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Objective:To preliminarily evaluate the effect of tension stimulation on the biological activity of and expression of fibrosis marker genes in keloid fibroblasts (KD-Fbs) .Methods:Three patients who were diagnosed with keloids and received surgical treatment were collected from the Department of Dermatology, Tangdu Hospital, the Fourth Military Medical University from January to March 2017. Human KD-Fbs were isolated from resected keloid tissues, and subjected to primary culture. The third- to sixth-passage KD-Fbs were divided into tension group and control group to be cultured in the tension-based chamber and control chamber respectively, and subjected to tension stimulation and normal culture respectively. Cell counting kit-8 (CCK8) assay was performed to assess the proliferative activity of KD-Fbs after 1-, 2-, 3- and 4-day culture, and the scratch assay to evaluate the migratory ability of KD-Fbs after 1- and 2-day culture. After 48-hour treatment, real-time quantitative PCR and Western blot analysis were performed to determine the mRNA and protein expression of fibrosis markers type Ⅰ collagen, fibronectin and α-smooth muscle actin (α-SMA) in KD-Fbs respectively. Two-independent-sample t test was used for comparisons between 2 groups. Results:CCK8 assay showed that the proliferative activity of KD-Fbs was significantly higher in the tension group than in the control group after 1-, 2-, 3- and 4-day culture ( t=3.05, 7.00, 16.65, 15.19, respectively, all P< 0.05) . After 1- and 2-day culture, the scratch assay showed that the migration rate of KD-Fbs was significantly higher in the tension group (48.65%±3.96%, 100.00%, respectively) than in the control group (9.36%±1.14%, 50.35%±4.23%, t=16.53, 20.35, respectively, both P< 0.01) . Real-time quantitative PCR showed that the mRNA expression of type Ⅰ collagen, fibronectin and α-SMA was significantly higher in the tension group (3.04±0.20, 2.16±0.10, 3.76±0.24, respectively) than in the control group (1.00; t=17.57, 21.01, 20.25, respectively, all P< 0.01) . As Western blot analysis revealed, changes in the protein expression of the 3 fibrosis markers were consistent with their mRNA expression changes (all P< 0.05) . Conclusion:Tension may participate in the fibrosis in keloids by promoting the expression of fibrosis marker genes, and enhancing the proliferative and migratory ability of KD-Fbs.
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Objective:To observe the effects of rosuvastatin calcium at different doses combined with the basic treatment in elderly patients with unstable angina pectoris ( UAP) , and investigate the influence on the serum inflammatory factors .Methods:Totally 82 ca-ses of elderly patients with UAP were divided into the low dose group and the high dose group randomly .In addition to the basic treat-ment, the low dose group was given rosuvastatin calcium tablets 10mg, once per night, and the high dose group was given rosuvastatin calcium tablets 20 mg, once per night .The serum inflammatory factors and blood lipid levels of the two groups before the treatment and after 3-months treatment were compared .The therapeutic effect , frequency and duration of attack and the incidence of adverse events of the two groups were evaluated .Results:After the treatment , the levels of TC , TG and LDL-C in both groups significantly decreased , and that of HDL-C increased (P<0.05), and the blood lipid indexes in the high dose group were better than those in the low dose group (P<0.05).The levels of TNF-α, IL-6 and TGF-β1 decreased significantly after the treatment (P<0.05), and level of these indexes in the high dose group were much lower than that in the low dose group (P<0.05).The total effective rate in the high dose group was 95.43％, which was much higher than that (77.50％) in the low dose group (P<0.05).The frequency and duration of angina pectoris in the high dose group were both lower than those in the low dose group (P<0.05).The incidence of adverse events in the two groups showed no statistically significant difference (P>0.05).Conclusion:High dose rosuvastatin calcium combined with the basic treatment can effectively improve the blood lipid levels and serum inflammatory factors in the elderly patients with UAP , which exhibits good clinical curative effect .
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OBJECTIVE:To study the effects of maggot oil on the healing of rat with acute skin trauma and infection and its mechanism,in order and to provide reference for further development of maggot oil. METHODS:SD rats were randomly divided into normal group,model group,maggot oil group and Jingwanhong treatment group (positive control),with 70 mice in each group. Except for normal group,acute skin trauma and infection model was induced in other groups by smearing Staphylococus aureus suspension at the wound. After modeling,normal group and model group were given normal saline,and maggot oil group and Jingwanhong treatment group were given relevant medicine 0.3 mL/100 g,at 9:00 and 17:00,for consecutive 15 d. Wound and wound healing time of rats were observed in each group. The content of hydroxyproline in wound was determined in 10 rats of each group after 1,2,4,6,8 d of administration. The content of water in wound was determined after 2,4,6,8 d after administration. 2 h after last administration,the content of lysozyme,the levels of inflammatory factors (TNF-α,IL-6),the expression of NF-κB p65(in cytoplasm and nucleus)and p-IκB-α(in cytoplasm)protein were determined in 10 rats of each group. RESULTS:Compared with normal group,wound edema of model group was obvious,and wound healing time was(14.3±2.1)d. After 4,6,8 d of medication,the content of hydroxyproline in wound of rats was decreased significantly in model group (P<0.05 or P<0.01). After 2,4,6,8 d of medication,the content of water in wound was increased significantly in model group(P<0.01). After 15 d of medication,the serum contents of lysozyme,TNF-α and IL-6 in rats were increased significantly in model group (P<0.01). The expression of NF-κB p65 (in cytoplasm) in wound was decreased significantly (P<0.01),while the expressions of NF-κB p65 (in nucleus) and p-IκB-α(in nucleus) protein were increased significantly (P<0.01). Compared with model group,above indicators of administration groups were improved significantly (P<0.05 or P<0.01). CONCLUSIONS:Maggot oil could protect tissue injury induced by acute skin wound infection,promote wound healing. The possible mechanism might play anti-inflammatory effect through promoting collagen production,increasing lysozyme content,regulating NF-κB signal pathway.
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OBJECTIVE:To discuss clinical efficacy and safety of alteplase combined with butylphthalide to the patients with acute ischemic stroke. METHODS:98 patients with acute ischemic stroke in our hospital were selected and divided into ob-servation group and control group according to random number table,with 49 patients in each group. Control group was addition-ally given Butylphthalide and sodium chloride injection 100 ml,ivgtt,bid,on the basis of routine treatment as controlling blood glucose,blood pressure,etc.;observation group additionally received Alteplase for injection 5 mg added into NS 10 ml,iv+Al-teplase for injection 45 mg added into NS 100 ml,ivgtt,qd,on the basis of control group. Both groups were treated for 2 weeks. Clinical efficacies of 2 groups were compared as well as cerebral infarction area,NIHSS score,ability score of daily liv-ing,the levels of IL-6,IL-8,IL-10,CRP,24 h urine protein,Scr and creatinine clearance rate before and after treatment. The occurrence of ADR was also recorded. RESULTS:1 patient of observation group and 2 patients of control group withdrew from the study due to severe hemorrhage. Total effective rate of observation group(95.83%)was significantly higher than that of con-trol group(80.85%),with statistical significance(P0.05). The cerebral infarction area and NIHSS score were reduced significantly in observation group after treatment,ability score of daily living were increased significantly in observation group after treatment,and the im-provement of observation group was significantly better than control group,with statistical significance (P0.05). CONCLUSIONS:Alteplase combined with butylphthalide show signifi-cant therapeutic efficacy,can effectively reduce the level of serum inflammatory factors,control brain tissue ischemia and cere-bral infarction area,and improve neurologic function and pro-tect renal function in patients with acute ischemic stroke.
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Objective: To study the acute toxicity of Xiaobai capsules in mice after intragastric administration .Methods: The mice were randomly divided into two groups , the treatment group and the control group .The treatment group was given Xiaobai cap-sules by gavage, 3 times daily.The acute toxicity was recorded, and the median lethal dose (LD50) and the maximum dose were deter-mined.Results:The maximum daily dose of Xiaobai capsules was 141.6 g· kg-1(equivalent to 211.3 times of the clinical dose).At the dose, the mice showed no toxicity without death in 14 days or changes in organs after the dissection .Conclusion:Xiaobai capsules have very low acute toxicity in mice after intragastric administration with high security .
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Objective To investigate the mRNA and protein expressions of nine integrin subunits in human keloid-derived mesenchymal stem cells (KD-MSCs).Methods Cultured KD-MSCs and normal skin-derived MSCs (NS-MSCs) served as the experiment group and control group respectively.Real-time quantitative PCR and Western blot were performed to measure the mRNA and protein expressions of nine integrin subunits in the two groups respectively.Statistical analysis was carried out by t test.Results Real-time quantitative PCR showed no significant difference in the mRNA expression level of any of the integrin units α2,α3,α5,αV,α10,α11 or β1 between KD-MSCs and NS-MSCs (all P ＞ 0.05).The mRNA expression level of integrin α8 was decreased,while that of integrin β3 was significantly increased in KD-MSCs compared with NS-MSCs (both P ＜ 0.01).Western blot revealed that the changes in protein expression levels of integrin units α8 and β3 were consistent with those in their mRNA expression levels in both KDMSCs and NS-MSCs (both P ＜ 0.01).Conclusions Integrin units α8 and β3 may be involved in the occurrence and development of keloid,and the receptors made up of them may play important roles in the pathogenesis of keloid.
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Objective To establish the rat model of type 2 diabetes mellitus (T2DM)-induced Alzheimer’s disease (AD).Methods The rat model of T2DM was established by continuous high fat and high glucose diet in aged rats. Then the rat model was screened and identified by using Morris water maze, cerebrospinal fluid microdialysis technique,ELISA,electrophysiological technique and pathologic method,respectively.Results Compared with the normal group and the T2DM group,the rats in T2DM+DM group had obviously learning and memory impairment;the level ofβ-AP in the hippocampus was significantly higher and the frequency of the spikes induced by Achin the hippocampus was notably lower.Conclusion The rat model of T2DM-induced AD can be successfully established by continuous high fat and high glucose diet in rats.
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Objective:To investigate the effect of EBV protein on the production of Immunoglobulin(Ig)by cultured B cells.Methods:The purified human umbilical blood B cells were treated respectively with UV and Heat-inactivated EBV and cultured in complete IMDM. CD5, CD3, CD4 and CD8 cells were measured by flow cytometric analysis. IgG and IgM in the supernatant were detected by ELISA. At the mean time, IgG and IgM in the supernatant of cultured EBV-transformed B lymphocytes were detected by ELISA,and compared with that of UV-inactivated EBV group at the same period.Results:In UV-inactivated EBV group,CD3, CD4 and CD8 T cells couldn’t be detected,CD5 + cells occupied about 42% of all detected cells at the 14th day; and at the 28th day, 47%.The IgG A value of the 10th、18th、22th and 26th day in UV-inactivated EBV group have a significant difference compared with that in the control group ( P
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Objective To prepare natural anti-keratin IgM monoclonal antibody. Methods Spleen cells of BALB/c mice raised in specific pathogen free conditions were directly fused with Sp2/0 myeloma cells. The hybridoma supernatants were tested by ELISA using pre-extracted keratin. The natural IgM obtained was further identified by immunochemistry and immunoblot methods. Results The cell fusion rate was about 60% without pre-immunization. About 14% supernatants reacted with the keratin antigen. Three hybridoma strains secreting natural IgM monoclonal antibody against keratin were obtained. The immunochemistry results showed that the natural anti-keratin IgM was able to bind to epidermis, sebaceous gland, hair follicule, and muscle tissues. Conclusion B lymphocytes in normal BALB/c mice spleen can produce natural antibody against kerain.
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Objective To investigate UV- or heat-inactivated Epstein-Barr virus(EBV)stimulating the production of anti-keratin autoantibody(AK auto Ab)in cultured human umbilical cord blood B cells. Methods Mononuclear cells were isolated routinely from umbilical cord blood, in which monocytes, NK cells and cytotoxicity T cells were eliminated by L-leucine methyl ester method, and T cells were removed by sheep red blood cells(SRBCs)treated with 2-amino ethyl-isothiouronium bromide (AET). The purified B cells were treated with UV- or heat-inactivated EBV respectively and then cultured in complete IMDM. CD5, CD3, CD4 and CD8 cells were detected by flow cytometry. IgG and IgM of AK auto Ab were measured by ELISA in the supernatant which came from the B cells treated by UV-inactivated EBV or EBV-transformed B cells respectively. Results In UV-inactivated EBV group CD5+B cells accounted for 43% and 47% of all cells detected on the 14th and 28th day, respectively. No CD3, CD4 and CD8 cells were detected during this period. In UV-inactivated EBV group the AK auto Ab of IgG and IgM increased significantly on the 18th and 26th day, respectively (P 0.05). On the 40th day the AK auto Ab of IgG and IgM were significantly higher in EBV-transformed B cell group than those in UV-inactivated EBV group. Conclusions UV-inactivated EBV is able to induce AK auto Ab production but heat-inactivated EBV does not, which suggests that EBV protein might be the effective agent in inducing the production of AK auto Ab.
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Objective To design and testify a novel strategy for acquiring mimetic epitope mapping by screening for a phage random peptide library using polyclonal anti keratin autoantibodies (AK auto Ab). Methods AK auto Ab were isolated and purified from pooled human sera by keratin affinity column in which keratin had been linked with CNBr Sepharose 4B,then biotinylated by the biotin ester. A 15 mer phage random peptide library was biopanned for 3 cycles and positive clones were identified by ELISA,competition assay and DNA sequencing. ResultsBy sequence comparison 23 positive clones were selected randomly and three epitopes were confirmed. Among the three epitopes SLSPMPTTNRR was the dominant epitope. The phages carrying positive clones reacted with AK auto Ab specifically and keratin could prevent interaction between AK auto Ab and positive phages. Conclusion The designed strategy is successfully applied in acquiring epitopes of polyclonal autoantibodies to keratin, which could provide a new approach for the discovery of epitope mapping which binds to natural autoantibodies.