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.
ObjectiveTo explore the immunosuppressive effect of XGD1 and its mechanism. MethodsDifferent concentrations of XGD1 were added to PHA or ConA induced human peripheral blood T lymphocyte.Seventytwo hours later modified MTT assay was employed to test the effect of XGD1 on T cell proliferation. Flowcytometry was used to examine the effect of XGD1 on the expression of IL2 receptor(IL2R) on the surface of T cells individually at 48 h and 72 h.And the effects of XGD1 combined with cyclosporine A(CsA) on the proliferation of Tlymphocytes and the expression of IL2R were also investigated. ResultsIn the concentration range of 0.2～25 μg/ml,XGD1 exerted marked inhibitory effect on PHA or ConA induced T cell proliferation,which was proportional to dose. Flowcytometry showed that XGD1 inhibited the expression of IL2R,and the percentage of IL2Rα positive cells after stimulation of PHA decreased from 47.67% to 25.03% in the presence of XGD1 (1 μg/ml).XGD1 and CsA had synergism in inhibition of T cell proliferation and IL2R expression. ConclusionThe study suggests that XGD1 has immunosuppressive effect. The suppressive effect of XGD1 on T cell proliferation is most probably mediated by decreasing IL2R expression.
Objective To explore the effects and mechanism of autonomic nervous control on the proliferation of human hepatocytes. And to examine the cellular localization of some related receptors expression in human hepatocytes. MethodsNorepinephrine (NE), and its agonist, antagonist, acetylcholine (Ach), and its antagonist have been added to human hepatocyte line L02 and hepatoma cell line Bel7402. Modified MTT assay was employed to test the effects of them on the proliferation of the two cell lines at 4 h, 24 h, 48 h and 72 h. Immuocytochemical staining was used to examine the cellular localization of alpha1Badrenoceptor (α1BAR), β2AR and epidermal growth factor receptor (EGFR) expression in human hepatocyte line L02. ResultsNE potentiated the proliferation of human hepatocyte and hepatoma cell, which was enhanced significantly with dose increased. The proliferative rate of 4 h were higher than that of the other time points (P＜0.05). There were no significant differences between the group of NE combined with propanolol and the group of NE alone. Metaproterenol had no significant effect. Ach significantly inhibited the proliferation of human hepatocyte. Its effect was enhanced with dose increased. Atropine significantly attenuated the inhibitory effect of Ach at 24 h and 48 h (P＜0.05). Scoline alone inhibited hepatocyte proliferation at 24 h, 48 h and 72 h (P＜0.05, P＜0.01). In immunocytochemical staining, there were positive responses to α1BAR, β2AR and EGFR in all cultures. It was observed that the responses to α1BAR, β2AR and EGFR were mainly both cytoplasmic and cell membrane localized. Conclusion NE, the sympathetic neurotransmitter, acts via α1BAR potentiate the proliferation of human hepatocyte and hepatoma cell in the presence of serum. Ach, the vagus neurotransmitter acting via mAchR and nAchR inhibits hepatocyte proliferation.
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.
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 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.
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.
Objective In vivo, the microenvironment of epidermal stem cells (ESCs) is complex, and estrogen might be involved in the micro environment. To investigate the biological effects of estrogen on the prol iferation and migration of ESCs in vitro. Methods hESCs were isolated from normal human foreskin and cultured. The second generation of hESCs were identified with flow cytometry after being marked with integrin β1, cytokeratin 19 (CK19), CK14, and CK10 antigens.hESCs of 2 × 106 cell density were cultured with ESCs special medium supplemented with 0.1 nmol/L Diethylstilbestrol in group A (estrogen group), with ESCs special medium supplemented with 10 nmol/L Raloxifene hydrochloride in group B (ER blocking agent group), and with ESCs special medium in group C (control group), respectively. The 100 μm “scratch” wounds were created by scraping confluent hESCs plated on Petri dishes with a sterile pipette tip in vitro. The migrating cells from the wound edge were quantified at 24, 48, and 72 hours after incubation. The rates of wound heal ing were calculated by SigmaScan Pro 5.0 software at 72 hours. The prol iferating effect of estrogen on hESCs was determined with MTT method at 24, 48, 72, 96, and 120 hours. Results Cultured primary hESCs could adhere to the wall showing ovoid in shape and grew into colonies. Flow cytometry showed the positive results for integrin β1, CK19, and CK14 (with positive rate of 96.63%, 95.47%, and 94.27%, respectively) and the negative result for CK10 (with positive rate of 1.32%). In group A, the number of cells crossing the wound edge was more than those of group B and group C at 24, 48, and 72 hours. The rates of wound heal ing were 69.00% ± 0.05% in group A, 35.00% ± 0.05% in group B, and 48.00% ± 0.06% in group C at 72 hours, showing significant differences among groups (P lt; 0.05). The prol iferating speed of hESCs was significantly higher in group A than in groups B and C (P lt; 0.01), and significantly higher in group C than in group B (P lt; 0.01) at 24, 48, 72, 96, and 120 hours. Conclusion The estrogen can promote the prol iferation and migration of hESCs in vitro. It may be involved in many biological activity of skin.
Objective To introduce the basic research and cl inical potential of the hair foll icle stem cells related signal transduction in prol iferation and differentiation. Methods The recent original articles about the hair foll icle stem cells were extensively reviewed. Results Many different signal pathways had been involved in the skin development and self-newals.The hair foll icle stem cells could play an important role in the skin self-renewal and regeneration which were modulated by several different signal pathways, which included bone morphogenetic protein/transforming growth factor β, Wnt, Notch and ectodysplasin A genes. Conclusion The hair foll icle stem cells may be a future approach to repair cutaneous wounds as a cell therapy．