OBJECTIVETo construct the eukaryotic expression recombinant pSG. SS. C3d3. YL-Fbeta and analyze the expression of mouse fertilin beta subunit in HEK293 cells.

METHODSThe cDNA fragment expressing the disintegrin domain of mouse fertilin beta was obtained by PCR, and then inserted into the eukaryotic plasmid pSG. SS. C3d3. YL to get recombinant plasmid pSG. SS. C3d3. YL-Fbeta, which was transfected into the HEK293 cell line to express the target protein Fbeta after identified by restriction enzyme digestion. And then Fbeta was detected by indirect immunofluorescence through confocal laser scanning microscopy, Western blot, immunohistochemistry staining and flow cytometry assay.

RESULTSThe recombinant vector pSG.SS.C3d3. YL-Fbeta could express Fbeta in HEK293 cells.

CONCLUSIONThe expression of Fbeta in eukaryotic cells provides a foundation for further researches on the effect of high F, expression on fertilization process.

ADAM Proteins ; biosynthesis ; genetics ; Animals ; Blotting, Western ; Cells, Cultured ; Eukaryotic Cells ; metabolism ; Fertilins ; Gene Expression ; Genetic Vectors ; Humans ; Male ; Membrane Glycoproteins ; biosynthesis ; genetics ; Mice ; Plasmids ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis ; Transfection

OBJECTIVETo explore changes of mitochondrial structure and functions, as well as the protection of ligustrazine in the process of myocardial hypertrophy.

METHODSNeonatal myocardial cells were isolated and cultured with angiotensin II (Ang II) for 72 or 96 h. The total protein content was detected using BCA method. The cell diameter was measured by inverted microscope, by which to reflect the proliferation situation of cardiomyocytes. The mitochondrial membrane potential (MMP) was measured by fluorescence microscope. The mitochondrial monoamine oxidase (MAO) activity was detected by spectrophotometer. The mitochondrial cytochrome oxidase (COX) activity and the mitochondrial damage percentage were detected by microplate reader, by which to reflect the damage of mitochondrial outer membrane's structure and the membranes' function. Also, cells were treated with ligustrazine and losartan and then the pharmacological effects on the mitochondrial structure and functions in the myocardial cells treated with Ang II were observed.

RESULTSAt 72 h and 96 h, when compared with the blank group, cells treated with Ang II had increased total protein content (P < 0.01) and enlarged diameter (P < 0.01). Treated with Ang II, the MAO activity and the outer membrane damage percentage of myocardial cells significantly increased (P < 0.01), and mitochondrial COX activity and the mitochondrial MMP significantly decreased (P < 0.01). Compared with the model group at the same time period, ligustrazine significantly reduced myocardial cells' total protein content and myocardial cell diameter, and significantly decreased myocardial cells' MAO activity, increased mitochondrial COX activity, improved the outer membrane damage percentage and inner membrane MMP at 72 and 96 h, all showing statistical difference (P < 0.01, P < 0.05).

CONCLUSIONSDuring the process of myocardial hypertrophy existed the damage to the mitochondrial structure and functions. Ligustrazine protected the mitochondrial structure and functions of the myocardial cells in reversing Ang II induced myocardial cell hypertrophy.

Angiotensin II ; adverse effects ; Animals ; Cardiomyopathy, Hypertrophic ; chemically induced ; metabolism ; pathology ; Cells, Cultured ; Electron Transport Complex IV ; metabolism ; Mitochondria, Heart ; drug effects ; enzymology ; Monoamine Oxidase ; metabolism ; Myocytes, Cardiac ; drug effects ; metabolism ; pathology ; Pyrazines ; pharmacology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the effects of sub-micro emulsion composition on cellular uptake and disposition of incorporated lipophilic drug.

METHODSSub-micro emulsions containing 10 % oil, 1.2 % lecithin and 2.25 % glycerol were prepared, and the fluorescent agent coumarin 6 was used as a model drug. The effects of oil types, co-surfactants and cationic lipid on uptake and elimination kinetics of 6-coumarin in HeLa cells were studied. The uptake mechanism of sub-micro emulsions was further investigated.

RESULTSOil type and Tweens had no influence on the cellular uptake. Modifications of surfactants with Span series increased the cellular influx, among which Span 20 with hydrophilic-lipophilic balance (HLB) value of 8.6 was the best enhancer. The intracellular drug level reached up to (46.09 ± 1.98)ng/μg protein which had significant difference with control group [(38.54 ± 0.34)ng/μg protein]. The positively charged emulsions significantly increased the uptake rate constant and elimination rate constant which were 4 times and 1.5 times of those in anionic groups, respectively. The uptake enhancement was also observed in cationic emulsions, cellular concentrations at plateau were (42.73 ± 0.84)ng/μg protein, which was about 3 times of that in anionic emulsions [(15.71 ± 0.74)ng/μg protein], when extracellular drug concentration kept at 100 ng/ml. Cationic emulsions delivered the payload mainly by direct drug transfer to contacted cells, while the negative ones depended on both drug passive diffusion and clathrin-mediated endocytosis of drug containing oil droplets which accounted for 20% of the intracellular drug.

CONCLUSIONInterfacial characteristic of sub-micro emulsions such as co-surfactants HLB as well as zeta potentials can influence lipophilic drug both in cellular uptake and elimination.

Anions ; Cations ; Coumarins ; pharmacokinetics ; Emulsions ; Endocytosis ; HeLa Cells ; Humans ; Surface-Active Agents ; pharmacokinetics ; Thiazoles ; pharmacokinetics

BACKGROUNDCerebral amyloid angiopathy (CAA) is one of the main causes of spontaneous intracranial hemorrhage (ICH). No established link is available between pathological scores of CAA and its outcome. This study aimed to identify the correlations between pathological severity and poor postoperative outcome in the Chinese population.

METHODSBetween May 2006 and April 2011, 367 consecutive patients who underwent surgery for CAA-related ICH in 71 hospitals throughout the mainland of China were enrolled in this study. Twelve months after surgery, we evaluated these patients' outcomes according to the modified Rankin Scale (mRS) and statistically correlated risk factors (demographics, medical history, pathological results, and surgical details) that are associated with a favorable (mRS < 3) and poor (mRS ≥ 3) outcome groups.

RESULTSRisk factors for poor postoperative outcome in 367 patients with CAA-related ICH included advanced age (OR 1.034, 95%CI 1.001 - 1.067, P = 0.042), CAA pathology severity (OR 2.074, 95%CI 7.140 - 16.25, P < 0.001), lobar hematoma (OR 0.225, 95%CI 0.104 - 0.486, P < 0.001), presence of intraventricular hemorrhage (OR 0.478, 95%CI 0.229 - 1.001, P = 0.050), and/or subarachnoid hemorrhage (OR 2.629, 95%CI, 1.051 - 6.577, P = 0.039).

CONCLUSIONSPoor postoperative outcome of patients with CAA-related ICH was more related to the severe pathological manifestation instead of other factors. Prior ischemia may present an early stage of CAA.

Aged ; Cerebral Amyloid Angiopathy ; pathology ; physiopathology ; China ; Female ; Humans ; Intracranial Hemorrhages ; pathology ; physiopathology ; Male ; Middle Aged ; Risk Factors

OBJECTIVETo investigate the value of magnetic resonance imaging (MRI) in the preoperative assessment of endometrial cancer.

METHODSNinety-four patients with histopathologically confirmed endometrial carcinoma were retrospectively enrolled in this study. MRI findings were compared with the pathologic findings in all cases. The depth of myometrial invasion and lymph node metastasis were evaluated by T2 weighted imaging (T2WI) combined with contrast enhancement or diffusion weighted imaging (DWI).

RESULTSAmong these 94 patients,62 had no or superficial myometrial involvement and 32 cases had deep myometrial involvement. Meanwhile, 24 groups of metastatic lymph nodes and 164 groups of non-metastatic lymph nodes were detected. The accuracy, specificity, sensitivity, negative predictive value, and positive predictive value of T2WI combined with contrast enhancement in discriminating no/superficial myometrial involvement from deep myometrial involvement were 88.3%, 90.3%, 84.4%, 91.8%, and 81.8%, whereas those of T2WI combined with DWI were 81.9%, 87.1%, 71.9%, 85.7%, and 74.2%, respectively. The accuracy, specificity, sensitivity, negative predictive value, and positive predictive value of T2WI combined with contrast enhancement in identifying metastatic lymph nodes were 89.4%, 96.8%, 54.5%, 90.9%, and 78.3%, whereas those of T2WI combined with DWI were 91.5%, 95.5%, 72.7%, 94.3%, and 77.4%, respectively.

CONCLUSIONST2WI combined with contrast enhancement is superior to T2WI combined with DWI in evaluation of the depth of myometrial invasion. However, DWI is more sensitive in identifying lymph node metastasis than T2WI combined with contrast enhancement.

Adult ; Aged ; Endometrial Neoplasms ; diagnosis ; pathology ; Female ; Humans ; Magnetic Resonance Imaging ; methods ; Middle Aged ; Retrospective Studies ; Sensitivity and Specificity

BACKGROUNDBiofilms have given new insights to the understanding of pathogenesis of chronic rhinosinusitis (CRS). However, the link between biofilms formation and local inflammatory response remains poorly defined in CRS with nasal polys. The aim of this study was to determine the potential association of the presence of biofilms in the nasal mucosal tissues with clinical features in Chinese patients, which had CRS with nasal polyps (CRSwNP).

METHODSA total of 19 patients with CRSwNP and 12 patients with non-CRS were subjected to endoscopic surgery and their nasal mucosal tissue specimens were examined histologically and by scanning electron microscopy (SEM). Their demographic and clinical features were recorded.

RESULTSThirteen (68.4%) out of the 19 specimens from patients with CRSwNP, but none from control patients, were positive for biofilms that displayed typical characteristics of bacterial and fugal structures. The presence of biofilms in the nasal mucosal tissues was associated with significantly greater values of purulent nasal discharge and preoperative Lund-Kennedy scores, higher levels of serum total IgE and percentages of subjects with endoscopic surgery (ESS) history in patients with CRSwNP, and more severe inflammation in the nasal mucosal tissues of patients with CRSwNP.

CONCLUSIONOur study demonstrated the presence of biofilms in the nasal mucosal tissues of many patients, contributing to the understanding of the pathogenic process of CRSwNP in Chinese patients.

Adolescent ; Adult ; Biofilms ; Chronic Disease ; Female ; Humans ; Male ; Microscopy, Electron, Scanning ; Middle Aged ; Nasal Mucosa ; pathology ; Nasal Polyps ; etiology ; microbiology ; pathology ; Rhinitis ; etiology ; microbiology ; pathology ; Sinusitis ; etiology ; microbiology ; pathology

OBJECTIVETo investigate the feasibility of magnetic resonance (MR) diffusion weighted imaging (DWI) in discriminating inflammatory from VX2 carcinoma metastatic lymph nodes in rabbit model.

METHODSTwenty New Zealand white rabbits were randomly divided into 2 groups. Complete Freund's adjuvant was injected into the bilateral dorsal footpads to set up ipsilateral lymphadenitis model (n = 10), and the other 10 rabbits received a subcutaneous implantation of VX2 tumor cell suspension (1.5 x 10(7) cells/mL) in both thighs to set up metastatic lymph node model. MR imaging scan covering the popliteal fossa and iliac fossa including short time inversion recovery echo-planar imaging DWI (STIR-EPI-DWI), T1-weighted imaging (T1WI) and T2-weighted imaging (T2WI) was performed 2 weeks after injection. T2WI signal intensity (SI), DWI SI, long/short axial ratio (LSR) and apparent diffusion coefficient (ADC) values of the lymph nodes were evaluated in all cases. Right after MR imaging scan, popliteal and iliac fossa lymph nodes were collected for hematoxylin-eosin staining.

RESULTSTotally 33 lymph nodes larger than 5 mm, including 22 inflammatory and 11 metastatic ones, were successfully isolated and taken into pathological analysis. LSR showed no significant difference between the inflammatory and malignant lymph nodes (P > 0.05). Both benign and malignant lymph nodes appeared iso-intense on T1WI and hyperintense on both T2WI and DWI images with an even lower T1WI and higher T2WI SI core at the hilum. Both T2WI and DWI SI showed no significant difference between two pathological groups (P > 0.01) in popliteal fossa. The mean ADC value of inflammatory nodes [(1.199 +/- 0.281) x 10(-3) mm2/s] was significantly higher than that of metastatic nodes [(0.858 +/- 0.090) x 10(-3) mm2/s, P < 0.01]. On ADC map, a high ADC value central area could be seen in most of the lymph nodes no matter benign or malignant. ADC value gave out the largest area under curve (AUC(ADC) = 0.955) compared with other three indexes (AUC(LSR) = 0.488; AUC(T2WI SI)= 0.727; AUC(DWI SI) = 0.822) and gave the best sensitivity and specificity in lymph node differential diagnosis compared with the other three indexes.

CONCLUSIONSHigh quality DWI image can be obtained using STIR-EPI-DWI sequence in rabbit model. DWI is a new promising technique for differentiating inflammatory from metastatic lymph nodes. Compared with routine MR sequence, DWI could provide more useful physiological and functional information for diagnosis.

Animals ; Diffusion Magnetic Resonance Imaging ; methods ; Inflammation ; pathology ; Lymph Nodes ; pathology ; Lymphatic Metastasis ; pathology ; Male ; Models, Animal ; ROC Curve ; Rabbits ; Random Allocation

OBJECTIVETo evaluate the clinical impact of whole body diffusion weighted imaging (WB-DWI) on diagnosis and staging of malignant lymphoma.

METHODSThirty-one patients with suspected lymphadenopathy were enrolled. WB-DWI was performed by using short TI inversion recovery echo-planar imaging sequence with free breathing and built-in body coil. Axial T2-weighted imaging images of the same location were used as reference. The results of WB-DWI were compared with pathological results and other imaging modalities. The mean apparent diffusion coefficient (ADC) values of different kinds of lymph nodes were compared.

RESULTSWB-DWI was positive in all 18 cases with lymphoma, 5 cases with metastatic lymph nodes and 4 of 8 cases with benign lymphadenopathy. The mean ADC value of lymphomatous, metastatic and benign lymph nodes was (0.87 +/- 0.17) x 10(-3), (0.98 +/- 0.09) x 10(-3) and (1.20 +/- 0.10) x 10(-3) mm2/s. There was significant difference in ADC value between benign lymph nodes and other two groups (P < 0.01). The sensitivity, specificity and accuracy of WB-DWI in diagnosis of lymphoma were 100% (18/18), 30.8% (4/13) and 71.0% (22/31). When an ADC value of 1.08 x 10(-3) mm2/s was used as the threshold value for differentiating malignant from benign lymph nodes, the best results were obtained with sensitivity of 87.8% and specificity of 91.3%. Sixteen of eighteen cases (88.9%) of lymphoma were accurately staged in accordance with clinical staging.

CONCLUSIONSWB-DWI is a sensitive, but less specific technique for diagnosis of lymphoma. It is difficult to differentiate lymphomatous from metastatic lymph nodes using WB-DWI. However, it is a valuable imaging modality for staging of patients with malignant lymphoma.

Adolescent ; Adult ; Aged ; Diffusion Magnetic Resonance Imaging ; methods ; Female ; Humans ; Lymph Nodes ; pathology ; Lymphoma ; diagnosis ; pathology ; Male ; Middle Aged ; Neoplasm Staging ; methods ; ROC Curve ; Whole Body Imaging ; methods ; Young Adult

OBJECTIVETo evaluate the clinical impact of body diffusion weighted imaging (DWI) on the diagnosis and preoperative N staging of cervical cancer.

METHODSTwenty-four patients (mean age 37.9 years old) with proved cervical cancer by cervical biopsy and 24 female patients with other suspected pelvic abnormalities received preoperative body DWI scan. Results of body DWI were compared with pathological findings. The apparent diffusion coefficient (ADC) values of normal cervix and different pathological types of cervical cancer were compared. ADC value of normal or inflammatory lymph nodes was also compared with that of metastatic ones. Student's t test was used for statistical analysis.

RESULTSThere were 5 adenocarcinomas and 19 epitheliomas showed with biopsy results, and DWI showed 21 cervical lesions out of them (87.5%). ADC values of the normal cervix (n = 24), epithelioma (n = 19), and adenocarcinoma (n = 5) were (1.73 +/- 0.31) x 10(-3), (0.88 +/- 0.22) x 10(-3), and (1.08 +/- 0.12) x 10(-3) mm2/s, respectively. Statistical analysis showed significant difference in ADC value between normal cervical tissue and either tumor tissues (both P < 0.01). In patients had lymphadenectomy (n = 24), totally 67 lymph nodes including 16 metastatic lymph nodes were pathologically analyzed, and DWI showed 66 (98.5%) out of them. ADC values of normal/inflammatory and metastatic lymph nodes were (1.07 +/- 0.16) x 10(-3) and (0.77 +/- 0.13) x 10(-3) mm2/s (P < 0.01). Receiver operating characteristic (ROC) curve of ADC value of metastatic lymph node showed that area under curve was 0.961.

CONCLUSIONSADC value in cervical carcinoma is lower than that in normal cervix, and ADC may have predictive value in subtype discrimination. ADC value may improve the preoperative characterization of lymph node metastasis. And at least abdominal and pelvic DWI scan is suggested for N staging evaluation in such patients.

Adult ; Diffusion Magnetic Resonance Imaging ; methods ; Female ; Humans ; Lymphatic Metastasis ; diagnosis ; pathology ; Neoplasm Staging ; methods ; Preoperative Care ; ROC Curve ; Uterine Cervical Neoplasms ; diagnosis ; pathology ; Whole Body Imaging ; methods

OBJECTIVEThe culture of human spermatogonial stem cells (SSC) has not been studied in detail yet. Here we tried to explore the optimized culture method of human SSC by using several different co-culture systems.

METHODSThe alpha6 +Thy-1 +c-kit- cells acquired by the immunomagnetic beads sorting technique were cultured in different co-culture systems. Their morphological, biological characteristics and survival rates were intensively observed by microscopic or immunocytochemical assay. The long-term survival rate of human SSC during culture period was evaluated by germ cell transplantation technique.

RESULTSThe alpha6 +Thy-1 +c-kit- cells could stably survive in the DMEM and DMEM/F12 mediums with fetal bovine serum (FBS) on feeder layer. The survival rates within 1 week were more than 90%. The long-time culture showed the cells were gradually attached on the surface of Sertoli cells by the manner of scattered single cell or accumulated masses. Part of the SSC became more tightly attachment with Sertoli cells or mounted among the Sertoli cells. They could survive or even proliferate for more than 3 months in vitro. Germ cells transplantation study showed that some alpha6 +Thy-1 +c-kit- cells labeled by PKH26 could resided on the basal membrane of seminiferous tubule of nude mice, appearing as single or coupled cells 2 months later after transplantation. The function evaluation of the cultured cells by counting the fluorescent cells in the seminiferous tubule showed 54.9% and 9.2% of SSC in the alpha6 +Thy-1 +c-kit- cells were still remained after cultured for 2 and 4 weeks, respectively.

CONCLUSIONHuman SSC could maintain survival in vitro for more than 3 months, but it was still need to seek for a more optimized and successful culture system for its efficient expansion and proliferation. Thus it will open up a wide prospect for the understanding of the biology of human SSC and the treatment of male sterility.

Adult ; Cell Culture Techniques ; Cell Survival ; Cells, Cultured ; Coculture Techniques ; Humans ; Male ; Sertoli Cells ; cytology ; Spermatogonia ; cytology ; physiology ; Stem Cell Transplantation ; Stem Cells ; cytology