Comparing with normoxia, the A values of PASMCs exposed to hypoxia increased after 12 hours ( P lt;0. 05) , significantly increased after 24 hours ( P lt;0. 01) . Compared with 2 hours’exposure to hypoxia, the A values increased after 12 hours( P lt; 0. 05) , markedly increased after 24 hours ( P lt; 0. 01 ) , which after 48 hours was similar with 24 hours. The result of PCNA immunohistochemistry was consistent with that of MTT. Conclusions The tissue explants adherent method is simple and convenient, and can easily obtain rat PASMCs with high purity and stability. Hypoxia canpromote the proliferation of PASMCs.
Objective To study the effects of the human keratinocyte growth factor 2 (hKGF-2) on the survival and differentiation of human neural stem cells (hNSCs). Methods The hNSCs at 17 passages preserved in liquid nitrogen were resuscitated and cultured for 7 days with normal methods to form neural spheres. The specific Nestin antigen and differentiated cells antigen were identified using immunohistochemistry technology. Some concentrated hNSCs were incubated in 12-well culture plate with 1 mL basic medium [(DMEM/F12 + N2 (1 ∶ 100) + epidermal growth factor (EGF) (20 ng/mL)] and divided into 7 groups, 6 wells each group. hKGF-2 (0, 10, 30, 60, 90, and 120 ng/mL) and bFGF (10 ng/mL) were added in groups A (control), B, C, D, E, F, and G, respectively. The neurospheres and the cell number were recorded for analyzing growth and multiplication of neural spheres. Some concentrated hNSCs were incubated in 6-well culture plate (cover glass coated with polylysine) with 3 mL DMEM/F12 medium and divided into 4 groups, 6 wells each group. N2 (1 ∶ 100), N2 (1 ∶ 100) + hKGF-2 (90 ng/mL), FBS (1 ∶ 20), and FBS (1 ∶ 20) + hKGF-2 (90 ng/mL) were added in groups A1, B1, C1, and D1, respectively. Then, the growth and multiplication of neural spheres were observed during culture; the separated neural spheres was identified and analyzed with indirect immunofluorescence and flow cytometry. Results Reanimated hNSCs could form neural spheres containing a lot of Nestin antigen; differentiated cells by induction expressed the specific antigens of neurofilament 200 (NF- 200) and glial fibrillary acidic protein (GFAP). At 7 days after culture, enlarged neural spheres were observed in each group. The neurospheres and the cell number of hNSCs increased with increased concentration of hKGF-2, showing a gradually increasing tendency; they were significantly higher in groups E, F, and G than that in groups A, B, C, and D (P lt; 0.05); significant differences were found among groups B, C, and D (P lt; 0.05), but no significant difference between groups A and B, and among groups E, F, and G (P gt; 0.05). After induction in vitro, the cell growth showed a progressive increase, significant difference was found among groups (P lt; 0.05); the percentage of NF-200 positive cells in group B1 was significantly higher than that in the other 3 groups (P lt; 0.05); the percentage of GFAP positive cells in group B1 was significantly lower than that in the other 3 groups (P lt; 0.05), but no significant difference among groups A1, C1, and D1 (P gt; 0.05). At 14 days after culture, cell growth reached the peak, which were mainly astero-cells. Conclusion The hNSCs are pure after incubated to 17 passages in vitro. hKGF-2 can promote the clone and the growth of differentiated cells, and increase the proportion of neuron.