Adult ; China ; epidemiology ; Hepatitis B ; prevention & control ; Hepatitis B Vaccines ; Humans ; Practice Guidelines as Topic ; Vaccination

Objective To observe the level of hypoxia-inducible factor-1 alpha (HIF-1α) and its downstream target gene cyclooxygenase-2 (COX-2) in LPS-treated intestinal epithelial cells,and to explore the possible intervention targets of Rheum emodin.Methods Human intestinal epithelial cells were cultured in vitro treated with LPS to establish the experimental model.The protein level trends of HIF-1α and COX-2 were measured by Western blot in LPS dose-dependent and time-dependent manners.The protein level trends of HIF-1α,COX-2,Phospho-IκB-α and Phospho-NF-κB p65 were measured in LPS plus various concentrations of Rheum emodin treated groups.The expression of HIF-1α mRNA were detected by PCR after cells treated with LPS or LPS plus Rheum emodin,respectively.The effect of Rheum emodin on the proliferation of intestinal epithelial cells was measured by MTT assay in each group.Data were analyzed with ANOVA,and P ＜0.05 was considered significant.Results LPS induced the protein level of HIF-1α in a dose-dependent and a time-dependent manners.With increasing concentrations of LPS,the protein level of HIF-1α increased to the peak when cells were treated with LPS at 10-30mg/mL,and then gradually decreased (P ＜0.05).Firstly the protein level of HIF-1α reached the peak at 0.5 h after treatment,and then decreased to the lowest level at 4 h,and finally returned to a high level (P＜0.05).The protein level trend of COX-2 went a similar way to that of HIF-1α (P ＜0.05).Rheum emodin inhibited the protein levels of LPS-induced HIF-1α,COX-2,Phospho-IκB-α and Phospho-NF-κB p65 with a significant dose-effect relationship (P ＜ 0.05).The PCR showed Rheum emodin inhibited LPS-induced increasing expression of HIF-1α mRNA.MTT assay showed different concentrations of Rheum emodin (0 μmol/L,20 μmol/L,40 μmol/L,60 μmol/L,80 μmol/L) had no significant effect on cell proliferation (0.95 ± 0.02,0.89 ± 0.03,0.88 ± 0.04,0.91 ± 0.03,0.83 ± 0.03,P ＞ 0.05).Although Rheum emodin produced biological effect at this concentration range,and it had no toxicity to intestinal cells.Conclusions LPS induces HIF-1α/COX-2 signaling pathway in a time-dependent and a dose-dependent manners in intestinal epithelial cells.Rheum emodin blocks the hypoxia pathway of LPS/HIF-1α/COX-2 and the inflammatory pathway of LPS/IκB-α/NF-κB/COX-2,which may play a protective effect on intestinal epithelial cells.

Objective To investigate the effect of human umbilical cord mesenchymal stem cells (UC-MSCs) on immune cells and inflammatory factors in septic rats.Methods 184 male Sprague-Dawley (SD) rats were divided into normal control group (n = 8), sham operation group (n = 48), sepsis model group (n = 64), and UC-MSCs treatment group (n = 64). An animal model of sepsis was produced by cecal ligation and puncture (CLP). In the UC-MSCs treatment group 1 mL UC-MSCs (2×106/mL) were injected intraperitoneally at 1 hour after the model establishment;the sham operation group and the sepsis model group were given the same amount of saline. Sixteen animals in each group of the sham operation group, sepsis model group, and UC-MSCs treatment group were observed for 72-hour survival rate. The percentages of CD4+ T cells and the ratio of helper T cells 1/2 (Th1/Th2) in whole blood cells were measured by flow cytometry at 12, 24, 48 and 72 hours after operation. The levels of tumor necrosis factor-α (TNF-α), high mobility group box 1 (HMGB1), interleukin-10 (IL-10) were measured by enzyme linked immunoabsorbent assay (ELISA).Results The 72-hour survival rate of the UC-MSCs treatment group was slightly higher than that of the sepsis model group [62.5% (10/16) vs. 50.0% (8/16),χ2 = 0.509,P > 0.05]. The percentage of CD4+ T cells and Th1/Th2 ratio in the sepsis model group were significantly higher than those in the sham operation group at 12 hours after operation, and decreased as the time prolonged to 48 hours. The levels of plasma inflammatory factors were significantly higher than those of sham operation group at 12 hours after operation, TNF-α and IL-10 were decreased at 48 hours after operation, while HMGB1 continued to increase until 72 hours after operation. Compared with those in the sepsis model group, the percentages of CD4+ T cells at 12 hours and 24 hours after operation [(49.66±0.91)% vs. (59.11±1.17)%, (41.80±0.89)% vs. (49.84±0.99)%], the levels of Th1/Th2 ratio at 12, 24, 48 hours after operation (0.745±0.065 vs. 1.254±0.115, 0.407±0.077 vs. 0.806±0.061, 0.280±0.057 vs. 0.454±0.049), and the levels of TNF-α and HMGB1 were significantly reduced at 12, 24, 48 and 72 hours after operation in the UC-MSCs treatment group [TNF-α(ng/L):52.60±6.60 vs. 58.03±6.53, 71.77±8.48 vs. 147.39±11.37, 111.83±10.76 vs. 271.36±19.04, 83.09±7.43 vs. 171.04±14.06; HMGB1 (ng/L): 149.12±9.89 vs. 187.33±12.79, 192.94±14.92 vs. 442.35±52.72, 1393.67±88.86 vs. 1950.90±126.66, 1875.84±111.67 vs. 2557.12±186.01], all with statistically significant differences (allP <0.05). The level of IL-10 was significantly higher at 12, 24, 48 and 72 hours after operation (ng/L: 65.46±5.51 vs. 33.32±4.17, 86.49±5.78 vs. 63.11±5.53, 142.73±9.94 vs. 106.81±6.36, 123.74±10.90 vs. 89.90±7.71, allP <0.01).Conclusion UC-MSCs can make CD4+ T cells in early sepsis, and Th1/Th2 ratio to normal, by reducing the levels of proinflammatory factors, and increasing the level of anti-inflammatory factor, and improve sepsis immune function status, but cannot improve the survival rate of animals.

Objective To explore the method of culture of rat adipose-derived stem cells(ADSCs) and differentiation induction into myoblasts. Methods Adipose tissues were obtained from SD rats,and were isolated by enzyme digestion and cultured into ADSCs.The expression of surface antigen CD90,CD105 and CD34 was detected by immunofluorescence and flow cytometry.ADSCs of the second passage with logarithmic growth were obtained,and culture media containing 5-azacytidine(5-aza) and basic culture media were employed for cells in induction group and control group,respectively.The induction lasted for 7 d,14 d,21 d,28 d and 35 d,respectively.Cell growth and cell morphology were observed by inverted phase contrast microscope,and immunofluorescence and flow cytometry were utilized to detect the expression of myoblast specific antigens desmin and myosin. Results ADSCs were successfully isolated and cultured,and were identified to be stem cells.On the 28th day of induction,cells in induction group displayed "swirl" morpholgy,and multinucleation was observed.It was revealed by immunofluorescence and flow cytometry that the highest expression rates of desmin and myosin were 52.57% and 50.04%,respectively on the 28th day of induction,while there was no expression before induction and in control group. Conclusion ADSCs can be isolated and cultured from rat adipose tissues,and can further differentiate into myoblasts after induction by culture media with 5-aza.The expression of myoblast specific antigen is the highest on the 28th day of induction.

OBJECTIVETo explore the characteristics of immune imbalance in patients with multiple organ dysfunction syndrome (MODS) induced by severe intra-abdominal infection and its relationship with changing of TCM sthenia-asthenia syndrome.

METHODSForty-six patients with MODS induced by severe intra-abdominal infection and treated with etiological and syndrome differentiation of integrative medicine were observed in succession. Patients' peripheral blood levels of interleukin-6/interleukin-10 ratio (IL-6/IL-10), human leukocyte antigen DR site (HLA-DR), helper T lymphocyte1/2 ratio (Th1/Th2), and the regulatory T lymphocyte (Treg) were measured on the 1st, 3rd and 7th day of the research respectively. And the distribution laws of TCM syndrome types, sthenia (S), asthenia (A), and mingled sthenia/asthenia (M), in patients were observed as well.

RESULTSIL-6/IL-10 ratio at all the testing time points showed insignificant difference in patients of types S and M, while in those of type A, it was more lowered on the 7th day than that on the 1st day. HLA-DR lowered to <30% on the 7th day in all patients of type A and showed significant difference to that on the 1st day (P <0.05), while HLA-DR <30% was not found in all patients of types S and M. Th1/Th2 ratio in patients of types S and A was insignificant different at the foremost 3 days, but lowered significantly on the 7th day, while in patients of type M, it was unchanged in all the 7 days of observation. Treg level was unchanged in the foremost 3 days in patients of types S and M, while in those of type A, it raised on the 3rd day, but no raising was found in the subsequent 4 days. Comparisons of various indexes detected at corresponding time points respectively among patients with various syndrome types showed that, for levels of IL-6/IL-8, HLA-DR, and Th1/Th2, the sequence was S>M>A; and for Treg, it was A>M>S.

CONCLUSIONIn the pathological process of MODS induced by severe intra-abdominal infection, the index IL-6/IL-10, reflecting the balance of the pro-/anti-inflammatory cytokines and the indexes HLA-DR, Th1/Th2 and Treg reflecting the immune function, all can exactly reflect the TCM asthenia-sthenia syndrome types. The sequence in patients of various syndrome types for levels of IL-6/IL-10, HLA-DR and Th1/Th2, is S> M>A, but for Treg it is the inverse, as A>M>S.

Adolescent ; Adult ; Aged ; Diagnosis, Differential ; HLA-DR Antigens ; immunology ; Humans ; Interleukin-6 ; immunology ; Interleukin-8 ; metabolism ; Medicine, Chinese Traditional ; Middle Aged ; Multiple Organ Failure ; complications ; drug therapy ; immunology ; Peritonitis ; complications ; drug therapy ; immunology ; Sepsis ; complications ; drug therapy ; immunology ; metabolism ; T-Lymphocytes, Regulatory ; immunology ; Th1 Cells ; immunology ; Th2 Cells ; immunology ; Yang Deficiency ; immunology ; Young Adult

OBJECTIVETo evaluate the dynamic change of Th1/Th2 cytokines in serum, peritoneal lavage fluid (PLF) and splenic macrophages (SM) in rats with severe peritonitis, and to observe the therapeutic effects of recombinant interleukin-12 (rIL-2) and Shenmai injection (SMI), a Chinese medicinal preparation.

METHODSSevere peritonitis (SP) model was induced by intraperitoneal injection of E. coli and B. frag, and mild peritonitis (MP) model was induced by cecal ligation and punching. Then the following experiments were done: (1) Survival rates of animals after every 6 hrs in the 72 hrs after modeling were recorded, serum and PLF levels of cytokines, including tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), and interleukin-10 (IL-10), 6 hrs, 12 hrs, 24 hrs and 48 hrs after modeling were measured. (2) Model rats were treated with rIL-12 or SMI, the survival rate was recorded and serum levels of TNF-alpha, INF-gamma, and IL-10 before and after treatment were measured, and (3) amount of these cytokines produced by SM were determined 6 hrs, 12 hrs and 24 hrs after treatment. The survival rates and levels of cytokines were then compared between the groups (model group treated with rIL-12 or SMI, untreated model group, and blank group).

RESULTSSerum and PLF levels of IFN-gamma, TNF-alpha at all the time points in SP rats were significantly lower than those in MP rats while those of IL-10 6 hrs and 12 hrs after modeling were significantly higher in the former than that in the latter (P < 0.05). IFN-gamma secretion of SM in SP rats was significantly higher than that in MP rats 6 hrs after modeling (P < 0.05). Administration of rlL-12 or SMI given before modeling could improve the survival rate of the model rats (P < 0.05) and cause significant increase of the serum level and SM secretion of IFN-gamma.

CONCLUSIONImbalance in promoting/antagonizing inflammatory cytokines and Th2 response dominance appear in SP rats early at the initiating stage, and SM secretion of inflammation promoting factor also reduces. Administration in time of rIL-12 and SMI, may increase the survival rate, and its mechanism may be related with their immuno-stimulating action.

Animals ; Cytokines ; metabolism ; Disease Models, Animal ; Drug Combinations ; Drug Therapy, Combination ; Drugs, Chinese Herbal ; pharmacology ; Interferon-gamma ; metabolism ; Interleukin-10 ; metabolism ; Interleukin-12 ; pharmacology ; Male ; Peritonitis ; drug therapy ; mortality ; Plant Extracts ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins ; pharmacology ; Sepsis ; drug therapy ; immunology ; mortality ; Severity of Illness Index ; Survival Rate ; Th1 Cells ; drug effects ; metabolism ; Th2 Cells ; drug effects ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

Objective To evaluate the therapeutic effect of oral and external application of Chinese medicine combined with Ilizarov external fixator and the accordion technique for severe chronic osteomyelitis of the tibia.Methods A total of 78 patients with severe chronic osteomyelitis of the tibia were randomized into a routine treatment group and a combined treatment group, 39 in each group. All the patients in the two groups received external fixation by use of Ilizarov external fixator and the accordion technique. All the patients in the combined treatment group received oralGuyu decoction. The patients who had large wound received vacuum-sealing drainage in the routine treatment group, and vacuum-sealing drainage combined with external application ofShengji-Yuhong plaster in the combined treatment group. All the patients were treated for 8 weeks and followed up for 2 years. The time to wound healing and fracture healing, and the drainage time were compared between the two groups. Functional and radiologic findings were evaluated according to Paley's criteria. The functions of the knee joint and ankle joint were evaluated using the Hospital for Special Surgery (HSS) knee score and the scoring system of Baird and Jackson, respectively.Results The time to wound healing (17.33 ± 6.21 dvs. 22.27 ± 8.12 d;t=3.018,P=0.004) and the time to fracture healing (32.25 ± 6.02 weeks vs. 36.37 ± 7.75 weeks;t=2.623,P=0.011), and the drainage time (17.01 ± 4.66 dvs. 21.51 ± 5.23 d;t=4.012, P<0.001) in the combined treatment group were significantly shorter than those in the routine treatment group. According to Paley's criteria, the patients who achieved a score of excellent or good for fracture healing (84.6%vs. 53.8%;χ2=7.282,P=0.007) and function (92.3%vs. 66.7%;χ2=6.369,P=0.012) in the combined treatment group were significantly more than those in the routine treatment group. The scores of the HSS knee score (84.56 ± 7.42vs. 78.81 ± 5.33;t=3.391,P=0.002) and the scoring system of Baird and Jackson (85.01 ± 8.21vs. 79.21 ± 6.78;t=3.402,P=0.024) in the combined treatment group were significantly higher than those in the routine treatment group.Conclusion OralGuyu decoction and external application ofShengji-Yuhong plaster combined with Ilizarov external fixator and the accordion technique can promote recovery of the joint function, fracture healing and wound healing.

Objective To investigate the feasibility of constructing tissue engineering urethral substituted graft using human lingual keratinocytes and natural derived scaffold.Methods From Oct.2009to Jan.2010,ten patients with anterior urethral stricture were enrolled in this study.A 0.5 × 0.8 cm lingual mucosa was harvested during the operation.Lingual keratinocytes were then isolated and cultured from the mucosa.AE1/AE3 antibody was used to identify the lingual keratinocytes.Keratinocytes were collected and seeded onto three types of scaffold including dehydrated BAMG,liquid stored BAMG and 4-layer SIS product,with the density of 1 × 107/ml at passage three.After being cultured for seven days in vitro,H&E staining and Electronic Scan Microscopy were used to evaluate the compound matrixes.Results No complication occurred in the patients after operation.The primary passage of confluence lingual keratinocytes appeared in a typical cobblestone shape after being cultured for 14 days in vitro.The proliferation rate of these cells increased rapidly during the three passages.However,it decreased significantly in the 4th passage.H&E and Electronic Scan Microscopy examinations showed that few cells grew on the surface of the liquid stored BAMG.Nevertheless,multiple keratinocytes layers could be seen in dehydrated BAMG and 4-layer SIS.Meanwhile,cellular infiltration could be observed in SIS sections.Conclusions Human lingual keratinocytes could be the alternative of seeding cells for constructing tissues for engineering the urethra.These cells exhibited good compatibility and are adhesive to the SIS or BAMG.The compound matrix,which used human lingual keratinocytes and natural derived scaffold,may meet the clinical need of urethral disease in the future.

Objective To investigate the incidence,sonographic appearance of the anatomic variation of carpal tunnel median nerve and its accompanying structures in healthy volunteers and explore the value of this variation in carpal tunnel syndrome.Methods A total of 360 hands of 180 healthy volunteers were included in the study.The full course of the median nerve in the forearm and carpal tunnel was examined with high-frequency ultrasound.The median nerve was first located in cross section at wrist and then with continuous cross-sectional scanning to observe the the full course of the median nerve in the forearm and carpal tunnel with high-frequency ultrasound.Results Anatomic variation of carpal tunnel median nerve and its accompanying structures were observed:① High division median nerve were found in 2 wrists (0.56 ％) ;②Bifid median nerve were found in 17 wrists (4.72％) ;③Persistent median artery were found in 22 wrists (6.11％),and 2 wrists (0.56％) were also found accompanied vein.Aanatomic variation of carpal tunnel median nerve accompanied with persistent median artery were observed in 16 wrists (4.44 ％).Conclusions High-frequency ultrasound was sensitive to diagnose the anatomic variation of carpal tunnel median nerve and its accompanying structures.Recognition of these variations can help us to make correct diagnosis of carpal tunnel syndrome.

Objective To investigate the feasibility of replacing urinary epithelial cells with oral keratinocytes by being seeded on bladder acellular matrix graft(BAMG). Methods Twenty-four male rabbits were randomly divided into 2 groups:experimental group and control group.A length of 2.0 cm and width of 0.8 cm penile urethral mucosal defect was induced in the anterior urethra 2.0 cm awav from the urinary meatus in all the rabbits.Oral keratinocytes were isolated from a small buecal mucosa(1.0 cm×0.4 cm)of the 12 rabbits in experimental group and seeded onto a culture dish with a feeder layer of 3T3 mouse fibroblasts inhibited by mitomycin(i3T3).Passage 2 oraI keratinocytes cuItured with i3T3 were expanded and seeded onto sterilized BAMG(2.2 cm×1.0 cm)to repair the de-fects of the urethra.Urethroplasty was performed with BAMG with no cell seeding in the controlgroup.Catheter examination and retrograde urethI ography was done in 1,2 and 6 months after graft-ing.The urethral grafts were harvested and analyzed by histological staining. Results Oral kerati-nocytes seeded onto a feeder layer of i3T3 had better morphous and amplification capability.The corn-patibility of the compound graft was assessed by HE staining and scanning electron microscope.Twelve rabbits in the experimental group could void without difficulty.Catheter examination and ret-rograde urethrogram revealed that a wide urethral caliber was maintained with no sign of strictures and fistula formation.Histological examination showed the grafted urethra was covered with smooth mu- cosa with no strictures at 1,2 and 6 months.The oral keratinocytes of the graft still existed even 6 months after grafting.On contrast,gross and urethrogram demonstrated urethral stricture in the con-trol group.And inflammatory reactions could be found in the area of the stricture through light micro-scope. Conclusions Oral keratinocytes have good compatibility with BAMG and the compound graft could be used for urethral reconstruction in the animal model.