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BACKGROUND@#Surface modification is used to modify the biomaterials for the regulation of cell culture using different approaches, such as chemical graft and mechanical treatment. However, those conventional methodologies often require precise fabrication in a high resolution involving either high cost or laborious steps to remove chemical residues that are toxic to the cells. @*METHODS@# A novel and simple method was proposed and evaluated to rapidly generate surface ceases on the gelatin methacrylate (gelMA) surface using the heating-hydration process. Human umbilical vein endothelial cells (HUVECs) were cultured on the gelMA surface. The surface binding was characterized using the RGD (Arg-Gly-Asp) antibodies and cell adhesion pattern captured by scanning electron microscopy. The effect of the heating-hydration parameters on the creasing formation was investigated. The morphology of HUVECs cultured on such micropatterned gelMA was characterized and compared. @*RESULTS@#It is found that the hydration solution, gelMA mixture, and hydration rate are the major factors that influence the cracking sizes in the range from 20 to 120 lm which resulted in capillary-like patterns on the gelMA surface. Low concentration of gelMA, high water concentration of cooling agent, and slow hydration rate result in the long creases, and heating of at least 60 min is required for complete dehydration. Strong fluorescence was around the creases with RGDstaining. Consequently, micropatterned gelMA demonstrated good biocompatibility with endothelial cells with more than 95% cell viability and continuous cell proliferation throughout 2 weeks as well as a good trace of neovascular formation. In comparison, normal gelMA surface did not exhibit RGD-fluorescent signals, and the cultured HUVECs on it were rounded with no spreading for network formation. @*CONCLUSION@#The heating-hydration approach can successfully and easily produce the micropatterned gelMA that allows rapid and effective vascularization to potentially improve the functionalities of the tissue-engineered construct.
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BACKGROUND@#Surface modification is used to modify the biomaterials for the regulation of cell culture using different approaches, such as chemical graft and mechanical treatment. However, those conventional methodologies often require precise fabrication in a high resolution involving either high cost or laborious steps to remove chemical residues that are toxic to the cells. @*METHODS@# A novel and simple method was proposed and evaluated to rapidly generate surface ceases on the gelatin methacrylate (gelMA) surface using the heating-hydration process. Human umbilical vein endothelial cells (HUVECs) were cultured on the gelMA surface. The surface binding was characterized using the RGD (Arg-Gly-Asp) antibodies and cell adhesion pattern captured by scanning electron microscopy. The effect of the heating-hydration parameters on the creasing formation was investigated. The morphology of HUVECs cultured on such micropatterned gelMA was characterized and compared. @*RESULTS@#It is found that the hydration solution, gelMA mixture, and hydration rate are the major factors that influence the cracking sizes in the range from 20 to 120 lm which resulted in capillary-like patterns on the gelMA surface. Low concentration of gelMA, high water concentration of cooling agent, and slow hydration rate result in the long creases, and heating of at least 60 min is required for complete dehydration. Strong fluorescence was around the creases with RGDstaining. Consequently, micropatterned gelMA demonstrated good biocompatibility with endothelial cells with more than 95% cell viability and continuous cell proliferation throughout 2 weeks as well as a good trace of neovascular formation. In comparison, normal gelMA surface did not exhibit RGD-fluorescent signals, and the cultured HUVECs on it were rounded with no spreading for network formation. @*CONCLUSION@#The heating-hydration approach can successfully and easily produce the micropatterned gelMA that allows rapid and effective vascularization to potentially improve the functionalities of the tissue-engineered construct.
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PURPOSE: Intestinal dysfunction is one of the most common complications in patients after abdominal surgery. Daikenchuto (DKT), a traditional herbal medicine, is recently employed to improve postoperative intestinal dysfunction. The aim of this meta-analysis was to assess the efficacy of DKT in improving intestinal dysfunction after abdominal surgery. METHODS: PubMed, Embase, and the Cochrane library were systematically searched to identify randomized controlled trails (RCTs) in adult patients undergoing abdominal surgery, who were randomly distributed to administrate DKT and placebo. The primary outcomes included the time to first postoperative flatus or bowel movement. We used random-effects models to calculate summary mean differences (MDs) with 95% confidence intervals (CIs). RESULTS: Nine RCTs totaling 1,212 patients (618 in DKT, 594 in control group) were included in our study. Compared with control group, DKT can effectively improve postoperative intestinal dysfunction by shortening the time to first postoperative flatus (MD, −0.41; 95% confidence interval [CI], −0.66 to −0.16; P = 0.001) with significant heterogeneity (I2 = 71%, P = 0.004), and bowel movement (MD, −0.65; 95% CI, −0.97 to −0.32; P < 0.001) without significant heterogeneity (I2 = 40%, P = 0.14). Sensitivity analyses by indication of surgery and type of surgery yielded similar results. CONCLUSION: These data provide limited evidence that DKT shows efficacy on improving intestinal dysfunction after abdominal surgery. However, the results should be interpreted cautiously, due to the heterogeneity of the studies included. Thus, the efficacy of DKT on improving postoperative intestinal dysfunction warrants further investigation.
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Adult , Humans , Flatulence , Herbal Medicine , Population CharacteristicsABSTRACT
Objective To analysis the effects of Ang-(1-7) on the blood brain barrier permeability after subarachnoid hemor-rhage .Methods SAH-rats were produced by two times injection of blood into cisterna magna .Evans blue was used to detect the the permeability of SAH-rats brains and brain water content .RT-PCR and Western blot were performed to measure the expression of adhesion protein ICAM-1 and VCAM-1 in brains of SAH-rats .The artificial hemorrhagic cerebrospinal fluid (BCSF) was used to stimulate vascular endothelial cells (HBMEC) ,and the proliferation and apoptosis of HBMEC cell were analyzed .Results Ang-(1-7) reduced the content of Evans blue and brain water in brains of SAH-rats in dose and time dependent manner with the most sig-nificant change under the treatment of 10 - 5 mol/L Ang-(1-7) for 24 h (P< 0 .05) .Under the above condition ,the mRNA and pro-tein levels of ICAM-1 and VCAM-1 in brains of SAH-rats were significantly up-regulated (P< 0 .05) ,while the content of Evans blue in HBMEC cells stimulated by BCSF was obviously reduced .Besides ,Ang-(1-7) was observed to increase the expression of ICAM-1 and VCAM-1 in BCSF-stimulated HBMEC cells ,enhance the proliferation of HBMEC cells but reduce their apoptosis . Conclusion Ang-(1-7) plays a protective role in the blood-brain barrier damage induced by subarachnoid hemorrhage .
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Objective To investigate the effect of mitogen activated protein kinase / extracellular signal-regulated kinase (MEK / ERK)1 / 2 signaling pathway on early brain injury (EBI)following experimental subarachnoid hemorrhage (SAH)in rats. Methods Sixty male SD rats were randomly divided into a control group and a 1,6,12,24,48,or 72 h group after SAH modeling. SAH + MEK inhibitor U0126 was used to intervene the 24,48,and 72 h groups (a total of 10 groups;n = 6 in each group). In addition to the control group,blood was injected into the cisterna magna of the rats to induce a SAH model in another 9groups. The blood samples were taken from infraorbital venous plexus. Enzyme-linked immune sorbent assay (ELISA)was used to detect the levels of interleukin-6 (IL-6),IL-1β,and tumor necrosis factor α(TNF-α)in each group. Evans blue content in brain tissue was used to evaluate the blood-brain barrier damage. Western blot was used to detect the levels of phosphorylated extracellular signal-regulated kinase (p-ERK1/ 2)and matrix metalloproteinase-9 (MMP-9)proteins in basilar artery tissue,and compared them. Results Compared with the control group at the same time points,there were significant differences in the levels of IL-6 and IL-1β at 6,12,24,48,and 72h after modeling in the SAH group (all P <0. 05). At 12,24, 48,and 72 h after modeling,the expression levels of p-ERK1/ 2 protein of the basilar artery tissue of the SAH group were 0. 73 ± 0. 09,0. 85 ± 0. 12,0. 94 ± 0. 09,and 0. 96 ± 0. 09,respectively,they were significantly higher than those of the control group (all P < 0. 05). At 48 and 72 h after modeling in the SAH group,the level of MMP-9 protein was significantly higher than that in the control group (1. 27 ± 0. 15 vs. 0. 68 ± 0. 08,2. 41 ± 0. 11 vs. 0. 71 ± 0. 14). At 72 h after modeling,the Evans blue content in brain tissue of the SAH group was significantly higher than that of the control group (15. 3 ± 2. 2 μg/ g vs. 2. 7 ± 0. 4 μg/ g). After giving the MEK inhibitor U0126 intervention,the levels of serum IL-6,IL-1β,and TNF-α at 24,48, and 72 h after modeling,and the expression levels of p-ERK1 / 2 and MMP-9 proteins at 48 and 72 h (p-ERK1 / 2:0. 76 ± 0. 07,0. 81 ± 0. 06;MMP-9:0. 92 ± 0. 14,1. 79 ± 0. 16),and the Evans blue content (8. 9 ± 1. 7 μg / g)in brain tissue at 72 h after modeling were significantly lower than those of the SAH group (P < 0. 05). Conclusion The MEK/ ERK1/ 2 signal pathway may be closely associated with the inflammatory reaction and blood-brain barrier damage after SAH,which suggests that the intervention of the MEK/ ERK1 / 2 signal pathway may be a potential target for the prevention of early brain injury after SAH.
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Objective To study the role of ERK signal pathway in the endochondral ossification of bone mesenchymal stem cells ,and to explore the mechanism of ERK signal pathway in persistent enhanced FGFR2 function on development of mice BMSCs by a knock‐in mouse model with the FGFR2S252W/+ .Methods Mice with neo‐FGFR2 gain‐of‐function mutation were mated with EII‐Cre mice .The genotype of generation mice were identified by PCR and divided into wild type group and mutant type group ac‐cording to their genotype .6 week‐old mice were sacrificed to receive bone mesenchymal stem cells .The western blot was used to compare the level of P‐ERK and ERK and the RT‐PCR was applied to detect the genes of Col2 ,Col10 ,OC ,OP in chondrogenic dif‐ferentiation medium of BMSCs .Then ,treatment of cultured BMSCs with PD98059 ,compare the changes of genes and utilize the in vitro culture of long bones detect the role of ERK signal pathway in the endochondral ossification by FGFR2 mutant .Results We successfully derive BMSCs from FGFR2S252W/+ mutant mice and found the activity of ERK signal pathway of FGFR2S252W/+ was en‐hanced .After been cultured in chondrogenic differentiation medium ,the expressions of the BMSCs mRNA of Col2 ,Col10 from mu‐tant group were decreased ,while the expressions of OC ,OP were increased .Those OC ,OP genes levels showed an increased treated by PD98059 .Using in vitro culture of long bones ,we found the retardation of total length growth of long bones has been rescued by PD98059 treatment ,suggesting that ERK signal pathways was responsible for the retarded long bone development in FGFRS252W/+mice .Conclusion The results indicate these effects are mediated by the ERK signal pathway .Furthermore ,the retardation of long bones has been recued by PD98059 treatment ,suggesting that ERK signal pathway is responsible for the retarded long bone devel‐opment in FGFR2S252W/+ mice .
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Objective To develop a sheet to assess infection risks in children with malignant tumors after chemotherapies.Methods A risk assessing sheet (to consult) was designed and used to consult 24 experts from 9 hospitals located in 6 provinces.After 3 rounds of expert consultation,a sheet (to try) was developed and tried by nurses in the clinic.And then a sheet (to use) was developed.Results The authority coefficient of experts was 0.79,the positive coefficient of experts was 1.00 and 0.96.The sheet (to try) developed after 3 rounds of expert consultation consisted of 30 items and was divided into 2 parts,one was about static items and the other was about dynamic items.Depending on the trying of the sheet (to try),the sheet (to use) was constituted by 8 fundamental immune state assessing items and 16 infection risk screening items.Conclusions Experts consulted in the study were quite reliable,representative and positive.The sheet (to use) seemed to have good pertinence and practicability.Further studies should be done to verify whether the sheet (to use) may help nurses to prevent children with malignant tumors from infections after chemotherapies.
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Objective To investigate the satisfaction degree of family members of children patients with the quality of nursing by follow-up analysis using telephone.Methods We used the high quality nursing questionnaire as the follow-up investigation.311 parents of the discharged pediatric patients were enrolled in this study,and answered the questionnaire within 1 ～2 weeks after their children were discharged from hospital.Meanwhile,we collected specific comments through asking questions by telephone.Then we analyzed and summarized the results by the content analysis.Results We found the total satisfaction rate was 96.65％ and there were three levels after qualitative analysis on the specific recommendations:evaluation of the hospital system,evaluation of professional knowledge and skills of the medical workers,evaluation of the attitude of the medical workers.Conclusions The parents of pediatric patients are satisfied with the high quality nursing which has pediatric features.However,what the parents focused on had already beyond the scope of the high quality nursing itself.We should expand the concept of quality service to every aspect of the hospital work,in order to meet the needs of the parents of pediatric patients in the department of pediatrics.
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Objective To establish the murine congenital infection model by MCMV and observe the pathological changes and infection status of brain tissue.Methods After anesthesia,mice who were pregnant 11-13.5 days (E11-13.5 d) were intra-amniotic injected one uterus by one with virus (MCMV K181 suspension,1 μl,1×103 PFU).The control group of the same period was intra-anmiotic injected with culture medium DMEM (1 μl).Carefully reset the uteruses and close the abdomen.After 5 days of separated feeding,kill the pregnant mice,take the fetus out of the uterus,anesthetize and kill them.Make frozen sections of these fetal brains.Some sections were stained using conventional HE method,to observe the pathological changes under the light microscope.Detect MCMV early antigen in the brain tissue by immunohistochemistry staining and immunofluorescence assay.Results The survival rates of the infected group were 71.9％.Compared with the control group,intra-amniotic inoculation of MCMV does not affect the rate of fetal survival,fetal absorption,fetal death and the average weight of the heads,but decrease their average weight of the bodies.The pathological changes are found in the brain tissue of the mouse in the infection group.Through enzyme immunohistochemistry assay,there are many MCMV infected cells in brain-ventricular zone,brain subependymal zone,cerebral cortex and hippocampus area in the infection group.Similar findings were observed by immunofluorescence method.Conclusion By intra-amniotic injection of MCMV suspension,murine model of MCMV congenital infection can be successfully established.This model could be used to study the mechanisms of encephalodysplasia caused by congenital CMV infection in vivo.
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Objective To investigate the influence of murine cytomegalovirus ( MCMV) infection on the expression of downstream differentiation related target genes of Wnt signaling pathway in neural stem cells (NSCs) in vitro and explore the molecular mechanism of fetal encephalodysplasia caused by CMV infection. Methods NSCs were separated from fetal BALB/c mouse and cultured in vitro. The NSCs infected by MCMV at a MOI (multiplicity of infection) of 5, 1 and 0.1, respectively, were cultured in differentiation medium. The dynamic expression of the downstream differentiation related target genes ( c-myc, cyclinD1, ngn-1 and ngn-2) of Wnt signal pathway in NSCs were measured by Western blot. Real-time RT-PCR was employed to measure the expression levels of the key differentiation genes ngn-1 in Wnt signal pathway of NSCs post infection. Results The protein levels of c-myc in the infected groups were significantly lower than that in the normal control at 0.5-5 d (P＜0.05) ; At 0. 5 d and 1 d post-infection (p. i. ) , the protein levels of cyclinDl in the infected groups were lower than that in the normal control (P＜0.05). At 2 d and 3 d p. i. , the cyclinD1 expression in the infected groups was higher than that in the control group (P ＜ 0. 05). However, at 4 d and 5 d p. i. , the cyclinD1 levels in the group of the MOI of 5 were lower than in other three groups (F＜0.05). The expression of ngn-1 protein in the infected groups was reduced importantly compared with normal control at 1 -5 d p. i. ( P ＜ 0.05 ). The expression of ngn-1 mRNA in the infected groups was lower than that in the control group at all time points (P ＜ 0. 05 ). The expression of ngn-2 protein decreased at first and then increased, which was opposite to the normal control. The peak of ngn-2 expression in groups of the MOT of 0.1 and 1 occurred later and were significantly lower than that in the normal control (P ＜0. 05). No distinct peak was seen in the group of the MOI of 5. At 1 d p. i. , the expression of ngn-2 of all infected groups was significantly lower than that in the normal control ( P ＜ 0. 05 ). At 2 d p. i. , the expression of in the group of the MOI of 5 was still lower (P ＜ 0.05). While at 3 d, 4 d and 5 d p. i. , the protein levels in all infected groups were higher than that in the normal control (P ＜ 0. 05). The protein expression of these genes increased following the increase of MOI. Conclusion MCMV inhibited the protein expression of c-myc and ngn-1 in differentiated NSCs, repressed the mRNA expression of ngn-1 and caused the perturbed expression of cyclinDl and ngn-2 in a MOI-dependent manner. These data suggest that inhibition of or interference with the protein expression of downstream differentiation related target genes of Wnt signaling pathway in NSCs by MCMV may be one of the important mechanisms, by which proliferation and differentiation of NSCs are inhibited and thus fetal brain is impaired after MCMV infection.
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Objective To investigate the expression of matrix metalloproteinases 1 (TIMP-1)products inhibitor on smooth muscle cell (SMC) proliferation in rat autologous vein graft.Methods Autogenous vein transplantation model was established in 30 Wistar rats. The vein grafts were harvested at different time after grafting. The change of TIMP-1 was detected by using hematoxylin and eosin, immunohistochemistry and in situ hybridization (ISH). Results 1. Changes of histopathology in vein grafts: Intimal hyperplasia (IH) could be seen in 7 - 14 days, the peak at 2 ~ 4 weeks after operation.2. ISH results: TIMP-1mRNA positive cells appeared at 24 hours, increased significantly in 72 hours and reached the peak at 1 ~ 2 week after operation. There was significant difference between day 1 and 2 week.TIMP-I expression was not detected in normal vessels (P <0. 01). 3. Immunohistochemistry results: There was trace TIMP-I expression in normal vessels. The expression of TIMP-1 appeared at 72 hours after vein graft, increased mostly in 1 week, reached the peek at 2 week and reduced later. There was significant difference between day 1 and 2 week (P < 0. 01). Conclusions 1. The activation of TIMP-1 exists in autogenous vein grafts. 2. The intima experienced hyperplasia in spite of increased secretion of endogenous TIMP-1 after autogenous vein grafts.
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Objective To investigate the biological function of M122 in pathogenesis of MCMV in developmental brain disorders and brain damage, screening for mouse brain cDNA library interacting with M122 was performed by a yeast two-hybrid system. Methods The reconstructed bait plasmid pGBKT7-M122 was transformed into yeast cells AH109 and screened on the nutrient deficiency medium SD/-Trp. After express of the bait protein in AH109 yeast strains was detected by Western blot analysis, yeast-two hybrid screening was performed by mating AH109 with Y187 containing mouse brain cDNA library plasmid. The diploid yeast cells were plated on the nutrient deficiency medium SD/-Trp/-Leu/-His/-Ade. The second screening was performed with SD/-Trp/-Leu/-His/-Ade containing X-α-gal. The plasmids in positive colonies were extracted and transformed into E. coli JM109 cells. After plasmid DNA in JM109 cells were extracted form positive colonies and sequenced, the results were analyzed by bioinformatic methods. The interactions between M122 protein and the protein obtained from positive colonies were further confirmed by repeating yeast-two hybrid. Then, autoactivations of the proteins obtained from positive colonies were detected.Results The reconstructed bait plasmid was transformed into yeast cells AH109 successfully. The bait protein expressed in the yeast cells AH109 stably. 24 proteins interacting with MCMV M122 were screened, including syntaxin 8 ( Stx8 ), phosphoglucomutase 2 ( Pgm2 ), potassium voltage-gated channel, shaker-related subfamily, beta member 1 ( Kcnab1 ), collagen, type ⅪⅩ, alpha 1 ( Col19a1 ), archain 1 ( Arcn1 ), cytidylate kinase( Cmpk), DnaJ(Hsp40) homolog, subfamily A, member 1 (Dnaja1), ATPase, Na+/K + transporting, beta 3 polypeptide( Atp1b3 ), SH3-domain GRB2-like ( endophilin ) interacting protein 1 ( Sgip1 ),ankyrin repeat domain 17 (Ankrd17), Smg-7 homolog, nonsense mediated mRNA decay factor(Smg7),sperm associated antigen 9 ( Spag9 ), FK506 binding protein 1a ( Fkbp1a), MYST histone acetyltransferase monocytic leukemia 4 ( Myst4), hyaluronan and proteoglycan link protein 1 ( Hapln1), autophagy-related 3 (Atg3), splicing factor, arginine/serine-rich 5 ( Sfrs5 ), zinc finger, C3HC-type containing 1 ( Zc3hc1 ),thioredoxin-related transmembrane protein 1 ( Txndc1 ), adaptor protein complex AP-1, gamma 1 subunit (Ap1g1), Cullin 1 ( Cul1 ), and so on. Three of them were formerly unknown proteins. M122 protein could interact with the proteins obtained from positive colonies in the yeast cells AH109. Ap1g1 and Cul1 were proved to have autoactivation. Conclusion A class of proteins in brain interacting with M122 has been obtained. It is presumed that these proteins are correlated with neuropathogenesis of the brain disorders caused by CMV, but the candidates still need further confirmation for the interaction.
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AIM: The influence of MCMV infection on differentiation and differentiation gene expression in neural stem cells ( NSCs) in vitro were investigated for studying the mechanisms of brain abnormalities caused by congenital cytomegalovirus infection. METHODS: NSCs were separated from fetal BALB/C mouse, and cultured and identified in vitro. The differentiation potency of NSCs was observed by immunofluorescence. The NSCs infected by MCMV at dosage of MOI( multiplicity of infection) equaled to 5, 1 and 0.1 .respectively, were cultured in differentiation medium. The morphological changes of infected cells were observed under inverted microscope. The ratios of NSCs and its differentiated cells were detected by flow cytometry. The expressions of nestin, GFAP and NSE, markers of NSCs and its differentiated cells, were studied by immunofluorescence( MOI = 1). The expression of early antigen ( EA ) of MCMV was detected to observe the infection process. Real - time RT - PCR method was employed to measure the expression levels of the key genes Neurog2, Myc and Ccnd1 in Wnt signal pathway of NSCs at early stage of differentiation culture. RESULTS: NSCs isolated from embryonic mouse brains proliferated to form neurospheres, strongly expressed nestin and differentiated into NF - 200 positive neurons or GFAP positive astrocytes. The infected NSCs did not adhere to the wall and appeared differentiation growths, but showed swollen gradually after differentiation culture. The nestin expression in the infected cells downregulated slowly and was higher than that in control groups ( P < 0.05). The GFAP and NSE expressions of the infected cells were lower than those in control groups ( P <0.05). The early antigen ( EA) of MCMV was always detected in the cells in infected groups. The ratios of nestin positive cells in infected groups were higher than those in control groups, but the ratios of GFAP and NSE positive cells of former were lower than that of the latter from 3rd to 9th d after differentiation culture(P < 0.05 ). The levels of Neurog2 mRNA and Myc mRNA in infected groups were markedly lower than those in normal control groups on 1st d and from 1st to 4th d after differentiation culture, respectively( P <0.05). The levels of Ccnd1 mRNA of infected groups were obviously lower than those in normal control groups from 12th h to 1st d( P <0.05 ). These changes in infected groups became more obvious as MCMV MOI increased. CONCLUSION: MCMV significantly inhibits differentiation of NSCs to neurons and astrocytes, and leads to the decrease in differentiated cells. MCMV inhibits or interferes with the gene expression of Newog2, Myc and Ccnd1 in Wnt signal pathway of NSCs. The effect that MCMV inhibits the expressions of differentiation and the differentiation genes in NSCs shows dose - dependent with MCMV MOI. The inhibitory effect of MCMV on the differentiation of NSCs might be induced by interfering with the expression of differentiation gene in NSCs, which is possibly the one of primary causes of brain development disorders induced by congenital CMV infection.
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Objective To investigate the influence of murine cytomegalovirus(MCMV) infection on differentiation and differentiation gene expression of neural stem cells (NSCs) in vitro for studying the mechanisms of brain abnormalities calmed by congenital cytomegalovirns infection. Methods NSCs were separated from fetal BALB/c mouse and cultured and identified in vitro. The differentiation potency of NSCs was observed by immunnfluorescence. The NSCs infected by MCMV at dosage of multiplicity of infection (MOI) equaled to 5, I and 0. 1, respectively, were cultured in differentiation medium. The morphological changes of the cells were observed by inverted microscope. The ratios of NSCs and its differentiated cells were detected by flow cytometry. The expression changes of nestin, GFAP and NSE, markers of NSCs and its differentiated cells, were studied by immunofluorescence ( MOI = 1 ). The expression of early antigen (EA) of MCMV was detected to observe the infection process. Real-time RT-PCR method was employed to measure the expression levels of the key differentiation genes Wnt-3 and Wnt-7a in Wnt signal pathway of NSCs at early phage of differentiation culture. Results NSCs isolated from embryonic mouse brains could proliferate to form neurnspheres and strongly express Nestin and differentiate into NF-200 positive neurons or GFAP positive astrocytes. The NSCs of the infected groups couldn't adhere to the wall and appear differentia-tion growth, but showed swollen gradually after differentiation culture. The nostin expression of the infected groups downregulated slowly and was higher than that of the control groups ( P < 0.05 ). The GFAP and NSE expression of the infected groups were lower than that of the control groups (P <0.05). The EA of MCMV could be always detected in the cells of the infected groups. The ratios of nestin positive cells of the infected groups were higher than that of the control groups, but the ratios of GFAP and NSE positive cells of the for-mer were lower than that of the latter from 3rd to 9th day after differentiation culture ( P < 0.05 ). The levels of Wnt-3 mRNA and Wnt-7a mRNA of the infected groups were markedly lower than that of the control groups from 1st to 2nd clay and from 12th hour to 2nd day after differentiation culture respectively ( P < 0.05 ) . These changes of the infected groups became more obvious as MCMV MOI increased . Conclusion MCMV could inhibit significantly NSCs differentiate to neurons and astrocytes and lead to the decrease of dif-ferentiated cells. MCMV could inhibit or interfere with the gene expression of Wnt-3 and Wnt-7a in Wnt sig-nal pathway of NSCs. The effect that MCMV inhibited the differentiation and the differentiation gene expres-sion of NSCs showed dose-dependent with MCMV MOI. The inhibitory effect of MCMV on the differentiation of NSCs might be induced by interfering the differentiation gene expression of NSCs, which is possibly the one of primary causes of brain development disorders caused by congenital CMV infection.
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Objective Through the investigation of the compliance and impact factors of prenatal check to the pregnant women,we aimed to supply the ideas and methods to improve antenatal care compliance. Methods This investigation used the questionnaire which was designed by the author to survey 100 pregnant women in class three level hospital in Guangzhou.The results underwent subsequent analysis. Results There were 68.4%of the pregnant women didn't finish the check.The average check times Wag only five.70.4%of them who were younger than 30 hadn't fulfilled seizure frequency rates,while 65.9%for who were older than 30(P＞0.05).The pregnant women with school-age≤9 and school-age＞9 who hadn't fulfilled seizure frequency rates were 83.3%and 50.0%.Irregular check rates were 59.3%、29.5%(P＜0.01).The floating population and the local population in the area of prenatal compliance results showed statistical difference.P＜0.01.The family whose income was higher than 3000 yuan per-month were better than those lower than 3000 yuan per-month about the antenatal check compliance(P＜0.05).More than 70% of pregnant women felt risk factors of pregnancy complications,their partners' support,local medical conditions and attitudes of medical staff had influence of their antenatal check compliance.Conclusions The compliance of the low education and low family income group is relatively low.For the affecting factors.how to improve the prenatal check compliance of such crowd is worthy of further study.
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Objective To explore the effects of murinecytomegalovirus(MCMV)infected mouse embryonic fibroblasts(MEF)on the proliferation and activation of regulatory T cell in vitro. Methods A co-culture system of T cell and MCMV infected MEF(T-MEF MCMV)was established.The viral load of supernatant was determined by plaque assay.The proliferation of T cells was observed with cell counting.The proportion of CD4+ CD25+ cells was measured by flow cytometry.The levels of Foxp3 protein were measured by Western blot.Reverse transcription polymerase chain reaction(RT-PCR)assay was utilized tO determine whether MCMV infection of MEF influenced the level of transforming growth factor(TGF)-β mRNA.The level of TGF-β protein in supernatant was measured by double-antibody sandwich enzyme linked immunosorbent assay(ELISA).The difference between T-MEF MCMV group and control group was assessed by one-way ANOVA.Results When T cells were co-cultured with MCMV infected MEF for 1 day and 3 days,the viral load in supernatant decreased.But when co-culture lasted for 6 days,the antiviral effect obviously diminished,as the viral load[(5.58±0.67)×105 PFU/mL]of the experimental group showed no statistic difference with MEF MCMV control group[(6.05±0.34)×105PFU/mL].When co-cultured with MCMV infected MEF for 3 days,T cell increased from pre-culture level of[(2.02±0.05)×106/mL]to(2.25± 0.13)×106/mL(P＜0.05).But when co-culture lasted for 6 days,the number of T eelI returned to (2.08±0.14)×106/mL,which had no statistic difference with that of co-culture for 3 days system. Both the expressions of Foxp3 protein and the proportion of CD4+ CD25+ Foxp3+ Treg cells in T-MEF MCMV group were up-regulated as the infection time extended,which were two times and three times of the control group level,respectively.The mRNA level(A value)of TGF-β in MCMV infected MEF increased from baseline of 1.09±0.13 to 3.15±0.54 on day 3 after infection.The expression of TGF-β in supernatant 3 days after infection was(3.85±0.32) μg/L,which was significantly higher than that before infection[(1.74±0.14)μg/L,P＜0.05].Conclusions Activated T cells have antiviral effect.However,the function of T cells is rapidly inhibited after activation,which may be due to the expression of Foxp3 mRNA induced by MCMV infected MEF and increased CD4+ CD25+ Treg proportion of the co-cultured T cells.TGF-β level is significantly increased after CMV infection,which may be an important mechanism of Treg proliferation.MCMV may manipulate Treg to evade specific immune elimination and,as a result,to cause CMV replication.
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Objective To explore the role of CD4+CD25+regulatory T cells(Treg)in the co-culture svstem consisting of T cells and mouse embryo fibroblasts(MEF)infected with murine cytomegalovirus (MCMV)in vitro.Methods A co-culture system of T cells and MCMV infected MEF(MEFMCMV)was established.The viral load in the supernatants was determined by plaque assay.TH1/TH2 differentiation-specific transcription factors T-bet/GATA-3 were assayed by Western blot.The levels of cytokines IL-4,IL-10and IFN-γ in the co-culture supenatants were measured by double-antibody sandwich ELISA.Results After 3 d incubation,the viral load in the supernatants of TdepTreg-MEFMCMC group,in which the T cells depleted Treg(TdepTreg)were co-cultured together with MEFMVMV,was significantly lower than that in MEFMCMV group.And in the co-cultivation of MEFMCMV and T cells without Treg the expressions of T-bet/GATA-3,IL-4,IL-10 and IFN-γ incteased significantly.Compared with the virus content in the TdepTreg-MEFMCMV group,the MCMV load in the"add-on Treg group"increased and the levels of T-bet/GATA-3 and IFN-γ were lower,and the expression of IL-4 didn't show any significant difference. Compared with the level of IL-10 in the TdepTreg-MEFMCMV group,the IL-10 level in the"add-on TREG group"didn't show any significant differece when the Treg ratio among total T cells was 1%～2%,and increased significantly when the Treg ratio was more than 5%.These eflfects were all correlated with Treg ratios in a dose-dependent manner.Conclusion MCMV infected MEF can induce the proliferation and activation of effector T cells,but Treg can inhibit the T cell-mediated protective effect on MCMV infection.
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To establish a method of directional differentiation and efficient production of neurons from embryonic stem cells (ES cells) in vitro, based on the 4-/4+ protocol described by Bain, a new method was established to induce ES cells differentiating into neurons by means of three-step differentiation using all-trans retinoic acid (ATRA) combined with astrocyte-conditioned medium (ACM) in Vitro. The totipotency of ES cells was identified by observation of cells' morphology and formations of teratoma in immunocompromised mice. The cells' differentiation was evaluated continuously by the detection of the specific cellular markers of neural stem cells, neurons and astrocytes, including nestin, NSE and GFAP using immunohistochemistry assay. The NSE positive cells' ratio of the differentiated cells was determined by flow cytometry. It was found that the transparent circular clusters surrounding embryoid bodies induced with combining induction protocol formed just after 24 h and gradually enlarged later. This phenomenon could not be observed in EBs induced only by ATRA. The NSE positive cells' ratio in the cells induced with ATRA and ACM was higher than that of the cells induced by ATRA at different time points of differentiation, and finally reached up to 73.5% among the total differentiated population. It was concluded that ES cells could be induced into neurons with high purity and yield by means of inducing method combining with ATRA and ACM.
Subject(s)
Astrocytes/cytology , Cell Differentiation , Cells, Cultured , Embryo, Mammalian , Neurons/cytology , Stem Cells/cytology , Tretinoin/pharmacologyABSTRACT
To establish a method of directional differentiation and efficient production of neurons from embryonic stem cells (ES cells) in vitro, based on the 4-/4+ protocol described by Bain, a new method was established to induce ES cells differentiating into neurons by means of three-step differentiation using all-trans retinoic acid (ATRA) combined with astrocyte-conditioned medium (ACM) in Vitro. The totipotency of ES cells was identified by observation of cells' morphology and formations of teratoma in immunocompromised mice. The cells' differentiation was evaluated continuously by the detection of the specific cellular markers of neural stem ceils, neurons and astrocytes,including nestin, NSE and GFAP using immunohistochemistry assay. The NSE positive cells' ratio of the differentiated cells was determined by flow cytometry. It was found that the transparent circular clusters surrounding embryoid bodies induced with combining induction protocol formed just after 24 h and gradually enlarged later. This phenomenon could not be observed in EBs induced only by ATRA. The NSE positive cells' ratio irthe cells induced with ATRA and ACM was higher than that of the cells induced by ATRA at different time points of differentiation, and finally reached up to 73.5 % among the total differentiated population. It was concluded that ES cells could be induced into neurons with high purity and yield by means of inducing method combining with ATRA and ACM.
ABSTRACT
<p><b>OBJECTIVE</b>To find the novel gene related to the multi-drug resistance in leukemia and explore the molecular mechanism of multi-drug resistance.</p><p><b>METHODS</b>The subtracted HL-60/VCR cDNA library was generated through the suppression subtractive hybridization using the wild HL-60 cells' cDNA as target and HL-60/ ATRA cells' as driver. A novel expression sequence tag (EST) sequence, which differentially expressed in HL-60/ ATRA cell, was screened by cDNA chip. Then a novel human gene, HV126 was assembled by the EST assembly tools. Bioinformatical databases and softwares were used to analyze and predict its function. Reverse transcription-PCR (RT-PCR) was used to detect the expression of HV-126 gene in leukemia cells before and after chemotherapy.</p><p><b>RESULTS</b>The full open reading frames (ORFs) of the novel EST assembled by overlapping dbEST sequences included a 1991 bp nucleic sequence, which was named HV126. The deduced amino acid sequence consisted of 365 amino acids. The sequence of the novel gene exhibited 43% homology to a known gene, which is a possible member of the death domain-flood family implicated in apoptosis and inflammation. The expression of HV126 was proved to be related to the drug sensitivity in leukemia cells by RT-PCR.</p><p><b>CONCLUSION</b>HV126, the novel gene, might have roles in regulating multi-drug resistance in leukemia. Further laboratory research should be done on cloning and making clear the gene function.</p>