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BACKGROUND:The use of mesenchymal stem cels in the field of tissue engineering for osteoarticular injury repair is a very promising tool since these cels are readily expandable and able to differentiate into chondrocytes. Abundant evidence suggests that microRNAs play critical roles in chondrogenic differentiation of mesenchymal stem cels. OBJECTIVE:To observe the chondrogenic effect of human bone marrow mesenchymal stem cels transfected with lentiviral vectors bearing miR-221-3p/222-3p inhibition, thereby provding new strategies for cartilage injury. METHODS: miRNA microarray technology was applied to detect microRNAs expression profiles at three different stages of chondrogenic differentiation induction after transforming growth factor-β3 treatment and verified by real-time fluorescence quantitative PCR (RT-qPCR). Human bone marrow mesenchymal stem cels were infected with lentivirus bearing miR-221-3p/222-3p inhibition. After co-suppressing the expression of miR-221/222-3p, cel counting kit-8 was used to determine the cel proliferation, the differentiation of bone marrow mesenchymal stem cels towards chondrocytes was verified by type II colagen protein expression through immunohistochemistry and glycosaminoglycan accumulation was also elevated by sarranine O staining. RT-PCR was used to detect type II colagen and aggrecan mRNA expression at 21 days of chondrogenic induction. RESULTS AND CONCLUSION: The expression of miR-221-3p/222-3p was inhibited after Lv-miR221-3p/222-3p inhibition co-transfected into bone marrow mesenchymal stem cels. microRNA microarray and RT-qPCR results showed that the expression of miR-221-3p/222-3p was declined significantly at the anaphase of chondrogenic differentiation. The expression levels of chondrogenic markers, Aggrecan and type II colagen were significantly increased in the miR-221-3p/222-3p inhibition group and cel proliferation was also inhibited significantly compared with non-transduced cels or transduced with the empty lentiviral vector group. miR-221-3p/222-3p knockdown in bone marrow mesenchymal stem cels could inhibit proliferation but promote chondrogenic differentiation of bone marrow mesenchymal stem cels.
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OBJECTIVE: Cisplatin is a widely used chemotherapeutic agent in the treatment of cancers in clinic; but it often induces adverse effects on ovarian functions such as reduced fertility and premature menopause. Mesna could attenuate the cisplatin-induced ovarian damages; however, the underlying mechanism is still unknown. This study aimed to figure out the underlying mechanism of the protection of mesna for ovaries against cisplatin therapy in cancers. METHODS: We performed female adult Sprague-Dawley rats into normal saline control (NS), low-dose cisplatin (CL), high-dose cisplatin (CH), CL plus mesna (CL+M), and CH plus mesna (CH+M) groups and detected anti-Mullerian hormone (AMH)-positive follicle, oxidative stress status and anti-oxidative capability in ovaries. RESULTS: AMH-positive follicles were significantly decreased after cisplatin administration, which was significantly reversed when mesna was co-administered with cisplatin. The end product of lipid peroxidation, malondialdehyde (MDA), was significantly increased, but the anti-oxidative enzymatic activity of superoxide dismutase (SOD) and glutathione (GSH) were significantly decreased in cisplatin groups when compared with NS group. In contrast, after co-administration of cisplatin with mesna, MDA was significantly decreased whereas the activity of SOD and the concentration of GSH were increased. Moreover, mesna did not decrease the anti-tumor property of cisplatin in HePG2 cell lines. CONCLUSION: Cisplatin damages the granulosa cells by oxidative stress to deplete the ovarian reserve and mesna could protect ovarian reserve through anti-oxidation. These results might highlight the mechanism of the protection of mesna for ovarian reserve and open an avenue for the application of mesna as a protective additive in cisplatin chemotherapy in clinical practise.
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Adulto , Animais , Feminino , Humanos , Ratos , Hormônio Antimülleriano , Cisplatino , Fertilidade , Glutationa , Células da Granulosa , Células Hep G2 , Peroxidação de Lipídeos , Malondialdeído , Menopausa Precoce , Mesna , Ovário , Estresse Oxidativo , Ratos Sprague-Dawley , Superóxido DismutaseRESUMO
Objective To study the associations between neo-adjuvant chemotherapy (NACT)sensitivity and expression of relative proliferation and apoptosis genes in cervical cancer tissues. Furthermore,the potential roles of relative genes expression as monitor in the chemotherapy against cervical cancer tissues were studied. Methods Fifteen pathologically proved Ⅱ A stage cervical cancer patients with HPV 16 positive were divided into effective group and non-effective group according to the clinical response to NACT, the changes of HPV16-E6, p53 genes and proteins expressions were measured by real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot. At the same time, MTT assay was used to detected proliferative activity to paclitaxel liposome and carboplatin (paraplatin) in HeLa cells. Results In effective group, the overall response rate was 73.3 % (11/15) and the HPV16-E6 mRNA and protein level after NACT was significantly decreased than that before NACT (t=3.359, t=3.614, P＜0.05), while p53mRNA and protein level increased significantly (t =5.852, t=2.838, P ＜0.05). However, there have no significant different between pre-NACT and Post-NACT of the two genes in the non-effective group. After using chemotherapy HeLa cells growth decreased. Conclusion HPV16-E6 and p53 expreesion level are useful paraneters in evaluating NACT response in cervical cancer tissues. The detection of the two genes expression is a method for predicting efficacy in NACT.
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BACKGROUND: Group pre-test has confirmed that amnion endothelial cell conditioned medium can induce human umbilical cord blood mesenchymal stem cells into dopaminergic neuron-like cells. In this process, neurotrophic factors and their receptors may play an important role. OBJECTIVE: To study the function of neurotrophic factors secreted by amniotic epithelial cells in the differentiation of human umbilical cord blood mesenchymal stem cells into neurons.METHODS: P1 human umbilical cord blood mesenchymal stem cells at 2×10~8 /L were incubated and assigned to 3 group. Control group was added with HG-DMEM medium. Induction group received human amniotic epithelial cell medium. Blocking agent group underwent blocking agent K252a fluid, and the incubated was conducted at 36 ℃ for 40 minutes, and then amniotic epithelial cell medium was added. Immunofluorescence chemistry was used to determine neuron specific enolase and dopamine transporter expression in human umbilical cord blood mesenchymal stem cells. Real-time quantitative PCR was employed to detect neuron specific enolase, dopamine transporter and tyrosine hydroxylase expression in human umbilical cord blood mesenchymal stem cells. RESULTS AND CONCLUSION: Nerve growth factor and brain-derived neurotrophic factor were observed in human amniotic supernatant. P1 human umbilical cord blood mesenchymal stem cells expressed Trka and Trkb. Forty-eight hours following induction, compared with the control group, positive expression of neuron specific enolase and dopamine transporter was significantly increased in the induction and blocking agent groups (P < 0.05), especially in the induction group (P < 0.05). Neuron specific enolase, dopamine transporter and tyrosine hydroxylase mRNA levels were significantly greater in the induction and blocking agent groups compared with the control group (P < 0.01), and each gene mRNA levels were significantly greater in the induction group than in the blocking agent group (P < 0.01). Results verified that neurotrophic factor in the human amniotic epithelial cells plays important effects on differentiation of human umbilical cord blood mesenchymal stem cells into neurons. The promotion effects are mediated by activating Trk receptor.
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BACKGROUND: Recent trials and clinical studies have shown that intracoronary transplantation of bone marrow-derived mesenchymal stem cells (MSCs) improves cardiac function following acute myocardial infarction (AMI). However, whether homing of MSCs into the infarcted myocardium or not is still unknown.OBJECTIVE: To study the homing of MSCs intracoronary administration in porcine myocardial infarction model using in vivo magnetic resonance imaging tracking.METHODS: Porcine MSCs were isolated and cultured by the whole bone marrow method. Following labeling by superparamagnetic iron oxide (SPIO), MSCs were treated with trypsinization to adjust the concentration at 10~(10)/L. Myocardial infarction was induced in all 10 pigs. At one week after modeling, the labeled MSCs were delivered via intracoronary infusion with standard over-the-wire (OTW) balloon angioplasty catheters. Prussian blue staining was used to evaluate labeling efficiency, and double echo steady state was used to scan four-chamber and cor biloculare at long axis view, which was considered as locating phase to obtain image of left ventricle at short axis view. RESULTS AND CONCLUSION: MSCs could be efficiently and safely labeled with SPIO. Intracoronary transplantation of MSCs is able to home the sites of myocardial injury and the border between infarcted and normal tissue. MRI can track SPIO-labeled MSCs delivered through intracoronary and were confirmed on pathology. After 5 weeks the injected labeled cells could still be detected with MRI.
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Objective To compare the sensitivities of the stimulation effect of human cord blood mesenchymal stem cells(CB-MSCs) and CB-MSCs of neuronal differentiation to lymphocytes(LCs) detected with carboxyfluorescein didcetate(CFSE) and MTT. Methods To prepare LCs from SD rat and divided into four group stimulating cells: 1. CB-MSCs;2. Dif-CB-HSCs;3. SH-SY5Y(positive control);4. Auto-LC(negative control).Stimulating cells were respectively Co-cultured with LCs. The proliferation of LCs was detected with MTT and CFSE ( n =3). Results CB-MSCs and Dif-CB-HSCs stimulated LCs to proliferate more weakly than positive control detected with MTT and CFSE. The quantity of proliferation of lymphocytes Co-cultured with CB-MSCs and Dif-CB-HSCs were higher than that of Auto-LC detected with CFSE. But MTT OD value of CB-MSCs and Dif-CB-HSCs was a little lower than that of Auto-LC. Statistical analysis results showed no significant difference.Conclusion CFSE can reflect proliferation status of lymphocytes better than MTT. CFSE shows more advantages in practical use.
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Background and purpose: Osteopontin (OPN) is a glycophosphoprotein that is expressed by numerous human cancer cells. The function of OPN in skeletal modeling and remodeling, bone resorption, angiogenesis and tumor cell metastasis and progression through binding with integrin and CD44 receptors were studied. Our purpose of the study was to detect the level of osteopontin(OPN) and CD44 variant isoforms(CD44v6) in multiple myeloma (MM) patients, and to explore the relationship between OPN and CD44v6 with the progress of MM. Methods: 32 MM patients were admitted to our hospital from Sep. 2007 to Dec. 2008. The patients were divided into two groups, group A (untreated and relapsed MM patients) and B (stable MM patients), and the control group including 15 subjects were the benign anemia patients or healthy people who suffered bone fracture. Bone marrow mononuclear cells (MNCs) and bone marrow stromal cells (BMSCs) from MM patients and subjects were investigated as potential OPN and CD44v6 producers. The level of OPN and CD44v6 of the conditioned media from MM patients and subjects were analyzed by ELISA. Results: The OPN level in group A (19 cases) was significantly higher than group B (13 cases) and control group (P<0.05). The CD44v6 level of 14 patients in group A was significantly higher than that of 10 cases in group B and control group (P<0.05); The OPN level of MM patients was correlated with the level of CD44v6 (r=0.52, P=0.000), the percentage of plasma cells in the bone marrow (r=0.74, P=0.000), M protein (r=0.53, P=0.014), and β2-microglubin (r=0.62, P=0.002). Conclusion: The increase of OPN and CD44v6 is associated with progress and pathogenesis of MM,and may be involved with tumor burden, stage and tumor invasion.
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BACKGROUND:In 1990s,overseas researchers use balloon occlusion method for establishing closed-chest animal models of myocardial infarction. But,ventricular fibrillation and thrombosis of intraoperative factors reduce the success rate of establishing the models. Currently,there are a few reports on establishing the large animal models. OBJECTIVE:We used balloon occlusion method for establishing closed-chest swine models of myocardial infarction,and explored ways to improve the success rate of modeling. DESIGN,TIME AND SETTING:The randomized controlled animal study of pathology observation was performed at the Department of Cardiology,First Affiliated Hospital of Kunming Medical College and Research Room of Pathology,Kunming Medical College from July 2008 to May 2009. MATERIALS:Fifteen Diannan small-ear pigs weighing 19-25 kg,aged 8-11 months,were divided into three groups:sham operation group,ischemia-reperfusion group,and ischemic postconditioning group,with 5 pigs in each group.METHODS:After the coronary occlusion and reperfusion period,the prophylactic use of lidocaine (1.0-2.0 mg/kg) infusion to control arrhythmia,and use of heparin to prevent and treat the thrombosis. A balloon catheter was positioned in the distal end of the first diagonal branch of the left anterior descending (LAD) artery under fluoroscopic guidance. In the sham operation group,the balloon was only placed to the LAD,did not block coronary artery. In the ischemia-reperfusion group,inflatable balloon occlusion was done for 60 minutes in the LAD after the balloon removed. In ischemic postconditioning group,after the balloon was inflated and occluded the LAD for 60 minutes,ischemic postconditioning was elicited by eight cycles of 30-second reperfusion,followed by 30-second reocclusion.MAIN OUTCOME MEASURES:Coronary angiography,electrocardiogram (ECG) and cardiac enzymes test was conducted to evaluate models of myocardial infarction. After three days,cardiac 2,3,5-triphenyltetrazolium chloride (TTC) staining and pathological examination was done to verily myocardial infarction.RESULTS:In the sham operation group,all pigs survived. In the ischemia-reperfusion group,4 pig models of myocardial infarction were successfully established,and one died of refractory ventricular fibrillation. In the ischemic postconditioning group,models of myocardial infarction after ischemia were successfully established. Following distal left anterior descending artery occlusion,the ECG leads V13 on the ST-segment elevation,the sick rational Q-wave formed;myocardial enzyme evolution of myocardial infarction in the human body was basically the same process. The site of myocardial infarction,basically the same parts,was located in apical,left ventricular anterior wall,and the former interval. TTC staining was normal myocardium brick red,myocardial infarct area appeared pale;pathological examination revealed a normal structure of myocardial infarct damage,cytoplasm condensed,dyeing deepening,transverse striations disappeared,nuclear enrichment,dissolution,fragmentation,many erythrocytes around the infarct area with abundant granulation tissue and a large infiltration of inflammatory cells.CONCLUSION:The described model presents a less invasion to the animals,and is the closest to the process of clinical practice.Intraoperative use of lidocaine and heparin for controlling arrhythmia and thrombosis of the models is successfully established as an effective manner. Ischemic postconditioning may be one of the factors for improving the modeling success rate.
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Objective To compare the renal effects of dopamine and dobutamine in patients with sepsis.Methods 90 patients with sepsis were admitted to this study.After resuscitation,each patient was randomly given different vasoactive agent.The changes in urine output,fractional excretion of sodium(FeNa),and creatinine clearance(CCr)were observed.Results The urine output and FeNa in dopamine group were increased significantly as compared with control group and dobutamine group[(3072±480),(2038±515)and(362±522)ml/24h,(3.80±1.09),(2.06±1.14)and(2.10±0.95)%](P＜0.05).Compared with control group and dopamine group,CCr increased significantly in dobutamine group[(79.2±39.1),(50.6±21.8)and(47.4±16.7)ml/min](P＜0.05).Conclusion Dopamine infusion markedly elevates urine output and FeNa,but has no effect on CCr.Dobutamine treatment misht significantly increase CCr,but has no effect on urine output.
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Objective To investigate the function of amnion endothelial cell in the differentiation of human umbilical cord blood stem cells into dopaminergic neurons.Methods Primary human amnion endothelial cells were separated and cultured in vitro;the conditioned medium(CM) was prepared through high speed centrifugation.The cord blood mesenchymal stem cells of P_1 passage were induced by the conditioned medium,and the mophology of cells was observed under the inverted phase contrast microscope.The expression of tyrosine hydroxylase(TH)and dopamine transportor(DAT) of the induced cord blood mesenchymal stem cells were detected by immunocytochemistry staining method and immunoblotting(Western blotting).Results The masculine rate of TH and DAT of the cord blood mesenchymal stem cells of P_1 passage which were induced by amnion endothelial cells conditioned medium was higher than that of the control group,with a significant difference(P
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Objective To investigate the effects of oestrogen-receptor(ER), progesterone-receptor(PR), vascular endothelial growth factor(VEGF) and kinase insert domaincontaining receptor(KDR) in uterine bleeding induced by intrauterine device(IUD). Methods Immunohistochemical method was used to detect the expressions of ER, PR, VEGF and KDR in endometrium from IUD bleeding group and normal control group, respectively. Microvessel density(MVD) was evaluated by using anti factor Ⅷ-related antigen (F8-RA) antibody to mark stromal microvessels. Results The expressions of ER, VEGF and KDR were significantly higher in IUD-induced endometrium bleeding group than those in control group(P0.05). MVD was significantly higher in IUD-induced endometrium bleeding than that in normal endometrium(P
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Objective To study the effects of amniotic epithelial cells conditioned medium on the differentiation of bone marrow stromal cells into neural cells. Methods Bone marrow stromal cells and amniotic epithelial cells were isolated and cultured in vitro,then the cell surface antigen was detected by flow cytometry and the expressions of nestin and ki67 were detected by immunofluorescence staining method.When the cells were co-cultured with amniotic epithelial cells conditioned medium,the morphological character of cells was observed by inverse phase-contrast microscope,and the expressions of NSE(neurone specific enolase),TH(tyrosine hydroxylase) and DAT(dopamine transporter) were detected by immunofluorescence staining method. Results Amniotic epithelial cells conditioned medium had obvious inductive effect on bone marrow stromal cell's neural differentiation.Conclusion The amniotic epithelial cells conditioned medium may have inductive effect neuron-like cell's differentiation and dopaminergic neuron-like cell's differentiation of bone marrow stromal cells in vitro.
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Objective To investigate the dynamic expression of nitric oxide synthase(NOS) in the apoptosis of primary cultured rat cortical neruons following hypoxia/reoxygenation(H/R) and the protective role of extract of ginkgo biloba(EGB). Methods The cortical neurons of E16-17 days fetal rat were primarily cultured.The apoptosis model of primary cultured cortical nurons following H/R was established by using W-G staning,electromicroscopy,TUNEL staining.The dynamic expression of NOS different H/R times was investigated with NADPH-diaphorase histochemical method. Results H/R can cause apoptosis of primary cultured rat cortical neurons.In the experiment of H-2R-0,H-4R-0, H-6R-0,H-8R-0 and H-2R 18,H-4R 18,H-6R 18 H-8R 18,the apoptosis cells occurred after 4 hour hypoxia.The increasing of apoptosis cell acted as time-dependence and the peak value was at H-8R 18.The expression of NOS increased both after 2 hour hypoxia and reoxygenation 18 hour after 8 hour hypoxia compared with the normal control group.EGB could inhibit the increasing and decrease the percentage of apoptosis.Conclusion The apoptosis of primary cultured rat cortical neurons could be induced by H/R.The increasing of NO might be one of the mechannisms of apoptosis.EGB could singnificantly inhibit the apoptosis by means of inhibiting the expression of NOS and reducing the production of NO.;
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GFAP.Conclusion RA is the best factor for neurons and astroglia,and RA+EGF+bFGF are the best for oligodendrocytes.