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Identification and characterization of ovarian cancer stem-like cells from primary tumor

Dongmei LAI ; Te LIU ; Yong HUANG ; Lihua WANG ; Jian ZHANG ; Weiwei CHENG

Chinese Journal of Obstetrics and Gynecology.2009;44(12):936-940. doi:10.3760/cma.j.issn.0529-567x.2009.12.013

Objective To study whether the primary ovarian cancer cells containing cancer stem cells and its characterization in serum-free culture condition.Methods The primary cancer cells were isolated from one stage Ⅲ, grade 2 serous adenocareinoma tissue.Cells were cultured in serum-free culture system supplemented with epidermal growth factor,basic fibroblast growth factor,leukemia inhibitory factor and insulin.or standard serum-containing system.Methyl thiazolyl tetrazolium assay,quantitative PCR analysis,flow-cytometric analysis and xenograft experiments in vivo were performed.Results The primary cancer cells could maintain and forill cell sphere in serum-free culture system.These cells had the properties of self-renewal,overexpression of stem cell marker genes Nanog,Oct-4,Sox-2,nestin,ABCG2,CD_(133) and CD_(117) By contrast with the difierentiated cells under standard serum-containing culture conditions.these sphere-forming cells were more resistant to cisplatin and paclitaxel after treated 48 and 72 hours(61% vs.31%,73% vs.29%,P<0.05).With Hoechst 33342 exclusion assay,only 21.83%of sphere-forming cells were positive with the dye,compared with 83.04% positive cells in differentiated cells (P<0.01).Only 500 sphere-forming cells resulted subcutaneous xenograft tumors.All of these xenografts were categorized as serous adenocareinomas, overexpression of CA_(125) and cytokeratin-7 which were original tumor phenotype of ovarian cancer. Conclusion The sphere-forming cells isolated from primary ovarian cancer tissues have the characterization of cancer stem cell and may be a more reliable model system for understanding the biology of primary human tumors.

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Clinical study on in vitro maturation of immature oocytes transferred from conventional in vitro fertilization

Peiyu WANG ; Junzhao ZHAO ; Wei ZHANG ; Jinju LIN ; Shiquan XIAO ; Yonggen WU ; Xuefeng HUANG

Chinese Journal of Obstetrics and Gynecology.2009;44(12):924-928. doi:10.3760/cma.j.issn.0529-567x.2009.12.010

Objective To investigate clinical effect of in vitro maturation(IVM)of immature oocytes transferred from conventional in vitro fertilization embryo transfer(IVF-ET) cycles.Methods From January 2008 to June 2009.medical documents of 155 infertile patients underwent IVF-ET in the Reproductive Medical Center of Fimt Affiliated Hospital of Wenzhou Medical College were analyzed retrospectively.If more than 20 oocytes were monitored after 5-7 days of ovulation induction or follicular developmental retardation were confirmed after 8-13 days of ovulation induction.according to patients' wish,IVM were transferred in 60 cycles(group A).In the mean time.IVF was continued in 95 cycles (group B).The mean dosage of gonadotropin,the cancel lation rate of cycles,the mean numbers of oocytes retrieved and maturation,the rate of fertilization and excellent quality embryos.pregnancy outcome and the incidence rate of ovarian hyperstimulation syndrome(OHSS)were compared and analyzed.Results The rates of embryo transfer were 92%(55/60)in group A and 63%(60/95)in group B,which showed significant differences (P<0.05).In group A,the mean dosage of the gonadotropin,the mean number of oocytes retrieved,the cleavage rate and OHSS rate were(1030±468)U,10±6,82.2%(231/281)and O,and were(1544±338)U,14±4,94.0%(502/534)and 35%(21/60)in group B,respectively,all data abeve exhibited statistieal difference between two groups(P<0.05).However,the rates of fertilization and excellent quality embryos had no significant differences between two groups(P>0.05).In group A,the rate of clinical pregnancy per transfer was 53%(29/55)and multiple pregnancy was 14%(4/29),and were 47%(28/60) and 32%(9/28)in group B,they all had no significant differences (P>0.05).Conclusion IVM of immature oocytes used in conventional IVF cycles not only obtained a high clinical pregnancy rate,but also reduced gonadotropin using dosage and avoided OHSS completely.

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Expression of aquaporin 3 and aquaporin 9 in placenta and fetal membrane with idiopathic polyhydramnios

Xueqiong ZHU ; Shanshan JIANG ; Shuangwei ZOU ; Yingchun HU ; Yuhuan WANG

Chinese Journal of Obstetrics and Gynecology.2009;44(12):920-923. doi:10.3760/cma.j.issn.0529-567x.2009.12.009

Objective To investigate the pathogenesis role of aquaporin 3 and aquaporin 9 in idiopathic polyhydramnios by detecting their expression and distribution in fetal membranes and placenta.Methods Twenty-one of term pregnancy women with idiopathic polyhydramnios were enrolled as patient group matched with 30 women with normal term pregnancy as control group.The expression and localization of aquaporin 3 and aquaporin 9 in fetal membranes and placenta were detected by real-time polymerase chain reaction and streptavidin peroxidase immunohistochemiscal staining.Results (1)The mRNA expressions of aquaporin 3 and aquaporin 9 were detected in amnion,chorion and placental tissue in both patient group and control group.Both aquaporin 3 and aquaporin 9 were demonstrated positive staining in the amnion epithelia,chorion cytotrophoblasts and placental trophoblast.(2)The ratio of aquaporin 3 and aquaporin 9 mRNA expressions in amnion in patient group comparing to those in control group were 5.00 and 3.25,while in chorion they were 2.03 and 2.08.When compared with those in amnion and chorion of control group,there was a significant difference(P<0.01).However,the relative change fold of aquaporin 3 and aquaporin 9 in placental trophoblast in patient group were decreased in comparison of those in control group,which also showed statistical difference(P<0.01).(3)The expression of aquaporin 3 and aquaporin 9 protein in anmion were 7.5 ±2. 0 and 11.1 ± 1.8 in patient group, while they were 5.3 ± 1. 6 and 5.6 ± 2. 3 in control group. In chorion, the expression of aquaporin 3 and aquaporin 9 protein was 7.5±2. 0 and 10. 0 ±1.6 in patient group, respectively, while in control group, they were 5.4 ±2.2 and 5.6±2. 1. When compared with those proteins in control group, it exhibited statistical difference (P<0.05). However, in placental trophoblast of patient greup,the expression of aquaporin 3 and aquaporin 9 protein were 3.5±1.4and 4. 0±2. 5, respectively, which were significantly decreased than 5.6±1. 3 and 7. 1±2. 9 in control group(P< 0. 05). Conclusions The alterations of aquaporin 3 and aquaporin 9 expressions in fetal membrane and placenta might be an adaptive response to idiopathic polyhydramnios. Further investigation should be needed to clarify the regulatory mechanism of aquaporin 3 and aquaporin 9 expressions.

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Correlation of toll-like receptor 3 and tumor necrosis factor-α with idiopathic fetal growth restriction

Ling LIU ; Shihong CUI ; Guomei CHENG ; Yajuan XU ; Xiaojuan WANG ; Lindong ZHANG ; Yiming CAI ; Yanni TIAN

Chinese Journal of Obstetrics and Gynecology.2009;44(12):909-914. doi:10.3760/cma.j.issn.0529-567x.2009.12.007

Objective To investigate the expression and the significance of toll-like receptor 3 (TLR-3)in placenta,tumor necrosis factor-α(TNF-α)in maternal and cord blood of idiopathic fetal growth restriction(IFGR),and their correlation with the pathogenesis of symmetric and asymmetric IFGR.Methods From April 2008 to April 2009,42 primiparae of singleton pregnancy and their IFGR babies,who delivered at term through cesarean section, in the Third Affiliated Hospital of Zhengzhou University were enrolled. All subjectects were divided into symmetric IFGR group (n=20) and asymmetric IFGR group (n =22). Another 42 non-IFGR pairs were randomly selected as the control group. The polink-2 plus polymerized horseradish peroxidase (HRP) immunohistochemical method and the enzyme linked immunosorbent assay (ELISA) were applied to detect TLR-3 and TNF-α levels. Results (1) The expression of TLR-3 protein were observed in all maternal placenta of the three groups. TLR-3 essentially expressed in syncytiotrophoblasts and hofbouer cells in the symmetric IFGR and control group, but expressed mostly in hofbouer cells and less in syneytiotrophoblasts in the asymmetric IFGR group. (2) The expression of TLR-3 in the syncytiotrophoblasts of the symmetric and asymmetric IFGR group was significantly lower than in the control group (111±14 and 118±11 vs. 156 ± 9, P<0. 01). The number of TLR-3 positive in Hofbourer cell in the symmetric IFGR group was lower than the control group (8. 9±2. 8 vs 17.5±2. 8, P <0. 01 ), but the number in the asymmetric IFGR group was higher (23.8±3.7) compared with the control group (P <0. 01). (3) The TNF-α levels in the maternal and cord blood of the symmetric and the asymmetric group were higher than that of the control group [maternal : (90±10) μg/L and ( 86±11 ) μg/L vs. (73±9) μg/L;cord blood: (92±12) μg/L and (96±8) μg/L vs. (79±9) μg/L;P<0.01]. (4) Neither symmetric nor the asymmetric IFGR group showed any correlations between the maternal and cord blood levels of TNF-α (P>0. 05). (5) Significant correlation was found between the TNF-α level of the cord blood and TLR-3 expression in the placenta in both the symmetric and asymmetric IFGR group(P<0. 05),but no relationship was found between the maternal blood TNF-α level and TLR-3 expression in the placenta (P>0. 05). Conclusions The variantions of TLR-3 expression in placenta and the increased expression of TNF-α in cord blood are associated with the genesis IFGR. The reduced expression of TLR-3 may related to symmetric IFGR, while the increased TLR-3 level in hofbouer cells may lead to asymmetric IFGR.

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Preparation of human papillomavirus 16 E7 peptide vaccine and its effectiveness in vitro and in vivo

Shujie LIAO ; Xiaoji HU ; Lingfei HAN ; Xuefeng JIANG ; Xi XIA ; Wei WANG ; Yunping LU ; Shixuan WANG ; Ding MA

Chinese Journal of Obstetrics and Gynecology.2009;44(12):903-908. doi:10.3760/cma.j.issn.0529-567x.2009.12.006

Objective To prepare the human papillomavirus (HPV) 16 peptide vaccine and explore the effect in vitro and in vivo. Methods (1) Prediction of the major histocompatibility complex (MHC) class I restricted T cell epitopes by bioinformatics target at transporter associated with antigen processing (TAP) and named by E7Pa, E7Pb, E7Pc separately. (2)In vivo, the C57BL/6 mice were divided into five groups with same amounts randomly after loading with TC-1 cells (HPV 16 positive tumor cells from C57BL/6 mouse), named as E7Pa + CpG,E7Pb + CpG,E7Pc + CpG (as experiment groups, and added 50 μg/ml E7Pa, E7Pb, E7Pc, respectively), CpG(as positive control group and added Con A with 12 mg/L final concentration) and blank control group (without any treatment). The T cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) assay at different time points;the lactate dehydrogenase (LDH) delivery method was used to test the cytolytie T lymphocyte (CTL) activity of mouse splenic lymphocyte in different ratio of effector cells and target cells (E:T);the related cytokines in tumor tissue and mouse peripheral blood were evaluated by real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively. The tumor volumes were measured to contrast the therapeutic effect in different groups. Results (1) Three peptide named E7Pa, E7Pb, E7Pc were successfully preparated which had high affinity and specificity. (2) After vaccination of 24, 48, 72,96 hours, MTT results shown that the proliferation rate in E7Pa + CpG group were(131±32)%, (302±15)%, (552±28)%, (731±24)% individually, which were much higher than those in blank control [(72± 15) %, (120 ± 57) %, (176 ±41)%, (288±29)% ;P<0.01], and the other groups i. e. E7Pb + CpG,E7Pc +CpG and CpG groups all proliferated much higher than those in blank control group with statistic signification (P<0. 05), but there was no significant difference between groups(P>0.05);the LDH delivery assay showed that when the ratio of E:T was 100:1, the activity of CTL in the E7Pa + CpG group was most powerful than the other groups with statistic signification (P<0. 01). Meanwhile, the ratio of E:T was concentration-dependent. Compared E7Pb + CpG, E7Pc + CpG or CpG groups with blank control group, there were significantly difference(P<0. 05) ,while there was no significant difference between groups(P >0. 05). The mRNA levels of interferon γ (IFN-γ), interleukin-2 (IL-2) in tumor tissue and peripheral blood in E7Pa + CpG group were significantly higher than those in blank control group (P<0. 01), which was the similar results when compared E7Pb + CpG, E7Pc + CpG or CpG groups with control group (P < 0. 05), and without significant difference between groups(P > 0. 05). The tumor volumes were suppressed obviously in all the experiment groups, especially at the 60th days, the volumes in ETPa + CpG group were much smaller than that in blank control group with statistic signification (P < 0. 01),which was the similar results that E7Pb + CpG, E7Pc + CpG or CpG groups had difference than blank control group with statistic signification (P < 0. 05), and without significant difference between groups(P >0. 05). Conclusion The HPV16 E7 peptide target at TAP combination with CpG as a vaccine could treat effectively the HPV16 E7 positive tumor in experiment.

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Prevalence of human papillomavirus infection in women in Tibet Autonomous Region of China

Qiong JIN ; Keng SHEN ; Hui LI ; Xianrong ZHOU ; Huifang HUANG ; Jinhua LENG ; Wenhua ZHANG ; Xiaoming GONG ; Xuemei CHENG ; Lang SUO ; Yanchun ZHU ; Jinghe LANG ; Chongmei LU ; Ping WANG ; Wenxin MO

Chinese Journal of Obstetrics and Gynecology.2009;44(12):898-902. doi:10.3760/cma.j.issn.0529-567x.2009.12.005

Objective To determine the prevalence of cervical type-specific human papillomavirus (HPV)infection as well as risk factors associated in Tibet Autonomous Region of China.Methods A cluster sampling study was performed in Lasa,Rikaze and Naqu of Tibet.An epidemiological questionnaire was applied and 3036 cervical specimens were obtained for liquid-based cytology and HPV DNA detection.Statistical analysis included Wald Chi-square and stepwise logistic regression model.Results The overall HPV prevalence of involved 3036 women was 9.19%(279/3036),of which 7.05%(214/3036)of the women were infected by high-risk types (including 14 sorts of types) and 2.14%(65/3036)by low-risk types(including 6 sorts of types).There were no significant differences of HPV prevalence between age groups(P=0.936),race(P=0.718)and areas(P=0.746),respectively.Twenty-one types of HPV were detected,of which HPV16(1.52%) was the most common type,followed by HPV33(1.42%).HPV58(1.22%),HPV52(1.15%),and HPV31(1.05%).HPV type distribution was varied by age.Of the 279 HPV infected women.14.3%(40/279)exhibited multiple HPV infections.Independent risk factors for HPV infection were smoking(P=0.027),number of sex partners(P=0.198)and early age of first intercourse(P=0.237).Conclusion The overall prevalence of HPV infection in Tibet Autonomous Region is lower than that in China or abroad,in which the most common genotype is HPV16 and the independent risk factors for HPV infection included early age of first intercourse,smoking,and number of Bex partners.

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Detection of human papillomavirus L1 capsid protein expression in liquid-based cytology samples with abnormal cytology

Wei XIAO ; Meilu BIAN ; Li MA ; Jun LIU ; Ying CHEN

Chinese Journal of Obstetrics and Gynecology.2009;44(12):887-891. doi:10.3760/cma.j.issn.0529-567x.2009.12.003

Objective To investigate the possibility of detection of the human papillomavirus (HPV)L1 capsid protein to predict the coarse of mild or moderate cervical intraepithelial neoplasia(CIN).Methods Immunocytochemical analysis using antibody against HPV L1 capsid protein was carried out on 274 samples obtained from women performed Tri Path Pap tests.positive for high-risk HPV DNA detected by hybrid capture Ⅱ (HC-Ⅱ)or cytologic diagnosed atypical squamous cells of undetermined significance (ASCUS)or more severe.For cytological diagnosed,there were ASCUS 105 cases,low-grade squamous intraepithelial lesions (LSIL) 119 cases,atypical squamous cells cannot exclude high-grade squamous intraepithelial lesion (ASC-H)9 cases,high-grade squamous intraepithelial lesions(HSIL)36 cases,and squamous cell carcinoma(SCC)5 cases.But for the pathologic diagnosed,there were chronic cervicitis 96 cases.CIN Ⅰ 55 cases,CIN Ⅱ 55 cases,CIN Ⅲ 32 cases,and SCC 6 cases.Results Of the 274 cases,HPV L1 capsid protein was positive in 69.8%(67/96) of cervicitis,83.5%(71/85)of CIN Ⅰ,41.8% (23/55)of CIN Ⅱ,3.1%(1/32)of CIN Ⅲ and 0(0/6)of SCC.Cytologic diagnosis revealed a higher expression rate in LSIL(75.6%.90/119) than that in ASCUS(63.8%,67/105)or in HSIL + SCC (9.8%,4/41;all P<0.01).Of 71 cases with ASCUS and ISIL without treated,none of HPV L1 positive cases(0/55)progressed in cytology,while 19%(3/16)of HPV L1 negative cases progressed to ASC-H,HSIL(P<0.01).Conclusion The expression rates of HPV L1 protein in liquid-based cell specimen is decreased as the cytopathology diagnosis severe degree.which may imply the histopathology diagnosis of cervix,predict the progression of cervical lesion,and help to treat the cases with ASCUS and LSIL.

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Clinical application of telomerase RNA component gene amplification assay in cervical lesions

Jing JIANG ; Lihui WEI ; Ruifang WU ; Guo ZHANG ; Na WULAN ; Jingran LI ; Yibing LI ; Zheng TU ; Yanqiu ZHOU ; Yun ZHAO ; Zhong CHEN

Chinese Journal of Obstetrics and Gynecology.2009;44(12):883-886. doi:10.3760/cma.j.issn.0529-567x.2009.12.002

Objective To investigate the significance of genomic amplification of the telomerase RNA component (TERC) gene to serve as a genetic biomarker in the screening of cervicallesions.Methods A total of 715 cases were recruited,with liquid-based cytology diagnosis as normal (n=347),atypical squamous cells of undetermined significance (ASCUS,n=180),atypical squamous cells cannot exclude a high-grade lesion (ASC-H,n=13),low-grade squamous intraepithelial lesions (LSIL,n=115),high-grade squamous intraepithelial lesions(HSIL,n=59)and atypical glandular cells(AGC,n=1).The remaining cervical cells in the cytological preserving fluid were analyzed using a two-color fluorescence in situ hybridization (FISH) probe targeted to chromosome 3q26 containing TERC gene.The TERC gene findings were compared to the cytological and histological detected results,as well as high-risk human papillomavirus (HPV) detected results.Results Genomic amplification of TERC gene was found in 5.8% of normal specimens,22.2% of ASCUS.30.8% of ASC-H,27.8% of LSIL,86.4% of HSIL and 1/1 of AGC.The positive rate was significantly lower in normal,ASCUS,ASC-H and ISIL.compared with HSIL(all P<0.01).Significantly more cells with genomic amplification of TERC gene were found in cervical intraepithelial lesion(CIN) Ⅱ-Ⅲ than CIN Ⅰ (77.8% vs.9.3%),as well as invasive cervical cancer (96.7% vs.9.3%).both P < 0.01.The rate of TERC gene amplification was higher in HPV positive patients (33.5%) than in HPV negative patients(5.2%,P<0.01).The sensitivity of TERC gene amplification was significantly higher than that of cytological screening (81.88% vs.36.96%,P<0.01) in the differentiation of CIN Ⅱ or higher and CIN Ⅰ or lower diseases,its specificity Was hisher than high-risk HPV test (93.32% vs.33.93%,P<0.01) and positive prediction value (81.29%) was similar with cytological method (86.44%,P>0.05);but its negative prediction value (93.56%) was lower than HPV test (97.06%,P<0.05).Conclusions The positive rates of TERC gene amplification increased as cervical diseases worsened.TERC gene amplification is related to HPV infection.The gain of chromosome 3q26 in cytological specimens is an effective molecular genetic biomarker in screening of CIN Ⅱ or higher and invasive cervical cancer.

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Regulation of synthesis function of porcine ovarian granulose cells by mitogen-activated protein kinase signaling pathway

Xuemin QIU ; Yang LIU ; Jing CONG ; Xiaoke WU

Chinese Journal of Obstetrics and Gynecology.2009;44(12):933-935. doi:10.3760/cma.j.issn.0529-567x.2009.12.012

Objective To investigate the role of mitogen-activated protein kinase(MAPK)signaling pathway regulating synthesis function of steroid hormone of porcine ovarian granulose cells.Methods Porcine ovarian granulose cells were cultured in medium added with dimethyl sulfoxide(DMSO)at concentration of 50μmol/L or with MAPK inhibitor PD98059 (PD) for 48 hours.Then,granulose cells in DMSO medium were added with activator of adenylate cyclase(20 μmoL/L)or blank agent for next 48 hours incubation,which were defined as observation group 1 and control group 1,similarly,granulose cells in PD medium were also added with activator of adenylate cyclase(20 μmol/L)or blank agent for next 48 hours,which were defined as observation group 2 and control group 2.The level of estradiol(E_2),progesterone (P)and testosterone (T) were detected by chemoluminescence.The mRNA expression of 17alpha-hydroxylase/17,20-lyase (CYP17) and aromatase P450 (P450 arom) were assessed by RT-PCR.Results (1)Hormone expression:the level of T,P and E_2 were(29.5±2.5)nmol/L,(80±5)nmol/L,(49±4) pmol/L in control group 1 and(42.3±3.4)nmol/L,(170±15)nmol/L,(75±6)pmol/L in control group 2,which showed significant difference(P<0.05).In the mean time,the level of T, P and E_2 were (106.2±7.6)nmol/L,(210±16)nmol/L,(130±11)pmol/L in observation group 2 and(47.2±3.5)nmol/L,(130±6)nmol/L,(81±6)pmol/L in observation group 1,which also reach statistical difference(P<0.05).(2)The expression of enzyme:the expression of CYP17 mRNA was inereased by 50% and P450 arom mRNA was decreased by 20% between control group 1 and 2. However, the mRNA expression of P450 arom and CYPI7 were upregulated remarkably, especially, the expression of CYP17 mRNA were increased by 125%. Conclusion MAPK signaling pathway plays an inhibitory role in regulating synthesis of steroid hormone of ovarian granulose cell.

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Expression of estrogen receptor, progesterone receptor and integrin αVβ3 in endometrium of patients with luteinized unruptured follicle

Lei JIANG ; Wangming XU ; Jing YANG ; Qingzhen XIE ; Liangfei AO

Chinese Journal of Obstetrics and Gynecology.2009;44(12):929-932. doi:10.3760/cma.j.issn.0529-567x.2009.12.011

Objective To investigate endometrium receptivity in patients with luteinized unruptured follicle(IUF)by measuring the expression of estrogen receptor(ER),progesterone receptor(PR)and integrin αVβ3 in the endometriunm Methods From May 2007 to Nov.2007,17 infertile women with LUF were selected as LUF group matched with 13 infertile cases with normal ovulation as control group.They all underwent frozen-thawed embryo transfer in Reproductive Medicine Center.Renmin Hospital of Wuhan University.Endometrial tissue in anterior and posterior wall of uterus of LUF group and control group were biopsied by a small curettage between 7 and 11 days after luteinizing hormone(LH)surge.The expression of ER,PR and integrin αVβ3 in endometriam were detected by immunohistochemistry staining.The level of estrogen and progesterone were measured by chemiluminescence assay.Then,the relationship between αVβ3 expression in endometrium and the level of estrogen/progesterone were analyzed in LUF patients.Results (1)There was no remarkable difference in the level of estrogen between LUF [(656±299)pmol/L]and control group[(727±275)pmol/L,P>0.05].However,the level of progesterone were(23±8)nmol/Lin LUF group and(35±10)nmol/L in control group,which reached statistical difference(P<0.01).(2) The expression of ER,PR in endometrium of LUF patients were 183.9±2.4 and 168±3.which were significantly higher than 109.4±6.3 and 106±4 in control group(P<0.01).The expression of integrin α Vβ3 in endometrium of 115±11 in LUF group were significantly lower than 191±9 in control group(P< 0.01).(4)In LUF group,the expression of αVβ3 in endometrium waft correlated positively with the level of progesterone(r=0.77,P<0.01)and irrelevant with the level of estrogen(r=0.01,P>0.05).Conclusion The higher expression of estrogen and progesterone and lower expression of integrin αVβ3 misht confer impaired receptivity of endometrium and interfere with embryo implantation.

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