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

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 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

Abstract: Objective To evaluate the key quality control and protective performance test of Halcyon medical linear accelerator. Methods WS 674-2020Specification for Testing of Quality Control in Medical Linear Accelerator（ According to the hereinafter WS 674-2020） ， referred to and the manufacturer´s manual the performance of the first Halcyon medical linear accelerator in Results ， ， Hunan Province was tested. The results showed that all ten indicators of the accelerator including dose deviation ， ， （ ， ， ， - repeatability linearity daily stability and symmetry the results were 0.10% 0.03% 0.04% 0.50% and 100.50% 100.80% ）， - - respectively met the requirements of WS 674 2020. The results of manufacturer quality control indicators such as dose rate ， ， stability in beam gantry rotation isocenter and mechanical position accuracy megavolt image parameters and cone beam computer tomography image parameters met the requirements of the manufacturer´s regulations. Due to the special structure and ， - ： function of the accelerator it is difficult to detect the parameter required by WS 674 2020 as below the radiation leakage - ， ， ， ， outside the M zone the uniformity the indicators related to the light field the offset of the radiation beam axis the zero scale Conclusion - position of the rotating motion scale and others. It is difficult to carry out complete testing according to WS 6742020 for Halcyon medical linear accelerator and it is urgent for the state to issue relevant testing standards to standardize and strengthen the quality control testing of various accelerators.

This study was designed to evaluate the effects of drilling through the growth plate and using adipose-derived stem cells (ADSCs) and bone morphogenetic protein-2 (BMP-2) to treat femoral head epiphyseal ischemic necrosis,which can be done in juvenile rabbits.Passage-four bromodeoxyuridine (BrdU)-labeled ADSCs were cultured,assayed with MTT to determine their viability and stained with alizarin red dye to determine their osteogenic ability.Two-month-old,healthy male rabbits (1.2 to 1.4 kg,n=45) underwent ischemic induction and were randomly divided into five groups (group A:animal model control;group B:drilling;group C:drilling & ADSCs;group D:drilling & BMP-2;and group E:drilling & ADSCs & BMP-2).Magnetic resonance imaging (MRI),X-ray imaging,hematoxylin and eosin staining and BrdU immunofluorescence detection were applied 4,6 and 10 weeks after treatment.Approximately 90％ of the ADSCs were labeled with BrdU and showed good viability and osteogenic ability.Similar results were observed in the rabbits in groups C and E at weeks 6 and 10.The animals of groups C and E demonstrated normal hip structure and improved femoral epiphyseal quotients and trabecular areas compared with those of the groups A and B (P＜0.01).Group D demonstrated improved femoral epiphyseal quotients and trabecular areas compared with those of groups A and B (P＜0.05).In summary,drilling through the growth plate combined with ADSC and BMP-2 treatments induced new bone formation and protected the femoral head epiphysis from collapsing in a juvenile rabbit model of femoral head epiphyseal ischemic necrosis.

OBJECTIVETo identify the distribution and differentiation of ABL kinase domain mutation in the Chinese Han nationality imatinib resistant chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+)ALL).

METHODSBone marrow or peripheral blood samples of 112 imatinib resistant CML patients and 21 Ph(+)ALL patients were obtained from the first affiliated hospital of Soochow university according to local law. Total RNA was extracted from the mononuclear cells using a TRIzol reagent. ABL kinase domain (KD) mutation was detected by direct sequencing.

RESULTSOf the 112 imatinib resistant CML patients, 54.46%(61 cases) had ABL KD mutation. Twenty-three mutants were identified in 20 amino acid sites and 23.21% (26 cases) ABL KD mutations were in P-loop region. ABL KD mutations were also detected in 71.43% (15 cases) imatinib resistant Ph(+)ALL patients, with 10 mutations in 8 amino acid sites. The most frequent mutation was T315I (28.57%), followed by E255K/V (19.05%) and Y253F/H (14.29%). The frequency of T315I was much higher in imatinib resistant Ph(+) ALL than that in imatinib resistant CML (P = 0.001). Ph(+)ALL with additional chromosomal aberrations also had a higher rate of ABL KD mutation than that of CML (P = 0.010). Ph(+)ALL gained ABL KD mutation faster than CML (P < 0.010).

CONCLUSIONChinese imatinib resistant CML and Ph(+)ALL patients had different characteristics in ABL KD mutation. The rate of ABL KD mutation in Ph(+)ALL with additional chromosomal aberrations was much higher than that of CML with additional chromosomal aberrations.

Adolescent ; Adult ; Aged ; Asian Continental Ancestry Group ; genetics ; Benzamides ; pharmacology ; Chromosome Aberrations ; Drug Resistance, Neoplasm ; genetics ; Female ; Humans ; Imatinib Mesylate ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; Middle Aged ; Mutation ; Philadelphia Chromosome ; Piperazines ; pharmacology ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; Protein-Tyrosine Kinases ; genetics ; Proto-Oncogene Proteins c-abl ; genetics ; Pyrimidines ; pharmacology ; Young Adult

BACKGROUNDInteractions of tumor cells with the microenvironment were deemed to promote the tumor invasion and metastasis. CXC chemokine receptor 4 (CXCR4) and extracellular matrix metalloproteinase inducer (EMMPRIN) had reported to participate in this process. However the roles of bone marrow microenvironment in leukemic infiltration were not well investigated.

METHODSA co-culture system between SHI-1 cells and bone marrow stromal cells (BMSCs) is used to simulate the interactions of leukemic cells with their microenvironment. The trans-matrigel invasion was used to detect the capability of SHI-1 cells invasion. The BMSCs and SHI-1 cells were mixed in a ratio of 1:10 and added to the millicell chamber coated with matrigel. Either the co-culture supernatant or the functional blocking peptide of CXCR4 and EMMPRIN were added to the trans-matrigel invasion system. The expressions of EMMPRIN in SHI-1 cells and BMSCs were detected by RT-PCR. The changes of the expression of matrix metalloproteinase-2, 9 (MMP-2, MMP-9), tissue inhibitor of metalloproteinase 2 (TIMP-2), and CXCR4 mRNA in SHI-1 cells were determined by real-time PCR. The concentration of stromal cell derived factor 1 (SDF-1) in serum free supernatant was measured by ELISA.

RESULTSBoth SHI-1 cells and BMSCs express EMMPRIN. SHI-1 cells could hardly invade the matrigel membrane; the coculture supernatant did not induce the invasion of SHI-1 cells. When contacting directly with BMSCs, SHI-1 cells invaded to the lower chamber of millicell were significantly increased. The functional blocking peptide of CXCR4 and EMMPRIN could significantly inhibit the invasion triggered by BMSCs. When co-culturing with BMSCs, the expression of CXCR4, MMP-2, MMP-9 and TIMP-2 mRNA in SHI-1 cells were significantly elevated in company with a significantly higher level of SDF-1 in the co-cultured serum-free supernatant.

CONCLUSIONThe interactions of leukemic cells and BMSCs play important roles in leukemic cell infiltration.

Basigin ; physiology ; Cell Communication ; Cell Line, Tumor ; Coculture Techniques ; Humans ; Leukemia, Monocytic, Acute ; pathology ; Mesenchymal Stromal Cells ; physiology ; Neoplasm Invasiveness ; Receptors, CXCR4 ; physiology

This study was purposed to construct three siRNA eukaryotic expression vector specific to mouse Qa-1 gene, to investigate its silencing effect on Qa-1 gene and to select the most efficient siRNA plasmid specific to mouse Qa-1 gene. Three siRNA peptides specific to mouse Qa-1 through siRNA Web design tools of Ambion company were chosed. Jingsai Company helped to complete the siRNA eukaryotic expression vector. The mouse NIH3T3 cells cultured in RPMI 1640 medium with 10% fetal bovine serum were divided into four groups: three groups of the cells were transfected with lipofectamine 2000 reagent and three different siRNA eukaryotic expression vectors, while one group cells were transfected with lipofectamine 2000 reagent alone as negative control. Cells were collected at 24, 48, 72 hours after transfection; the RNA level of Qa-1 was detected by RT-PCR, and the expression position was examined with flow cytometry analysis by using anti-Qa-1 monoclonal antibody. The results indicated that the constructed three siRNA eukaryotic expression vectors were found to be specific to mouse Qa-1 gene. The sequence analysis showed that the sequence was identical to what chosed from web tools. NIH3T3 cells in vitro were adhered in culture that cell shape appeared to change after transfection. RT-PCR and flow cytometry analysis by using anti-Qa-1 monoclonal antibody approved that both Qa-1 RNA and the expression of Qa-1 on cell surface decreased. The decreased levels in the three groups were different. At 24, 48 and 72 hours, the expression of Qa-1 on NIH3T3 cells decreased as in the following: H2-T231: 60.9%, 81.9%, 43.6%; H2-T232: 64.5%, 73.9%, 61.1%; H2-T233: 61.9%, 71.2%, 47.5%. H2-T232 was most efficient one in all three time points. It is concluded that all three siRNA eukaryotic expression vectors selected can successfully suppress the expression of the Qa-1, and from them H2-T232 is most efficient.
Animals ; Base Sequence ; Cells, Cultured ; Gene Silencing ; Genetic Vectors ; Histocompatibility Antigens Class I ; genetics ; Mice ; NIH 3T3 Cells ; Plasmids ; RNA, Small Interfering ; genetics ; Transfection

OBJECTIVETo investigate the influence of tissue inhibitor of metalloproteinase 2 (TIMP-2) on the infiltrative patterns of human monocytic leukemic cell line SHI-1 in nude mice.

METHODS1) 1 x 10(7) TIMP-2 gene transduced SHI-1 (SHI-1-TIMP-2) and SHI-1 transduced MSCV gene (SHI-1-MSCV) cells were inoculated via tail vein into 6-week nude mice, which pretreated by splenectomy, cytoxan intraperitoneal injection, and sublethal irradiation(referred as SCI nude mice). 30 days after inoculation, half of the mice were sacrificed, and the infiltration patterns were investigated by histological exam and human CD45 immunohistochemistry, other mice were observed for survival time. 2) Leukemic cells inoculated subcutaneously into the axillary area of mice without any pre-treatment. On day 23 and 30, mice were sacrificed to measure the volume of neoplasm. TIMP-2 protein expression and the micro vein density were detected by immunohistochemistry.

RESULTSIn SCI nude mice inoculated via caudal vein with SHI-1-TIMP-2 cells, the survival time was shorter and infiltration (including in central nervous system) was higher than that in those inoculated with SHI-1-MSCV cells. However, in inoculated subcutaneously group, the neoplasm though grew rapidly at first, over expression of TIMP-2 limited the tumor growth and angiogenesis.

CONCLUSIONThe functions of TIMP-2 are diversity; the role of TIMP-2 in tumor infiltration and metastasis was worthy of further investigation.

Animals ; Cell Line, Tumor ; DNA, Complementary ; genetics ; Genetic Vectors ; Humans ; Leukemia, Experimental ; genetics ; pathology ; Leukemic Infiltration ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Tissue Inhibitor of Metalloproteinase-2 ; genetics ; Transfection

BACKGROUNDThis work was carried out to evaluate the clinical efficacy and the complications of S-ROM modular hip arthroplasty combined with transverse subtrochanteric shortening for Crowe type IV congenital dislocation of the hip (CDH).

METHODSA total of 28 consecutive patients with Crowe type IV CDH received treatment using this surgical technique from June 2003 to June 2010. The follow-up was conducted at 3 days, 1, 6, and 12 months after the operation and later annually at the outpatient of our hospital. Sequential pelvic plain film and normotopia film of the affected hip joint were taken. The limp and the Trendelenburg sign were also assessed, the ischiadic nerve injury was also evaluated by electromyogram, and Harris hip scores were recorded.

RESULTSAfter operation, both the alignment and the position of the transverse osteotomies were good. None of the patients had presented complications of joint infection, prosthesis loosening, joint dislocation, or nerve injury.

CONCLUSIONSS-ROM modular hip arthroplasty combined with transverse subtrochanteric shortening was a satisfactory and safe technique for the Crowe type IV congenital hip dislocation within a mean follow up of 53 months. Transverse subtrochanteric shortening could effectively prevent the distraction injury of sciatic nerve.

Adult ; Arthroplasty ; methods ; Female ; Hip Dislocation, Congenital ; diagnostic imaging ; surgery ; Humans ; Male ; Middle Aged ; Osteotomy ; methods ; Radiography