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Objective:To investigate the regulatory effects and mechanism of mannose-binding lectin(MBL) on autophagy during the differentiation of 3T3-L1 adipocytes, and provide the feasibility for targeting autophagy to prevent obesity and related pathological conditions in natural immunity.Methods:3T3-L1 preadipocytes were cultured in vitro and induced to differentiation. Cell differentiation and lipid accumulation were analyzed by oil red O staining and CCK-8 was used to detect the effect of different concentrations of MBL (0, 1, 5, 10 μg/ml) on cell proliferation ability at different differentiation stages. Western blot was used to analyze the expression of MBL(10 μg/ml) on the key autophagy factors LC3B, Beclin1 and p62 protein at different stages of differentiation, and the changes of lipid droplet accumulation under the intervention of MBL were observed by oil red O staining. The protein and mRNA expression of autophagy key factors under the intervention of different concentrations of MBL were detected by Western blot and qRT-PCR. And autophagy flow analysis based on autophagic degradation was used to further illustrate the autophagic activity. The expression and phosphorylation of adenosine monophosphate-activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR) signaling molecules were analyzed by Western blot. Results:The results of oil red O staining showed that 3T3-L1 preadipocytes could achieve complete differentiation after 10 days of induction. CCK-8 showed that the concentration of MBL (1-10 μg/ml) in the experimental group had no effect on cell proliferation at different differentiation stages. During the differentiation of 3T3-L1 preadipocytes, Western blot and qRT-PCR showed that the expression of autophagy-related proteins and mRNA levels was enhanced in the MBL treated group, and presented a concentration-dependent relationship. Oil red O staining showed that the lipid droplets in adipocytes at different stages of differentiation are reduced to varying degrees under the intervention of MBL. Fluorescence microscopy results further confirmed that MBL enhanced the autophagy activity of adipocytes by increasing the synthesis of autophagosomes. Moreover, under the intervention of MBL, the phosphorylation level of AMPK was significantly up-regulated, while the phosphorylation level of mTOR was significantly down-regulated, also showing a concentration-dependent relationship.Conclusions:MBL accelerates the autophagy process during the differentiation of 3T3-L1 adipocytes through AMPK/mTOR signaling pathway, reduces lipid accumulation, providing a possible functional pathway for the treatment of obesity and related metabolic diseases.
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Objective:To investigate the regulatory effects and mechanism of mannan-binding lectin (MBL) on adipogenic differentiation of 3T3-L1 preadipocytes.Methods:3T3-L1 preadipocytes were induced to differentiate into adipocytes in vitro, and stimulated with different concentrations of MBL (0, 1, 10, 20 μg/ml). Firstly, changes in cell proliferation ability were detected by CCK-8. Then lipid accumulation was analyzed by Oil red O staining and intracellular triglyceride content determination. Further, the expression of adipogenic differentiation-related factors PPARγ and C/EBPα at protein and mRNA levels were detected by Western blot and qRT-PCR, respectively. Finally, Western blot was used to analyze the expression and phosphorylation of Akt, a signal molecule related to adipogenic differentiation. Results:MBL at the concentrations of 0, 1, 10 and 20 μg/ml had no effect on the proliferation of 3T3-L1 preadipocytes. The level of triglyceride in MBL treatment groups decreased in a dose-dependent manner on 3 d after 3T3-L1 preadipocyte differentiation. Results of Oil red O staining showed that the number of lipid droplets in MBL treatment groups reduced significantly, and the absorbance values also decreased significantly in a concentration-dependent manner. Western blot and qRT-PCR results showed that the expression of PPARγ and C/EBPα at both protein and mRNA levels in MBL treatment groups decreased significantly in a dose-dependent manner, and the phosphorylation level of Akt was significantly down-regulated as well.Conclusions:MBL regulates the adipogenic differentiation of 3T3-L1 preadipocytes via Akt signaling pathway.
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Objective To evaluate the clinical efficacy and adverse reactions of S-1 chemotherapy plus high-low oxygen radiotherapy synchronously in treatment of patients with locally advanced pancreatic cancer. Methods Sixty-four patients with locally advanced pancreatic cancer in Rizhao People's Hospital and the Affiliated Hospital of Qingdao University from January 2013 to October 2015 were randomly divided into study group and control group by using envelope method, in which the study group was treated with oral administration of S-1 plus high-low oxygen radiation synchronously and the control group with intravenous gemcitabine chemotherapy. The efficiency, disease control rate, clinical benefit rate, distant metastasis rate, survival rate and adverse reactions of the two groups were compared. Results The effective rates of the study group and the control group were 70.4 %(19/27)and 32.1 %(9/28),the difference was statistically significant (χ2=8.04,P<0.01), and the disease control rates were 88.9 % (24/27) and 67.9 % (19/28) (χ2= 3.56, P >0.05). The clinical benefit rates of the study group and the control group were 77.8 % (21/27) and 57.1 % (16/28) (χ2=2.66,P >0.05), and the distant metastasis rates were 63.0 %(17/27) and 71.4 %(20/28) (χ2=0.45, P > 0.05). The 1-year survival rates of the study group and the control group were 63.0 % (17/27) and 32.1 % (9/28), and the 2-year survival rates were 37.0 % (10/27) and 10.7 % (3/28), the differences were statistically significant (χ2= 5.24, P < 0.05; χ2= 5.28, P< 0.05). While there were no significant difference in the incidence of serious adverse reactions between the two groups (P> 0.05). Conclusion The treatment of locally advanced pancreatic cancer with S-1 plus high-low oxygen radiotherapy synchronously is better than that with intravenous gemcitabine chemotherapy in terms of effective rate, 1-year survival rate and 2-year survival rate,with no increase of adverse reactions.
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Objective To evaluate the efficacy of low dose 5-fluorouracil (5-Fu) combined with triamcinolone in the treatment of keloids, comparing with results with use of triamcinolone treatment alone.Methods Patient records from 2012 to 2013 were reviewed.45 patients (56 keloids) were completely randomized into 2 groups.Low dose of 5-Fu combined with triamcinolone were used to treat keloids in group A, while triamcinolone alone was used in group B.The therapeutic effects were evaluated by the certification of excellent remission, remission, inefficacy and total efficacy.The results were analyzed with statistics.Results In group A (27 keloids), the excellent remission cases were 16 (59.3%), the remission cases were 9 (33.3%), and the inefficacy cases were 2 (7.4%).The total percentage of efficacy in group A was 92.6%.In group B (29 keloids), the excellent remission cases were 9 (31.0%), the remission cases were 12 (41.4%), and the inefficacy cases were 8 (27.6%).The total therapy efficacy percentage of group B was 72.4%.Both the total percentage of efficacy and the excellent remission percentage in group A were apparently higher than those of group B, and the differences between the two groups were of statistical significance (P<0.05).The recurrence rates of group A were 3.7% (one case) while that of group B were 24.1% (7 cases).The differences between the two groups were also of statistical significance (P<0.05).Conclusions Low dose 5-Fu combined with triamcinolone is superior to intralesional triamcinolone therapy alone in the treatment of keloids.
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Objective To analysis the correlation of obstructive sleep apnea-hypopnea syndrome (OSAHS) and perihematoma edema of hypertensive cerebral hemorrhage.Methods One hundred and forty-four patients with hypertensive cerebral hemorrhage were collected and 78 of these patients were suffered from OSAHS.The patients were divided into two groups,control and OSAHS group,according to whether were accompanied by OSAHS or not.Both of the groups received the routine treatments including dehydration,reducing blood press,protecting the cerebral cells and so on.Cerebral CT scan was taken on admission.Night polymonography (PSG) was done within 24 hours of admission.Twenty-four hours and 4 days after admission,cerebral CT scan was taken again.The volumes of cephalophyma and perihematoma edema were calculated according to the results of CT scan.The changes of cephalophyma and perihematoma edema were dynamic observed.Results No difference in patients' age,sex,body mass index,serum glucose,blood lipid and so on,was observed between the two groups.The relative edema index became significantly different until 4 days after admission (0.40 ± 0.45,0.96 ± 1.35 in control and OSAHS group respectively,t =4.149,P =0.000).Similarly,the alternation edema index of OSAHS was obviously higher than that of control group only in 4 days after admission.While the analysis of the correlation between different degree OSAHS groups and edema indexes showed that at 24 hours after admission the edema volumes for different degree OSAHS groups were consistent (1.05 ± 0.65,0.84 ± 0.48,1.20 ± 0.54,1.10 ±0.40 in control,slight,moderate and severe groups respectively,F =1.061,P =0.374).At 24 hours and 4 days after admission,the edema volumes were positively correlated with the degree of OSAHS.Alternation edema index was significantly correlated with apnea hypopnea index according to the result of Pearson' s correlation analysis (r =0.652,P =0.000).Conclusion OSAHS complication can promote the progression of perihematoma edema of hypertensive cerebral hemorrhage,and the degree of edema aggravation is positive correlated to the degree of OSAHS.
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Objective To evaluate the efficacy and adverse effects of low doses gemcitabine chemotherapy combined with synchronous high-low oxygen radiotherapy in patients with locally advanced pancreatic cancer.Methods Fifty-six patients with locally advanced pancreatic cancer were randomly divided into two groups by envelop method:radio-chemotherapy group or chemotherapy group.Patients in radio-chmotherapy group were treated with low doses of gemcitabine chemotherapy ( 600 mg/m2 ) combined with high-low oxygen radiotherapy synchronously,paients in chmotherapy group were treated with full doses of gemcitabine chemotherapy ( 1000 mg/m2).The short-term effect,distant metastasis rate,clinical benefit rate,survival rate and adverse events of two groups were observed.Results There was one patient achievedcomplete relief and 15 patients achieved partial relief in radio-chemotherapy group with an overall effective rate of 66.7％ (16/24) ; there were 9 patients achieved partial relief in chemotherapy group with an overall effective rate of 36.0％ (9/25),the difference between the two groups was statistically significant (X2 =4.6082,P =0.0318 ).The clinical benefit rates were 83.3 ％ ( 20/24 ) and 60％ ( 15/25 ),respectively,the difference between the two groups was not statistically significant ( P =0.070).The distant metastasis rates were 66.7％(16/24) and 72％ (18/25),respectively,the difference between the two groups was not statistically significant (P =0.6855).The 12,24 months survival rates were 62.5％ vs 32％,37.5％ vs 12％,the difference between the two groups was statistically significant ( P =0.0325,0.0380).The incidence of serious adverse events was 45.8％ and 4 0 ％ without statistical difference.Conclusions Low doses of gemcitabine chemotherapy combined with high-low oxygen radiotherapy synchronously is better than full doses of gemcitabine chemotherapy with regard to total effective rates and 12,24 months survival rates,with no obvious increase in the incidence of serious adverse events.
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BACKGROUND:Recently,application of stem cells and growth factor to promoting lung regeneration in repair of emphysema lesion has been a hot focus in study.Thus,it is worth to pay attention on whether stem cells carrying relevant foreign growth factor gene can repair emphysema lesion.OBJECTIVE:To evaluate the effidency of adenovirus vector mediated green fluorescence protein(Ad-GFP)transfecting bone marrow mesenchymal stem cells(BMSCs)and its effect on the cell proliferation,to explore oriented migration of intravenously administrated BMSCs transfected with Ad-GFP in the lung tissues of pulmonary emphysema rats.METHODS:MSCs were separated and purified from the bone marrow of rats by density gradient centrifugation and by adherence.At different multiplicity of infection(MOI),transfection efficiency was observed by laser confocal microscopy.At 48 hours of transfection,MTT method was used to evaluate the proliferation of MSCs.A total of 16 Wistar rats were randomly divided into emphysema model group and control group(n=8).Model rats were established by exposure to cigarette smoke.MSCs,transfected with Ad-GFP,were grafted into the body of rats via tail vein.Lungs derived at 24 hours after implantation,and frozen sections were made.Migration and survival of MSCs in the lung tissues were observed by fluorescence microscopy.RESULTS AND CONCLUSION:MSCs from Wistar rats were successfully cultured,grew well and infected by Ad-GFP.The highest transfection effincincy(88.42 %)could be achieved at MOI of 200.Green fluorescent protein labeling had little effect on proliferation of MSCs by different MOI(P＞0.05).At 24 hours posttransplantation,the green fluorescence-positive tissue was Found in the lung tissues of emphysema model group and control group.Compared with control group,the expression of GFP in lung tissues was higher in emphysema model group(P＜0.05).These suggested that introduction of target gene cannot affect proliferation and homing property of BMSCs.
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After sequencing, we amplified and cloned foot-and-mouth disease virus (FMDV) O/QYYS/s/06 whole genome by three fragments. These three fragments were cloned into vector P43 one by one to construct recombinant plasmid P43C, which carried the full-length cDNA of FMDV O/QYYS/s/06. Then, plasmid P43C and plasmid T7 expressing T7 RNA polymerase were co-transfected into BHK-21 cells. After 48 h, we harvested the culture broth from transfected BHK-21 cells and inoculated into 2-3 day-old sucking mice. After four generation passage, the virus harvested from sucking mice was confirmed to be type O FMDV by the indirect hemagglutination test, sucking mice's neutralization test and sequencing. The results showed that we have successfully constructed the full-length cDNA clone of FMDV O/QYYS/s/06 strain.
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Animais , Camundongos , Animais Recém-Nascidos , Clonagem Molecular , DNA Complementar , Genética , DNA Viral , Genética , Febre Aftosa , Virologia , Vírus da Febre Aftosa , Classificação , Genética , Virulência , Transcrição Gênica , TransfecçãoRESUMO
Objective To evaluate the efficacy of inspiring carbogen and low concentration oxygen combined with late coupe accelerated hyperfraction radiotherapy on esophageal carcinoma.Methods 74 patients with esophageal carcinoma were randomly divided into two groups:the trial group and the centrol group,which consisted of 37 patients respectively.6MV X-ray was employed in the two groups.The schedule of the trial group was as following:conventional radiotherapy of 1.8～2.0Gy per day was employed during the first phase to a total dose of 38～40Gy,followed by late course accelerated hyperfraetion radiotherapy(twice fractions per day,interval between two fractions more than 6 hours,1.3～1.4Gy per fraction,middle total dose of 64.2Gy).Carbogen and low concentraetion oxygen was inspired during the course of radiation.Results 71 patients were enrolled.At the end of radiation at a total dose of 38～40Gy,complete remission rate(CRR)in the trial group was 31%,whereas that in the control group was 19%(P＞0.05),when the whole radiotherapy was finished,the CRR was 57%and 31%(P＜0.05)respectively,and one month after radiotherapy.the CRR was 71%and 33%respectively(P＜0.01).Six months after radiotherapy,the CRR WaLa 74%and 36%respectively(P＜0.01).Thelocal controlrate of sixmonthswas 91% and 72% respectively in the two groups(P＜0.05).Conclusions Inspiring carbogen and low concentration oxygen combined with late course accelerated hyperfraction radiotherapy may sigllificantly improve short-term efficacy and local control rate in esophageal carcinoma patients in Ⅰ～Ⅲstage.Furthermore,its side effects can be tolerated,and its elongating life time of patients may be prognostic.