Objective To study the isolation method and the culture method of primary human endometrial cells and to iden‐tify the purity and biological activity .Methods We digested human endometrial tissue by collagenase at first ,and then to separate , purify and culture the human glandular stromal cells(ESC) and endometrial epithelial cells(EEC)by differential centrifugation ,dab‐ble screen filtration and adhesion purification technology .Ultimately ,we identified the isolated cells with cytokeratin and vimentin immunocytochemical staining and immunofluorescence method .Results Stromal cells showed a parallel growth .The cells were spindle or polygonal ,and the vimentin antibody showed a positive immunohistochemical staining ,the purity was more than 95% .At the same time ,glandular epithelial cells grew in whorls .The cells were polygonal or tadpole shaped ,and the cytokeratin antibody immunohistochemical staining ,the purity were up to 90% .Conclusion The successful isolation and culture of high quantity ,viabili‐ty and purity by collagenase digestion and dabble screen filter method of endometriall cells make a strong operability .The laboratory which has the basic cell culture conditions can develop the experiment .

OBJECTIVETo investigate the immunological characteristics of human tonsil mesenchymal stem cells (TMSCs).

METHODSHuman tonsil tissues were obtained from the children patients with chronic tonsillitis. TMSCs were separated, cultured, and were detected the expression profiles of HLA-I, HLA-II, CD80, CD86 by flow cytometry. The measurement of immunogenicity, the effect on phytohemagglutinin (PHA) induced peripheral blood mononuclear cell (PBMCs) proliferation and mixed lymphocytes reaction (MLR) were performed to identify the immunological characteristics of TMSCs. The co-cultures of TMSCs + PBMCs + PHA and TMSCs + MLR were established, respectively, and the concentration of kynurenine, which is the metabolin of indoleamine 2, 3-dioxygenase, in the culture supernatant were examined. Then we added 1-methyl-L-tryptophan into the co-culture of TMSCs + PBMCs + PHA and TMSCs + MLR, respectively, and tested the proliferation of PBMCs. Each experiment was repeated three times, and there were six samples in each group. Statistical significance was assessed by analysis of variance (ANOVA), and a P value less than 0.05 was considered statistically significant.

RESULTSTMSCs expressed HLA-I, were negative for HLA-II and co-stimulatory molecules CD80 and CD86. The stimulation index in the group of TMSCs + allogeneic PBMCs was 1.38 ± 0.26, whereas the stimulation index in the group of allogeneic PBMCs was 1.22 ± 0.28, and there was no significant difference between the two groups (P > 0.05), indicating that TMSCs could not initiate the proliferation of allogeneic PBMCs. The stimulation indexes in the group of TMSCs + allogeneic PBMCs + PHA were 1.49 ± 0.29 and 1.23 ± 0.22, respectively, whereas the stimulation index in the group of allogeneic PBMCs + PHA was 4.60 ± 0.81, and the difference between the two groups had a statistical significance (P < 0.05) suggesting that TMSCs could inhibit PHA-induced PBMCs proliferation. The stimulation indexes in the group of TMSCs + MLR were 1.29 ± 0.23 and 1.26 ± 0.27, respectively, however, the stimulation index in the group of MLR was 3.04 ± 0.66, and the difference between the two groups had a statistical significance (P < 0.05), demonstrating that TMSCs could suppress MLR-induced PBMCs proliferation. The levels of kynurenine were (26.0 ± 2.3) μmol/L and (23.5 ± 4.5) μmol/L in the culture of TMSCs + PBMCs + PHA and TMSCs + MLR, respectively, thus elevating significantly. After adding of 1-methyl-L-tryptophan, TMSCs-mediated-proliferation suppression of PBMCs restored to normal levels.

CONCLUSIONTMSCs possess low immunogenecity and immunosuppressive function, may be used in allogeneic transplantation.

Cell Proliferation ; Cells, Cultured ; Child ; Coculture Techniques ; Flow Cytometry ; Humans ; Immunosuppression ; Kynurenine ; analysis ; Leukocytes, Mononuclear ; Lymphocyte Culture Test, Mixed ; methods ; Mesenchymal Stromal Cells ; cytology ; immunology ; Palatine Tonsil ; cytology ; Tryptophan ; administration & dosage ; analogs & derivatives

ObjectiveTo evaluate the clinical value of prenatal ultrasonography combined with maternal serology screening for chromosomal abnormality in 15 to 20+6 gestational weeks.MethodsSix hundred and twenty-eight pregnant women (628 fetuses) in 15 to 20+6 gestational weeks were selected to undergo prenatal ultrasonography, who were in critical risk of trisomy 21 or trisomy 18 by maternal serology screening. Transabdominal ultrasonography were performed and fetal nasal bone and nuchal fold were detected. Those who had nasal bone hypoplasia, thickened nuchal fold (NF>6 mm) and other abnormal fetal soft markers underwent amniocentesis for karyotyping analysis. ResultsThere were 6 cases of nasal bone hypoplasia (0.96%, 6/628), including one case of thickened nuchal fold, two cases of echogenic bowel, two cases of choroid plexus cysts and 1 case of echogenic cardiac focus. All these 6 cases underwent amniocentesis and 2 were trisomy 21 (33.3%, 2/6). The other 4 cases had no significant chromosomal abnormality.ConclusionsPrenatal ultrasonography may improve the detection rate of chromosomal abnormality for those pregnant women who are in critical risk of chromosomal abnormality prompted by serology screening. But invasive procedures are still needed to verify the chromosomal abnormality.

AIM:To evaluate the correlation between microRNA-1284 (miR-1284) and gastric cancer, and to investigate the underlying mechanism.METHODS: The expression of miR-1284 was examined by real-time PCR in 63 gastric cancer ( GC) tissue samples and 63 non-malignant adjacent tissue samples.The correlation between miR-1284 and the clinicopathological feature of GC was analyzed.Lentiviral vector containing miR-1284 was constructed and transfected into GC SGC-7901 cells.After transfection, the expression of miR-1284 was examined by real-time PCR.The cell activity was evaluated by CCK-8 assay.The cell cycle and apoptosis were determined by flow cytometry.The ability of cell migra-tion was measured by wound-healing assay.The potential target gene of miR-1284 was predicted by online bioinformatic softwares.The expression of JAG1 mRNA was examined by real-time PCR.The protein levels of JAG1, Notch1 and NF-κB were analyzed by Western blotting.RESULTS:Compared with non-malignant adjacent tissue samples, the results of real-time PCR showed significant downregulation of miR-1284 in 42 GC tissue samples ( P<0.05 ) .The expression level of miR-1284 was not significantly associated with age and gender of the patients, tumor size, TNM staging and lymph node metastases (P>0.05), but significantly associated with histologic grading (P<0.05).Compared with LV-NC-GFP group and control group, after transfection of miR-1284 in LV-miR-1284 group, the expression of miR-1284 was significantly in-creased (P<0.05), the percentages of apoptotic cells and the cells in G0/G1 phase were significantly increased (P<0.05), the cells activity and ability of migration were significantly decreased (P<0.05), and the expression of JAG1, Notch1 and NF-κB was significantly decreased (P<0.05).CONCLUSION:The inhibitory effect of miR-1284 on gastric cancer may be associated with the regulation of its targeting gene JAG1.

Background and purpose:It has beenreported that miR-1284 is associated with gastric cancer lymph node metastasis in the research of microRNA microarray in human gastric cancer tissues. But the specific role of miR-1284 in gastric cancer has not been reported. The aim of this study was to investigate the effect of miR-1284 over-expression on the gene expression profiling and invasion/metastasis of human gastric cancer SGC-7901 cells. Methods:Gastric cancer SGC-7901 cells of LV-miR-1284 group were transfected with lentiviral vectors of miR-1284, cells of LV-NC-GFP group were transfected with lentiviral vectors without miR-1284, and cells of control group were not transfected with lentiviral vectors. The expression of miR-1284 was detected by the real-time fluorescent quantitative PCR. Differential expression genes were detected by the microRNA chip. Target genes of miR-1284 were predicted by the bioinformatics. Invasive ability was detected by the Transwell invasion assay. Metastasis ability was detected by subcutane-ously transplanted tumor model of nude mice.Results:Compared with LV-NC-GFP and control groups, the expressions of miR-1284 and 20 genes were up-regulated, and the expression of 17 genes was down-regulated in LV-miR-1284 group. One hundred and thirty-eight target genes of miR-1284 were predicted by the bioinformatics website. Compared with invasive cell number of LV-NC-GFP group (168.67±4.55) and control group (170.33±3.08), the ability of invasion ofcells was weakened in LV-miR-1284 group (70.00±2.37). Compared with the liver metastasis rate of LV-NC-GFP group (85.71%) and control group (85.71%), the ability of metastasis of cells was weakened in LV-miR-1284 group (14.29%). Conclusion:The ability of invasion and metastasis of SGC-7901 cells is suppressed by over-expression of miR-1284. The mechanism may be related to regulating the expression ofSUMO1 andJUNgenes.

AIM:To study the effect and the molecular mechanism of CDX 2 over-expression on the prolifera-tion, growth and cell cycle of human gastric cancer cell line SGC-7901.METHODS:The SGC-7901 cells in LV-CDX2-GFP group were transfected with the recombinant lentivirus vector LV-CDX2-GFP, the cells in LV-GFP group were trans-fected with the negative control lentiviral vector for the negative control , and the cells in blank control group were without any treatment.The cell proliferation was detected by CCK-8 assay.The cell cycle distribution was analyzed by flow cytome-try.The expression of CDX2, Bax, Bcl-2, cyclin D1 and survivin was determined by semi-quantitative RT-PCR and Wes-tern blotting .RESULTS:Compared with LV-GFP group and blank control group , the proliferation activity of the SGC-7901 cells was significantly lower (P<0.05), the G0/G1 phase proportion increased (P<0.05), the mRNA and protein levels of Bcl-2, cyclin D1 and survivin were reduced (P<0.05), and the mRNA and protein levels of Bax were up-regula-ted (P<0.05) in LV-CDX2-GFP group.No statistically significant difference of the above indexes was observed (P>0.05) between LV-GFP group and blank control group .CONCLUSION:Over-expression of CDX2 mediated by lentivirus inhibits the proliferation and growth of human gastric cancer SGC-7901 cells and arrestes the cell cycle at G 0/G1 phase, which may be related to down-regulation of Bcl-2, cyclin D1 and survivin and up-regulation of Bax .

Objective To clone and express the partial encoding sequence of Mr 70 000 heat shock protein of Cryptosporidium andersoni (CaHSP70) in Escherichia coli and identify the recombinant protein. Methods Total RNA was extracted from oocysts of C.andersoni isolated from Xuzhou, Jiangsu (XZ-BOV). The CaHSP70 gene was amplified by RT-PCR. The PCR product was cloned and then subcloned into pET28a vector, and the recombinant plasmids were transformed into E.coli BL21(DE3) subsequently. The expressed protein induced by IPTG was purified and identified by SDS-PAGE and Western blotting, and was further analyzed by relevant bioinformatics softwares. The specific IgG antibodies in mice immunized by rCaHSP70 were detected by Western blotting and ELISA respectively. Results The deduced amino acid sequence showed to be identical with that of C. andersoni Mr 70 000 heat shock protein (HSP70). The recombinant protein expressed in the form of inclusion body was about Mr 43 000. It could be recognized by anti-His G labeled HRP antibodies and all the sera from mice infected with C. andersoni and children infected with C. parvum as well as sera from mice immunized with rCaHSP70 respectively. The rCaHSP70 possibly had multiple domains and potential antigenic determinants. Phylogenetic analysis showed that XZ-BOV and C. andersoni were in the same clade. ELISA showed that the level of specific antibodies against rCaHSP70 in immunized BALB/c and C57BL/6 mice was significantly higher than that of mice before immunization. Conclusion The recombinant plasmid pET28a-CaHSP70 has been constructed. The purified rCaHSP70 exhibits high antigenicity and seems a potential candidate antigen for immunodiagnosis of cryptosporidiosis.

Objective To investigate the relationship between cervical intraepithelial neoplasia (CIN) and high-risk (HR)HPV infection among late pregnant women.Methods From Aug.2007 to Feb.2010,168 women at 13 to 32 gestational weeks undergoing prenatal examination in Beijing Obstetrics and Gynecology Hospital went through three stage cervical disease screening,including 21 women with cervicitis and 147 women with C1N (42 women with CIN Ⅲ,37 women with CIN Ⅱ and 68 women with CIN Ⅰ ).Hybrid capture assay version Ⅱ ( HC- Ⅱ ) test was used to measure HR-HPV DNA load,and the logarithmic transtormation (log10) was performed.All 168 women were followed up to postpartum 3 -6 months.HR-HPV infections rates of cervicitis and different CIN,the rate of HR-HPV infection turned naturally negative at postpartum of 3 to 6 months,and HR-HPV load at pregnancy and 3 -6 months postpartum were observed.Results ( 1 ) HR-HPV infection rate:CIN Ⅲ,Ⅱ,Ⅰ and cervicitis pregnant women's HR-HPV positive infection rates were 98％ (41/42),86％ ( 32/37 ),76％ ( 52/68 ) and 62％( 13/21 ) respectively,which reached statistical difference (P =0.002).(2) HR-HPV naturally negative:the rate of pregnant women with different levels of CIN who turned HR-HPV naturally negative within 3 -6 months of postpartum were CIN Ⅲ 5％ (2/41),CIN Ⅱ 47％ (15/32),CIN Ⅰ 52 ％ (27/52) and cervicitis 10/13,which also reached statistical difference among those four groups (P =0.000).(3) HR-HPV load:pregnant women with different grade of CIN and cervicitis HR-HPV DNA load were CIN Ⅲ 2.02 ng/L(1.53,2.67 ng/L),CIN Ⅱ 1.94 ng/L ( 0.75,2.75 ng/L),CIN Ⅰ 2.04 ng/L (0.08,2.95 ng/L) and cervicitis 1.98 ng/L( -0.07,2.47 ng/L).There was no significantly different HPV load in women with cervicitis and different CIN (P =0.719).At 3 -6 months postpartum,HR-HPV load was CIN Ⅲ1.55 ng/L(0.90,2.10 ng/L),which was significantly higher than the amount of CIN Ⅱ 0.09 ng/L(-0.69,1.74 ng/L),CIN Ⅰ 0.48 ng/L( -0.56,2.2 ng/L) and cervicitis -0.46 ng/L ( -0.78,1.40 ng/L,P =0.036).Conclusions With the increasing of CIN grade,the rate of HR-HPV infection in pregnant women was increased,however,the rate of HR-HPV turning negative naturally at 3 -6 months postpartum decreased.With different CIN grade during pregnancy,HR-HPV DNA load did not change significantly,but HR-HPV DNA load increased at 3 -6 months of postpartum.HR-HPV DNA loads with the same grade of CIN and cervicitis during pregnancy higher than that of postpartum among pregnant women.

Objective To investigate the value and safety of biopsy guided by colposcopy in diagnosis of cervical diseases in pregnant women.Methods From Aug.2007 to Feb.2009.17 828 pregnant women who receive antenatal examination underwent cervical cytological screening thinprep cytology test(TCT)in Beijing Obstetrics and Gynecology Hospital.If abnormal cytological results were found,those preguant women were administered by eolposcopic examination and biopsy after they signed informed consent.Results (1)TCT:the abnormal TCT results of 1502 preguant women(8.425%,1502/17 828) were found in 17 828 cases.(2)Colposeopie examination:two hundred and four pregnant women underwent colposcopic examination.The rate of satisfied colposcopic imaging wag 92.6%(189/204),colposcopic examination identified 125 cages with cervical inflammation or cervical intraepithelial neoplasia (CIN)Ⅰ,25 cases with CIN Ⅱ and 54 cases with CIN Ⅲ or microinvasive squamous carcinoma (MIVC) of squamous cervical carcinoma(SCC).(3)The results of biopsy guided by colposcopy:among 204 cases,it was found 33 cases with cervical inflammation or wart,95 cases with CIN Ⅰ,28 CIN Ⅱ,36 cases with CIN Ⅲ and 12 cases with MIVC. (4) The rate of concordance: compared with biopsy pathologic examination, colposcopy examination found 113 cases with cervical inflammation and CIN Ⅰ , the rate of concordance was 90. 4%(113/125). And 54 cases with CIN Ⅲ or SCC diagnosed by colposcopy examination, however biopsy pathologic examination confirm 23 cases with CIN % and 10 cases with SCC at stage Ⅰ a, the concordance rate was 61% (33/54). (5) Complication: eight (3.9%, 8/204) pregnant women underwent cervical wound suturing due to continuous bleeding after colposcopy exam or biopsy. No other complications were recorded. Conclusions It is necessary that TCT should be performed in pregnant women without cytological screening within one year. Colposcopic examination and biopsy were indicated if pregnant woman with abnormal cytological result were found. Pregnant women with cervicitis or CIN Ⅰ diagnosed by colposcopy should be followed up. If pregnant woman was suspected with CIN Ⅱ or advanced disease, biopsy guided by colposcopy should be performed.

Objective To evaluate the maternal and fetal outcomes of planned delay in treatment for cervical microinvasive squamous cancer during pregnancy.Methods A prospective study of pregnant women was done from August 1,2007 to May 31,2010.Pregnant women who had not been carried out cervical cytological screening within one year were got thin-prep cytology test (TCT) screening at their initial prenatal visit.Patients with abnormal cytological results were performed colposcopic examination and directed biopsy.Women with cervical microinvasive cancer were followed up every 8 to 12 weeks.If lesion progression were suspected,compared with previous image,repeated biopsy directed by colposcopy should be performed.Once worsening invasive cancer was confirmed,the pregnancy should be terminated timely.All patients should be reevaluated 6 to 12 weeks postpartum with repeated colposcopic examination and biopsy.All mothers were performed cold knife conization (CKC) at 6 to 12 weeks postpartum.Results We totally diagnosed 17 cases cervical microinvasive squamous carcinoma during pregnancy.The positive rate is 6.2/10 000 (17/27 230).After informed consent,15 pregnant women decided to delay treatment until fetal maturation.The mean gestational age of initial diagnosis was (19.3 ± 5.9) weeks.The women were followed up 2 to 4 times during pregnancy.Only 1 patient was verified lesion progression by directed biopsy at 34 weeks and delivered by cesarean section.The progression rate during pregnancy was 1/15.The mean delivered time was (37.1 ± 1.8) weeks (ranged from 34 to 40 weeks).The mean diagnosis-to-delivery interval was (18.4 ± 5.2) weeks.All patients were delivered by cesarean section and all newborns had good outcomes.Finally we confirmed 1 case with cervical cancer stage Ⅰ a2,11 cases with stage Ⅰ al,3 cases with cervical intraepithelial neoplasia (CIN) Ⅲ by pathological diagnosis after CKC during 6 to 12 weeks postpartum.All cases were disease free after follow-up ranged from 22 to 48 months.Conclusions It is necessary to perform TCT screening for pregnant women who have not been carried out cervical cytology screening within 1 year.If cervical microinvasive squamous cancer were suspected during pregnancy,in order to achieve fetal maturity it is acceptable for the women who desired pregnancy to delay treatment under closely monitoring until postpartum.