[Expression of inhibin B betaB subunits in the testicular tissues of azoospermia patients with different pathological alterations].

OBJECTIVE: To investigate the expression of inhibin B betaB subunits in human testicular tissues. METHODS: Eighty-three cases of the azoospermia underwent testicular biopsy. In accordance with the pathological alterations of spermatogenesis, the samples were divided into four groups: Sertoli-cell-only syndrome (n = 21); hypospermatogenesis (n = 20), maturation arrest (n = 24) and almost normal spermatogenesis (n = 18). Immunohistochemical staining for inhibin B betaB subunits was conducted on the paraffin-embedded sections of different spermatogenesis to localize inhibin B betaB subunits in the seminiferous tubules. RESULTS: Immunohistochemically, positive products of inhibin B betaB subunits were found in both the seminiferous tubules and interstitial tissues of the testis as brown or yellow particles in the cytoplasm. Leydig cells and early intermediate spermatogenic cells showed a very strong positivity; Sertoli cells in the seminiferous tubules were mostly positive; peritubular myoid cells showed a weak positive staining; but no positive expression of inhibin B betaB subunits was found in late spermatids and mature sperm. CONCLUSION: Inhibin B may be produced by both Sertoli cells and early spermatogenic cells in the seminiferous tubules.

[The inhibition effects of EPA on the proliferation of human vascular endothelial cells].

OBJECTIVE: To investigate inhibition effects and the mechanism of EPA on the proliferation of human umbilical vascular endothelial cells (HUVEC). METHODS: Different concentrations of EPA were added to the cultured HUVEC in vitro. The time cause and does response for the inhibition the cells proliferation in all groups were measured by the MTT assay. Light absorption values and cytostasis ratios in all groups were compared. One-way ANOVA in the SPSS 13.0 version statistical software was used. The effect of EPA on cell cycle, proliferative index (PI) and apoptosis of HUVEC in vitro were observed by Flow Cytometry. chi2-test of R x C contingency table was used as a method for statistical analysis. RESULTS: When the concentration of EPA was equal to or more than 0.15 g/L, MTT assay showed a significant difference of light absorption value in the cultured cell after EPA exposure compared with control, the suppressing effects enhanced as the treatment time increased. The peak time of the inhibition of the cell proliferation induced by EPA was at 60 hours and the effect was last until 72 hours. The proliferative index in the treatment group was 23.9%, which was lower than that in the control group (26.9%). No apoptosis was found in the cell in each group. CONCLUSIONS: EPA plays an important role of inhibition of proliferation of cultured HUVEC in vitro. No apoptosis was induced by the exposure HUVEC to EPA, therefore, it suggests a potential application for clinical trial.

[Observation on the effect of acupuncture of "Wuyi" (ST 15) and multiple acupoints on the size of lacteal gland in rats with cyclomastopathy].

OBJECTIVE: To compare the therapeutic effects of acupuncture of "Wuyi" (ST 15) and multi-acupoints for hyperplasia of mammary glands so as to choose the key acupoints for cyclomastopathy. METHODS: A total of 48 FEMALE Wistar rats were randomized into normal control, model, "Wuyi" (ST 15) and multi-point groups with 12 cases in each group. Cyclomastopathy model was established by intraperitoneal injection of benzestrofol (0.5 mg/kg per day) for 25 days. followed by administration of flavolutan (4 mg/kg per day, i.p.) for 5 days. Bilateral ST15 were punctured and stimulated manually for ST15 group; while for multi-point group, bilateral ST15, "Hegu" (LI 4), "Zusanli" (ST 36) and "Shanzhong" (CV 17) were punctured, once daily, 27 sessions altogether. After decapitation, the rats' second pairs of mammary glands were removed to be fixed routinely, embedded in paraffin, sectioned (5 microm), and stained with H & E method for observing the structural changes under microscope and with norphometry. RESULTS: Compared with control group, the diameter and area of acinar lumina (AL) of mammary glands in model group increased significantly (P < 0.01), whereas those in both ST15 and multi-point groups were significantly lower than those 2 indexes in model group (P < 0.01). No significant differences were found between ST15 and multi-point groups in the diameter and area of AL. CONCLUSION: "Wuyi" (ST 15) can effectively suppress estrogenic hormones-induced hyperplasia of mammary glands and is thus a key acupoint for treatment of cyclomastopathy.

[Effects of amniotic membrane on proliferation of retinal Müller cells: an in vitro experiment with rabbits].

OBJECTIVE: To observe the effects of amniotic membrane on the proliferation of retinal Müller cells. METHODS: (1) Human amniotic membrane was collected form normal lying woman undergoing cesarean section. Müller cells were obtained from the retina of New Zealand rabbit, cultured, and put into 96-well culture plate. Homogenate of human amniotic membrane at different concentrations: 100, 200, 400, and 800 microg/ml respectively were added into the culture fluid for 96 h. The proliferation rate of the Müller cells was measured with MTT method. (2) Müller cells were inoculated in 6-well culture plate with cover slips. Amniotic homogenate of different concentrations was added for 48 hours, and immunohistochemistry was used to detect the expression of proliferating cell nuclear antigen (PCNA). (3) Amniotic homogenate conditioned culture medium on different concentrations was added into the culture fluid of Müller cells for 48 hours. The change of cell cycles was observed by flow cytometry (FCM). RESULTS: Homogenate of human amniotic membrane increased the proliferation rate of the Müller cells dose-dependently and the proliferation rate reached the peak (62.5%) when the concentration homogenate of human amniotic membrane was 800 microg/ml. FCM showed that the amniotic homogenate increased the rate of cells in S phase and decreased the rate of those in G2/M phase and G0/G1 phase concentration-dependently. The proliferation rate of the Müller cells treated with the amniotic homogenate of the same concentration increased time-dependently and peaked 48 h later, and the proliferation rate 96 h later was not significantly different from that 48 h later (F=0.233, 0.007, P=0.492, 0.729). The positive rates of PCNA when the concentrations of the amniotic homogenate were 800 microg/ml and 400 microg/ml respectively (0.84+/-0.07, 0.79+/-0.06) were significantly higher than that of the control group (0.64+/-0.12). CONCLUSION: Amniotic membrane promotes the proliferation on retinal Müller cells dose and time-dependently in vitro.

[Variation of nestin expression in SD rat eyes in different stages of postnatal development].

OBJECTIVE: To observe the position and quantity of nestin expression in SD rat eyes in different stages of postnatal development. METHODS: Immunocytochemical method was used to identify nestin expression in the eyes of SD rats of 1 to 30 days old. RESULTS: Nestin expression was detected in the retina and extraocular muscles of SD rats. The expression varied with the time of postnatal development, distributing in the entire retina layers in earlier stages and confined in the nerve fiber layer in later stages. The quantities of nestin expression in the extraocular muscles decreased gradually with growth. CONCLUSION: Nestin expression in the retinas and extraocular muscles of SD rats decreases during the postnatal development.

[Inducement effect of matrine on phenotype changes of rat hepatic oval cells].

BACKGROUND & OBJECTIVE: Atypical dysplasia of oval cells plays a crucial role in the pathogenesis of hepatocellular carcinoma (HCC). To prevent oval cells from carcinogenesis or induce them to differenciate into hepatocytes will become an effective way for chemical treatment of HCC. This study was to construct a rat hepatic oval cell proliferation model, and observe the inducement effect of matrine on phenotype changes of hepatic oval cells. METHODS: Hepatic oval cell proliferation model was constructed with SD rats by 2-acetaminofluorene administration and 2/3 partial hepatectomy, and divided into model group, low dose matrine group, high dose matrine group, and control group. Ultrastructure of oval cells was observed by electron microscopy. The expression of Thy-1 and alpha fetoprotein (AFP) was detected by immunohistochemistry. The expression of gamma-glutamyl transpeptidase (gamma-GT) and adenosine triphosphatase was detected by enzyme-histochemisty. RESULTS: The oval cells in high dose matrine groups were larger and contained more rough endoplasmic reticula than those in control group. The expression index of Thy-1 was 8.15+/-2.64 in model group, 5.27+/-1.32 in low dose matrine group, 3.83+/-0.35 in high dose matrine group, and 1.63+/-0.22 in control group; it was significantly lower in high dose matrine group than in model group (P<0.05). The number of AFP-positive cells was 15.36+/-4.42 in model group, 9.75+/-2.41 in low dose matrine group, 7.33+/-1.38 in high dose matrine group, and 2.51+/-0.93 in control group; it was significantly lower in high dose matrine group than in model group (P<0.05). The inhibition rate of gamma-GT was significantly higher in high dose matrine group than in low dose matrine group (55.37% vs. 33.35%, P<0.05). CONCLUSION: Matrine can inhibit the proliferation of oval cells, induce ultrastructure changes, and suppress the expression of Thy-1, AFP, and gamma-GT.

[The effects of nerve growth factor-induced apoptosis on hepatic stellate cells of fibrotic rat liver].

OBJECTIVE: To evaluate the effects of nerve growth factor (NGF) on the proliferation and apoptosis of hepatic stellate cells (HSCs) of fibrotic rat. METHODS: HSCs of fibrotic rat were incubated in medium with different concentrations of NGF (50 ng/ml, 100 ng/ml, and 150 ng/ml respectively) or without NGF for 24, 48 and 72 hours respectively. The proliferation rate of the HSCs was determined by MTT assay. Apoptotic HSCs were detected by TUNEL method. The apoptotic rate of HSCs was studied by flow cytometry. The conformation of HSCs was observed by microscopy. RESULTS: (1) MTT method indicated that the proliferation rates of the HSCs induced with different concentrations of NGF for 24 hours was significantly reduced compared with that of the control group (0.77 +/- 0.03, all P < 0.05). The proliferation rate of the HSCs treated with NGF of the concentration of 100 ng/ml for 24 hours was the lowest (0.63 +/- 0.02, P < 0.05). (2) The proliferation rate of the HSCs treated with NGF of the concentration of 100 ng/ml, the most appropriate concentration, increased time-dependently, and the proliferation rate at the 72 th hour was the lowest in comparison with that of the control group (0.48 +/- 0.03 vs 0.89 +/- 0.01, P < 0.05). (3) After stimulation of the HSCs by NGF of the concentration of 100 ng/ml for 24 hr, TUNEL method showed a significant increased apoptotic rate of HSCs compared with the control group (10.2% +/- 1.2% vs 1.6% +/- 0.1%, P < 0.05). Meanwhile, flow cytometry showed an apoptotic rate of 6.2% +/- 0.2% among the HSCs in the experimental group, but no apoptosis in control group. (4) Microscopy did not show significant morphological change in the HSCs with the proliferation inhibited. CONCLUSION: NGF can induce apoptosis of activated HSCs. The mechanism may be the interference with the apoptotic pathway.

[Establishment of an undifferential orchitis model in rats and changes of spermatogenic epithelium].

OBJECTIVE: To explore the changes of rat testicular spermatogenic epithelium stimulated by bacterial lipopolysaccharide (LPS) in vivo. METHODS: Twenty Wistar rats were divided into two groups: control group and experimental group. The control group was treated with pyrogen-free saline (1 ml/kg) and the experimental group was injected ip with saline containing LPS (1 mg/kg) once every two days. Two groups were operated after ten days in order to investigate the testicular pathological changes by HE staining and the expression of proliferating cell nuclear antigen( PCNA), alpha-catenin in spermatogenic epithelium by immunohistochemistry assay. RESULTS: The testes of the experimental group showed inflammatory changes. The positive expression of PCNA in seminiferous epithelium was significantly lower than that of control group. The number of positive cells in every seminiferous, in which only spermatogonia were stained in experimental group were 59 +/- 5 and it showed significant decrease compared with the control (P < 0.01). Furthermore, the percentage of such seminiferous tubules was 0.673 +/- 0.054 and increased apparently (P < 0.01). The expression of alpha-catenin in testicular tissue of the experimental group declined (P < 0.01), and cellular positive granular light density was 0.150 +/- 0.014. CONCLUSION: The ability of spermatogonium proliferation and the function of conglutination of cells under inflammatory condition of the testes declined, which may be one of the etiologies of male infertility.

[Experimental choroidal neovascularization is inhibited by subretinal administration of Endostatin].

OBJECTIVE: Endostatin is an endogeneous angiogenesis inhibitor. The purpose of this study was to investigated the effect of Endostatin on the eyes of rats with experimental choroidal neovascularization (CNV). METHODS: Experimental CNV was induced by laser photocoagulation. Animals were given subretinal injections of recombinant human Endostatin 20 microl (5 g/L) or 0.9% chlorine sodium. The intensity of fluorescein leakage from the photocoagulated lesions was studied 13 days after photocoagulation. The area of CNV at each rupture site was measured using high molecular weight FITC-dextran (MW 2 x 10(6)) for high resolution angiography in RPE-choroid-sclera flat mounts. In addition, 8 eyes in each group were removed and fixed 14 days after photocoagulation, cut into thin sections. The sections were examined by light microscopy. Immunolocalization of Endoglin (CD105) and factor VIII on sections of CNV lesions was studied by immunohistochemical evaluation. RESULTS: After Endostatin injection, fluorescein leakage from the CNV lesions decreased significantly compared with the control eyes. The average area of CNV at sites of the Bruch's membrane rupture showed significant difference in eyes injected with Endostatin compared with control eyes. Endothelial cells demonstrated strong immunoreactivity of CD105 and factor VIII in CNV lesions of control eyes. CD105-positive cell were not detected in normal chorioretinal tissues. CONCLUSIONS: The development of CNV can be inhibited by injection of Endostatin, which suggest that Endostatin may be beneficial in treating CNV and that further studies can be considered to evaluate this possibility.

[The establishment of testicular fibrosis model in Wistar rats].

OBJECTIVES: To establish the testicular fibrosis model in rats. METHODS: Wistar rats were divided into control group(n = 12) and model group(n = 40) randomly. Testicular fibrosis model was built with the classical method of establishing experimental autoimmune orchitis (EAO) combined with injecting Bacille Calmette-Guerin (BCG) into left testis. RESULTS: The incidence rate of EAO and the rate of testicular fibrosis were 100%, 11.1% and 100%, 81.5% at 80, 140 days after the first infection, respectively. CONCLUSIONS: The model of rat testicular fibrosis was established successfully.

Effects of Yigan Decoction on proliferation and apoptosis of hepatic stellate cells.

AIM: To investigate the effects of Chinese herb Yigan Decoction on proliferation and apoptosis of the hepatic stellate cells (HSC) in vitro. METHODS: The study in vitro was carried out in the culture of HSC lines. Various concentrations of Yigan Decoction were added and incubated. Cell proliferation was detected with MTT colorimetric assay. Cell apoptosis was detected by electron microscopy, flow cytometry and TUNEL. RESULTS: The proliferation of HSC was inhibited by Yigan Decoction, which depending on dose and time significantly. The HSC proliferation rates of groups at the end concentrations 144 and 72(g.L(-1)) were 21.62% and 40.54% respectively, significantly lower than that of normal control group(P<0.01). The HSC proliferation rates of groups at the end concentrations 36, 18 and 9(g.L(-1)) were 54.05%, 45.95% and 51.35% respectively, lower than that of control group (P<0.05). When the end concentration was 4.5 g.L(-1), the proliferation rate was 83.78%, which appeared no significant differences compared with control group. At the same concentrations of 18 g.L(-1), the inhibitory effects of Yigan Decoction at 24 h, 48 h and 72 h time point were observed, the effects were time-dependent, and reached a peak at 72 h. Meanwhile, it was showed that the inducing effects of Yigan Decoction on HSC apoptosis were dose-dependent and time-dependent. The apoptosis index(AI) was detected by TUNEL. After Yigan Decoction had been incubated for 48 h at the end concentration of 18 g.L(-1), the AI (14.5+/-3.1)% was significantly higher than that of control group (4.3+/-1.3)% (P<0.01). When visualized under transmission electron microscopy, some apoptotic stellate cells were found, i.e. dilated endoplasmic reticulum, irregular nuclei, chromatin condensation and heterochromatin ranked along inside of nuclear membrane. By flow cytometry detection, after HSC was treated with Yigan Decoction at different concentrations of 36, 18 and 9(g.L(-1)) for 48 h, AI (%) were 13.3+/-3.2, 10.7+/-2.7 and 10.1+/-2.5 respectively, which were significantly higher than that of control group(4.1+/-1.9) (P<0.01). At the same concentration of 18 g. L(-1) for 24h, 48 h and 72 h, AI (%) were 9.3+/-1.8,10.7+/-2.7 and 14.6+/-4.3 respectively, which were significantly higher than that of control group (P<0.01). CONCLUSION: Yigan Decoction could significantly inhibit HSC proliferation and increase the apoptosis index of HSC dose-dependently and time-dependently, which may be related to its mechanism of antifibrosis.

Effect of mifepristone on estrogen and progesterone receptors in human endometrial and endometriotic cells in vitro.

OBJECTIVE: To investigate the expressions of estrogen (E) receptor and progesterone (P) receptor in human eutopic and ectopic endometrium and the effect of mifepristone (RU486) on them. DESIGN: Prospective study. SETTING: University hospital. PATIENT(S): Twenty-two patients with ovarian endometriosis and 13 patients with uterine leiomyoma were recruited. INTERVENTION(S): Samples of ovarian endometrioma cyst tissue and endometrium were obtained from the 22 patients. A sample of endometrium was obtained from the 13 patients. MAIN OUTCOME MEASURE(S): Expressions of E and P receptors were determined using immunocytochemical method. RESULT(S): P receptor expression in endometrial epithelial cells with endometriosis was significantly higher than that without endometriosis in the early secretory phase. Estrogen receptor and epithelial P receptor expressions in endometriotic cells were significantly lower than those of endometrial cells during the proliferative phase, similar with the latter during the early secretory phase and significantly higher during the late secretory phase. RU486 down-regulated the expressions of E and P receptors in both the eutopic and the ectopic endometrial cells, and in some cases this down-regulating effect was more apparent when the concentration of RU486 was higher. CONCLUSION(S): Different steroid receptor expressions indicate different hormonal regulation between endometriotic and endometrial cells. The down-regulating effect on E and P receptors may be one of the therapeutic mechanisms of RU486 on endometriosis.