Objective:For severe skin defects which are deep to dermis, engineered skin with epidermis and dermis (bilayered) is required. Based on the success of engineering epidermis with GT/PCL electrospun membranes, our study was to investigate whether this membrane could be also used for engineering bilayered skin graft.Methods:From 2013 to 2019, we first prepared three GT/PCL electrospun membranes with different proportion (70∶30; 50∶50; 30∶70) in our laboratory; the biocompatibility of the membrane was evaluated in vitro by seeding fibroblasts or keratinocytes on the membranes. Then the outcome of GT/PCL membranes repairing skin defects in the nude mouse was investigated.Results:Cell attachment and proliferation were significantly improved with increase of gelatin. Histological analyses showed that bilayered skin engineered with GT/PCL (70∶30) group could form relatively better structure after 3 weeks of cultivation in vitro. Further in vivo transplantation studies revealed that scaffolds were not degraded in all three groups, indicating that these materials were not suitable for engineering bilayered skin although they had good biocompatibility.Conclusions:The higher gelatin membranes possess better biocompatibility. Further in vivo transplantation studies reveal that bilayered skin engineered with GT/PCL membranes is able to repair skin defects in the nude mouse.

Objective: To study the effects of three different granular fats on proliferation, differentiation and migration of adipose-derived stem cells. Methods: Ten patients were selected for lumbar liposuction. The adipose tissue was obtained with different sized side-hole fat aspiration devices: 10 ml of Macrofat (n=10), 10 ml of Microfat (n=10) and 10 ml of Nanofat (n=10). Enzyme digestion method was used to separate and extract adipose-derived stem cells(ADSCs). The differences of vascular matrix components in these 3 kinds of fat sources were analyzed. XTT assay was used to detect proliferation and growth ability. The migration ability of the cell injury model was observed in vitro, and the multi-directional differentiation ability was compared by the analysis of adipogenic and osteogenic induction. The experimental data were analyzed using SPSS 13.0 software. One-Way ANOVA was used to compare the difference of multiple groups. P<0.05 was considered as statistically significant. Results: The detection of adipose-derived stem cells was by flow cytometry and it showed Macrofat, Microfat and Nanofat was(4.23±0.37)×10⁵, (2.29±0.33)×10⁵ and(1.56±0.16)×10⁵. The content of fat-derived stem cells in Macrofat was the highest, followed by Microfat, and the content of Nanofat was the least (F=209.533, P<0.001). XTT assay showed that the proliferative ability of adipose-derived stem cells in the first two generations of Macrofat was the highest, followed by Microfat, while the proliferation of Nanofat was the lowest (the absorbance in 3 groups increased in a time-dependent manner). There was no significant difference in the proliferative capacity of the third generation of 3 groups (the absorbance of these 3 groups were compared at each time point). The cellular trauma model showed that the first generation of Macrofat-derived stem cells had the best migration ability, followed by Microfat, while the Nanofat had the weakest migration ability(Compared with the remaining area of wounds at 0 h, 12 h, 24 h point between the three groups, F=306.370, 1409.907, P<0.001). From the second generation, the migration ability of each group of ADSCs gradually self-repaired, and the migration ability at 12 h was similar to that of the first generation (F=11.410, P<0.001), but there was no significant difference in 24 h (F=0.070, P=0.933). Oil red O and alizarin red staining showed that the first generation of Macrofat and Microfat had better fat-forming and osteogenic differentiation ability than Nanofat(F=523.532, 620.022, P<0.001). However, there was no significant change after the second generation (F=2.144, 0.866, P=0.137, 0.432). Conclusions In the process of adipose tissue extraction of Nanofat, the production and activity of adipose-derived stem cells was impacted. However, in the process of culture and passage, the cell activity, proliferation ability, migration ability and differentiation ability can be achieved through self-repair, evenclose to the level of Macrofat or Microfat.

Objective To investigate the effect of adipose-derived stem cells (ADSCs) on pho toaging skin after laser pretreatment with GaAlAs.Methods ADSCs were isolated from healthy wistar rats,ADSCs were isolated and cultured to establish an adipose-derived stem cell culture system.ADSCs were pretreated with GaAlAs laser at a wavelength of 650 nm 4 J/cm2.A rat model of pho toaging aging was established.Different doses of ADSCs and low energy laser ADSCs were pretreated with ADSCs for the treatment of photoaging skin,and the morphological changes of epidermis and dermis were observed before and after treatment with low energy laser pretreatment.Results When the concentration of ADSCs was 103/100 μl,there was no significant difference in epidermal thickness and dermal thickness between ADSCs treated group and GaAlAs pretreatment group (P＞0.05).The thickness of epidermis in the GaAlAs pretreatment group was significantly lower than that in the ADSCs group (P＜0.05) at 104/100 μl.When the concentration of ADSCs was 5 × 104/100 μl,the epidermal thickness of the GaAlAs pretreatment group decreased significantly and the thickness of the dermis increased significantly,which was significantly different from that of the ADSCs group (P ＜ 0.05).Conclusions GaAlAs laser pretreatment can enhance ADSCs anti-skin photoaging ability.

Objective To analyze the curative effect of traditional Chinese medicine(TCM)combined with early enteral nutrition(EN)for treatment of patients with severe acute pancreatitis(SAP). Methods 70 SAP patients were randomly divided into TCM plus EN group(36 cases)and conventional therapy group(34 cases). Both groups received routine treatment. Additionally,TCM+EN group received early EN and TCM decoction treatment〔the ingredients of decoction including radix bupleuri,radix paeoniae alba,radix scutellariae,fructus aurantii immaturus, magnolia bark,raw rhubarb(rhubarb was added at last during cooking the decoction)and natrii sulfas exsiccatus (dissolved in water to be administered)each 10 g,the decoction was concentrated to 150 mL and then administered via a stomach tube to the patient,afterwards the tube was clipped for 2.5-3 hours,twice a day,4-7 days constituting a therapeutic course〕. After treatment,the time for patients' symptoms improvement,the situation of intestinal recovery, the length of stay in hospital,the total medical cost,serum C-reactive protein(CRP),aspartate aminotransferase (AST), lactate dehydrogenase (LDH), amylase (AMY), acute physiology and chronic health evaluationⅡ(APACHEⅡ)score and complications,intensive care unit(ICU)transfer rate and case fatality rate in two groups were observed. Results The time for symptoms improvement of abdominal tenderness(day:1.68±1.01 vs. 3.89±1.07), abdominal distension(day:2.17±1.48 vs. 4.24±3.23),abdominal pain(day:3.12±1.14 vs. 4.94±3.21)and the intestinal recovery of exhaust defecation time(day:3.48±0.92 vs. 5.32±3.30)of SAP patients after treatment in the TCM+EN group were faster significantly than those in the conventional therapy group(all P<0.05). The length of stay in hospital(day:15.50±1.75 vs. 19.35±1.69)and total cost(wan yuan:1.812±0.424 vs. 3.292±1.081) of TCM+EN group were less than those of conventional therapy group(P<0.05 or P<0.01). After treatment,the levels of serum CRP,AST,LDH,AMY,APACHEⅡscore in TCM+EN group and conventional therapy group were all lower than those before treatment,and on day 10,the degree of descent was more prominent in TCM+EN group〔CRP(mg/L):98.972±43.384 vs. 122.392±71.621,AST(U/L):75.952±55.668 vs. 126.391±47.431, LDH (μmol?s-1?L-1):1.48±0.21 vs. 2.61±1.46,AMY(U/L):146.362±58.792 vs. 226.392±37.692,APACHE Ⅱscores:6.978±3.352 vs. 13.652±7.621,P<0.05 or P<0.01〕. There was no death in TCM+EN group,while in the conventional therapy group,there was 1 case dead. ICU transfer rate in TCM+EN group was less than that in the conventional therapy group(2.78% vs. 11.76%),but there was no statistical significant difference between the two groups(χ2=0.99,P>0.05). Among the 70 patients with SAP,the cause of the disease due to gallstone accounted for 55.72%,hyperlipidemia for 37.14%,alcoholic for 4.28%and other 2.86%. Conclusion The use of TCM combined with early EN for treatment of patients with SAP can enhance the curative effect of SAP,reduce the hospitalization time and the total cost of patients,and decrease complications and mortality,that is conducive to the faster recovery of patients.

Objective To study the correlation between genetic polymorphisms of interleukin (IL)-1A/1B and susceptibility to tuberculosis (TB).Methods Genetic polymorphisms of IL-1A and IL1 B in 1032 TB patients and 1008 non-TB patients were analyzed using PCR-MassARRAY method.The correlation between genetic polymorphisms of IL-1A/1B and susceptibility to TB was statistically analyzed.Results Two tag SNPs of IL-1A and three tag SNPs of IL-1B were screened for the study.There were differences in the allele frequencies of rs2853550 and rs3783526 between TB group and non-TB group (P=0.047and P =0.034,respectively).IL-1 B SNP1 rs2853550 (P =0.025,OR =1.302,95 ％ CI =1.034-1.640,TC vs.CC) was found to be highly associated with TB,while the other SNPs showed no significant correlations with TB.Furthermore,IL-1B SNP1 rs2853550 [P=0.019,OR=1.308,95％ CI=1.045-1.638 for (TC+TT) vs.CC] in the dominant model conferred significant risk for TB,but IL-1A SNP2 rs3783526 [P=0.000,OR=0.764,95％ CI =0.591-0.988 for GG vs.(AA+GA)] in the recessive model showed protective effects against TB.The haplotype ‘TG’ in the IL-1B block showed a higher risk for TB compared with the common ‘ CA’ haplotype (P=0.032,OR=1.265,95％ CI=1.020-1.567).The diplotypes containing ‘ GA’ haplotype in IL-1A block and ‘ TG’ haplotype in IL-1B block were major risk factors for TB (for onecopy,adjusted P=0.014,OR=1.403 and 95％ CI=1.072-1.836; adjusted P=0.013,OR=1.339 and 95％ CI=1.063-1.688,respectively),but the diplotype with ‘CG’ in IL-1B block played a protective effect against TB (for two-copy,P=0.006,OR=0.664 and95％ CI=0.494-0.891).Conclusion The genetic polymorphisms of IL-1B rs2853550 might be closely associated with TB,but the GG genotype of IL1 A SNP rs3783526 might have the characteristic of anti-TB.

Objective To study the relationship between the genetic polymorphisms of carboxylesterase 1 gene (CESI) and the susceptibility to antituberculosis drug-induced hepatotoxicity (ATBDIH).Methods Genetic polymorphisms of CES1 in 473 tuberculosis patients with or without hepatotoxicity (200∶ 273) after antituberculosis chemotherapy were analyzed by PCR-MassArray.Results In4 tags of CES1 single nucleotide polymorphism (SNP),the frequency of the rs1968753 allele had statistical difference between the hepatotoxicity group and the no-hepatotoxicity group ( P =0.0236 ).The characteristics of anti-hepatotoxicity had been shown relationship with rs8192950 ( P =0.044,OR =0.649,95％ CI =0.426-0.989,AC/AA ) and rs1968753 ( P =0.048,OR =0.556,95％ CI =0.311-0.995,GG/AA).The diplotypes with ‘ CGC' haplotype exhibited significant protection against hepatotoxicity at one copy (P=0.048,OR=0.654,95％CI=0.430-0.996).Conclusions The genetic polymorphisms of CESI might have significant association with ATBDIH.SNP rs8192950 AC genotype and rs1968753 GG genotype might be the candidates for risk prediction of ATBDIH.

Objective To investigate the incidence and distribution of aberrational karyotype in acute myeloid leukemia (AML), and study the significance of the grouping by Southwest Oncology Group/Eastern Cooperative Oncology Group (SWOG/ECOG) in the prognosis of AML Methods The chromosome was prepared with brief culture of bone marrow, and the karyotype was analysed by G banding technique. All the patients were grouped according to the criterion of SWOG/ECOG, and the survival function of different groups was observed by the Kaplan-Meier method.Results 56 (67.47 %) out of 83 patients had clonal chromosome aberrations. Among those 56 patients, AML with translocation (15;17) and with translocation (8;21) presented in 30 patients(53.57 %), and the other kinds of aberrational karyotypes shared the left proportion. Among the 74 followed-up patients, 42 patients were dead. Among three groups with favorable, intermediate and adverse prognosis respectively, there is a significant difference (P <0.001). The complete remission rate of favorable group is higher than that of both intermediate and adverse (P <0.05). There is no difference between intermediate and adverse groups(P>0.05). Conclusion Cytogenetic aberration is one of the important factors affecting the effect on prognosis. The criterion of SWOG/ECOG can predict prognosis objectively.

Objective To study the therapeutic effects of Bizhi pills in mycobacterium tuberculosis infection.Methods KunMing mice were infected by intratail-vein injection with mycobacterium tuberculosis H37Rv, then treated with saline(A)or Rifampin(B) or Bizhi pills(C).The lungs,hvers and spleens were taken to observe their pathological changes,weighted and performed mycobacterial culture after 4 months of treatment.Results After 4 months of treatment, indexes of the lung in group C were markedly lower than that in group A (P＜0.05),but higher than that in group B,although the differences were not significant.The lesion of lung in group C were similar as that in group B,and were lessen than that in group A.No obvious lesion of lung was observed in 8 mice of group B and C,but the differences were not significant.Swell of spleen was observed in 9 mice in group A,2 mice in group B and5 mice in group C,the differences were significant(P＜0.01).The numbers of bacteria in the lung and spleen of the female mice of C group were remarkably less than that in group A, and decreased 4 times and 10 times respectively compared with group A(P＜0.01),the numbers of bacteria in the lung and spleen of the male mice of C group were less than that in group A, and decreased 3 times and 2 times respectively.However,the numbers of bacteria in the lung and spleen of the male mice of B group was decreased 14 times and 7 times respectively(P＜0.01).Conclusions The therapeutic efficacy of Bizhi pills in mice with mycobacterium tuberculosis infection is significant, but it is not as good as Rifampin.The therapeutic efficacy of Bizhi pills in female mice of mycobacterium tuberculosis infection is better than that in male mice.

Objective To develop a diagnosis model for active pulmonary tuberculosis. Methods The proteomic fingerprinting of 264 sera from active tuberculosis patients and controls were analyzed using the surface-enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS) and protein-chip technology. The peaks were detected and filtrated by Ciphergen PrnteinChip(R) Software (version 3.1.1). Using the Biomarker Pattern 5.0 software, a diagnostic model was developed for diagnosis of active tuberculosis. Re-sults Fifty protein peaks were significantly different between the patients with active pulmonary tuberculosis and the controls with overlapping clinical features (P<0.01). Five protein peaks at 4360, 3311, 8160, 5723, 15173 m/z were chosen for the system classifier and the development of diagnosis model 1. The model differenti-ated the patients with active pulmonary tuberculosis from the controls with a sensitivity of 83.0%, and a speci-ficity of 89.6%. The diagnostic accuracy was up to 86.4%. Three protein peaks at 5643, 4486, 4360 m/z were chosen for the system classifier and the development of diagnosis model 2. The model differentiated the pa-tients with active pulmonary tuberculosis from the controls with a sensitivity of 96.9%, and a specificity of 97.8%. The diagnostic accuracy was up to 97.3%. Conclusion It might be a new diagnostic test for the de-tection of sera from the patients with active pulmonary tuberculosis using SELDI-TOF-MS and protein chip.

Objective To study the therapeutic effects of Ag85A plasmid DNA vaccines in a mouse model of multi-drug resistant-(MDR-) Mycobacterium tuberculosis infection. Methods BALB/c mice were infected with Mycobacterium tuberculosis clinical strain HB361 with isoniazid and rifampin resist-ance by intratail-vein injection and were subsequently divided into 6 groups. At the third day after infection, the mice were treated with saline (group A), vector pVAX1 (greup B), rifampin (group C), vaccae (group D), Ag85A plasmid DNA vaccines (group E),rifampin and Ag85A plasmid DNA vaccines (group F) for 60 d. The lungs and spleens from the mice were taken and their pathological changes, weight and number of myeobacterial colony were examined at the third week after the end of treatment. Results At third week af-ter the end of treatment, the gross pathological observation and histopathological examination in lung showed that the lung lesions were limited, the profile of the alveoli was relatively clear, and normal structure could be seen in 2/3 areas of the lung sections in group D, E and F. The extent of lung lesion was 50% in group D,20% in group E and F. The pathological changes in group A, B, and C were more severer than those in group D, E and F. Compared with group A, the colony-forming units (CFU) in the lungs from mice in group D,E and F decreased 52%, 68%, 78%, respectively. The CFU in the spleens from mice in group D,E and F decreased 48%, 65%, 79%, respectively. Conclusion Ag85A plasmid DNA vaccines alone or Ag85A plasmid DNA vaccines along with chemotherapy have significant therapeutic effects on the mouse model of MDR-Mycobacterium tuberculosis infection.