INHIBITORY EFFECTS OF MIFEPRISTONE ON THE GROWTH OF HUMAN GASTRIC CANCER CELL LINE MKN-45 IN VITRO AND IN VIVO

Objective To explore the effects of mifepristone on the growth of human gastric cancer cell line MKN-45 and its possible mechanisms. Methods In situ hybridization was used to detect the expression of progesterone receptor (PR) mRNA in MKN-45 cells. Proliferation, cell cycle distribution, and the expression of Bcl-xL and vascular endothelial growth factor (VEGF) of MKN-45 cells incubated with various concentrations of mifepristone (1, 5, 10, and 20 μmol/L) were analyzed using MTT reduction assay, flow cytometry, reverse transcription-polymerase chain reaction (RT-PCR), and enzyme-linked immunoab-sorbent assay (ELISA), respectively. After transplantation of MKN-45 cells underneath the skin of athymic mice, mifepristone was administrated with the dose of 50 mg/(kg·d) for 6 weeks to evaluate the tumor growth. Apoptosis and the expression of proliferating cell nuclear antigen (PCNA) in xenografted tumors were detected using transmission electron microscopy and immunohistochemical staining, respectively. Results PR mRNA was highly expressed in cultured MKN-45 cell. Mifepristone dose-dependently inhibited the pr-oliferation of MKN-45 cells, and the inhibitory rate was dramatically increased from 7.21% to 47.23%. The inhibitory effect was accompanied by a dose-dependent increase in the percentage of cells in G 0 /G 1 phase, and with a concurrent decrease in the proportion of S- and G 2 /M-phase cells and the proliferative index from 57.65% to 24.54%. Meanwhile, mifepristone down-regulated the expression of Bcl-xL and VEGF in a dose-dependent manner. In vivo, mifepristone effectively inhibited the growth of xenografted tumors in nude mice (55.14% for inhibitory rate), induced apoptosis, and down-regulated PCNA expression in gastric cancer. Conclusion Mifepristone exerts significant growth inhibitory effects on PR-positive human MKN-45 gastric cancer cells via multiple mechanisms, and may be a beneficial agent against the tumor.

Fertilization of in vitro matured human oocytes by intracytoplasmic sperm injection (ICSI) using ejaculated and testicular spermatozoa

Aim: To evaluate the fertilization competence of spermatozoa from ejaculates and testicle when the oocytes were matured in vitro following intracytoplasmic sperm injection (ICSI). Methods: Fifty-six completed cycles in 46 women with polycystic ovarian syndrome were grouped according to the semen parameters of their male partners. Group 1 was 47 cycles that presented motile and normal morphology spermatozoa in ejaculates and Group 2 was the other nine cycles where male partners were diagnosed as obstructive azoospermia and spermatozoa could only be found in testicular tissue fragment. All female patients received minimal stimulation with gonadotropin. Immature oocytes were matured in vitro and inseminated by ICSI. The spermatozoa from testes were retrieved by testicular fine needle aspiration. Results: A total of 449 and 78 immature oocytes were collected and cultured for 48 hours, 75.5 % (339/449) and 84.6 % (66/78) oocytes were matured in Groups 1 and 2, respectively. The percentage of oocytes achieving normal fertilization was significantly higher in Group 1 than that in Group 2 (72.9 % vs. 54.5 %, P < 0.05). There were no significant differences in the rates of oocytes cleavage and clinical pregnancies in these two groups [87.4 % (216/247) vs. 88.9 % (32/36); 21.3 % (10/47) vs. 44.4 % (4/9)]. A total of 15 babies in the two groups were healthy delivered at term. Conclusion: It appears that IVM combined with ICSI using testicular spermatozoa can produce healthy infants, while the normal fertilization rate of in vitro matured oocytes after ICSI using testicular spermatozoa was significantly lower than using the ejaculated spermatozoa.

Formation of Nucleolar Channel System in Human Endometrium in vitro is Independent of Progesterone

Objective To determine whether formation of the nucleolar channel system (NCS) in human endometrium depends on the presence of progesteronal steroids. Materials & Methods Tissues of late proliferative endometrium were obtained from 5 normally cycling women of reproductive age. Half of each tissue was cultured in the DMEM medium containing diethylstilbesterol (25 μg/mL) plus medroxyprogesterone acetate (25 μg/mL) (E + P culture). As a control, the other half was cultured in the medium alone. After 100 h incubation, the tissues were assessed for the formation of NCS with transmission electron microscope.Results NCS was observed in the endometrial epithelium treated with E + P or the medium alone. Moreover, giant mitochondria and glycogen accumulation were both seen in epithelia derived from both types of cultures.Conclusion Progesterone would be not indispensable for the formation of NCS in human endometrium. Transition of proliferative endometrium to the secretory stage in vitro could occur even in the absence of both estrogen and progesterone.

IMPROVING P-gp EXPRESSION IN HUMAN MONONUCLEAR CELLS IN VITRO TRANSFECTED BY MULTIDRUG RESISTANCE-1 mRNA

Objective To evaluate the expression and function activity of P-glycoprotein (P-gp) in human mononuclear cells (MNCs) in vitro transfected by multidrug resistance-1(MDR1) mRNA. Methods Two MDR1 cDNA vectors, pT7TS_MDR1 and pGEM5Zf(+)_MDR1, were constructed and transcripted in vitro. Vector pGEM5Zf(+)_MDR1 only contained the coding region of mdr1 cDNA, and pT7TS_MDR1 also included Xeponus β-globin 5’ and 3’ untranslated region. MNCs were prepared from peripheral blood of parvicellular lung cancer patient. The two human mdr1 mRNAs were then transferred into human MNCs in vitro by DOTAP. And the expression efficiency and pump function of P-gp were measured with flow cytometry. Results Expression of P-gp significantly elevated in both transferred cells compared with untransferred cells (P < 0.01). And pT7TS_MDR1 showed higher capability in elevating the expression of P-gp than pGEM5Zf(+)_MDR1 (P < 0.01). The P-gp function was elevated in both pT7TS_MDR1 and pGEM5Zf(+)_MDR1 groups. The survival ratio of MNCs in erythrocyte-lysis-solution (ELS, 86.07%) and lymphocyte-isolation-solution (LIS, 83.67%) had no significant difference. The CD34+ cells content of the MNCs used for transfection was 2.65% and 1.01% in ELS and LIS group, respectively (P < 0.01).Conclusions It is a feasible approach to improve P-gp expression in human MNCs by transfection of MDR-1 mRNA. And the ELS may be more suitable for purifing MNCs for mRNA transfection than LIS.

POSTOPERATIVE EVALUATION OF TENSION-FREE VAGINAL TAPE PROCEDURE

Objective To study the outcome of tension-free vaginal tape (TVT) for the treatment of stress urinary incontinence (SUI) in women with cystocele. Methods Forty-two patients with SUI confirmed by urodynamics underwent the TVT procedure under local anesthesia. A prolapse repair was done simultaneously. Results Mean TVT operation time was 26.29 minutes. Mean blood loss was 29.86 mL. Eighty-eight percent of the patients were able to micturate spontaneously within 12 hours and residual urine was less than 100 mL. And 12% of the patients had to use indwelling catheter for 3-11 days. Average hospital stay was 2.91 days. Eighty-eight percent of patients were discharged within 2 days. All patients were followed up (an average of 10.26 months). According to subjective and objective assessment of the outcome, 39 patients (93%) were cured, another 3 patients (7%) were significantly improved and none was failed. There were no major complications such as bladder injury occurred. Conclusion TVT procedure is a minimal invasive, effective, and safe surgery for treatment of SUI.

Distribution of CD 4^+ Lym phocyte Th1/Th2 Subsets in Peripheral Blood of Women with Idiopathic Prem ature Ovarian Failure

Thepathogenesisof prem ature ovarian failure (POF) rem ains unknow n. Accu- m ulated evidences indicate that abnorm ality in the im m une system m ay be one of the m ajor reasonsand theim balanceof Thelp typeI(Th1) and Thelp typeII(Th2) m ay play an importantrolein theprocess. Study of thedistribution and functionalstatusof Th1/Th2 m ay behelpfulforevaluating thepathogenesisof POF.13 patientsw ith id- iopathic POFand 7 w om en of reproductiveagew ith norm alcyclew ereenrolled in this study.The percentage of Th1 and Th2 cellsfrom peripheralblood of thepatientsand the controls w as studied using FITC labelled CD4 m Ab to separate CD4+ cells by FACS, in com bination w ith in situ hybridization using Dig-labelled IL2, IL6 cDNA probes.Results:The patients w ith POF had significantly higher percentage of Th1 cellsascompared w ith the controls(49.76±9.22,20.06±7.10respectively, P< 0.001).The ratio of Th1/Th2 in thepatientsw ith POFw assignificantly higherthan thatof thecontrols(1.15±0.17, 0.63±0.09, P< 0.001). Conclusions: Thefindings of thisstudy suggestthatpatientsw ith POFhaveincreased Th1 cellactivation, w hich m ay be related to the pathogenesisof POF. Corresponding author: WANG Yi-li E-m ail: w yl@ irix. xam u. edu. cn