BACKGROUND:In vitro isolation and purity technique of stem cells mostly depends on the identification of cell surface marker,such as monoclonal antibody adherent spreading method,flow cell sorting method and immunomagnetic beads sorting method,but the operation was complicated and the price was high.OBJECTIVE:To observe the biological characteristics of human amniotic fluid-derived embryonic mesenchymal stem cells,which were isolated and cultured using the two-step method.DESIGN,TIME AND SETTING:The opening study was conducted at the Stem Cell Research Room of Xinjiang Medical University from March 2008 to March 2009.MATERIALS:Totally 10 amniotic fluid specimens were obtained from pregnant women who underwent prenatal diagnosis following 16-22 weeks of gestation or voluntarily induced abortion.With ultrasonic guidance,amniocentesis was performed to collect 20-40 mL amniotic fluid.METHODS:Human amniotic fluid-derived embryonic mesenchymal stem cells were isolated and cultured using the two-step method.Amniotic fluid was first centrifuged and incubated till spindle-shape cells were seen,with the presence of flbroblast-tike cell colonies.Supematant was moved to a new 25 cm~2 culture flask for further culture till spindle-shape fibroblast-like mesenchymal stem cell colonies.When 70% confluence,cells were digested,and incubated in α-MEM,supplemented with basic fibroblast growth factor,served as the first passage.MAIN OUTCOME MEASURES:Morphological changes in human amniotic fluid-derived embryonic mesenchymal stem cells of primary culture and subculture were measured.Karyotype,cycle,growth curve and colony formation ability of human amniotic fluid-derived embryonic mesenchymal stem cells were measured.Surface antigen and cytokine were examined using flow cytometry,immunofluorescence and RT-PCR.RESULTS:Human amniotic fluid-derived embryonic mesenchymal stem cells were successfully isolated and subcultured.During metaphase,primarily cultured amniotic fluid cells presented scattered spindle cells and flbroblast-like mesenchymal stem cell colonies every 7 days.Passaged cells completely adhered in 12 hours.Following 1 or 2 days of latent period,cells proliferated rapidly.About 90% confluence was observed following 6 or 7 days of culture.Cell arranged regularly,showing whirlpool-shape,radiated shape.Cells were spindle-shape,with unclear boundary.Chromosome karyotype of human amniotic fluid-derived embryonic mesenchymal stem cells was normal diploid.Growth curve showed "S" shape,but the two-step method reached a peak at (6.1±0.5) days,which was significantly rapid compared with the one-step method (7.2±0.6) days (P=0.035).Flow cytometry analyses showed that P3 cells at S phase took up (14±2.3)% using the two-step method,which was more than the one-step method (9.0±1.4)% (P=0.031).Low-density human amniotic fluid-derived embryonic mesenchymal stem cells were incubated for 7 days prior to cells formed scattered cell colonies.However,colony forming efficiency using the two-step method (15.0±2.3)% were significantly more than the one-step method (10.0±1.8)% (P=0.021).Flow cytometry results showed that human amniotic fluid-derived embryonic mesenchymal stem cells expressed CD44,CD29 and CD105,but were negatively for CD45,CD34,HLA-DR.Immunofluorescence suggested that Oct-4-positive cells were observed in amniotic fluid.However,the proportion of Oct-4-positive cells using two-step method (1.2±0.3)% was significantly greater than the one-step method (0.9±0.2)% (P=0.041).RT-PCR suggested that human amniotic fluid-derived embryonic mesenchymal stem cells obtained using the two methods expressed Oct-4.CONCLUSION:Human multipotent mesenchymal stem cells are present in human amniotic fluid.The two-step culture protocol could be a kind of high performance and simple protocol which may not interfere with the normal prenatal diagnosis procedure.

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Objective The aim of this study was to investigate the distribution and clinical significance of CD8+ T cells in bladder cancer tissues in situ.Methods Immunohistochemistry were used to examine the distribution of CD8+ T cells in bladder cancer tissues,which were obtained from January 2003 to December 2009 from 302 patients.Among all the patients,262 were male while 40 were female;mean age is 60 years;tumor size ≤ 3 cm was in 235 and tumor size ＞ 3 cm was in 67;Unifocal tumor was in 214 and multifocal tumors were in 88.Amount of tumor stage Ta-T1 was 212 and T2-T4 was 90.Sixteen patients have lymph node metastasis.Histological low grade was diagnosed in 175 and histological high grade was diagnosed in 127.According to the differences between anatomic structure and cellular composition,bladder tumor tissues can be classified to two localization patterns:(1) intratumoral regions,defined as tumor cell nests;(2) stromal regions,defined as stromal areas that lack direct contact with tumor cells.Therefore,we divided 302 bladder cancer patients into two groups based on the median frequency of intratumoral CD8+ T cells (median,3/× 400 high resolution) and stromal CD8+ T cells (median,37/× 400 high resolution),respectively.x2 analysis was used to evaluated the correlation between CD8+ T cell density and clinicalpathological variables.Kaplan-Meier analysis and Cox proportional hazards regression models were applied to estimate overall survival (OS).Results CD8+ T cells were predominantly located in the intratumoral regions (mean,14 ± 2/× 400 high resolution) rather than in associated stromal regions (mean,50 ± 3/× 400 high resolution,P ＜ 0.05).The density of intratumoral CD8+ T cells was inversely associated with age (P =0.026),tumor size (P ＜ 0.05) and tumor stage (P ＜ 0.05),and could represent a favorable prognostic predictor of OS (HR =0.427,P =0.003).However,the density of stromal CD8+ T cells was positively associated with age (P =0.004) and histological grade (P ＜ 0.01),and could represent an adverse prognostic predictor of OS (HR =2.206,P =0.009).Conclusions Our findings suggest that intratumoral/ stromal CD8+ T cells could potentially serve as favorable/ adverse prognostic markers for bladder cancer patients,respectively.

Objective:To observe the impact of the medicated serum of Shumai Capsule and its decomposing on vascular smooth muscle cells(VSMC) proliferation induced by angiotensinⅡ(AngⅡ) and level of NO,SOD,MDA in cell culture supernatant.Methods:Tissue explant method was used for cultivating vascular smooth muscle cells,serum pharmacology was used,AngⅡ10-7mol/L as a stimulating factor,medicared serum divided into Shumai Capsule group,Huoxue Huayu decomposing group(group 1) and Bupi Yishen decomposing group(group 2),MTT colorimetric was used to test OD values,biochemical detection kit was used to test NO,SOD,MDA levels.Results:The OD value of Shumai Capsule and group1 had significant difference from AngⅡ group and group2(P

PD-L1 is a member of the B7 protein family, most of whose members so far were identified as dimers in a solution and crystalline state, either complexed or uncomplexed with their ligand(s). The binding of PD-L1 with its receptor PD-1 (CD279) delivers an inhibitory signal regulating the T cell function. Simultaneously with the Garboczi group, we successfully solved another structure of human PD-L1 (hPD-L1). Our protein crystallized in the space group of C222(1) with two hPD-L1 molecules per asymmetric unit. After comparison of reported B7 structures, we have found some intrinsic factors involved in the interaction of these two molecules. Based on these results, we tend to believe this uncomplexed hPD-L1 structure demonstrated its potential dimeric state in solution, although it could just be an evolutionary relic, too weak to be detected under present technology, or still a functional unit deserved our attentions.
Antigens, CD ; chemistry ; immunology ; B7-H1 Antigen ; Crystallography, X-Ray ; Evolution, Molecular ; Humans ; Protein Multimerization ; Protein Structure, Quaternary ; Protein Structure, Secondary ; T-Lymphocytes ; chemistry ; immunology

Enolase is a conserved cytoplasmic metalloenzyme existing universally in both eukaryotic and prokaryotic cells. The enzyme can also locate on the cell surface and bind to plasminogen, via which contributing to the mucosal surface localization of the bacterial pathogens and assisting the invasion into the host cells. The functions of the eukaryotic enzymes on the cell surface expression (including T cells, B cells, neutrophils, monocytoes, neuronal cells and epithelial cells) are not known. Streptococcus suis serotype 2 (S. suis 2, SS2) is an important zoonotic pathogen which has recently caused two large-scale outbreaks in southern China with severe streptococcal toxic shock syndrome (STSS) never seen before in human sufferers. We recently identified the SS2 enolase as an important protective antigen which could protect mice from fatal S.suis 2 infection. In this study, a 2.4-angstrom structure of the SS2 enolase is solved, revealing an octameric arrangement in the crystal. We further demonstrated that the enzyme exists exclusively as an octamer in solution via a sedimentation assay. These results indicate that the octamer is the biological unit of SS2 enolase at least in vitro and most likely in vivo as well. This is, to our knowledge, the first comprehensive characterization of the SS2 enolase octamer both structurally and biophysically, and the second octamer enolase structure in addition to that of Streptococcus pneumoniae. We also investigated the plasminogen binding property of the SS2 enzyme.
Amino Acid Sequence ; Binding Sites ; Crystallography, X-Ray ; Humans ; Models, Molecular ; Molecular Sequence Data ; Phosphopyruvate Hydratase ; chemistry ; metabolism ; Plasminogen ; metabolism ; Protein Multimerization ; Protein Structure, Quaternary ; Solutions ; Species Specificity ; Streptococcus suis ; enzymology

Objective: To understand the epidemic status and influencing factors of Mongolian children with ASD in central and eastern Inner Mongolia, so as to provide data support for formulating prevention and intervention strategies and improving the overall epidemiological investigation of ASD in Inner Mongolia. Methods: Sixteen kindergartens and primary schools were selected from Chifeng City, Ulanqab City, Tongliao City, Hulunbuir City and Xilingol League cities in Inner Mongolia by means of random cluster sampling. Firstly, 7 108 children aged 3-14 were initially screened with the Kirschner Autism Behavior Scale(CABS), and then the children with ASD positive were given the autism behavior test scale (ABC). According to the diagnostic criteria, the professionals, including chief physicians and associate chief physicians from the major of child psychiatry, diagnosed ASD with the total score of ABC scale ≥62. Univariate and Logistic regression multivariate analysis were carried out among Mongolian children to find out the influencing factors related to the occurrence of Mongolian ASD in Inner Mongolia. Results: The prevalence of Mongolian children was 0.37%. Mongolian ASD group and Mongolian normal children series in the household register, habitual twitch, hyperactivity, bite lips, families have extreme introverts, mothers age, father s cultural level, cultural degree of mother, father mother mild character, irritable, neonatal diseases, fetal gestational age distribution had statistical significance( χ 2/Z= 12.58 , 16.68, 14.93, 64.43, -3.76, -2.86, 4.57, 11.12, 12.33, 16.66, P <0.05). Conclusion Measures such as shaping a healthy growth environment, adjusting parental style, paying attention to the level of early childhood language development, and preventing neonatal diseases might lower the risk of ASD in children.

Objective: To understand the prevalence and influencing factors of children with ASD in central and eastern Inner Mongolia, and to provide theoretical basis for disease prevention and prevalence of ASD. Methods: Sixteen primary schools and kindergartens were selected from 5 cities in central and eastern Inner Mongolia through random cluster sampling. A total of 15 817 children aged 3-14 years were selected. Children who were positive using Clancy Autism Behavior Scale were further diagnosed according to the teacher s nomination form and the Autism Behavior Checklist, as well as the diagnostic criteria of the fifth edition of the American Diagnostic & Statistical Manual of Mental Disorders by 2 professionals. Results: The prevalence of ASD was 0.27% (42/15 817), with prevalence in urban areas (0.16%, 15/9 231) higher than that of rural areas (0.41%, 27/6 586) ( χ 2=8.89, P <0.01). Logistic regression analysis showed that maternal education and language development were negatively associated with ASD in urban children [ OR =0.29(95% CI =0.12-0.69) and 0.18(95% CI =0.05-0.60), P <0.05]. ASD in rural children were positively associated with enuresis and introverted family members [ OR =7.09(95% CI =1.60-32.27) and 8.63(95% CI =3.10- 24.01 ), P <0.05]. Conclusion High prevalence of ASD is found in urban area of central and eastern Inner Mongolia. Unhealthy habits, neonatal diseases, low parental education, delayed language development and poor exercise performance are primary factors associated with ASD in both urban and rural areas.

Leukocyte immunoglobulin-like receptors (LILRs), also called CD85s, ILTs, or LIRs, are important mediators of immune activation and tolerance that contain tandem immunoglobulin (Ig)-like folds. There are 11 (in addition to two pseudogenes) LILRs in total, two with two Ig-like domains (D1D2) and the remaining nine with four Ig-like domains (D1D2D3D4). Thus far, the structural features of the D1D2 domains of LILR proteins are well defined, but no structures for the D3D4 domains have been reported. This is a very important field to be studied as it relates to the unknown functions of the D3D4 domains, as well as their relative orientation to the D1D2 domains on the cell surface. Here, we report the crystal structures of the D3D4 domains of both LILRB1 and LILRB2. The two Ig-like domains of both LILRB1-D3D4 and LILRB2-D3D4 are arranged at an acute angle (∼60°) to form a bent structure, resembling the structures of natural killer inhibitory receptors. Based on these two D3D4 domain structures and previously reported D1D2/HLA I complex structures, two alternative models of full-length (four Ig-like domains) LILR molecules bound to HLA I are proposed.
Amino Acid Sequence ; Antigens, CD ; chemistry ; Crystallography, X-Ray ; Histocompatibility Antigens Class I ; chemistry ; Humans ; Immunoglobulins ; chemistry ; Leukocyte Immunoglobulin-like Receptor B1 ; Membrane Glycoproteins ; chemistry ; Models, Molecular ; Peptides ; chemistry ; metabolism ; Protein Binding ; Protein Structure, Tertiary ; Receptors, Immunologic ; chemistry ; Signal Transduction

Objective To investigate the intervention effect of Xuebijing Injection on the differentiation of bone marrow hematopoietic stem cells (HSC) in septic mice. Methods Fifty-four male C57BL/6N mice were randomly divided into three groups: normal control group, model group and Xuebijing group, each group with 18 mice. The mouse models of sepsis were duplicated by intra-peritoneal injection of 10 mg/kg E. coli lipopolysaccharide (LPS) method. Starting from the day of modeling, Xuebijing Injection 20 mL/kg was intravenously injected into the tail vein in Xuebijing group, once a day for consecutive 4 days; the normal control and model groups were intravenously injected with normal saline at the same dose and site for 4 days. The bone marrow cells of the femur and tibia of the mice were isolated after 4 days of various treatments in the three groups, and the proportions of bone marrow HSC Lin-Sca-1+c-Kit+ (LSK) and hematopoietic progenitor cells Lin-Sca-1-c-Kit+ (LS-K) of each group were detected by flow cytometry. Results Finally, 14 mice were included in the normal control group, 17 in the model group, and 12 in the Xuebijing group. With the prolongation of time, the body weight of the normal control group gradually increased, the body masses of the model group and the Xuebijing group were decreased first and then increased, reaching a peak at 96 hours after the model was established, but they were still significantly lower than the body mass of normal control group (g: 19.81±0.27, 19.58±0.39 vs. 22.23±0.30, both P < 0.05). Compared with the normal control group, the proportions of LSK, LS-K, long-term HSC (LT-HSC), and megakaryocyte-erythroid progenitor cells (MEP) were all significantly increased in the model group [LSK: (16.62±1.28)% vs. (12.89±0.83)%, LS-K: (44.77±1.77)% vs. (30.34±0.90)%, LT-HSC: (6.88±0.48)% vs. (1.83±0.24)%, MEP: (13.89±1.26)% vs. (9.38±0.66)%, all P < 0.05], the proportion of multipotential progenitor cells (MPP) was significantly decreased [(2.41±0.34)% vs. (5.99±0.59)%, P < 0.05]. Compared with the model group, the LSK and myeloid progenitor (CMP) of the Xuebijing group were significantly reduced [LSK: (12.25±0.69)% vs. (16.62±1.28)%, CMP :(0.31±0.05)% vs. (0.55±0.13)%, both P < 0.05], and LS-K, LT-HSC, MEP showed a decreasing trend [LS-K: (42.75±2.48)% vs. (44.77±1.77)%, LT-HSC:(5.98±0.70)% vs. (6.88±0.48)%, MEP: (10.94±1.36)% vs. (13.89±1.26) %], but the differences were not statistically significant (all P > 0.05). There were no significant differences in the proportions of short-term HSC (ST-HSC) and granulocyte-macrophage progenitor cells (GMP) among the three septic groups (all P > 0.05). Conclusion Xuebijing Injection can improve the differentiation function of bone marrow cells in septic mice, which may be possibly related to the inhibition of pathological proliferation of bone marrow hematopoietic stem cells and progenitor cells in septic mice by Xuebijing Injection.