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 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.

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 investigate and assess the best seeding method for constructing three dimensional urethral tissue in vitro. Methods High speed agitation decellular method was used for preparing the porcine acellular corporous spongiosum matrix (ACSM). Before seeding, the matrix was sterilized via soaking compound iodine solution. Rabbit tongue epithelial cells and cavernosal smooth muscle cells were isolated and cultured. Three different groups of seeding method was used in this study. Group A (sandwich seeding group): The smooth muscle cells and epithelial cells were seeded onto the different side of ACSM by static method. Group B (injection seeding group): The smooth muscle cells were injected into the scaffold. Then, epithelial cells were seeded onto the urethral surface of ACSM by static method. Group C (agitation seeding group): The smooth muscle cells were seeded into the scaffold by agitation method. Then, epithelial cells were seeded onto the urethral surface of ACSM by static method. After being seeded, all matrixes were cultured in vitro for 14 d. HE and immunoassay staining were used to examine the results of seeding. Results Looser matrix was obtained after using high speed agitation decellular method. Compound iodine solution could not only sterilize efficiently but also reserve the original structure of biomaterial. An intact epithelial cellular layer onto the surface of scaffold could be observed in HE staining section after 14 d culturing in vitro.Few smooth muscle cells could be found in big space of biomaterial in group A. In group B, smooth muscle cells were restrained in some regions of the matrix. Smooth muscle cells were well distributed into the scaffold in group C. Conclusions After using high speed agitation decellular method, an ideal matrix with three dimensional structure can be obtained. Combined with agitated seeding method, three dimensional urethral tissue can be constructed.

Objective To investigate the feasibility of constructing tissue engineered corpora cavernosa smooth muscle by seeding human umbilical artery smooth muscle cells (HUASMCs) in acel-lular collagen matrices.Methods Acellular corporal collagen matrices (ACCM) were obtained from the penis of adult rabbits by a cell removal procedure.HUASMCs were isolated from human umbilical cords through explant techniques and cultured in vitro.Subsequently,HUASMCs were seeded to ACCM and cultured in vitro.After that,the seeded ACCMs were implanted subcutaneously in 9 BALB/C athymic mice.Animals were killed 10,20 and 40 days after implantation.The implants were retrieved and morphological examinations were performed to evaluate characteristics of the engineered tissues.Additionally,organ bath studies were performed to address the contractility of the engineered tissues.Results The deeellularization process successfully extracted all cellular components; colla-gen fibers maintained their original porous morphology and structure.ACCM could be reseeded with cultured HUASMCs in vitro,and HUASMCs had the potential of attachment and proliferation on the three-dimensional ACCM scaffolds.Histologic analyses of the explants from all time points demon-strated a progressive regeneration of corpus cavernosum smooth muscle,with structures very similar to those of the native corpus cavernosum,The maximum contraction force induced by phenylephrine and electrical stimulation was (3.64+0.18)g and (2.50+0.21)g.Conclusion HUASMCs can be seeded on 3-dimensional ACCM scaffolds and will develop a tissue similar to that of the native corpus eavernosum smooth muscle.

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.

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 discuss the curative effect of different operative methods for the treatment of urethrocutaneous fistula (UCF) after urethroplasty.Methods Clinical data of 54 cases of UCF from January 2003 to July 2011 were collected.Simple suture,advancement skin flap,pedicle penile skin flap urethroplasty,tongue/buccal mucosa urethroplasty were performed according to the size,location,number and whether there was urethral stricture of UCF.The treatment effect was recored and analyzed.Results The success rate of UCF repair was 85％ (46/54).There were 4 patients cured after second surgery.The success rates of repair of UCF with simple suture,advancement skin flap,pedicle penile skin flap urethroplasty,tongue/buccal mucosa urethroplasty were 95％,76％,82％,and 83％,respectively.Postoperative recurrence was observed in 5 patients in 6 months after the surgery and 4 of them received the re-operation.Conclusion According to the principle and the individual circumstance,taking personalized operative methods may improve the success rate of the repair of all kinds of UCF.

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.