Objective To study anti-tumor effects on human ovarian cancer xenografted tumors in mice by constructing adenoviral expression vector containing canstatin gene and hTERT gene core promoter (AdhTERT-Can). Methods AdhTERT-Can vector was constructed and identified by means of enzyme cutting, electrophoresis and sequencing. Then, transfected into HO8910PM cells by means of lipofectamine and confirmed by green fluorescence protein(GFP) expression under laser confocus microscope. The mRNA expression of canstatin gene was tested by RT-PCR. Human ovarian cancer xenografted tumor models in nude mice were established and randomly divided into AdhTERT-Can group received viral supematant solution of AdhTERT-Can by tail vein injection, Ad-Can groups received viral supernatant solution of Ad-Can by tail vein injection, and control groups received phosphate buffer solution (PBS). The volume of tumors were measured and compared in each group to evaluated the anti-tumor efficacy. Results All the constructed vectors of AdhTERT-Can were verified by enzymed digestion. There were green fluorescence from 60% of HO8910PM cells transfected by AdhTERT-Can under laser confocus microscope, and the mRNA expression of canstafin gene in HO8910PM cells were also verified by RT-PCR The growth of tumor in AdhTERT-Can group was significantly inhibited compared with those in Ad-Can groups and control groups from the 8th day (P <0.01 ). However, there were not significant difference between Ad-Can group and PBS group (P> 0.05). On the 30th day, the tumors showed liquefaction necrosis and cystic degeneration in each group, especially in AdhTERT-Can group. Conclusions The recombinant adenovirus vector of AdhTERT-Can has been constructed successfully and could steady express in ovarian cancer cell lines HO8910PM. The results shown that it could inhibit significantly the growth of human ovarian cancer xenografted tumors in mice and shown to be the target of gene therapy.

Objective To study telomerase activity in cervical cancer and it′s precursor lesion Methods Thirty six cervical cancer and 16 cervical intraepithelial neoplasia (CIN) specimens were measured for telomerase activity using TRAP ELISA, and 11 normal cervix, 6 chronic cervicitis and 8 adjacent normal tissue specimens as controls Results Mean telomerase activity in CIN, cervical cancer, and controls were 0 398?0 293, 1 580?0 819, 0 050?0 012 There was statistically significant difference among three groups ( P

Objective To observe the effect of DNA methyltransferase 1 (DNMT1 ) gene silencing by RNA interfering technology on the proliferation and apoptosis of HeLa cells. Methods Recombinant plasmid pshRNA-DNMT1-A, B and C were respectively transfected into HeLa cells by lipofectamine 2000, while cells transfected plasmid vector pSilencer3.1-HI and cells untreated as control groups. RT-PCR was adopted to select the recombinant plasmid which showed the most optimal inhibition effect. RT-PCR and western blotting was used to detected the mRNA and protein expression of DNMT1 in HeLa cells transfected for 24, 48 and 72 hours. Cell counting kit-8 (CCK-8 ) assay was used to investigate the proliferation of the HeLa cells after transfection, while apoptosis was detected by flowcytometry(FCM ) method. Results Three DNMT1-targeted short hairpin RNA (shRNA) A,B and C were successfully inserted into the plasmid vector PShRNA, and the coding sequences of the obtained shRNA were consistent with the designed fragments. The results indicated that both recombinant plasmid pshRNA-DNMT1-A and B could effectively knock down the expression of DNMT1 gene in human cervical cancer cells, of which pshRNA-DNMTI-B was the better choice. While no effect of pshRNA-DNMTI-C was seen. BT-PCR results showed that the relative mRNA expression of DNMT1 gene in Helm cells transfected with pshRNA-DNMT1 for 24, 48 and 72 hours were 0.406±0.057,0.191±0.036 and 0.104±0.015, which were significantly lower than that in Helm cells transfected by empty vector and non-transfected cells (0.520±0.020, 0.537±0.041, respectively, P ＜ 0.05 ). The western blotting analysis manifested that the relative expression of DNMT1 protein of Helm cells transfected by pshRNA-DNMT1 for 24, 48 and 72 hours were 0.197±0.024, 0.075±0.015, 0.040± 0.013, which were significantly lower than that in transfected cells by empty vector and non-transfected cells (0.273±0.010, 0.283±0.016, respectively, P ＜0.05). The CCK-8 results showed that the cell survival rates of HeLa cells transfected by pshRNA-DNMT1 for 24, 48, 72, 96 and 120 hours were 70.8%, 64.8%, 51.6%, 45.3% and 38.0%, there were statistically different compared with cells transfected by empty vector and non-transfected cells at different time-points (P ＜ 0.01 ). The results of FCM indicated that the apoptesis rate of HeLa cells trandected with pshRNA-DNMTI for 24, 48 and 72 hours were (17.7± 1.3 ) %, (35.3±1.3 ) %, (47.6±1.6 ) %, which were significantly higher than empty vector transfected cells and non-transfected cells [(4.9±0.5 ) %, (5.1±0.7 ) %, respectively, P ＜ 0.05]. Conclusions DNMT1 can be successfully silenced by RNA interfering in cervical Helm cells. Downregulation of DNMT1 can inhibit cervical cancer cells proliferation and induce cell apoptosis.

ObjectiveTo investigate the expression of G protein-coupled ER (GPER) and ER in the activation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) induced by 17β-estradiol (17β-E2 )in endometrial carcinoma cells,Ishikawa and HEC-1A.Methods Expressions of GPER,Erα and Erβ protein in Ishikawa and HEC-1A cells were detected by immunohistochemical SP method.Levels of GPER,Erα and Erβ were examined by western blot in Ishikawa and HEC-1A cells after treated with 1 ×10-6 mol/L 17β-E2 at different time (0,15,30,60,120 minutes).ResultsGPER was positive expressed in Ishikawa and HEC-1A cells.Erα and Erβ were both positive expressed in Ishikawa cells.While,Erα was weakly expressed and Erβ was almost negatively expressed in HEC-1A cells.Western blot analysis showed that 1× 10-6 mol/L 17β-E2 treatment,the Ishikawa and HEC-1A cells GPER protein level for 15 minutes markedly increased (P ＜ 0.05 ),which Ishikawa 30 minutes,when cells reached the highest level (0.192 ± 0.004),HEC-1A cells for 15 minutes and reached the highest level (0.184 ±0.006) ; Ishikawa and HEC-1A cells,Akt,activation of 15 minutes from the treatment start was significantly increased (P ＜ 0.05 ),which Ishikawa cells for 30 minutes and reached the highest level (0.666 ± 0.021 ),HEC-1A cells for 15 minutes and reached maximum (0.788 ± 0.035); Ishikawa and HEC-1 A cells,Erα and Erβ protein expression did not change significantly ( P ＞ 0.05 ).Conclusion GPER likely involved in non-nuclear activation of PI3K/Akt signaling pathways in endometrial carcinoma cells,Ishikawa and HEC-1A.

Objective To investigate the effects of signal pathway inhibitors PD98059 and LY294002 on cell proliferation, apoptosis, expressions of phosphorylated extracellular signal-regulared kinase (p-ERK) and phosphorylated protein kinase B ( p-Akt) in endometrial carcinoma xenografts. Methods Human endometrial carcinoma Ishikawa cells were cultured in vitro. The effects of PD98059 and LY294002 on proliferation, apoptosis, and cell cycle distribution of endometrial cancer cells were detected by monotetrazolium ( MTT) assay and fluorescence-activated cell sorting technique. The models of xenografted tumor were established by the subcutaneous inoculation in 24 nude mice, and then they were randomly divided into 4 groups ( n = 6) , normal saline group, PD98059 group (PD group) , LY294002 group ( LY group) or PD98059 + LY294002 group ( PD + LY group) by intraperitoneal injections, respectively. The anti-tumor efficacy was evaluated by measuring tumor volume and tumor growth status. The histopathological change of tumor specimens was observed using HE staining and terminal deoxynucleotidyl transferasemediated dUTP-digoxigen in nick and labeling method (TUNEL) testing and the expression levels of p-ERK and p-Akt were detected by immunohistochemistry method. Results ( 1) The proliferation of Ishikawa cells were suppressed after treated by PD98059 and ( or) Y294002, in which A570 values of cells decreased showing both time-dependent and concentration-dependent manner ( LY294002: Fgroup = 9. 801, P = 0. 002; Ftime = 10. 398, P = 0. 001. PD98059: Fgroup= 8. 213, P = 0. 015; Ftime = 6. 839, P = 0. 036). Cell cycle distribution analysis revealed that percentage of Ishikawa cells at G0/G1 phase(Ftime =35.049, P= 0.004; Fgroup = 32. 024, P ＜0. 01) increased and percentage of S phase cells (Ftime = 7. 789, P = 0. 049; Fgroup = 30. 132, P ＜0. 01) decreased significantly. The percentage of apoptotic cells increased significantly among PD group, LY group and PD + LY group, in which there were significant difference [(63. 3 ±0.5)% vs (30. 7 ± 20. 1) % vs(40. 8 ± 1. 3) % ; F = 621. 059, P ＜ 0. 01]. (2) Compared with the control group, the increasing of transplanting tumor volume in the treated groups were obviously ( F = 23. 545 , P ＜ 0. 01) , and the inhibited rate of the tumor was higher in PD + LY group than that in PD group or LY group [(68 ± 9 ) % vs ( 32 ± 16 ) % or ( 38 ± 17 ) % ; F = 10. 283 , P ＜ 0. 05]. ( 3 ) HE staining shown that there were different degrees of necrosis for endometrial carcinoma cell in different groups. The apoptosis of tumor cells were significantly increased in treated groups by TUNEL testing [(13. 7 ± 1. 5)% , ( 14. 1 ± 1. 2)% , (29. 0 ± 1. 8 ) % ; F = 320. 344, P ＜ 0. 01]. Immunohistochemistry results demonstrated that the expressions of p-ERK and p-Akt in treated groups were lower than that in control group, of which LY + PD group was the lowest one. Conclusion The signal pathway inhibitors PD98059 and LY294002 could inhibit the growth of human endometrial carcinoma in vivo and in vitro, in which may induce cell apoptosis.

Objective To study the effect of estradiol supplementation during the luteal phase on mouse endometrial expression of leukaemia inhibitory factor and pinopodes in controlled ovarian stimulation cycles.Methods Female mice were randomly divided into four groups:group A[controlled ovarian stimulation(COS)group],group B(COS group with progesterone for luteal-phase-support),group C(COS group with progesterone and estradiol for luteal-phase-support),and group D of natural cycle group.Pinopodes were investigated by scanning electronic microscopy(SEM)in the uterine endometrium of pregnant mice on pregnancy days(pa)3-5.LEukaemia inhibitory factor(LIF)protein Was determined by immunohistochemistry in the uterine endometrium of pregnant mice on pd 3-5.Results (1)In groups B,C,and D,there were small developed pinopodes in the endometrial surface of pregnant mouse on day 3;there were large fully developed pinopodes in endometrial surface,which Was smooth with well defined borders resembling a mushroom on day 4.The regressing pinopodes were observed on day 5.In group A,there were small developed pinopodes in endometrial surface of pregnant mouse on day 3.The regressing pinopodes were seen on day 4.(2)In the pregnant mice of groups C and D,the level of LIF protein on days 3-5 ( 138.5±20. 3,143.1±19. 0) was significantly higher than group A ( 103. 2 ± 5.0, P < 0. 05 ), and strong immunostaining of LIF protein was found on day 4 of gestation. In group B, the level of LIF protein on days 3-5 ( 123.5±10. 8)was significantly higher than group A (P <0. 05), but significantly lower than groups C and D ( P <0. 05 ). Strong immunostaining of LIF protein was found on day 4 of gestation. In group A, weak immunostaining of LIF protein peaked on day 3 of gestation. In groups B, C, and D, the level of LIF protein on day 4 was significantly higher than group A on day 3 ( F = 55.76, P < 0. 01 ). Conclusions Estradiol supplementation during the luteal phase can improve the expression of LIF and pinopodes in mouse endometrium in controlled ovarian stimulation cycles and redress the harmful effect on implantation window by COS. Therefore, estradiol supplementation can improve the endometrial receptivity.

Objective To investigate the influence of pertussis toxin(PTX)on G protein-coupled estrogen receptor(GPER)-mediated activation of phosphatidylinositol 3-kinase(PI3K)/protein kinase B (Akt)signaling activated by 17 β-estradiol(17β-E2)in endometrial carcinoma cells.Methods Expressions of GPER protein were detected by immunohistochemical SP method in Ishikawa and HEC-1A cells.Changes of levels of GPER,ERα and ERβ protein and the activation of Akt protein were observed by western blot in the two cells after they were treated by PTX for 30 minutes at different concentrations(0,0.1,0.5,1.0 μg/ml),and then co-stimulated with with 1 × 10-6 mol/L 17β-E2 respectively at different time (Ishikawa 30 minutes,HEC-1A 15 minutes).Results(1)Immunohistochemical SP method showed that GPER was positive stained in cell cytoplasm of Ishikawa and HEC-1A cell.(2)After co-treated with PTX at different concentrations(0,O.1,0.5,1.0 μg/ml)and 10-6 mol/L 17β-E2,in Ishikawa cell,the ratio of pAkt/Akt was 0.74 ±0.54,0.34 ±0.06,0.18 ±0.03,0.07 ±0.15,the gray values of GPER was 0.872 ± 0.490,0.395 ± 0.054,0.145 ± 0.014,0.034 ± 0.008,and with increasing concentration of PTX,the ratio of p-Akt/Akt and the expression of GPER decreased gradually(P ＜ 0.05),which was most obviously when the concentration was 1.0 μg/ml(F =63.729,P =0.0001;F =160.284,P =0.0001);ERα and ERβ protein had no significant change among different groups(P ＞0.05).In HEC-1A cell,the ratio of pAkt/Akt was 0.73 ±0.09,0.26 ±0.14,0.11 ±0.03,0,the Gray values of GPER is 0.927 ±0.134,0.485 ± 0.022,0.194 ± 0.004,0,and with increasing concentration of PTX,the ratio of p-Akt/Akt and the expression of GPER decreased gradually(P ＜ 0.05),which were also completely inhibited when the concentration was 1 μg/ml(F =1039.321,P =0.0001;F =109.646,P =0.0001),ERα protein had no significant differences(P ＞ 0.05)among different groups.ERβ was negatively expressed.Conclusion The results proposed that the activation of PI3K/Akt signaling in Ishikawa and HEC-1A cells could be inhibited after blocking the role of GPER by PTX.

Objectives To investigate the effects of plasma from the patients with preeclampsia on proliferation and apoptosis of human umbilical vein endothelial cells(HUVEC),and to explore the relationship between cell damage and lysophosphatidic acid(LPA)receptors.Methods Sixty patients with preeclampsia were recruited from October 2011 to June 2012 in the First Affilated Hospital of Zhengzhou University.Among them,thirty cases were defined as the mild preeclampsia group and thirty cases were defined as the severe preeclampsia group.The other thirty healthy pregnant women were recruited in the healthy pregnant women group.The levels of plasma LPA in the three groups were measured.The HUVEC were cultured in vitro with plasma from the three groups,and a blank control group was set up as well.Proliferation and apoptosis of HUVEC were measured by MTT assay and flow cytometry.Immunohistochemistry of biotin streptomyces protein peroxidase(SP)method was used to measure the protein expression level of Edg 2,4,7.Results(1)The plasma LPA levels in the healthy pregnant woman group,mild preeclampsia group and severe preeclampsia group were(3.38 ± 2.08)μmol/L,(6.12 ± 0.22)μmol/L,(9.10 ± 0.17)μmol/L,respectively.The plasma levels of LPA in patients with preeclampsia were significantly higher than that in the healthy pregnant women(P ＜ 0.01).(2)The proliferation rate of HUVEC in the mild and severe preeclampsia groups [(65.2 ± 2.7)％ and(51.9 ± 2.8)％] were significantly lower than that in the healthy pregnant women group and the control group [(84.3 ± 3.1)％ and(100.0 ± 0.0)％,P ＜ 0.01].(3)The early apoptosis rate,middle-late apoptosis rate and total apoptosis rate of HUVEC in the mild and severe preeclampsia groups [total apoptosis rate were(30.4 ±2.0)％ and(43.4 ±2.5)％] were significantly higher than those in the healthy pregnant women group and the control group [total apoptosis rate were(18.6 ± 1.6)％ and(8.0 ± 1.5)％,P ＜ 0.01].(4)The expression positive rates of Edg 2,4,7 proteins in the four groups were as following:mild preeclampsia group 83％,80％ and 73％;severe preeclampsia group 97％,93％ and 90％;healthy pregnant women group 40％,40％ and 37％,and the control group 10％,10％ and 7％ respectively.The positive rates of HUVEC in the mild and severe preeclampsia groups were significantly higher than those in the healthy pregnant women group and the control group(P ＜ 0.01).Conclusions The plasma of patients with preeclampsia could inhibit proliferation and promote apoptosis of HUVEC,and induce the expression of Edg 2,4,7 proteins.It suggested that the increase of lysophosphatidic acid in plasma could be one of the reasons of endothelial cell damage in patients with preeclampsia.

BMP6 is a potent protein for future treatment strategies of bone regeneration as it is a very important regulator of bone homeostasis. Active BMP6 is a dimer containing multidisulfide bonds and is a highly hydrophobic protein prone to aggregation. To obtain soluble and active BMP6 in Escherichia coli, we compared the effects of four N-terminal fusion tags (TRX, GST, MBP and CBD) and N-terminal His6-tag. The expression and solubility were tested under the different conditions (expression hosts, temperatures and inductor concentrations). A series of experiments leads to the finding that the placement of MBP before the BMP6 is best in availing the soluble expression of the protein. Our study alsodemonstrates that in E. coli BL21trxB(DE3) cytoplasm, which is a thioredoxin reductase mutant strain, soluble homodimeric BMP6 can be formed. The overexpressed MBP-BMP6 fusion protein is purified by chromatography, and shown to be functionally active.
Bone Morphogenetic Protein 6 ; biosynthesis ; genetics ; Carrier Proteins ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Humans ; Maltose-Binding Proteins ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; metabolism ; Solubility ; Transformation, Bacterial