The human ovarian mucinous cystadenocarcinoma (hOMC) cells were co-cultured with antisense oligodeoxynucleotide (antisense ODN), nonsense ODN, and follicle-stimulating hormone (FSH) at different concentrations for the purpose of observing the effects of antisense ODN to FSH receptor (FSHR) on the proliferation and apoptosis of cultured hOMC cells in vitro. The inhibitory rates of growth were measured by using MTT method on the 2nd, 4th, 6th, 8th and 10th days after the interference of antisense ODN, nonsense ODN, and FSH, respectively. The apoptotic rates and the cell cycles were determined by means of flow cytometry, the apoptosis indexes were detected by using TUNEL, and the expression of caspase-3 was measured by using SP immunohistochemistry. Compared with that in the control group, the proliferative activity of hOMC cells was increased obviously in FSH groups (P<0.05 or P<0.01), decreased distinctly in antisense ODN groups (P<0.05 or P<0.01), and unchanged in nonsense ODN groups, respectively. Meanwhile, antisense ODN could significantly antagonize the FSH-promoted cell proliferative activity (P<0.01). Compared with those in the control group, the apoptotic rates and the expression of caspase-3 were dramatically increased in the mid-and high-dose antisense ODN groups (P<0.05 or P<0.01), while the number of cells in G(1)/G(0) phase was significantly decreased and that in S phase distinctly increased (P<0.01). There was no change in nonsense ODN groups (P>0.05). It was suggested that FSH may improve the development of hOMC cells. However, antisense ODN could inhibit proliferative activity and the FSH-promoted proliferative activity in hOMC cells, at the same time, antisense ODN could inhibit hOMC cell growth by inducing apoptosis.

Objective By analyzing the changes in behavior and the myelin basic protein (MBP) of the offspring in adult that treated with Poly(I∶C) during pregnancy,and to understand the role of white matter abnormalities in the abnormal behavior of the offspring induced by infection in maternal hosts.Methods Two models maternal female rats were given Poly(I∶ C) with 5 mg/kg and 10mg/kg respectively during the early pregnancy,and control maternal female rats was administered 5 mg/kg saline.The prepulse inhibition test,passive avoidance test and active avoidance test were used to evaluate schizophrenia like behaviors for each groups offspring in 8 weeks,and the expression of MBP was detected by immunohistochemical staining methods.Results The results of prepulse inhibition test showed that significant differences of PP2,PP4 and PP8 results existed among control group,single-dose model group and double-dose model group (F=10.381,P=0.001,F=10.313,P=0.001,F=15.233,P=0.000).Compared with the control group,the two model groups showed significantly lower,the double-dose model group was lower than single-dose model group (P＜0.05).In passive avoidance test,there were significant differences of T1 and T2 results existed among control group,single-dose model group and double-dose model group (F=23.555,P=0.000,F=17.524,P=0.000).The T1 results of two model groups were significantly higher than control group,the double-dose model group was significantly higher than single-dose model group (P＜0.05) ; the T2 results of two model groups were lower than control group,the double-dose model group was lower than single-dose model group(P＜0.05).The results of passive avoidance test indicated that significant differences existed among control group,single-dose model group and double-dose model group in whole period of testing and total conditioned response rate(F=8.631,P=0.000,F=6.986,P=0.001),the two model groups were significantly lower than control group,double-dose model group was significantly lower than single-dose model group (P＜0.05).MBP results of two model groups were significantly lower than control group,two model groups had no significant difference (P＞ 0.05).Conclusion The adult offspring that were treated with Poly (I∶C) exit abnormal behavior and damaged white matter,and there is a correlation between the degree of abnormal behavior and drug dose.

Objective To understand the heterochro-Matin-associated protein 1(HP1-γ) expression during the carcinogenesis and progresston of esophageal carcmoma,and preliminarily investigate the supertortty of using laser scanning cytometer to analyze immunohistochemistry results compared to traditional scoring methods.Methods A tissue microarray containing different grades of esophageal carcinoma was selected and the HP1-γ expression was detected by immunohistochemistry.The results of immunohistochemistry were analyzed quantitatively by using laser scanning cytometer.The correlation of results analyzed by using laser scanning cytometer and traditional scoring methods was analyzed by chi square test.Results The HP1-γ was primarily expressed in the nucleus.The positive rate of HP1-γ in normal esophagus,moderate-severe atypical hyperplasia，in situ carcinoma and squamous cancer was 37.5% (3/8),100%(21/21),100%(7/7) and 23.7% (9/38),respectively,with the difference being statistically significant among normal esophagus，oderate-severe atypical hyperplasia plus in situ carcinoma and squamous cancer(P<0.01).There was a high correlation between the results analyzed by laser scanning cytometer and those by traditional scoring methods under a light microscope(P<0.01).Conclusion HP1-γ may play a resistant role in the transformation from normal esophageal cells to malignant cells.Compared to the traditionally artificial scoring methods,there are many advantages such as high resolution,high objectivity and accuracy when using laser scanning cytometer to analyze the immunohisto chemistry results.

OBJECTIVETo investigate the therapeutic effect of IL-6 mAb on experimental autoimmune myocarditis (EAM) in rats, and search the mechanism of the role of IL-6, helper T cells 17 (Th17) and regulative T cells (Treg) in EAM pathogenesis.

METHODSThirty-four Lewis rats were divided into three groups randomly, i.e. control group (n = 6), EAM group (n = 12), and IL-6 mAb intervention group (n = 16). Rats in EAM group and IL-6 mAb intervention group were injected intracutaneously with myosin to establish EAM model. Rats in IL-6 mAb intervention group were injected intraperitoneally with 1 mg IL-6 mAb on 1st, 7th to 20th day after cardiac myosin immune injection. Myocardial inflammation was examined by HE stain, Masson stain, and TdT assay (TUNEL reaction) on 21st and 84th day after IL-6 mAb therapy in order to assess the therapeutic role. Spleen cells were analyzed by flow cytometry to illustrate Th17 and Treg cells? number and function. The serum concentration of IL-6, IL-10, IL-17, and TGF-beta in each group was measured by ELISA, concentration of STAT3, RORgammat, and Foxp3 mRNA in each group was determined with RT-PCR. Spleen cells derived from EAM were stimulated by IL-6 mAb in vitro, and the concentration of IL-10, IL-17 and TGF-beta was measured by ELISA.

RESULTSInflammation score, fibrosis score, and apoptosis index in IL-6 mAb intervention group were significantly decreased as compared with those in EAM group (P < 0.01). The number of Th17 and Treg cells in EAM group on the 21st day (experimental acute peak stage) were increased, and those in intervention group on the 21st day were significantly inhibited (P < 0.01). The concentration of serum IL-6, IL-10, IL-17 and TGF-beta in intervention group on the 21st day was decreased dramatically in comparison with that in EAM group on the same day (P < 0.01). The levels of peripheral blood STAT3, RORgammat, Foxp3 mRNA in intervention group on the 21st day was decreased significantly as compared with that in EAM group (P < 0.01). The expression of IL-10, IL-17 and TGF-beta was increased significantly (P < 0.01) by stimulation of IL-6 mAb on spleen cells derived from EAM in vitro.

CONCLUSIONSIL-6 mAb could neutralize IL-6, and ameliorate myocarditis and reduce heart autoimmune responses. IL-6 mAb has significantly protective effects on EAM by suppressing Th17 and Treg cells.

Animals ; Antibodies, Monoclonal ; therapeutic use ; Autoimmune Diseases ; drug therapy ; immunology ; Disease Models, Animal ; Forkhead Transcription Factors ; metabolism ; Interleukin-10 ; metabolism ; Interleukin-17 ; metabolism ; Interleukin-6 ; immunology ; Male ; Myocarditis ; drug therapy ; immunology ; Nuclear Receptor Subfamily 1, Group F, Member 3 ; metabolism ; Rats ; Rats, Inbred Lew ; STAT3 Transcription Factor ; metabolism ; Th17 Cells ; immunology ; Transforming Growth Factor beta1 ; metabolism

Objective To explore the changes of pubescent immune response in the schizophrenia offspring induced by poly(I:C) during pregnancy and the effects on white matter.Methods The obtained pregnant rats were randomly divided into model group(n=6) and control group (n=5), receiving either poly (I:C) at a dose of 10 mg/kg diluted in 0.9％ NaC1 solution or vehicle solution alone (sterile pyrogen-free 0.9％ NaC1) on gestation day 9 (GD9).Immunohistochemical technique(IHC) was applied to detect the changes of microglias and astrocytes in the prefrontal cortex(PFC) and hippocampus(HC) of partly offsprings in the two groups at the sixth week,as well as Luxol fast blue(LFB) for the changes of white matter.The other offsprings of each group were selected for behavioral assessment at the eighth week.Results The results of prepulse inhibition test showed that PP2, PP4 and PP8 of model groups were significantly lower than that of the control group at young adult(P＜0.01).In passive avoidance test, and the T1 results of model group were significantly higher than those of the control group, the T results of model group were lower than those of control group (P＜ 0.01).Immunohistochemical results indicated that the number of microglias in the model group((264±33)/mm2, (271 ±38)/mm2) was significantly increased in PFC and HC than that in the control group((140±29)/mm2, (169±37)/mm2, P＜0.05) ,which was accompanied with significant morphological changes, while the OD value of astrocyte protein expression in the frontal lobe and hippocampus had no obvious difference between the model group and control group(P＞0.05).The OD value of LFB staining for myelin in the model group(0.29±0.02) was significantly decreased compared with that in the control group(0.33±0.03)(P＜0.01).Conclusion The young adult offsprings with prenatal infection present obvious schizophrenia-like behavior, meanwhile, the microglias activation and demyelination changes in white matter are observed,which provides more evidence for the relationship between immune response and white matter in the pathogenesis of schizophrenia.

AIM: To explore the protective effect of phytosterol ester (PSE) on aortic aging in rats.ME-THODS: The female SD rats (12 months old, n=42) were randomly divided into control group, model group and PSE group.During the experiment, the rats in control group, model group and PSE group were treated with basic feed, high-fat diet (HFD) and HFD with 2% PSE (W/W) for 6 months, respectively.The morphological changes of the aorta were observed by HE staining and Masson staining, and the absolute area of smooth muscle cells and collagen fiber in the vascular wall were measured by image analysis.The levels of advanced glycosylation end products (AGEs), malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) in the plasma were detected.The expression of silent information regulator 1 (SIRT1) and peroxisome proliferator-activated receptor γ (PPARγ) at mRNA and protein levels in the vascular tissue was determined by real time PCR and Western blot, respectively.RESULTS: PSE significantly lowered plasma TC and LDL-C, and increased plasma HDL-C level (P<0.05), but had no effect on plasma TG level.PSE significantly attenuated the thickening of intima and media of aging aortic, and decreased the migration of vascular smooth muscle cells (VSMC) and the amount of VSMC and collagen fiber in the aorta (P<0.05).PSE significantly reduced the contents of AGEs and MDA (P<0.05), but had no effect on the activity of SOD and CAT in the plasma.PSE also down-regulated the expression of PPARγ and up-regulated the expression of SIRT1 (P<0.05).CONCLUSION: PSE is able to attenuate the senescence process in the aorta by reducing the production of reactive oxygen species in plasma, and activating SIRT1, or inhibiting the expression of PPARγ in vascular tissues.

Objective To investigate the clinical features,karyotype,and the prenatal diagnosis for his sibling of a Chinese patient with rare ring chromosome 20 syndrome induced intractable epilepsy.Methods The clinical data of the patient diagnosed in Peking University People's Hospital were collected.The clinical manifestations,chromosome karyotype were summarized.Results The proband,a boy,started to show intermittent tonic seizures or atypical absence seizures and psychomotor retardation from the age of 11 months.Several anti-epilepsy drugs and globulin had been tried without effect.Common karyotype analysis and epilepsy-related genes analysis revealed no abnormality.However,abnormal karyotype 46,XY,r(20)(p13q13.3) in his peripheral blood lymphocytes was found by high resolution chromosome karyotype analysis with 550 G-banding,and the diagnosis of ring chromosome 20 syndrome,type Ⅱ was confirmed.The mother of the patient underwent amniocentesis at the midterm of the second pregnancy.The cultured amniocytes karyotypes were normal.The second child(a boy) of the family was 1 year old without epilepsy and the psychomotor development was normal.Conclusions Ring chromosome 20 syndrome is a rare human chromosome abnormality.The syndrome is associated with epileptic seizures,behavior disorders and mental retardation.Since karyotype testing is not a routine investigation for the patient with epilepsy,the diagnosis of ring chromosome 20 syndrome is usually delayed or misdiagnosed.The karyotype analysis should be considered for the etiological study of the patients with intractable epilepsy with unknown origin.

Objective:To explore the method of establishing a modified demyelination and myelination regeneration model induced by dicyclohexanone oxalyl dihydrazone (CPZ) in mice with multiple sclerosis (MS), and to analyze the image markers of demyelination and myelination regeneration in mouse MS model.Methods:After the intragastrically administered with sodium carboxymethyl cellulose (CMCNa) for one week, a total of 30 C57BL/6 male mice were randomly divided into the control group ( n=10), the demyelination group ( n=10), and the remyelination group ( n=10). The mice of the control group were immediately performed MR scanning and pathological specimen obtaining; the mice in the demyelination group were administered with intragastrical CPZ-CMCNa once a day for 6 weeks for inducing demyelination, then received MR scanning and specimen obtaining with the same protocols used in control group; the mice in the remyelination group were administered with intragastrical CPZ-CMCNa once a day for six weeks for demyelination, then CPZ was withdrawn and normal diet was given for another four weeks. Then MR scanning and specimen obtaining were performed with the same protocols used in the other two groups. Regions of interest (ROIs) were set at the rostrum of corpus callosum (rCC), the bilateral normal appearing white matters (NAWM) of the rostrum of corpus callosum, and the bilateral cerebral cortex (Cx). The normalized T 2WI (T 2-normalized), fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD), and radial diffusivity (RD) values were compared among the three groups by one-way ANOVA. Results:The demyelination and remyelination mice model of MS were successfully established. The T 2-normalized values of rCC in control group, demyelination group and remyelination group were 0.47±0.03, 0.72±0.04, 0.54±0.04, respectively, with statistically significant difference found ( F=90.511, P<0.05). Post-hoc multiple comparisons showed significant differences among those groups ( P<0.05). There was no significant difference of T 2-normalized value in NAWM and Cx among the three groups ( P>0.05). Moreover, there were significant differences in the FA values (0.36±0.04, 0.29±0.03, and 0.32±0.05), the MD values [(0.572±0.015), (0.598±0.034), and (0.626±0.043)×10 -3 mm 2/s], the AD values [(0.79±0.04), (0.77±0.06), and (0.83±0.04)×10 -3 mm 2/s], and the RD values [(0.46±0.02), (0.51±0.03), and (0.53±0.05)×10 -3 mm 2/s] of rCC of the control group, the demyelination group, and the remyelination group (all P<0.05). Significant difference was found in FA values between the demyelination group and the control group ( P<0.05), and in MD values between the remyelination group and the control group ( P<0.05), as well as in AD values between the remyelination group and the demyelination group ( P<0.05). There were also significant differences in RD values between the remyelination group and the control group, and the demyelination group and the control group (all P<0.05). However, no significant difference was found in all diffusion tensor imaging (DTI) metrics of NAWM and Cx among the three groups (all P>0.05). The LFB-eosin staining showed that the myelin sheath of rCC was lost in the demyelination group, and the rCC was partially regenerated and repaired in the remyelination group. Conclusion:The modified CPZ-CMCNa model can selectively induce demyelination and remyelination of rCC, and the changes of demyelination and remyelination of rCC in the modified CPZ-CMCNa model can be quantitatively detected by T 2WI combined with DTI, which might provide related theoretical basis for the study on dynamic changes of MS lesions.

OBJECTIVETo investigate the prevalence and subtypes of microdeletions in azoospermia factor (AZF) region in infertile men from Sichuan in order to correlate genotypes with phenotypes.

METHODSMultiplex-PCR was used to detect sequence tagged sites (STS) of AZF microdeletions in 1011 infertile men including 713 cases of non-obstructive azoospermia and 298 cases of severe oligospermia.

RESULTSThe overall prevalence of microdeletions was 10.48% (106/1011), and the deletion rates were 11.08% (79/713) in non-obstructive azoospermia and 9.06% (27/298) in severe oligospermia. Complete AZFa or AZFb deletions were associated with azoospermia, whereas AZFc deletion (60.38%) was the most frequent deletion. The deletions were associated with variable spermatogenic phenotypes, and 37.50% of the patients with a deletion had sperms in the ejaculate. A mild decline in sperm concentration was found in two cases with partial AZFb deletion and one case with partial AZFb-c deletion.

CONCLUSIONDeletions of the AZFc region were most commonly found in our patients. All cases with complete AZFa or AZFb deletions and a proportion of cases with AZFc deletion were associated with azoospermia. Our study has provided more insight into the genotype-phenotype correlation, and confirmed that Yq microdeletion screening has a significant value for the diagnosis for male infertility.

Adult ; Azoospermia ; genetics ; Chromosome Deletion ; Chromosomes, Human, Y ; Genetic Association Studies ; methods ; Humans ; Infertility, Male ; genetics ; Male ; Phenotype ; Young Adult

Oligonucleotide microarray is developed on the basis of hybridization on the solid substrate. The pre-activated glass substrates and the terminal modification of the oligonucleotides are the two important factors in the process of fabrication for microarray. In order to compare the hybridization signal intensity of the different terminal modified oligonucleotide probes, the eight kinds of oligonucleotides were designed according to the sequence of HLA-DRB1-12, including the amino modified oligonucleotides with PEG spacer and the one without spacer, the phosphorothioate modified oligonucleotides with PEG spacer and the one without spacer. They were modified on 5' terminal and 3' terminal, respectively. In addition, the oligonucleotides probes with the internal spacer of different number of PEG were designed to observe the relationship between the spacer of PEG and the hybridization efficiency. These probes were respectively fixed on the bromoacetylation activated and glutaraldehyde activated slides to manufacture the two kinds of microarray which hybridized with the fluorescence labeled PCR product of HLA-DRB1-12 gene. The results from the study demonstrated that the signal intensity of 3' amino-modified probes with the internal spacer of different number of PEG on the bromoacetylation activated slides was stronger than the others. It is concluded that the 3' amino-modified oligonucleotide with an internal PEG spacer and the bromoacetylation activated slide enhanced the hybridization efficiency and were worthy to be proposed for the fabrication of HLA microarray or other kinds of microarrays for detecting fluorescence labeled PCR product in the future study.
HLA-DR Antigens ; genetics ; HLA-DRB1 Chains ; Humans ; Oligonucleotide Array Sequence Analysis ; methods