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1.
Andrologia ; 53(10): e14194, 2021 Nov.
Article in English | MEDLINE | ID: mdl-34328658

ABSTRACT

To explore a new method of in vitro culture and purification of rat corpus cavernosum endothelial cells (CCECs). Male Sprague-Dawley rats' penile tissue were digested with elastase or collagenase combined with mechanical extrusion to isolate and culture the CCECs. The fixed-point digestion method was used to purify the primary cells. High-purity CCECs were successfully isolated. Following the digestion of the primary CCECs by elastase or collagenase coupled with mechanical extrusion, the cells were paving stone- and cobblestone-shaped over 10 days. The cell purity yielded in the second generation (P2) CCECs after using the fixed-point digestion method was significantly high. Compared with primary CCECs extracted by elastase digestion combined with the mechanical extrusion method, CCECs cultured by collagenase digestion yielded higher purity and a more stable morphology after fixed-point digestion and purification. Immunofluorescence staining of the third generation CCECs and the expression results of endothelial cell-associated marker antibodies CD31 and VWF were positive, and flow cytometry showed the purity of CCECs was 96.9%. Enzymatic digestion combined with mechanical extrusion and fixed-point digestion is a simple, economical method for in vitro culture and purification of CCECs, which is conducive to studying the pathophysiological mechanisms of endothelial dysfunction and erectile dysfunction.


Subject(s)
Endothelial Cells , Erectile Dysfunction , Animals , Cells, Cultured , Digestion , Humans , Male , Penis , Rats , Rats, Sprague-Dawley
2.
Reprod Sci ; 28(2): 595-602, 2021 02.
Article in English | MEDLINE | ID: mdl-33095425

ABSTRACT

At present, infertile patients with maturation arrest (MA) are difficult to obtain mature sperm. Spermatogenesis and its molecular mechanism are still not clear. Patients with MA and normal spermatogenesis (NS) were collected. iTRAQ-based proteomic approach was performed to reveal the different proteins between them. To validate the confidence of proteome data, the individual samples were analyzed by Western blotting (WB), quantitative polymerase chain reaction (qPCR), and immunofluorescence. The miR-449a and CEP55 were determined by Luciferase assay. Mouse GC-1 cells were transfected with CEP55 siRNAs, miR-449a mimic, or inhibitor, and cell proliferation was determined. Compared with NS, 27 proteins were differentially expressed in MA, and CEP55 protein was the most significant difference. WB and qPCR showed that CEP55 levels were significantly elevated in NS than MA. In transfected cells, overexpression of miR-449a and knockdown of CEP55 both downregulated CEP55 expression and decreased cell proliferation. miR-449a suppresses mouse spermatogonia proliferation via inhibition of CEP55.


Subject(s)
Azoospermia/metabolism , Cell Cycle Proteins/metabolism , Cell Proliferation , MicroRNAs/metabolism , Spermatogonia/metabolism , Adult , Azoospermia/genetics , Azoospermia/pathology , Cell Cycle Proteins/genetics , HEK293 Cells , Humans , Male , MicroRNAs/genetics , Proteome , Proteomics , Signal Transduction , Spermatogonia/pathology , Young Adult
3.
BMC Urol ; 18(1): 8, 2018 Feb 06.
Article in English | MEDLINE | ID: mdl-29409493

ABSTRACT

BACKGROUND: Scrotal hemorrhage after testicular sperm aspiration (TESA) is uncommon in clinical operation. Phosphodiesterase-5 inhibitors (PDE5i) are commonly given to men who have difficulty providing a sperm sample for assisted reproductive technique such as in vitro fertilization. In this study, we examine the incidence of scrotal hemorrhage after TESA in men who received a PDE5i. METHODS: In this retrospective study, 504 men with TESA operation in Center for Reproductive Medicine, Nanfang Hospital, Southern Medical University were collected. Men in the drug group had taken orally PDE5i before TESA. Men in the control group only operated TESA. The testis volume, coagulation function were measured. Sonographic examination with Doppler imaging was performed when scrotal hemorrhage appeared. RESULTS: A total of 504 men with a mean age of 28.63 ± 4.22 years were included in the analysis. Of these, 428 did not receive a PDE5i prior to TESA and 76 received a PDE5i prior to TESA. Measures of coagulation function were not different between the groups. The incidence of hemorrhage was 0.0% in the control group and the drug group was 5.3%. The incidence of hemorrhage between two groups was different significantly (P = 0.000). CONCLUSION: In summary, the results of this study suggest that a PDE5i administration increases the risk of scrotal hemorrhage in men undergoing TESA, although the study design does not allow drawing a conclusion of cause and effect. Given the potential risk of scrotal hemorrhage after the ingestion of PDE5i, it may be wise not to administer it to men in whom a TESA may be performed.


Subject(s)
Hemorrhage/chemically induced , Phosphodiesterase 5 Inhibitors/adverse effects , Scrotum/drug effects , Sperm Retrieval/adverse effects , Testis/drug effects , Adult , Follow-Up Studies , Hemorrhage/diagnosis , Humans , Infertility, Male/diagnosis , Infertility, Male/therapy , Male , Retrospective Studies , Scrotum/pathology , Testis/pathology
4.
BMC Med Genet ; 18(1): 98, 2017 09 06.
Article in English | MEDLINE | ID: mdl-28874128

ABSTRACT

BACKGROUND: Chromosomal disorders in non obstructive azoospermia (NOA) may have an important influence on spermatogenesis, which may be reflected by the serum inhibin B levels. Till now, few studies have concerned the relationship of genetic causes and inhibin B in NOA. METHODS: In this retrospective study, 322 men with NOA in Center for Reproductive Medicine, Nanfang Hospital, Southern Medical University were collected. The level of follicle stimulating hormone (FSH), inhibin B, Y chromosome microdeletion test (YCMD) and karyotype were measured. RESULTS: Abnormal karyotypes were present in 38.5% of NOA, and YCMD were present in 18.0%, there was a high correlation between karyotypes and YCMD (χ2 = 11.892, P < 0.001). The level of inhibin B in chromosomal abnormality from lowest to highest was 46,XX (or 45,X), 47, XXY, mosaics, polymorphisms, inversion and translocation. And the level of inhibin B within Non-AZF a&b region deletion was higher than AZF a&b microdeletion. CONCLUSION: According to the level of inhibin B, spermatogenesis in chromosomal abnormality from lowest to highest was 46,XX (or 45,X), 47, XXY, mosaics, polymorphisms, inversion and translocation. And spermatogenesis within Non-AZF a&b region deletion was better than AZF a&b microdeletion.


Subject(s)
Azoospermia/genetics , Inhibins/blood , Adult , Azoospermia/blood , Chromosome Deletion , Chromosomes, Human, Y , Follicle Stimulating Hormone , Humans , Karyotyping , Male , Retrospective Studies , Sex Chromosome Aberrations , Spermatogenesis/genetics
5.
Cancer Med ; 6(3): 689-697, 2017 03.
Article in English | MEDLINE | ID: mdl-28205361

ABSTRACT

In previous studies, it has been shown that the granulocyte macrophage-colony stimulating factor (GM-CSF) or interleukin-2 (IL-2) surface modified MB49 bladder cancer stem cells (MCSCs) vaccine could induce a specific antitumor immunity and against bladder cancer in mice model respectively. However, whether combined administration of GM-CSF and IL-2 could produce specific immune responses to cancer stem cells (CSCs) was uncertain. MCSCs were established and characterized. GM-CSF and IL-2 MCSCs vaccines were prepared and bioactivity was evaluated. The therapeutic, protective, specific, and memorial immune response animal experiments were designed. Tumor-specific cytotoxic T lymphocytes assay, enzyme linked immunosorbent assay, flow cytometry assay were performed to indentify whether vaccine caused an antitumor immunity. Streptavidin (SA)-GM-CSF and SA-IL-2 MCSCs vaccines were prepared successfully. Such vaccines inhibited the volume of tumor and prolonged the survival of the mice in animal experiments. The express of IgG or IFN-c, the portion of dendritic cells, CD8+ and CD4+ T cells were highest in the combined vaccines group than the SA-GM-CSF vaccine group, the SA-IL-2 vaccine group, the MCSCs group and the PBS group. The combined of GM-CSF and IL-2 vaccines could induce better antitumor immunity than a vaccine alone.


Subject(s)
Cancer Vaccines/administration & dosage , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Interleukin-2/metabolism , Neoplastic Stem Cells/immunology , Urinary Bladder Neoplasms/therapy , Animals , Cancer Vaccines/immunology , Cell Line, Tumor , Combined Modality Therapy , Immunotherapy , Mice , Neoplasm Metastasis , Neoplastic Stem Cells/transplantation , Treatment Outcome , Urinary Bladder Neoplasms/immunology , Xenograft Model Antitumor Assays
6.
Asian J Androl ; 18(6): 904-907, 2016.
Article in English | MEDLINE | ID: mdl-26486062

ABSTRACT

Patients with extremely severe oligozoospermia (ESO) and cryptozoospermia (CO) are suitable using intracytoplasmic sperm injection (ICSI) as an infertility treatment. However, some andrologists are confused to distinguish ESO and CO in clinic diagnose. This study was designed for the first time to evaluate and compare patients with ESO and CO to determine whether these are useful clinical distinctions. A total of 270 infertile men in our center were classified into four groups as Group nonobstruction azoospermia (NOA, n = 44), Group ESO (n = 78), Group CO (n = 40), and Group obstruction azoospermia (OA, n = 108). Comparisons of the volume of bilateral testes, the level of follicle stimulating hormone (FSH) and inhibin B were obtained in four groups. Then comparisons of fertilization rates, cleavage rate, and excellent embryos rate were obtained when couples performed ICSI. All indexes (volume of bilateral testis, level of FSH and inhibin B) in Groups ESO and CO were no difference, while Groups OA versus NOA, OA versus ESO, and OA versus CO were significant differences (P < 0.05). The rates of fertilization were no differences in Groups ESO and CO while Groups OA versus ESO, OA versus CO were significant differences (P < 0.05). Therefore, the spermatogenic functions in patients with CO and ESO were similar, better than NOA but worse than OA. However, it would be helpful to evaluate their spermatogenesis using testicular biopsies, especially accompanied azoospermia in clinical practice.


Subject(s)
Azoospermia/pathology , Oligospermia/pathology , Sperm Injections, Intracytoplasmic , Spermatogenesis/physiology , Testis/pathology , Adult , Azoospermia/blood , Female , Follicle Stimulating Hormone/blood , Humans , Inhibins/blood , Male , Oligospermia/blood , Organ Size/physiology , Pregnancy , Pregnancy Rate , Sperm Retrieval , Young Adult
7.
Stem Cell Res Ther ; 6: 224, 2015 Nov 14.
Article in English | MEDLINE | ID: mdl-26566931

ABSTRACT

INTRODUCTION: In previous study the streptavidin interleukin-2 (SA-IL-2)-modified MB49 vaccine was effective against bladder cancer in a mouse model. However, a small portion of tumors regrew because the vaccine could not eliminate MB49 bladder cancer stem cells (MCSCs). Accordingly, we developed a SA-IL-2-modified MCSCs vaccine and evaluated its antitumor effects. METHODS: MCSCs were isolated and identified in cancer stem cells (CSCs) characters, with high expression of CSCs markers, higher resistance to chemotherapy, greater migration in vitro, and stronger tumorigenicity in vivo. The SA-IL-2 MCSCs vaccine was prepared and its bioactivity was evaluated. The protective, therapeutic, specific and memory immune response in animal experiments were designed to identify whether the vaccine elicited antitumor immunity and acted against metastatic bladder cancer. RESULTS: MCSCs had higher level of CD133 and CD44, less susceptibility to chemotherapy, more pronounced migration and greater tumorigenic ability. The successfully prepared SA-IL-2 MCSCs vaccine inhibited the tumor volume and prolonged mice survival in animal experiments. The expression of IgG, the population of dendritic cells, CD8(+) and CD4(+) T cells were highest in the experimental group than in the four control groups. CONCLUSIONS: The SA-IL-2 MCSCs vaccine induced an antitumor immune response and was used to eliminate MCSCs to prevent tumor regrowth.


Subject(s)
Cancer Vaccines/immunology , Cancer Vaccines/therapeutic use , Neoplastic Stem Cells/immunology , Urinary Bladder Neoplasms/therapy , Animals , Cell Line, Tumor , Dendritic Cells/immunology , Female , Immunoglobulin G/blood , Immunologic Memory , Interleukin-2/immunology , Lung Neoplasms/immunology , Lung Neoplasms/secondary , Lung Neoplasms/therapy , Mice , Mice, Inbred C57BL , Mice, Nude , Streptavidin/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocytes, Cytotoxic/immunology , Urinary Bladder Neoplasms/immunology , Urinary Bladder Neoplasms/secondary
8.
Zhonghua Nan Ke Xue ; 18(5): 432-5, 2012 May.
Article in Chinese | MEDLINE | ID: mdl-22741443

ABSTRACT

OBJECTIVE: To analyze the embryo development potential after intracytoplasmic injection of sperm from azoospermia patients with different spermatogenic functions. METHODS: We performed ICSI with sperm retrieved from azoospermia patients with different spermatogenic functions using percutaneous epididymal sperm aspiration (PESA) and testicular sperm aspiration (TESA). Then we recorded and analyzed the rates of normal fertilization, cleavages, excellent embryos and pregnancies. RESULTS: No statistically significant differences were found between the PESA and TESA groups in the rates of normal fertilization ([74.9 +/- 19.6] vs [66.3 +/- 22.7]%, P > 0.05), cleavages ([96.7 +/- 8.6] vs [92.8 +/- 19.8]%, P > 0.05), excellent embryos ([43.5 +/- 26.2] vs [35.0 +/- 29.4]%, P > 0.05) and pregnancies (44.0 vs 52.0%, P > 0.05). The normal fertilization rates in the patients with normal spermatogenesis, mild spermatogenic dysfunction (SD), moderate SD and severe SD were (77.8 +/- 18.4), (68.4 +/- 18.5), (73.5 +/- 19.8) and (51.4 +/- 27.9)%, respectively, with significant difference between the normal spermatogenesis and mild SD groups (P < 0.05) as well as between the severe SD and the other groups (P < 0.05); the cleavage rates were (96.7 +/- 9.2), (96.5 +/- 15.0), (93.9 +/- 12.1) and (93.7 +/- 11.1)%, respectively, with no significant difference among the four groups; the excellent embryo rates were (47.1 +/- 25.8), (40.3 +/- 27.6), (36.2 +/- 23.1) and (15.0 +/- 24.6)%, respectively, with significant difference between the severe SD and the other groups; the pregnancy rates were 54.8, 50.0, 13.6 and 10.0%, respectively, with significant differences among the four groups (P < 0.001). CONCLUSION: ICSI by PESA or TESA is an effective approach to azoospermia. There are no significant differences between PESA and TESA in the rates of normal fertilization, cleavages, excellent embryos and pregnancies. The severity of spermatogenic dysfunction affects fertilization and initial development of embryos, which were shown in the rates of normal fertilization, excellent embryos and pregnancies but not that of cleavages.


Subject(s)
Azoospermia/therapy , Embryonic Development , Sperm Injections, Intracytoplasmic , Adult , Azoospermia/physiopathology , Epididymis , Female , Humans , Male , Pregnancy , Pregnancy Rate , Sperm Retrieval , Spermatogenesis , Young Adult
9.
Zhonghua Nan Ke Xue ; 17(8): 694-8, 2011 Aug.
Article in Chinese | MEDLINE | ID: mdl-21898990

ABSTRACT

OBJECTIVE: To study the differentially expressed genes in asthenospermia to gain a deeper insight into the molecular mechanisms of the disease. METHODS: We analyzed the differentially expressed genes in asthenospermia using GATHER, PANTHER and ToppGene online bioinformatics tools. RESULTS: Our bioinformatics mining and analyses revealed that the differentially expressed genes in asthenospermia played important roles in the cellular protein and macromolecular metabolism, protein modification, cell death, cell apoptosis and apoptosis induction. CONCLUSION: Asthenospermia patients experience a decline in sperm activity and the basic life activities of sperm simultaneously, and are also prone to cell apoptosis or death. Such differentially expressed genes as KIF3B, MYO15A, KIF6, KIF26B, KIF3A, DNHD2, DMN, DYNC2H1, STARD9, MYOHD1, and TPM1, which are involved in cytoskeletal structure, microtubule movement and cell movement, may be associated with asthenospermia, and therefore deserve further studies.


Subject(s)
Asthenozoospermia/genetics , Computational Biology , Spermatozoa , Asthenozoospermia/metabolism , Databases, Genetic , Gene Expression Profiling , Humans , Male , Oligonucleotide Array Sequence Analysis , Spermatozoa/metabolism
10.
Zhonghua Nan Ke Xue ; 17(3): 203-7, 2011 Mar.
Article in Chinese | MEDLINE | ID: mdl-21485539

ABSTRACT

OBJECTIVE: To investigate the mRNA and protein expression levels of cysteine-rich secretory protein 2 (CRISP2) in the sperm of asthenospermia patients, and explore their relationship with sperm motility and related molecular mechanism. METHODS: We collected 78 semen samples from adult male patients with asthenospermia and another 70 from healthy volunteers as controls. We extracted total RNA and total protein from the sperm following purification of the sperm by Percoll gradient centrifugation, and detected the relative expressions of CRISP2 mRNA and protein in the two groups by RT-PCR, SYBR Green real-time PCR and Western blot. RESULTS: The expression of CRISP2 mRNA was down-regulated by 4.3 times and that of the CRISP2 protein by 1.71 times in the asthenospermia patients, significantly lower than in the normal control group (P < 0.05). CONCLUSION: The down-regulation of CRISP2 mRNA and protein expressions in the sperm of asthenospermia patients may be closely related with decreased sperm motility, which suggests that CRISP2 may serve as a potential molecular target for the research of asthenospermia.


Subject(s)
Asthenozoospermia/metabolism , Glycoproteins/metabolism , Spermatozoa/metabolism , Adult , Asthenozoospermia/genetics , Case-Control Studies , Cell Adhesion Molecules , Glycoproteins/genetics , Humans , Male , Sperm Motility , Spermatozoa/physiology
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