The two-dimensional model of cell culture is an important method in the study of testicular development and spermatogenesis but can not effectively mimic and regulate the testicular microenvironment and the whole process of spermatogenesis due to the lack of relevant cell factors and the disruption of a three-dimensional spatial structure. In the past 20 years, the development and optimization of the in vitro model such as testis organotypic culture and in vivo model such as testis transplantation achieved a transformation from two- to three-dimension. The maintenance and optimization of the testicular niche structure could mimic the testicular microenvironment and cell types including Leydig, Sertoli and germ cells, which showed similar biological behaviors to those in vivo. Besides, the cell suspension or tissue fragment floats in the gas-liquid interface so that the development of somatic and germ cells is well maintained in vitro whilst the feedback linkage between grafted testis tissue and hypothalamus-pituitary of the host rebuilt in the in vitro model provides an endocrinological basis for spermatogenesis, which serves as an effective methodology to better understand the organogenesis and development of the testis as well as testicular function regulation, advancing the concept of treatment of male infertility. Al- though each of the methods may have its limitations, the progress in the processing, freezing, thawing, and transplantation of cells and tissues will surely promote their clinical application and present their value in translational medicine.
Cell Culture Techniques ; Germ Cells ; physiology ; Humans ; Infertility, Male ; therapy ; Male ; Spermatogenesis ; physiology ; Testis ; growth & development

With the active encouragement of the Chinese government, all domestic clinical research institutes pay more attention to the human research protect program (HRPP) during the process of clinical trials, and actively follow the regulations of medical ethical practice. We could make fully preparation for the accreditation by the correlated international organizations only by further analyzing the Association for Accreditation of Human Research Protection Program (AAHRPP) from a whole and in each accreditation field at different levels, thus having a clear understanding the difference in acknowledging the difference between China's hospitals and America's hospitals.
Accreditation ; China ; Clinical Trials as Topic ; legislation & jurisprudence ; Humans ; Public Policy ; United States

OBJECTIVETo study the effect of CEA gene regulation on the anti-tumor activity of oncolytic adenovirus H101 to esophageal carcinoma, and to explore the intrinsic factors influencing H101 sensitivity.

METHODSStable human esophageal cancer cell line EC9706 cells with lower (EC9706-SCEA) and higher CEA expression (EC9706-CEA) were chosen, thawed and cultured, and then to analyse the influence of CEA expressed at different levels on cell growth. The cytotoxic effect of H101 was assayed by in vitro and nude mouse in vivo.

RESULTSThe cell growth experiment showed that the population doubling time of EC9706-SCEA, EC9706-CEA and EC9706 cells were (30.9 ± 2.0) h, (31.1 ± 2.5) h and (29.1 ± 2.6) h, respectively, showing no significant difference among them (P > 0.05). The cytotoxic activity of H101 was higher on EC9706-SCEA than on other four groups, when MOI was ≥ 0.01 PFU (P < 0.05). The mouse experiment showed that H101 inhibited the growth of transplanted tumors in all experimental groups. Its effect on CEA-silenced tumors (inhibition rate was 61.5% to 74.5%) was significantly higher than that on CEA-overexpression tumors (32.3% to 38.5%) and control EC9706 transplanted tumors (35.5% to 44.8%). There was a significant difference between them (P < 0.05).

CONCLUSIONSThe results in vitro and in vivo experiments show that H101 can enhance the cytotoxic effect on EC9706 cells with lower CEA expression. To silence the expression of CEA may provide a novel strategy for target gene therapy of esophageal carcinoma.

Adenoviridae ; physiology ; Animals ; Carcinoembryonic Antigen ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Esophageal Neoplasms ; metabolism ; pathology ; therapy ; Female ; Gene Silencing ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Oncolytic Virotherapy ; Oncolytic Viruses ; physiology ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Tumor Burden

OBJECTIVETo investigate the intra-vaginal ejaculation latency time (IELT) of varicocele patients, the influence of spermatic vein ligation on IELT, and the relationship of Visual Analogue Score (VAS) with IELT.

METHODSWe selected 112 males who had regular sexual life after spermatic vein ligation and conducted follow-up visits for 6 months. According to preoperative IELT, we divided the patients into an IELT < or = 2 min group and an IELT > 2 min group, and compared their IELT, VAS and Chinese Index of Sexual Function for Premature Ejaculation-5 (CIPE-5) scores before and 6 months after operation.

RESULTSFollow-up was accomplished in 81 of the patients, 18 in the IELT < or = 2 min group and 63 in the IELT >2 min group. Compared with the baseline, IELT was significantly prolonged postoperatively in both the IELT < or = 2 min group ([1.26 +/- 0.37] vs [4.53 +/- 1.69] min, P < 0.01) and the IELT >2 min group ([5.14 +/- 2.03] vs [7.69 +/- 4.51] min, P < 0.05); the postoperative CIPE-5 scores were remarkably improved in the former (11.27 +/- 3.52 vs 15.64 +/- 2.37, P < 0.05) but insignificantly in the latter group (20.42 +/- 4.65 vs 21.83 +/- 5.49, P > 0.05); the postoperative grades of the CIPE-5 scores showed significant differences in both groups (chi2 = 6.353, P = 0.042 and chi2 = 3.910, P = 0.048); the postoperative VAS was markedly increased (3.18 +/- 0.92 vs 1.56 +/- 0.83 and 3.24 +/- 0.95 vs 1.74 +/- 0.79, P < 0.05), with significant differences in the grades of VAS in both groups (chi2 = 4.433, P = 0.035 and chi2 = 10.088, P = 0.001). The variation of VAS was negatively correlated with that of IELT in both groups (r = -0.572, P < 0.01 and r = -0.465, P < 0.05).

CONCLUSIONVaricocele may be one of the causes of premature ejaculation, and some of the varicocele patients with IELT < or = 2 min may benefit from spermatic vein ligation. Improved VAS is negatively correlated with prolonged IELT. The relationship between varicocele and premature ejaculation deserves further studies.

Adult ; Ejaculation ; physiology ; Follow-Up Studies ; Humans ; Ligation ; Male ; Retrospective Studies ; Varicocele ; surgery ; Young Adult

Objective To optimize the supercritical carbon dioxide(SC-CO2)extraction of lipids from tempeh(TE-C)and further improve the lipid classes ratio. Methods The experimental parameters of SC-CO2 extraction including extraction temperature, pressure,and moisture content of tempeh were optimized using a Box-Behnken design combined with response surface methodology (RSM),according to the weighted extraction ratio of TE-C and lipid classes after the experimental results of single factors. Detailed chemical compositions of TE-C obtained by optimum conditions of SC-CO2 extraction were analyzed by high performance liquid chroma?tography with an evaporative light-scattering detector(HPLC-ELSD)and high performance liquid chromatography-atmospheric pres?sure chemical ionization mass spectrometry(HPLC-APCI-MS). Results TE-C was composed of three lipid classes:fatty acids(Ⅰ), diacylglycerols(Ⅱ)and triacylglycerols(Ⅲ). The optimum SC-CO2 extraction conditions of TE-C were 50℃extraction temperature, 25 MPa pressure,1.99%moisture content of tempeh and 1.5 hour extraction time. Conclusion The optimum value of RSM for SC-CO2 extraction was(5.97±0.15)g/100 g.

This paper reported that the activation of oncogenes in human fetal esopha geal epithelium treated by alternariol (AOH). It was found that NIH/3T3 cells were transformed via transfeetion of DNA extracted from human fetal esophageal epithelium which was cultured and treated by 10?g/ml AOH in a short term in vitro. The efficiency of primary loci was 0.17 focus per ?g of DNA. In the secondary transfection, the efficiency was 0.58 focus per ?g of DNA (P

OBJECTIVETo study the influence of DNA polymerase beta (polbeta) gene silencing by small interfering RNA on biological behavior of human gastric cancer cell line BGC-823.

METHODSThe siRNA eukaryotic expression vectors targeting polbeta gene were constructed and transfected into BGC-823 cells by liposome. Stable cell lines were screened with G418. The expression levels of polbeta mRNA and protein were detected by real time PCR and Western blot in the cells of each group. The proliferation of each group was detected by flow cytometry and tumorigenicity was determined in nude mice.

RESULTSThe siRNA expression vector targeting polbeta gene was successfully constructed. The expression levels of polbeta mRNA and protein were significantly reduced in the experimental group transfected with siRNA expression vectors targeting polbeta, and the silencing effect of pRNAT-U6.1-sipolbeta2 (suppression degree was 83%) was stronger than that of pRNAT-U6.1-sipolbeta1 (depression degree is 56%). Compared with irrelevant siRNA control group, empty vector control group and untransfected group, the ratio of G0/G1 cells was increased, proportion of S phase cells and cell proliferation were decreased in the experimental group 1 cells transfected with pRNAT-U6.1-sipolbeta1 (P < 0.05). On the contrary, the ratio of G1/G0 was decreased, proportion of S phase cells and cell proliferation was increased in the experimental group 2 cells transfected with pRNAT-U6.1-sipolbeta2 (P < 0.05).

CONCLUSIONThe siRNA expression vectors targeting DNA polymerase beta gene can significantly inhibit the expression of polbeta mRNA. Neither high nor extremely low expression of polbeta is beneficial to maintain the cellular physiological functions. The expression of polbeta silenced to a proper level by siRNA may play an important role in inhibiting tumorigenesis.

Animals ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; DNA Polymerase beta ; genetics ; metabolism ; Gene Silencing ; Genetic Vectors ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; Random Allocation ; Stomach Neoplasms ; metabolism ; pathology ; Transfection ; Tumor Burden

OBJECTIVETo construct an expression vector of siRNA targeting human MTA1 gene and observe its gene-silencing effect in esophageal carcinoma cells.

METHODSThe siRNA sequences targeting MTA1 gene were designed and synthesized with two complementary oligonucleotide strands. The oligonucleotide strands were annealed and recombined into pRNAT-U6.2 vector, which was identified by sequencing following transformation and amplification. The siRNA expression vector pRNAT-U6.2-MTA1 was transfected into human esophageal carcinoma EC9706 cells via liposome. RT-PCR and Western blotting were used to detect expression levels of MTA1 mRNA and protein in the transfected EC9706 cells, respectively.

RESULTSThe double-stranded oligonucleotide fragments of the siRNA targeting MTA1 gene were cloned into pRNAT-U6.2 vector, which was validated by sequence analysis. RT-PCR and Western blotting indicated that MTA1 mRNA and protein expressions were significantly decreased in the transfected cells, especially in those transfected with the siRNA targeting the sequence of GACCCTGCTGGCAGATAAA (481-499), which induced almost complete silencing of MTA1 protein expression.

CONCLUSIONThe siRNA expression vector pRNAT-U6.2-MTA1 for silencing MTA1 gene expression in the esophageal carcinoma cells has been successfully constructed, which may facilitate further study for decreasing the invasive and metastatic potentials of malignant tumors by MTA1 gene silencing.

Base Sequence ; Blotting, Western ; Cell Line, Tumor ; Cloning, Molecular ; Esophageal Neoplasms ; genetics ; metabolism ; pathology ; Genetic Vectors ; genetics ; Histone Deacetylases ; biosynthesis ; genetics ; Humans ; Molecular Sequence Data ; RNA Interference ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; Repressor Proteins ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

Objective To investigate the effects of gonadotropin releasing hormone agonist (GnRH- a) and antagonist (GnRH-ant) on cyclophosphamide (CTX)-induced ovarian damage in rats.Methods Seventy-two Sprague-Dawley female rats were divided randomly into six groups,which received normal saline (NS),CTX,GnRH-a+NS,GnRH-a+CTX,GnRH-ant+NS,and GnRH-ant+CTX respectively.Levels of serum follicle-stimulating hormone (FSH),luteinizing hormone (LH) and estradiol (E_2) were measured successively by the enzyme-linked immunosorbent assay method,and half of the rats were killed in the first week and between the fourth and the fifth week after stop of medication,respectively to compare the weight of the ovaries and the number of the primordial follicles and the growth follicles.Results (1) Throughout experiment,the serum levels of FSH,LH and E_2 of the control group fluctuated slightly,while those in the CTX group kept rising.During medication treatment,compared with the control group[(118?16) ?g/L, (350?35) ?g/L] and the CTX group[(113?15) ?g/L,(289?42) ?g/L],the concentrations of LH [(42 ?8)-(47?7) ?g/L,(31?5)-(36?7) ?g/L] and FSH [(124?45)-(136?32)?g/L,(178 ?54)-(198+27)?g/L] in the GnRH-a groups and the GnRH-ant groups were maintained at low levels significantly and the levels of LH in the GnRH-ant groups were significantly lower than that in the GnRH-a groups,but the levels of FSH in the GnRH-ant groups were significantly higher than that in the GnRH-a groups(P0.05),but the levels of FSH,LH and E_2 of the GnRH-ant+CTX group rose obviously and were similar to the levels of the CTX group,especially the FSH,and the levels of LH and FSH of the GnRH- ant + CTX group [(156?12) ?g/L,(520?44) ?g/L] and the CTX group [(178?18) ?g/L,(546?36) ?g/L] were significantly higher than that of the other four groups [(121?15)-(132?13) ?g/L,(335 ?35)-(359?26) ?g/L] at the 4~(th)-5~(th) week after stop of treatment(P0.05),but the number of all kinds of follicles declined significantly in the GnRH-ant+CTX group[(195?15),(36?12)] and the CTX group [(212?11),(36?9)] compared to the other four groups[(302?15)-(690?43),(44?12)-(58?11),P

This study was aimed to investigate the effect of chemokine-like factor superfamily 8 (CKLFSF8) on proliferation and expression of epidermal growth factor receptor (EGFR) of HL-60 cells. Expression of CKLFSF8 mRNA on HL-60 cell line was assayed by RT-PCR; the target gene was transfected into the cells by lipid vector, cell proliferation was determined by MTT assay, while expression of EGFR in HL-60 was determined by immunocytochemical technique. The results indicated that expression of CKLFSF8 existed in HL-60 cells. After transfection, cell proliferation was inhibited (P < 0.05) and the expression of EGFR in HL-60 cells was also discovered to be inhibited (P < 0.05). It is concluded that the proliferation and expression of EGFR in HL-60 cells can be inhibited by transfection of CKLFSF8. The novel chemokine may provide a new approach in the treatment of leukemia.
Cell Proliferation ; Chemokines ; metabolism ; Gene Expression Regulation, Neoplastic ; HL-60 Cells ; Humans ; MARVEL Domain-Containing Proteins ; RNA, Messenger ; metabolism ; Receptor, Epidermal Growth Factor ; metabolism ; Transfection