Relationship between Stat5b Expression in Cervical Tissues and Lesions / 中国医科大学学报

Objective To investigate the expression of signal transducer and activator of transcription 5 b (Stat5 b) and its phosphorylation (p-Stat5 b) in cervical lesions, its relationship with different degrees of cervical lesions, and its role in the pathogenesis and development of cervical lesions. Methods We obtained 80 specimens, including normal cervical tissues, low-grade squamous intraepithelial lesions (LSIL), high-grade squamous intraepithelial lesions (HSIL), and cervical cancer tissues. To analyze the correlation between Stat5 b and the degree of cervical lesions, immunohistochemical staining was performed to detect Stat5 b and p-Stat5 b expression. Results Increased Stat5 b expression had a positive correlation with the development of cervical lesions. The percentages of Stat5 b expressed in normal tissues, LSIL, HSIL, and cervical cancer tissues were 15.0％, 0.0％, 50.0％, and 80.0％, respectively, with significant differences among the groups (P ＜ 0.05). Conversely, the expression of p-Stat5 b had a negative correlation with the development of cervical lesions.Expression of p-Stat5 b in normal tissues, LSIL, HSIL, and cervical cancer tissues was 80.0％, 45.0％, 5.0％, and 0.0％, respectively, with statistically significant differences among the groups (P ＜ 0.05). Conclusion The expression of Stat5 b is significantly different in different degrees of cervical lesions, suggesting that Stat5 b signaling molecules may be involved in the development of cervical lesions.

Changes of cytokines in serum and cerebrospinal fluid of newborns infected with human cytomegalovirus / 中华实用儿科临床杂志

Objective@#To investigate the molecules of cytokine in the serum and cerebrospinal fluid of newborns infected with human cytomegalovirus (HCMV) by using protein chip technology and to analyze the changes of specific cytokine in serum and cerebrospinal fluid caused by HCMV infection, in order to provide a reliable index for predicting nervous system injury caused by HCMV infection.@*Methods@#Serum and cerebrospinal fluid in 4 newborns with HCMV infection and central nervous system injury (HCMV-infected group), and 4 newborns without HCMV infection and central nervous system infection (control group) were collected in Shengjing Hospital of China Medical University from June 2016 to December 2017, and protein chip was used to screen the differentially expressed cytokines in newborns serum and cerebrospinal fluid.The samples were further expanded to collect cerebrospinal fluid from 30 newborns HCMV infection group and 30 newborns in the control group, and the expression of differentially proteins was verified by adopting enzyme linked immunosorbent assay(ELISA) method.@*Results@#The results of protein chip analysis showed that newborns in HCMV infection group, compared with the control group, had 3 differentially expressed cytokines in the cerebrospinal fluid sample: adipocyte complement-related protein of 30 kD(Acrp30), interleukin-1 alpha(IL-1α), and matrix metallo protein-3(MMP3) (all P<0.05). Newborns in the HCMV-infected group, compared with the control group, had no differential cytokine expression in the serum.The results of ELISA showed that expression of Acrp30 was significantly higher in the cerebrospinal fluid of newborns with HCMV infection and central nervous system injury [(39.76±2.01) ng/L vs.(7.75±0.10) ng/L, t=87.09, P<0.001], and MMP3 expression was higher than that of control group [(1.40±2.13) ng/L vs.(0.18±0.45) ng/L, t=3.07, P=0.003], while the expression of IL-1α was significantly lower than that of the control group [(2.36±0.99) ng/L vs.(2.91±0.78) ng/L, t=2.39, P=0.020], and the differences were statistically significant.@*Conclusions@#The changes of cytokine in cerebrospinal fluid of HCMV infected newborn children may provide a reliable index for predicting injury degree of central nervous system in HCMV, and may further assist clinicians to give timely and appropriate treatment to newborns, and further assist clinicians to improve the prognosis for newborns.

Changes of cytokines in serum and cerebrospinal fluid of newborns infected with human cytomegalovirus / 中华实用儿科临床杂志

Objective To investigate the molecules of cytokine in the serum and cerebrospinal fluid of newbo_rns infected with human cytomegalovirus(HCmV)by using protein chip technology and to analyze the changes of spe_cific cytokine in serum and cerebrospinal fluid caused by HCmV infection,in order to provide a reliable index for pre_dicting nervous system injury caused by HCmV infection. Methods Serum and cerebrospinal fluid in 4 newborns with HCmV infection and central nervous system injury(HCmV_infected group),and 4 newborns without HCmV infection and central nervous system infection(control group)were collected in Shengjing Hospital of China medical University from June 2016 to December 2017,and protein chip was used to screen the differentially expressed cytokines in newbo_rns serum and cerebrospinal fluid. The samples were further expanded to collect cerebrospinal fluid from 30 newborns HCmV infection group and 30 newborns in the control group,and the expression of differentially proteins was verified by adopting enzyme linked immunosorbent assay( ELISA)method. Results The results of protein chip analysis showed that newborns in HCmV infection group,compared with the control group,had 3 differentially expressed cytokines in the cerebrospinal fluid sample:adipocyte complement_related protein of 30 kD(Acrp30),interleukin_1 alpha(IL_1α), and matrix metallo protein_3(mmP3)(all P<0. 05). Newborns in the HCmV_infected group,compared with the control group,had no differential cytokine expression in the serum. The results of ELISA showed that expression of Acrp30 was significantly higher in the cerebrospinal fluid of newborns with HCmV infection and central nervous system injury[(39. 76 ± 2. 01)ng/L υs.(7. 75 ± 0. 10)ng/L,t=87. 09,P<0. 001],and mmP3 expression was higher than that of control group[(1. 40 ± 2. 13)ng/L υs.(0. 18 ± 0. 45)ng/L,t=3. 07,P=0. 003],while the expression of IL_1α was significantly lower than that of the control group[(2. 36 ± 0. 99)ng/L υs.(2. 91 ± 0. 78)ng/L,t=2. 39, P=0. 020],and the differences were statistically significant. Conclusions The changes of cytokine in cerebrospinal fluid of HCmV infected newborn children may provide a reliable index for predicting injury degree of central nervous system in HCmV,and may further assist clinicians to give timely and appropriate treatment to newborns,and further as_sist clinicians to improve the prognosis for newborns.

The study of detection methods of cytomegalovirus infection in newborns / 中国小儿急救医学

Objective To evaluate the diagnostic value of fluorescent quantitative polymerase chain reaction(FQ-PCR) assay in human cytomegalovirus (HCMV) infection by detecting quantitatively HCMV DNA in peripheral blood mononuclear cell ( PBMC) of newborns,to evaluate the choice of detection methods for neonatal HCMV infection,and to provide a reasonable diagnosis basis for the clinic. Methods The urina-ry HCMV-DNA levels in 102 neonates with suspected HCMV infection were detected by FQ-PCR. The HCMV-DNA in PBMC was detected by FQ-PCR,and serum HCMV-IgM antibody was detected by chemilu-minescence immunoassay ( CLIA) . Then the sensitivity, specificity, coincidence rate and other indicators in the three kinds of detection methods were compared. Results Among 102 cases of suspected HCMV-infec-ted newborns,56 cases were symptomatic and 46 cases were non-symptomatic. The positive rate of HCMV-DNA in urine[87. 3％(89/102)] was significantly higher than that of PBMC HCMV-DNA [58. 8％ (60/102)] and serum HCMV-IgM antibody [40. 2％ (41/102)](all P<0. 01). For symptomatic HCMV-infec-ted newborns, PBMC HCMV-DNA quantitative detection sensitivity ( 71. 4％) was higher than serum HCMV-IgM antibody (57. 1％), and the specificity (56. 5％) was higher than urine HCMV-DNA quantifi-cation (8. 7％). The area under receiver operating characteristic(ROC) curve of PBMC HCMV-DNA quan-tification and HCMV-IgM antibody detection were 0. 642 (P=0. 014) and 0. 659 (P=0. 006),respectively;therefore PBMC HCMV-DNA and HCMV-IgM antibodies were of great importance in diagnosing symptom-atic HCMV infection in neonates. The area under the ROC curve of urinary HCMV-DNA quantification was 0. 461 ( P =0. 496 ) , and there was no significant difference between symptomatic and non-symptomatic HCMV infections in neonates. Conclusion HCMV-DNA detection in PBMC has higher sensitivity compared with HCMV-DNA detection in urine and higher specificity compared with IgM antibody detection in serum. It can be used to detect the early infection of HCMV in newborns. The rate of detection of HCMV infection can be improved by combination of the three methods.

Effects of camel milk on intestinal mucosal immune function in mice / 中华微生物学和免疫学杂志

Objective To investigate the effects of camel milk on immune cells in lamina propria (LP) of intestinal mucosa in mice. Methods Six male C57BL/6 mice(6-8 weeks) were randomly divided into two groups as follows: camel milk treatment group and double distilled water (DDW) control group. Samples of cells in LP of intestinal mucosa were collected. Cell counts and percentages of immune cells in LP were analyzed by flow cytometry. Levels of IL-4,IL-10,IL-17 and IFN-γ in the supernatants of cell cul-ture were measured by ELISA. Results Compared with the DDW control group, the camel milk treatment group showed increased percentage and absolute number of CD4+T cells as well as IFN-γ-secreting CD4+T cells in LP of intestinal mucosa(P<0.05). Moreover,significantly enhanced expression of IFN-γ and sup-pressed secretion of IL-4 were found in the camel milk treatment group (P<0.05). Conclusion Camel milk can promote the proliferation of CD4+T cells and enhance the secretion of IFN-γ,indicating that camel milk regulates the proliferation and cytokine secretion of immune cells in LP of intestinal mucosa in healthy mice.

Screening and identification of proteins interacting with HCMV UL132 protein / 吉林大学学报(医学版)

Objective:To screen the proteins interacting with the human cytomegalovirus(HCMV)UL132 protein from the human fetus brain cDNA library by using Yeast Two-Hybrid System, and to elucidate the possible mechanism of UL132 protein in congenital cytomegalovirus infection.Methods:The HCMV UL132 fragment was amplified by polymerase chain reaction,the amplified HCMV UL132 fragment and expression vector pGBKT7 were digested and purified,and the HCMV UL132 fragment was linked to the vector pGBKT7.The pGBKT7-UL132 was constructed and transformed to yeast AH109, then the Human Fetal Brain DNA Library DNA was transformed into AH109 yeast.Using HCMV UL132 as abait, a human fetus brain cDNA was screened and the proteins interacting with UL132 protein were searched, the positive clone was sequenced and analyzed by bioinformatics methods.Results:The bait expression vector pGBKT7-UL132 was successfully constructed.The results of double enzyme digestion showed that there were two visible bands of 800 and 7 000 bp, respectively.After transformation of library plasmid, the transformation efficiency was calculated, and the transformation efficiency was 6.6×103 cfu· μg-1.There were 95 blue clones by X-gal coloration reactionsequencing and there were 10 clones interacting with the protein encoded by UL141 protein.The BLAST analysis showed that 7 of them were highly homologous with CAML.Conclusion:CAML might be one interaction protein with HCMV UL132 in Human Fetus Brain cDNA Library,suggesting that the interaction may be associated with the invasion and proliferation of the HCMV.

Screening and Analysis of Proteins Interacting with HCMV UL145 from a Human Fetal Brain cDNA Library / 中国医科大学学报

Objective To screen a human fetal brain cDNA library for proteins that can interact with HCMV UL145 using a yeast two-hybrid system.Methods A bait plasmid (pGBKT7-UL145) was constructed.Using HCMV UL145 as bait,a human fetal brain cDNA library was screened and proteins interacting with UL145 were identified using bioinformatic methods to sequence and analyze the positive clones.Results Three clones interacting with HCMV UL145 were found,and identified as FOXG1.Conclusion Several proteins interacting with HCMV UL145 in the human fetal brain cDNA library were identified as FOXG1,indicating that this protein may play an important role in the course of HCMV infection.

Correlation between Severity of Cervical Lesions and Methylation of Human Papilloma Virus Type 16 DNA / 中国医科大学学报

Objective To quantifiably measure the methylation frequency of 18 CpG sites in the 3′region of L1 gene and long control region(LCR) gene of HPVl6 DNA,and study the relationship between HPVl6 DNA methylation and severity of cervical lesions. Methods A total of 10 cases Normal/low?grade squamous intraepithelial lesion(Normal/LSIL),10 cases of high?grade squamous intraepithelial lesion(HSIL),and 10 cases of cervical cancer(CC)were recruited for the study. The relationship between severity of cervical lesions and HPV16 DNA methylation was analyzed by bisultlte?pyrosequencing. Results The methylation rate was highest in Normal/LSIL at position 7 089 located in 3′?L1,followed by CC. The low?est was found in HSIL. The difference in methylation percentage among the three lesions was significant(P=0.006). In 7 134,the proportion meth?ylation was also different among three groups(P=0.01),difference in methylation percentage between Normal/LSIL and CC,as well as Normal/LSIL and HSIL was significant(P=0.038,0.017). Conclusion The methylation status of CpG sites 7 089 and 7 134 in the 3′region of L1gene is asso?ciated with the severity of cervical disease. The quantification of HPV DNA methylation can be used for cervical disease screening in clinical samples.

Polymorphism of Human Cytomegalovirus （HCMV） UL146 Gene / 中国医科大学学报

Objective To investigate the polymorphism of human cytomegalovirus（HCMV）UL146 gene in clinical strains,and to evaluate its clinical diagnostic and therapeutic value of gene.Methods The UL146 gene of clinical strains was examined by quantitative polymerase chain reaction（Q-PCR）or general polymerase chain reaction（PCR）.Positive samples of PCR amplification were sequenced and analyzed.Results High variability of UL146 gene was found among 28 HCMV strains.According to phylogenetic analysis,all sequences of UL146 in clinical strains could be divided into three types and four subtypes.Chemokine ELRCXC region was highly conserved in all sequences.Conclusion HCMV-UL146 genes showed a high degree of polymorphism,and its encoded chemokine ELRCXC region was highly con-served.The relationship between HCMV-UL146 gene′s polymorphism and different clinical symptoms of HCMV infection was unclear.

Screening of Protein Interacting with HCMV UL130 Protein by Yeast Two Hybrid from Human Fetus Brain cDNA Library / 中国医科大学学报

Objective To screen the human proteins interacting with human cytomegalovirus（HCMV）UL130 from human fetus brain cDNA library by GAL4 two-hybrid system 3 technique and analyze the corresponding coding sequences.Methods The ＂bait plasmid＂（named as pGBKT7-UL130）was constructed.By using HCMV UL130 as the bait,a human fetus brain cDNA library was screened and the proteins interacting with UL130 protein were searched.The positive clones were sequenced and analyzed by bioinformatic methods.Results Nine clones interacting with HCMV UL130 were identified,two of them were synaptosome-associated protein（SNAP）.Conclusion Some proteins interacting with HCMV UL130 in human fetus brain cDNA library were successfully screened.SNAP might play an important role in HCMV infection pathogenesis.

Screening of protein interacting with the transcript of UL128 gene showed two protein patterns by yeast two-hybrid from human fetus brain cDNA library / 中华微生物学和免疫学杂志

Objective Using yeast two-hybrid system to screen the proteins which can interact with the human cytomegalovirus (HCMV) UL128 which have two difference transcription structure from human fetus brain cDNA library, and compare the difference with structure and function of interacting proteins. Methods Two fragments of UL128 were amplified by 3'RACE and 5'RACE technology, the length are 519 bp and 642 bp, respectively. The "bait plasmid" (named as pGBKT7-UL128-519 bp and pGBKT7-UL128-642 bp) was constructed successfully. Using pGBKT7-UL128-519 bp and pGBKT7-UL128-642 bp as a bait, a human fetus brain cDNA was screened and the proteins interacting with UL128-519 bp and UL128-642 bp encoded protein were searched, and the positive clones were sequenced and analyzed by bioinformatic methods. Results EFEMP2 interacting with HCMV UL128-519 bp were identified, THY-1 interacting with HCMV UL128-642 bp were identified. Conclusion EFEMP2 and THY-1 proteins interacting with HCMV UL128-519 bp and UL128-642 bp in human fetus brain cDNA library were successfully screened, but same proteins weren't found from the proteins interacting with UL128-519 bp and UL128-642 bp protein, UL128-519 bp and UL128-642 bp protein may be play an different effect in the process of infect by HCMV.

Study on Dsg3 induced activation of T cells in first degree relatives of patients with pemphigus vulparis / 中华微生物学和免疫学杂志

Objective To investigate The Th1/Th2 and Tc1/Tc2 polarization in the peripheral blood of first degree relatives of pemphigus vulgaris(PV) and healthy control individuals, and to approach the mechanism of the Dsg3-specific autoimmunity in PV. Methods The peripheral blood mononuclear cells (PBMC) from first degree relatives and healthy control was stimulated for72 h with Dsg3 and without Dsg3. Th1/Th2, Tc1/Tc2 was assessed by four-color flow cytometry. Results The mean frequency of Dsg3-spe-cific Th2 cells for PV antibody positive first degree relatives was 10.13%±3.72%, compared with stimula-tion without antigen 7.28%±3.58%, the difference was significant (P<0.05). The percentage Dsg3-spe-cific Th2 was markedly higher in the PV antibody positive first degree relatives group than that in the control group(10.13%±3.72% vs 6.10%±2.82%, P<0.05) , Tc2 was markedly higher also (20.01%± 10.43% v514.91%±8.06%, 20.01%±10.43% vs 9.58%±5.49%, P<0.05). Conclusion When Dsg3 stimulated PBMC were used to stimulate autologous T cells an increased amount of Th2 and Tc2 was observed, it is implied that the imbalance of Th1/Th2, Tc1/Tc2 might play an important role in the initia-tion of PV.

Amino acid sequences analysis of human cytomegalovirus UL149 proteins binding peptides / 中华微生物学和免疫学杂志

Objective To identify the peptide that have strong ability binding to HCMV-UL149 encoded protein,and to analyze the characteristics of the amino acid sequence of UL149-binding peptides.Methods Expressed UL149 proteins of three genotypes were used to screen the binding peptide in the random peptide display library,then the encoding sequence of binding peptides in the selected clones were sequenced.The amino acid sequences of the binding peptides were analyzed for their homology,and were com pared with those of the known protein in protein banks.Results The homologous amino acid sequence W/A/F/V-D/E-D/E-G-W/F/I/L were found within the binding peptides selected by proteins of all the three UL149 genotypes proteins,and no difference between three groups was found.The alignment with amino acid sequences of the known proteins in protein banks showed that the binding peptides of UL149 putative protein have homologous amino acid sequences with immunoglobulin heavy chain variable region(IgHV),the serine/threonine protein kinases,compliment factor H,zinc finger protein,MHC Ⅰ molecule,eukaryotic translation initiation factor,nuclear factor and so on.Conclusion The UL149 encoding proteins have binding ability to proteins mentioned above,and might interfere with the immunity responds to HCMV infection through multiple mechanisms.

Cloning and Expression of Heat Shock Protein 60 Gene from Chlamydia trachomatis / 中华皮肤科杂志

90%inasinglestep,andtheproteinyieldwas2.5mg/L.ConclusionPurifiedChsp60kDantigenisobtained,andtheantigenmightbeappliedtodetectantibodyinpatientsinfectedwithChlamydialtrachomatis,andwillcontributetostudytheroleofChsp60inimmunopathogenesis.

Preparation of Blood-Deficient Model and Effects of Tangkuei Blood-Supplementing Decoction on Hemopoiesis / 中成药

Objective: To prepare a blood deficient model and study the effect of Tangkuei Blood Supplementing Decoction on hemopoiesis in this model. Methods: This model was made in mice by i.p. an accumulate doses of 60mg/kg acetylphenylhydrazine (APH) and 160mg/kg cyclophosphamidum (CY). The RBCs, WBCs, reticulocytes and bone marrow nucleated cells (BMNC) were counted, the micro structure of bone marrow was observed. The swimming time, the body temperature and the plasma cAMP, cGMP levels were measured. The effects of Tangkuei Blood Supplementing Decoction by p.o. administration on promoting the hemopioetic function were observed with the model mice. Results: Tangkuei Blood Supplementing Decoction could remarkably increase RBC, WBC, BMNC, improve the proportion of reticulolytes in peripheral blood and the micro struture of bone marrow, prolony the swimming time, raise the body temperature and the specific value of cAMP/cGMP. Conclusion: The model exhibits the main features of blood deficiency, and can be used as one of models of blood deficiency. Tangkuei Blood Supplementing Decoction can obviously improve the dual deficiention of qi and blood of model mice by supplementing qi and blood.