Progress on the effect of mesenchymal stem cell derived exosomes on multiple organ dysfunction in sepsis / 中华危重病急救医学

Mesenchymal stem cell derived (MSC) exosomes are extracellular vesicles with a diameter of about 50-200 nm. Exosomes contain a large number of biologically active substances including mRNAs, miRNAs, cytokines, chemokines, proteins, lipids, etc. MSC exosomes exert biological effects through paracrine and endocrine pathways in vivo. Uncontrolled inflammation and multiple organ dysfunction are the key roles in the progression of sepsis, moreover, heart, lungs, kidneys and brain are the general target organs to be damaged. MSC exosomes regulate the expression of cytokines, the production of inflammatory cell, the levels of inflammatory response and the recovery of damaged tissues or organ function. Therefore, studying the application of MSC exosomes is significant for the clinical treatment of sepsis. This article reviews the systemic inflammation regulation by MSC exosomes and its protective function on targeted organs such as heart, lungs, kidneys, brain, liver, etc. to provide evidences for the treatment of sepsis.

The role of high mobility group box 1 in the injury of Caco-2 epithelial barrier induced by lipopolysaccharide / 中华急诊医学杂志

Objective:To investigate the role and mechanism of high mobility group box 1(HMGB1) in the injury of Caco-2 intestinal epithelial barrier induced by lipopolysaccharide (LPS).Methods:The Caco-2 cellular monolayer barrier was established with Transwell chamber. After the Caco-2 monolayer model was established, the transepithelial electrical resistance (TEER) values were measured. When the TEER value reached 500 Ω·cm 2, the cells were divided into 3 groups: control group, LPS treatment group, and LPS+ ethyl pyruvate (EP) treatment group. The concentration of LPS and EP were 100 μg/mL, 50 μg/mL, separately. Then TEER values were measured at 12, 24, 48 and 72 h, and FITC-dextran permeability was detected at 24 h. The cells were seeded on 6-well plates. After cell density reached 80%, treatments were given as the above. The real-time polymerase chain reaction (RT-PCR) and Western blot were used to measure the changes in the protein and mRNA expressions of Occludin, HMGB1, and nuclear factor-κB (NF-κB). Results:Compared with the control group, the TEER values (Ω·cm 2) reduced at 12, 24, 48 and 72 h in the LPS treatment group [(514.22±12.59) vs (304.96±9.69), (521.65±13.35) vs (276.21±7.82), (523.99±8.18) vs (206.64±15.85), (491.21±6.72) vs (156.33±10.83), all P<0.05]. The FITC-dextran permeability increased significantly at 24 h [(2.58±0.07) vs (1.04±0.06), P<0.05]. The expression levels of Occludin protein and mRNA were decreased (all P<0.05), while the expression levels of HMGB1 and NF-κB protein and mRNA were significantly increased (all P<0.05). Compared with the LPS treatment group, the TEER values (Ω·cm 2) increased significantly at 12, 24, 48 and 72 h in the EP treatment group [(519.00±5.66) vs (304.96±9.69), (504.69±8.57) vs (276.21±7.82), (453.65±10.74) vs (206.64±15.85), (385.28±7.57) vs (156.33±10.83), all P<0.05]. The FITC-dextran permeability decreased at 24 h [(1.23±0.11) vs (2.58±0.07), P<0.05]. The expression level of Occludin protein and mRNA were increased ( P<0.05), while the expression levels of HMGB1 and NF-κB protein and mRNA were significantly decreased (all P<0.05). Conclusions:LPS can injure intestinal barrier directly in vitro and reduces the expression of tight junction proteins between cells. The mechanism may be related to the increased expression of HMGB1 and NF-κB protein.

Potential regulatory role of long non-coding RNA-microRNA-mRNA axis in sepsis / 中华危重病急救医学

Sepsis is a life-threatening multiple organ dysfunction disease with high mortality and has become leading causes of death affecting intensive care unit (ICU) patients. Both long non-coding RNA (lncRNA) and microRNA (miRNA) are involved in the pathophysiological process of sepsis and can regulate the inflammatory response, both of which could be used as important diagnostic indicators and therapeutic targets of sepsis. The interaction among lncRNA, miRNA and messenger RNA (mRNA) plays an important role in sepsis and multiple organ dysfunction. This paper reviewed the regulatory relationship of lncRNA, miRNA and mRNA, as well as the regulatory role of lncRNA-miRNA-mRNA axis in inflammatory immune response and multiple organ dysfunction syndrome in sepsis, to provide new targets and strategies for the treatment of sepsis and organ dysfunction.

Role of high mobility group box 1 in intestinal mucosal barrier injury in rat with sepsis induced by endotoxin / 中华危重病急救医学

Objective:To investigate the role and mechanism of the high mobility group box 1 (HMGB1) in intestinal mucosal barrier injury in rat with sepsis induced by endotoxin lipopolysaccharide (LPS).Methods:The rats were given intraperitoneal injection of LPS to reproduce a model of sepsis. The effect of HMGB1 inhibitor EP solution (40 mg/kg) on sepsis was observed, and phosphate buffer (PBS) control group was set up. Seventy-two hours after modeling, abdominal aortic blood was obtained, and enzyme-linked immunosorbent assay (ELISA) was used to measure the plasma levels of D-lactic acid and diamine oxidase (DAO) of mucosal barrier permeability. The pathological changes of the intestinal mucosal were observed with light microscope and the Chiu score was recorded. The intestinal mucosal ultrastructural changes were observed with electron microscopy. Real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and Western Blot were used to measure the mRNA and protein expressions of Occludin, inflammatory factor HMGB1 and its downstream signal molecule nuclear transcription factor-κB p65 (NF-κB p65) in the rat small intestine.Results:The results of histopathology and ultrastructure of the small intestine showed that in the LPS group, the intestinal mucosa tissue swelled obviously, part of the glands were incomplete, the infiltration of neutrophils increased, themicrovillus cells were absent, arranged indisorder, and the number of tight connections significantly reduced compared with the PBS control group. The levels of D-lactic acid and DAO indicating mucosal barrier permeability, the levels of inflammatory factor HMGB1 and its downstream signaling molecule NF-κB p65 mRNA and protein expressions in the LPS group were significantly higher than those in the PBS control group, and the mRNA and protein expression of Occludin in the small intestine was significantly lower than that in the PBS control group, suggesting that the intestinal mucosal barrier function in septic rats was damaged, permeability increased, and the structure was damaged. After the administration of the HMGB1 inhibitor EP, the intestinal mucosal barrier damage was significantly improved. The performance was as follows: the Chiu score of the small intestine tissue and the plasma D-lactic acid and DAO levels in the EP intervene group were significantly lower than those in the LPS group [Chiu score: 1.60±0.48 vs. 3.40±0.48, D-lactic acid (mmol/L): 3.30±0.22 vs. 5.30±0.16, DAO (U/L): 23.66±0.97 vs. 30.47±1.11, all P < 0.05]. Occludin mRNA and protein expression levels were significantly higher than those in the LPS group [Occludin mRNA (2 -ΔΔCt): 0.82±0.05 vs. 0.37±0.08, Occludin protein (Occludin/β-actin): 1.04±0.09 vs. 0.75±0.11, both P < 0.05], while the mRNA and protein expression levels of HMGB1 and NF-κB p65 were significantly lower than those in the LPS group [HMGB1 mRNA (2 -ΔΔCt): 1.63±0.10 vs. 3.57±0.10, HMGB1 protein (HMGB1/β-actin): 1.40±0.07 vs. 1.87±0.07; NF-κB p65 mRNA (2 -ΔΔCt): 1.47±0.09 vs. 2.62±0.13, NF-κB p65 protein (NF-κB p65/β-actin): 1.24±0.14 vs. 1.60±0.13, all P < 0.05]. Conclusions:Intestinal mucosal barrier function of septic rats was damaged, permeability increased, and structure was damaged. The mechanism may be that the expression of inflammatory factor HMGB1 was up-regulated and promoted the activation of its downstream signaling molecule NF-κB, thereby mediated the inflammatory cascade reaction and caused damage to the intestinal mucosa.

Trend in proportion and clinicopathological characteristics of young women with stage Ⅰa2 to Ⅱa2 cervical cancer / 中华妇产科杂志

Objective@#To analyze the 13 years trend in proportion, risks factors and clinicopathological characteristics of young women with stage Ⅰa2 to Ⅱa2 cervical cancer by using multi-center data of cervical cancer in China.@*Methods@#The clinicopathological data of 46 313 patients with cervical cancer treated from 37 hospitals in China were obtained from January 2004 to December 2016. Using clinical and pathologic data, each patient's stage was reclassified by the 2018 International Federation of Gynecology and Obstetrics (FIGO) staging system. A total of 19 041 patients were selected according to the following criteria: FIGO stage Ⅰa2 to Ⅱa2, underwent type B or C radical hysterectomy and pelvic lymphadenectomy. All the patients were divided into two groups: the study group of 1 888 patients aged 35 years or younger and the control group of 17 153 patients aged over 35 years. The 13 years trend in proportion of young women with stage Ⅰa2 to Ⅱa2 cervical cancer, risks factors and clinicopathological characteristics of two groups were retrospectively analyzed.@*Results@#(1) The total number of hospitalized patients with stage Ⅰa2 to Ⅱa2 cervical cancer increased annually. However, a downward trend of patients aged 35 years or younger was observed (P<0.01) . The constituent ratio of patients aged 35 years or younger was significantly greater during 2004—2010 than that during 2011—2016 [12.6% (820/6 484) and 8.5% (1 068/12 557) , respectively; χ2=82.101, P<0.01]. (2) Compared with patients aged over 35 years, patients aged 35 years or younger had an earlier age at menarche, a later age at marriage, lesser gravida and parity (all P<0.01). The positive rate of high-risk HPV infection was not statistically different between two groups (all P>0.05). (3) The proportions of stage Ⅰ, exophytic type and non-squamous histological type in patients aged 35 years or younger were clearly higher than those in patients aged over 35 years (83.4% vs 68.5%, P<0.01; 63.2% vs 56.2%, P<0.01; 13.9% vs 12.0%, P<0.05, respectively). Whereas the poor differentiation ratios of the two groups had no statistical significance (P>0.05). (4) As for the postoperative pathological risk factors, the rate of surgical margin involvement in patients aged 35 years or younger was lower than that aged over 35 years (1.1% vs 1.8%, P<0.05), and the rate of depth of stromal invasion >1/2 in patients aged 35 years or younger was lower than that in patients aged over 35 years (40.1% vs 50.9%, P<0.01). In addition, there were no significant difference in parametrial margin involvement, tumor size and lymph vascular space invasion between two groups (all P>0.05).@*Conclusions@#The trend in proportion among hospitalized patients for stage Ⅰa2 to Ⅱa2 cervical cancer in young women is decreasing yearly. Compared with cervical cancer in middle-aged and elderly women, cervical cancer in young women have an earlier age at menarche, a higher proportion of stage Ⅰ patients and non-squamous histological type. In terms of the postoperative pathological risk factors, the rate of surgical margin involvement and depth of stromal invasion >1/2 in young women with cervical cancer are lower than in middle-aged and elderly women.

Trend in proportion and clinicopathological characteristics of young women with stageⅠa2 toⅡa2 cervical cancer / 中华妇产科杂志

Objective To analyze the 13 years trend in proportion, risks factors and clinicopathological characteristics of young women with stage Ⅰa2 to Ⅱa2 cervical cancer by using multi-center data of cervical cancer in China. Methods The clinicopathological data of 46 313 patients with cervical cancer treated from 37 hospitals in China were obtained from January 2004 to December 2016. Using clinical and pathologic data, each patient′s stage was reclassified by the 2018 International Federation of Gynecology and Obstetrics (FIGO) staging system. A total of 19 041 patients were selected according to the following criteria: FIGO stage Ⅰa2 to Ⅱa2, underwent type B or C radical hysterectomy and pelvic lymphadenectomy. All the patients were divided into two groups: the study group of 1 888 patients aged 35 years or younger and the control group of 17 153 patients aged over 35 years. The 13 years trend in proportion of young women with stage Ⅰa2 to Ⅱa2 cervical cancer, risks factors and clinicopathological characteristics of two groups were retrospectively analyzed. Results (1) The total number of hospitalized patients with stageⅠa2 toⅡa2 cervical cancer increased annually. However, a downward trend of patients aged 35 years or younger was observed (P<0.01). The constituent ratio of patients aged 35 years or younger was significantly greater during 2004—2010 than that during 2011—2016 [12.6% (820/6 484) and 8.5% (1 068/12 557), respectively; χ2=82.101, P<0.01]. (2) Compared with patients aged over 35 years, patients aged 35 years or younger had an earlier age at menarche, a later age at marriage, lesser gravida and parity (all P<0.01). The positive rate of high-risk HPV infection was not statistically different between two groups (all P>0.05). (3) The proportions of stageⅠ, exophytic type and non-squamous histological type in patients aged 35 years or younger were clearly higher than those in patients aged over 35 years (83.4% vs 68.5%, P<0.01; 63.2% vs 56.2%, P<0.01; 13.9% vs 12.0%, P<0.05, respectively). Whereas the poor differentiation ratios of the two groups had no statistical significance (P>0.05). (4) As for the postoperative pathological risk factors, the rate of surgical margin involvement in patients aged 35 years or younger was lower than that aged over 35 years (1.1% vs 1.8%, P<0.05), and the rate of depth of stromal invasion >1/2 in patients aged 35 years or younger was lower than that in patients aged over 35 years (40.1% vs 50.9%, P<0.01). In addition, there were no significant difference in parametrial margin involvement, tumor size and lymph vascular space invasion between two groups (all P>0.05). Conclusions The trend in proportion among hospitalized patients for stageⅠa2 toⅡa2 cervical cancer in young women is decreasing yearly. Compared with cervical cancer in middle-aged and elderly women, cervical cancer in young women have an earlier age at menarche, a higher proportion of stage Ⅰ patients and non-squamous histological type. In terms of the postoperative pathological risk factors, the rate of surgical margin involvement and depth of stromal invasion>1/2 in young women with cervical cancer are lower than in middle-aged and elderly women.

Glucosamine, chondroitin sulfate combined with bone health exercise can promote bone mineral density and muscle strength in postmenopausal women:a random community trial / 中国组织工程研究

BACKGROUND: Glucosamine and chondroitin sulfate are important compositions of cartilage tissues. Animal experiments have shown that glucosamine and chondroitin sulfate can treat osteoporosis synergistically, but the effect of their combinations with regular exercise on the bone and joint health of menopausal women is little reported. OBJECTIVE: To observe the effects of the combination of glucosamine, chondroitin sulfate and bone health exercises on skeletal and joint health in postmenopausal women by a random community trial. METHODS: From January to June 2016, 206 eligible postmenopausal women were selected from Bishan District of Chongqing and randomly divided into two groups by random digital method. The trial group received glucosamine and chondroitin sulfate (3 capsules/day, and bone health exercises (at least 3 times/week), while the control group did not receive special treatment. After 6 months of intervention, the bone mineral density, muscle strength of habitual knee, incidence of fracture, osteoarthritis and fall, and levels of symptom and function were compared between the two groups. The peak torque of extension and flexion of habitual knee at 0 and 60 (°) /s angular velocity was detected using Isomed 2000 Dynamometer. RESULTS AND CONCLUSION: (1) The bone mineral density and the peak torque of extension and flexion of habitual knee at 0 and 60 (°) /s angular velocity in the trial group were significantly higher than those in the control group at 6 months after treatment (P ＜ 0.05). (2) The incidence of fall in the trail group was lower than that in the control group at 6 months (17.00％ vs. 5.94％, P ＜ 0.05). The incidence of fracture and osteoarthritis had no significant difference between two groups (P＞ 0.05). (3) The self-ranking of joint pain, holding power of muscle, walking distance and joint stiff had significant differences between two groups (P ＜ 0.05). (4) These results indicate that glucosamine and chondroitin sulfate supplements combined with regular bone health exercises can improve bone mineral density and muscle strength, and prevent joint pain and stiff in postmenopausal women to some extent, thus preventing fall, fracture and osteoarthritis of postmenopausal women.

Role of edaravone in alleviating prognosis of rats following traumatic brain injury / 中华创伤杂志

Objective To investigate the effects of edaravone on improving the prognosis of TBI rats.Methods A total of 150 SD male rats were divided into normal control group (10 rats),TBI group (70 rats) and edaravone group (70 rats).In the edaravone treatment group,the rats were injected intraperitoneally once a day continously for 2 weeks with the injection dose of 5.4 mg · kg-1 · d-1.At 6 hours,12 hours,24 hours,48 hours,72 hours,1 week and 2 weeks after injury,the neurobehavioral and motor function scores of rats were monitored respectively,with 10 rats monitored at each time point.Serum and cerebrospinal fluid samples were collected and the levels of β-endorphin and gonadotropin-releasing hormone (GnRH) were determined by radioimmunoassay (RIA).Results In the edaravone group,the neurobehavioral and motor function scores were higher than those of the TBI group at 6 hours,12 hours,24 hours,48 hours,72 hours,1 week and 2 weeks after injury.At 48 hours after injury,the neurobehavioral scores of the TBI group and the edaravone treatment group were (8.2 ±0.9) points and (10.3 ±0.7) points,respectively (P ＜ 0.05),and the motor function scores were (5.9 ± 1.0) points and (6.9 ± 1.2) points respectively (P ＜ 0.05).Meanwhile,the contents of β-endorphin in blood and cerebrospinal fluid of the normal control group were (50.2 ± 9.5) pg/ml and (16.2 ± 2.8) pg/ml,and the contents of GnRH were (75.2 ± 11.2) pg/ml and (36.2 ± 10.8)pg/ml,respectively.The levels of β-endorphin and GnRH in serum and cerebrospinal fluid were significantly increased at 6 hours,12 hours,24 hours,48 hours,72 hours,l week and 2 weeks after injury.The levels of β-endorphin and GnRH in the edaravone group were lower than those of TBI group.At 72 hour after injury,the levels of β-endorph in serum in TBI group and edaravone group were (165.2 ± 8.5) pg/ml and (109.5 ± 6.3) pg/ml respectively (P ＜ 0.05),and the levels of β-endorph in cerebrospinal fluid were (63.3 ± 3.1) pg/ml and (38.2 ± 2.3) pg/ml respectively (P ＜ 0.05).At 72 hour after injury,the levels of GnRH in serum in TBI group and edaravone group were (203.7 ± 17.1)pg/ml and (110.4 ± 19.2)pg/ml respectively (P ＜0.05),and the levels of GnRH in cerebrospinal fluid is (153.0 ± 13.4) pg/ml and (93.2 ± 10.5) pg/ml respectively (P ＜ 0.05).Conclusion During acute and recovery periods after TBI,continuous treatment with edaravone can obviously reduce the levels of β-endorphin and GnRH,which is beneficial to alleviate the secondary brain injury after TBI in rats,promote the recovery of nerve and function,and improve the prognosis.

In Vitro Angiogenesis Effect of Xuefu Zhuyu Decoction () and Vascular Endothelial Growth Factor: A Comparison Study / 中国结合医学杂志

<p><b>OBJECTIVE</b>To compare the angiogenesis behaviors of vascular endothelial growth factor (VEGF) and Chinese medicine Xuefu Zhuyu Decoction (, XZD) treatments.</p><p><b>METHODS</b>Human microvascular endothelial cells (HMEC-1) were treated with various concentrations of either XZD-containing serum (XZD-CS) or VEGF for 24, 48, and 72 h, respectively. Cell viability, proliferation, migration, adhesion, and in vitro tube formation assays were used to assess their angiogenic effects.</p><p><b>RESULTS</b>VEGF promoted all cellular phases involved in angiogenesis including cell viability, proliferation, migration, adhesion, and tube formation (<0.05 or <0.01). Unlike the continuous promotion effects of VEGF at the above stages, XZD inhibited cell viability and proliferation (<0.05 or <0.01) and only promoted tube formation in the early phase of angiogenesis (<0.01).</p><p><b>CONCLUSIONS</b>These two medications promote different angiogenesis behaviors, which might be an important reason for their distinct therapeutic profile in clinical usage.</p>

Effects of bone marrow mesenchymal stem cells on the expressions of inflammatory factors in rats with multiple organ dysfunction syndrome / 中华急诊医学杂志

Objective To investigate the effects of bone marrow mesenchymal stem cells (BMSC) on the expression of inflammatory factors in rats with multiple organ dysfunction syndrome (MODS). Methods BMSC extracted from the 4-week-old Sprague-Dawley (SD) rats was cultivated and identified in vitro, then the 4th passage of which was used in the experimental study. Sixty SD rats were randomly(random number) divided into three groups (n=20 in each group): Sham group (SG), MODS group (MG) and BMSC group (BG). Rats in the MG was injected by 1 mg/kg lipopolysaccaride (LPS) via great saphenous vein, rats in the SG injected with the same volume sterile phosphate buffer saline and rats in the BG infused by 1×106/cells BMSCs through the tail vein at 2 h after LPS injection. The survival rate, tissue pathological changes of the lung, liver and heart by hematoxylin and eosin (HE) staining, organ dysfunction measurement by blood gas analysis and biochemical indicators as well as the related inflammatory factors by protein microarray and enzyme linked immunosorbent assay (ELISA), were detected 72 h post operation. Multi-group quantitative data was analyzed by one way ANOVA, paired-comparisons by LSD-t test and the comparisons of survival curves in the three groups by Log-rank test. The value of P<0.05 was considered statistically significant. Results The survival rate in SG, MG and BG was 100％, 60％ and 80％, respectively. The survival curves showed that the survival rate of SG was higher than the MG and BG (SG vs. MG, χ2=9.798, P=0.0017; SG vs. BG, χ2=4.333, P=0.0374), but there was no significant difference comparing the BG to the MG (χ2=2.408, P=0.1207). The tissue congestion and edema, and inflammatory cells infiltration in the lung, liver, and heart of the MG were observed by HE staining, while these changes reduced in the BG. Compared with the SG, the levels of pH and PaCO2 and lactic acid (Lac) increased significantly (all P<0.01), the level of total bilirubin (TB) significantly increased [(0.801±0.501)U/L vs. (2.533±0.382)U/L, P=0.003], while the albumin(ALB) level decreased significantly[(35.471±4.015)U/L vs. (23.202±4.872)U/L, P<0.01], and creatine kinase (CK) level increased significantly in MG [(315.670±41.402) vs. (708.250±219.201), P=0.042]. After BMSC treatment, the organ function improved significantly (all P<0.05). Gamma interferon (IFN-γ) and monocyte chemotactic protein 1 (MCP-1) were the differential expression factors in protein chips. The results of ELISA were similar to the protein chips: compared with the SG, IFN-γ and MCP-1 expressions in the MG increased significantly (P<0.01). Compared with MG, the expressions of IFN-γ and MCP-1 decreased significantly in the BG (P<0.01). Conclusion BMSC administration could modulate the inflammatory response of MODS rats by inhibiting the levels of IFN-γ and MCP-1, and improve the organ function and the survival rate.

Role of HMGB1-RAGE / TLRs-NF-κB signaling pathway on bone mesenchymal stem cells transplantation therapy for lipopolysaccaride-induced coagulation disorder rats / 中华危重病急救医学

Objective To determine the effect of bone mesenchymal stem cells (BMSCs) in transplantation therapy for lipopolysaccharide (LPS)-induced coagulation disorder and the underlying mechanism of high mobility group protein B1-receptors for advanced glycation end products / Toll-like receptors-nuclear factor-κB (HMGB1-RAGE / TLRs-NF-κB) signaling pathway.Methods BMSCs of female Sprague-Dawley (SD) rats ageing 4-5 weeks old were extracted and cultivatedin vitro, and the fourth-passaged BMSCs phenotype was identified by flow cytometry for transplantation in the following experimental study. The rats were randomly divided into normal saline (NS) control group, LPS group, and BMSC group according to the random number table with 15 rats in each group. Coagulation disorders model was reproduced by injection of 1 mg/kg LPS via saphenous vein, and the rats in the NS control group was injected with equal volume NS. Those in the BMSC group were infused BMSC 0.5 mL containing 1×106 cells via tail vein at 2 hours after LPS injection, and the rats in other groups were injected with equal volume NS. Abdominal aorta blood was collected at 1, 3 and 7 days post operation. Coagulation indexes such as platelet count (PLT), platelet volume distribution width (PDW), mean platelet volume (MPV), plateletcrit (PCT), platelet large cell ratio (P-LCR), activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), international normalized ratio (INR), and fibrinogen (FIB) were determined. The mRNA levels and contents of HMGB1, RAGE, TLR2/4 and NF-κB were determined by real-time reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively.Results ① The cells culturedin vitro were spindle shaped or flat. The fourth-passaged BMSCs phenotype was successfully identified by flow cytometry technology. ②Coagulation indexes: compared with NS control group, PLT, PCT and FIB in LPS group were significantly decreased, PDW, MPV, P-LCP, and INR were significantly increased, and APTT, PT, and TT were significantly prolonged from the first day. Furthermore, those in LPS group were gradually ameliorated with prolongation of LPS induction time. The coagulation function abnormality induced by LPS was reversed by BMSCs with significant difference at 1 day as compared with LPS group [PLT (×109/L):398.8±17.9 vs. 239.1±15.8, PCT (％): 0.35±0.04 vs. 0.23±0.06, FIB (g/L): 1.7±0.6 vs. 0.8±0.1, PDW (％):12.4±1.6 vs. 16.2±1.5, MPV (fl): 11.0±1.6 vs. 13.7±1.1, P-LCP (％): 13.0±2.1 vs. 15.3±2.7, INR: 1.52±0.17 vs. 1.82±0.19, APTT (s): 66.3±4.1 vs. 89.5±4.5, PT (s): 18.3±0.7 vs. 25.1±1.9, TT (s): 87.5±7.8 vs. 115.0±9.7, allP < 0.05], till 7 days. ③ HMGB1-RAGE / TLRs-NF-κB signaling pathway related molecules: compared with NS control group, the mRNA expressions and contents of HMGB1, RAGE, TLR2/4 and NF-κB were significantly increased in LPS group from the first day. However, the mRNA expressions and contents of the molecules in LPS group were gradually decreased with prolongation of LPS induction time. After BMSC intervention, the mRNA expressions and contents of molecules at 1 day were significantly lower than those of LPS group [HMGB1 mRNA (2-ΔΔCt): 10.77±0.04 vs. 24.51±3.69, HMGB1 content (μg/L): 0.48±0.01 vs. 0.95±0.06; RAGE mRNA (2-ΔΔCt): 11.57±1.11 vs. 18.08±0.29, RAGE content (μg/L): 0.73±0.04 vs. 1.37±0.06; TLR2 mRNA (2-ΔΔCt): 2.60±0.22 vs. 12.61±0.27, TLR2 content (μg/L): 0.81±0.03 vs. 1.59±0.09; TLR4 mRNA (2-ΔΔCt): 2.95±0.52 vs. 4.06±0.11, TLR4 content (μg/L):0.80±0.09 vs. 1.18±0.11; NF-κB mRNA (2-ΔΔCt): 1.29±0.06 vs. 7.79±0.25, NF-κB content (μg/L): 1.22±0.24 vs. 2.42±0.26, allP < 0.05], till 7 days.Conclusion BMSCs administration could ameliorate the coagulation function in LPS-induced coagulation disorder rats and these might be associated with HMGB1-RAGE / TLRs-NF-κB signaling pathway inhibition.

Bone marrow mesenchymal stem cells modulated the inflammatory response by regulating the expression of IL-4 and RAGE products in the rats with MODS / 中华危重病急救医学

Objective To investigate the underlying mechanism of bone marrow mesenchymal stem cells (BMSC) modulating the inflammatory response during the multiple organ dysfunction syndrome (MODS), especially the expression of inflammatory cytokines, which will provide new theoretical and experimental basis of MODS in clinic. Methods BMSC of Sprague-Dawley (SD) rat (female, 4 weeks) was extracted and cultivated, and the 4th passage were used in experimental study. According to the random number table, 60 female SD rats were divided into three groups (n = 20 per group): sham group, MODS group, BMSC group. MODS model in rats was induced by lipopolysaccaride (LPS, 1 mg/kg) via femoral vein injection. Sham group was injected with the sterile phosphate buffer saline (PBS) in the same volume. BMSC group, in which BMSC infusion was started at 2 hours after 0.5 mL LPS stimulation (1×106/cells) through the tail vein. The survival rate was observed after 72 hours in each group. Abdominal aortic blood was collected for routine blood and biochemical examination at 72 hours after operation. Protein microarray was used to detect the related 34 inflammatory cytokines. Signal ratio was defined as the differentially expressed factors when it was more than 2.0 or less than 0.5. And enzyme linked immunosorbent assay (ELISA) was be applied to validate the significant inflammation factor. Meanwhile, the heart, kidney, intestine tissue was harvested, then their pathological changes were observed by hematoxylin eosin (HE) staining.Results 20, 12, 16 rats lived in sham group, MODS group and BMSC group respectively at 72 hours after operation. Compared with the sham group, the indicators (routine blood, liver and kidney function, myocardial enzyme) were apparently unusual, and the heart, kidney, intestine tissue were injured obviously in the MODS group. After BMSC administration, the organ function was improved and tissue damaged was alleviated significantly. Protein microarray showed that interleukin-4 (IL-4) and receptor for advanced glycation end products (RAGE) were significantly different in 34 goal cytokines. The signal ratio change of IL-4 was 0.397, 1.124, 2.826 respectively, and the signal ratio of RAGE was 6.197, 1.552, 0.250, respectively in MODS/sham group, BMSC/sham group, BMSC/MODS group. ELISA validated the result that the expression level of IL-4 decreased significantly (ng/L:3.59±1.21 vs. 29.10±5.78) and the expression level of RAGE increased significantly (ng/L: 1.09±0.04 vs. 0.11±0.03) in MODS group as compared with sham group (bothP < 0.05). Compared with the MODS group, the level of IL-4 was obviously higher than that in BMSC group (ng/L: 9.59±2.21 vs. 3.59±1.21,P < 0.01), and RAGE decreased significantly (ng/L: 0.29±0.07 vs. 1.09±0.04,P < 0.05).Conclusions BMSC administration can regulate the expression of IL-4 and RAGE in the rats subjected to MODS. Moreover, BMSC can promote the restoration of tissue and organ function, thus improve the survival rate. BMSC may be the target in cell therapy for the inflammatory disease.

Immune cytotoxic effect of trichloroethylene in Jurkat T cells / 中国职业医学

OBJECTIVE: To explore the immune cytotoxic effect and the maximum non-effect dose of trichloroethylene( TCE) on Jurkat T cells in vitro. METHODS: i) Naive and activated Jurkat T cells were treated with different concentrations of TCE( 0. 10, 0. 50, 1. 00, 2. 00, 5. 00, 10. 00 mmol / L). Phorbol-12-myristate-13-acetate and ionomycin were used as agonist. No TCE was used in the control group and dimethyl sulfoxide( DMSO) was used as the solvent group. The morphology of Jurkat T cells was observed using a light microscope and the survival rate of Jurkat T cells was investigated using CCK-8 essay after cells were cultured for 24,48 and 72 hours. ii) Nave and activated Jurkat T cells were treated with different concentrations of TCE( 0. 00,0. 02,0. 20,2. 00 mmol / L). The apoptosis of cells was detected using flow cytometry and the level of interleukin-2( IL-2) in supernatant was detected using enzyme linked immunosorbent assay after cells were cultured for 24,48 and 72 hours. RESULTS: i) Cytotoxic effect was observed after cells were exposed to 10. 00 mmol / L TCE for 24 hours. Cells dispersed,cell volume diminished,cell membrane ruptured,cytoplasm condensed and increased outflow of intercellular organelles. The effect of interaction between exposure dose and exposure time was statistically significant on cell survival rate( P < 0. 01). Compared with the control and DMSO groups at the same time points,there were no significant differences in the 0. 10,0. 50,1. 00 and 2. 00 mmol / L TCE treatment groups in cell survival rates in three different time points( P > 0. 05),while the cell survival rates of 5. 00 and 10. 00 mmol / L TCE treatment groups were significantly decreased( P < 0. 01). ii) When TCE concentration was 0. 00-2. 00 mmol / L,there were no significant differences in the main effect of exposure dose and interactions of between exposure dose and cell type or exposure time on cell apoptosis rate( P > 0. 05). Compared with the same time points and groups of naive Jurkat T cells,the levels of IL-2 of activated Jurkat T cells were significantly increased( P < 0. 01). In the three different time points,the level of IL-2 of activated Jurkat T cells increased in accordance with the TCE exposure dose,showing a dose-effect relationship( P < 0. 01). The level of IL-2 of activated Jurkat T cells increased in accordance with TCE exposure time,showing a time-effect relationship( P < 0. 01). CONCLUSION:s TCE at the level of 2. 00 mmol / L had no observed effect in Jurkat T cells. High doses of TCE( ≥5. 00 mmol / L) showed cytotoxic damages to naive and activated Jurkat T cells and low doses of TCE( ≤2. 00 mmol / L) could stimulate activated Jurkat T cells secrete IL-2 in a dosedependent and time-dependent manner.

Investigation on invasion situation of cardinal ligament in stage ⅠA2- ⅡA2 cervical cancer and its risk factors analysis / 重庆医学

Objective To investigate the invasion situation of cardinal ligaments(CL) in stage Ⅰ A2 - ⅡA2 cervical cancer and to find the high risk clinicopathological factors affecting its invasion .Methods The retrospective analysis was performed on the clinicopathological data in 2 982 patients with cervical cancer treated by operation extracted in 5 hospitals from January 2004 to De‐cember 2014 .Results (1) Totally 855 cases according with the research condition were included .The CL invasion was found in 20 cases(2 .3% ) .No CL invasion occurred in the stage ⅠA2 ,the CL invasion rate in the stage ⅠB1 was 1 .7% ,in the stage ⅠB2 was 1 .2% ,in the stage ⅡA1 was 5 .7% and in the stage ⅡA2 was 2 .9% .With the increase of FIGO staging ,the CL invasion rate had no significant increase(P=0 .269) .(2) The infiltration rate of left CL and right CL had no statistical difference among the stageⅠA2- ⅡA2(P=1 .000) .And the invasion rate of the left CL with different FIGO stages had no statistical difference(P=0 .286) , while the right one had statistical difference(P=0 .005) ,in which the highest was in the stage ⅡA1 (4 .3% ) .(3)The univariate a‐nalysis found that lymph‐vascular invasion ,cervical stroma infiltration depth ,pelvic lymph node metastasis ,uterus body infiltration and vaginal fornix infiltration were the risk factors of CL invasion(P<0 .01);the further multivariate analysis determined that the invasion of uterine body(OR=11 .858) ,pelvic lymph node metastasis(OR=6 .359 ,) ,vaginal fornix infiltration(OR=6 .012 ,) were the risk factors for CL invasion(P<0 .05) .Conclusion The invasion rate of CL invasion in early stage cervcial cancer is low .The invasion of uterine body ,pelvic lymph node metastasis and vaginal fornix filtration are the risk factors for CL invasion in the stageⅠA2- ⅡA2 cervical cancer .

Bone marrow-derived mesenchymal stem cells in the treatment of myocardial infarction / 中国医学科学院学报

Bone marrow-derived mesenchymal stem cells (BMSCs) have the ability to regenerate myocadial tissue. BMSCs transplantation can produce cardiac-like cells,promote angiogenesis, and secrete a variety of growth factors and cytokines. This article summarizes the molecular mechanisms of myocardial repair, explores the potential regenerative capacity of cardiac muscles after myocardial infarction,and reviews the previous studies on BMSC transplantation for treatment of cardiac muscle after myocardial infarction, with an attempt to provide new insights in clinical decision-making.

Propranolol for proliferating hemangioma: therapeutic efficacy and expressions of vascular endothelial growth factor-A and hypoxia-inducible factor 1α in patients before and after treatment / 中华皮肤科杂志

Objective To evaluate the clinical efficacy of propranolol in treating proliferating infantile haemangiomas,and to measure the expression levels of vascular endothelial growth factor-A (VEGF-A) and hypoxiainducible factor 1α (HIF-1α) in sera and urine of patients during the treatment.Methods Thirty infants with proliferating haemangiomas were treated with propranolol at doses of 0.5-2 mg/kg per day.The radius of haemangiomas was measured,and blood and urine samples were obtained from these patients before,and at 4 and 12 weeks after the beginning of treatment.Clinical efficacy was estimated according to a four-graded scale as well as the feedback from parents of these patients.Enzyme-linked immunosorbent assay (ELISA) was performed to determine the serum and urine concentrations of VEGF-A and HIF-1α.Thirty check-up infants collected from the Department of Child Health Care served as the healthy controls.Statistical analysis was done by two-way analysis of variance followed by the least significant difference (LSD) test.Results After 12 weeks of treatment,clinical response was excellent in 2 patients,good in 11,moderate in 14,and poor in 3.The serum levels of VEGF-A and HIF-1α were (268.174 ± 95.056) μg/L and (10.809 ± 1.686) mg/L respectively in the control group,sequentially decreased in the patients from baseline to 4 and 12 weeks after the beginning of treatment (VEGF-A:(385.692 ± 136.146) vs.(264.853 ± 122.12) vs.(211.345 ± 104.035) μg/L; HIF-1α:(31.462 ± 7.458) vs.(21.454 ± 5.489) vs.(12.052 ± 3.623) mg/L).The trend in expression changes of VEGF-A and HIF-1α in urine samples was similar to that in blood samples in these patients.Positive correlation was observed between the expression level of VEGF-A and HIF-1α in sera (r=0.730,P＜ 0.05) and urine (r=0.667,P＜ 0.05) of these patients.Moreover,the levels of serum VEGF-A,urine VEGF-A,serum HIF-1α and urine HIF-1α were all negatively correlated with the time course following propranolol administration (r =-0.390,-0.689,-0.806,-0.683,P ＜ 0.05,0.01,0.05,0.01 respectively).Conclusion Propranolol is effective for the treatment of proliferating infantile haemangiomas,likely by reducing serum and urinary concentrations of VEGF-A and HIF-lα in children.

Chondrogenesis of synovial mesenchymal stem cells co-cultured with chondrocytes on the three-dimensional scaffold / 中国组织工程研究

BACKGROUND:Articular chondrocytes with the ability of autocrine and paracrine can provide the growth factors and microenvironment for synovial mesenchymal stem cels differentiating into the chondrocyte. The three-dimensional scaffold could provide space for cels adhesion, proliferation and differentiation. OBJECTIVE: To study the ability of chondrogenesis by co-culturing synovial mesenchymal stem cels and chondrocytes under the three-dimensional condition. METHODS:The synovial membrane and articular cartilage were harvested from rat knee joint. The synovial mesenchymal stem cels and chondrocytes were obtained through the method of enzyme digestion. The passage 3 synovial mesenchymal stem cels and passage 2 chondrocytes were co-cultured in the chitosan/I colagen composite scaffolds at the ratio of 1:2. Then, the cels/scaffold composite was harvested to be examined morphologicaly, histologicaly and immunohistochemicaly after being cultured 21 days. The confocal laser was also employed to detect the cels distribution in the scaffold. RESULTS AND CONCLUSION: After being cultured 72 hours, it could be observed from the cels/scaffold composite examined through the scanning electron microscope that the cels adhered on the surface of the scaffold and extracelular matrix surrounding the cels was seen on the scaffold. After being cultured 21 days, it could be found through the confocal laser scanning that the cels were wel-distributed on the scaffold, and cels decreased gradualy. Type II colagen was positive in the extracelular matrix immunohistochamicaly. It suggested from this study that the synovial mesenchymal stem cels could be co-cultured with chondrocytes in the chitosan/I colagen composite scaffolds and have the ability of chondrogenesis differentiation.

Chondrogenic differentiation of rat synovial-derived mesenchymal stem cells / 中国组织工程研究

BACKGROUND:Compared with other sources of mesenchymal stem cells, synovial-derived mesenchymal stem cells have significant characteristics of chondrogenesis and cloning. Therefore, synovial-derived mesenchymal stem cells are one of the most promising seed cells in cartilage tissue engineering. OBJECTIVE:To isolate and culture synovial-derived mesenchymal stem cells of Sprague-Dawley rats, identify the multipotential differentiation and the potential ability of chondrogenic differentiation in three-dimensional culture condition. METHODS:The synovium tissue was harvested from Sprague-Dawley rats. The synovial-derived mesenchymal stem cells were isolated with typeⅠcol agen enzyme digestion method and cultured in vitro. The passage 3 cells were detected with giemsa staining, the cellcycle, adipogenic and osteogenic differentiation were determined. The passage 3 cells were centrifuged as pel ets and cultured in the chondriogenic medium for 21 days. And the pel ets were examined by toluidine blue staining, typeⅡcol agen immunohistochemical staining and RT-PCR. RESULTS AND CONCLUSION:The mesenchymal stem cells isolated from the synovium tissue of rats have the characteristics of mesenchymal stem cells, and exhibit fibroblast-like morphology after cultured in vitro. The multilineage differentiation potentials were also revealed. After the cellwere cultured in chondrogenic medium for 21 days, chondroid tissue was found, type II col agen and aggrecan could be detected positively by toluidine blue staining, typeⅡcol agen immunohistochemical staining, and expressed by RT-PCR examination. Therefore, synovial mesenchymal stem cells have a chondrogenic differentiation potential.

Correlation between cervical lesion development and histone acetylation modification that regulates RAR-β2 expression / 中国肿瘤临床

Objective:To investigate the relationship between cervical lesion development and histone acetylation that regulates RAR-β2 expression. Methods:Immunohistochemistry and Western blot analysis were performed to detect AcH3, RAR-β2, and involu-crin expression in normal cervical tissues as well as in tissues with cervical intraepithelial neoplasia (CIN)Ⅱ-Ⅲand squamous cell cer-vical carcinoma. The relationship among histone acetylation level, RAR-β2 expression, and cervical lesion severity were analyzed. Re-sults:AcH3, RAR-β2, and involucrin expression were reduced or absent with the progression of cervical lesions;significant differences were noted between the groups (P<0.05). Histone acetylation level and RAR-β2 expression were positively correlated (r=0.797, P<0.05). AcH3 and RAR-β2 expression, which were both associated with the cervical lesions, were negatively correlated [r=-0.547(AcH3), r=-0.585(RAR-β2), P<0.05]. Conclusion:Histone acetylation modification is associated with the regulation of RAR-β2 expression. This pro-cess is also likely to participate in the carcinogenesis of cervical carcinoma.

Chondrocyte supernatant induces chondrogenesis and pellet cultivation of rat synovial mesenchymal stem cells / 中国组织工程研究

BACKGROUND:Synovial mesenchymal stem cells have the ability of multilineage differentiation in vitro, which are expected to be seed cells for the treatment of cartilage defects in cartilage tissue engineering. Appropriate growth factors are critical for the chondrocyte differentiation of synovial mesenchymal stem cells. OBJECTIVE:To study the role of secreted factors by chondrocytes to induce chondrogenesis of synovial mesenchymal stem cells. METHODS:The synovial mesenchymal stem cells and chondrocytes were harvested from rat knee joints and cultured through the digestion method. The supernatant was col ected from chondrocytes, and centrifuged, filtered and cryopreserved. The third passage synovial mesenchymal stem cells centrifuged as pel ets were cultured in the chondrocyte supernatant for 21 days. And the cells morphology was examined and the type II col agen and aggrecan were detected through immunohistochemistry and RT-PCR. RESULTS AND CONCLUSION:The synovial mesenchymal stem cellpel ets cultured in the chondrocyte supernatant became cartilage-like tissue after 21 days. The type II col agen was detected positively in the matrix of synovial mesenchymal stem cellpel et immunohistochemical y. RT-PCR examination showed that the type II col agen and aggrecan expressed in the synovial mesenchymal stem cellpel et cultured in the chondrocyte supernatant. It suggested that synovial mesenchymal stem cellcould be induced to differentiate into chondrocytes depending on soluble factors secreted by chondrocytes.