Enhanced proliferation and neuronal differentiation of neural stem cells by bone marrow stromal cells

AIM:To evaluate whether bone marrow stromal cells (BMSCs) could provide a supportive microenvironment for proliferation and differentiation of neural stem cells (NSCs). METHODS:The proliferation and differentiation of NSCs were compared, in media without growth factors or in BMSCs-conditioned media. RESULTS:The proportion of neurons to total NSCs was significantly increased when NSCs were cultured in BMSCs-conditioned media (41.1%±3.2% vs 23.3%±16.5%, P<0.05), while the proportion of astrocytes was significantly decreased (33.8%±4.9% vs 65.0%±10.4%, P<0.01), as compared with the NSCs cultured in media without growth factors. The proportion of proliferated cells was also significantly increased (74.7%±4.7% vs 51.4%±12.3%, P<0.01). CONCLUSION:These finding indicates that BMSCs support the proliferation and neuronal differentiation of NSCs and suggests that their addition may be useful to improve outcomes of NSCs transplantation.     

Triptolide induces apoptosis of rat synoviocytes in collagen-induced arthritis

AIM: To determine whether triptolide induce apoptosis of synovial cells in collagen-induced arthritis (CIA) in rats. METHODS: The male Wistar rats were used to make CIA models by immunized with Bovine collagen Ⅱ (BCⅡ) in Freund's complete adjuvant (FCA). A total of 20 CIA rats were randomly divided into 2 groups, triptolide group (10 rats) and CIA control group (10 rats). Triptolide group were administered with triptolide at 40 μg/kg body weight intramuscularly every three days. CIA control group and another 10 age-matched normal rats were given normal saline instead. The rats were sacrificed on the 31st day after the triptolide administration. The pieces of synovium of the rat knee joints were harvested. The synovium was examined by HE staining and electron microscope. The apoptosis was tested by TUNEL and flow cytometer. RESULTS: The earlier phase of apoptotic synoviocytes were observed under the electron microscope. The flow cytometry showed that the percentage of the apoptotic cells was (3.98±1.16)% in the triptolide group, (1.83±0.82)% in the CIA control group, and (0.87±0.24)% in the normal group (P<0.01: triptolide vs control group). While the percentage of the cells in DNA synthesis phase was (3.3±1.2)% in the triptolide group, (8.0±1.4)% in the CIA control group, and (3.4±0.7)% in the normal group. There is significantly different in the apoptosis changes between the triptolide group and the CIA control group (P<0.01: triptolide vs CIA control group). The TUNEL labeling demonstrated that the percentage of the apoptotic cells was (4.5±1.0)% in the triptolide group, (2.2±1.0)% in the CIA control group, and (1.0±0.4)% in the normal group. The difference of apoptotic rate between the triptolide group and the CIA control group is significant (P<0.01). CONCLUSION: This study demonstrates that triptolide can induce apoptosis in CIA rats, which may be one of the mechanisms that triptolide treats the rheumatoid arthritis.     

Myelin-associated glycoprotein inhibits the differentiation and neurite growth of neural stem cells

AIM: To observe the characterization in neural cells derived from the hippocampus of embryonic rats and to examine the effect of myelin-associated glycoprotein (MAG) on the proliferation, differentiation and neurite growth of neural stem cells (NSCs). METHODS: The hippocampus cells of embryonic rats were isolated and cultured in vitro. The expressions of nestin and doublecortin, the marks of NSCs, were observed by immunocytochemical method. The rate of proliferating cells was examined by BrdU immunocytochemistry. The average neuronal neurite length and the percentage of differentiated neurons were detected by immunocytochemistry staining. RESULTS: The hippocampus cells of 16 days old embryonic rats had the characteristics of NSCs. The percentage of differentiated neurons (β-tubulin Ⅲ-positive cells) was 18.17%±2.79% and the average neuronal neurite length was (136.27±33.66)μm, seven days after the differentiation initiated in vitro in control group. After NSCs were treated with MAG-Fc (200 μg/L), the percentage of differentiated neurons and the average neurite length were decreased, respectively, to 10.05%±3.42% (P<0.01) and (84.87±24.94)μm (P<0.01). The results from proliferation test indicated that the rate of BrdU incorporation did not changed after the treatment of MAG-Fc with different dosages (P>0.05). CONCLUSION: MAG-Fc inhibits the differentiation and neurite growth of the NSCs, but has no effect on the proliferation.     

Effect of γ radioactive stent on proliferation and apoptosis of smooth muscle cells

AIM: To observe effect of γ radioactive ［103Pd］ stent on the proliferation and apoptosis of smooth muscle cells, the mechanism of radioactive stent preventing in-stent restenosis was explored. METHODS: Fifty male New Zealand rabbits were randomized into stent group and ［103Pd］ stent group. Control group was set up. The materials were harvested on 3, 7, 14, 28, 56 days after operation and the following investigation were carried out, including pathomorphology, immunohistochemistry, apoptosis (TUNEL) and in situ hybridization studies.RESULTS: ① The severity of the stenosis in ［103Pd］ stent group was less severe than that in stent group. It was most obvious on 56 th day (P<0.01). ② The expression of proliferating cell nuclear antigen(PCNA) of ［103Pd］ stent group was lower than that in stent group on 3 to 28 days. It was most obvious on 7th day, 16.35%±0.79% vs 24.36±0.55% (P<0.01). ③ TUNEL method showed that the ［103Pd］ stent group had much more apoptosis of VSMC than that in stent group. The highest rate of apoptosis appeared on day 7, 14.72%±0.53% vs 12.42%±1.13% (P<0.01). ④ By calculating the ratio of PCNA/apoptosis (P〖KG*6〗∶〖KG-*2〗A), a much lower ratio was seen in ［103Pd］-stent groups than that in stent group at 3 to 28 days. There was significant statistic difference (P<0.05). ⑤ For bcl-2/bax ratio, the result in ［103Pd］-stent group was lower than that in stent group at 3 to 28 days. There was significant statistic difference (P<0.05).CONCLUSION: γ radioactive stent inhibits the proliferation and accelerates apoptosis of injured media vascular smooth muscle cells. It decreases the ratio of proliferation to apoptosis and relieves the severity of restenosis.     

CD45+CD86+ cells isolated from para-aortic lymph nodes in a murine abortion-prone model

AIM: To address whether the analysis of CD45+CD86+ cells isolated from para-aortic lymph nodes (PLNs) is valuable in assessment of the status of local immunity at the feto-maternal interface. METHODS: CBA/J×DBA/2, virgin CBA/J, and CBA/J×BALB/c mice were used as an abortion-prone model (group A), non-pregnant controls (group N), and fertile controls (group F), respectively. The percentage of CD45+CD86+ cell in the CD45+ cell group (CD45+CD86+ percentage for short) and the absolute number of these cells were determined with flow cytometry (FCM), using mononuclear cells isolated from PLNs collected on day 5.5, 9.5, and 13.5 of gestation, respectively, and mononuclear cells from placentas on day 13.5 of gestation. To clarify the identity of these CD86+ cells, FCM was also performed with CD3, CD19 and DX5 as markers for T cells, B cells, and NK cells, respectively. RESULTS: Both resorption rate and absolute number of resorption were significantly higher in group A (29.3%, 1.8±1.0) than those in group F (4.8%, 0.3±0.5, P<0.01, respectively). Similarly, both cell percentage and absolute number of CD45+CD86+ cells in PLNs collected on day 13.5 of gestation were significantly higher in group A than that in group F (27.5%±14.0% vs 12.3%±7.1%, and 1 362±687 vs 615±353, P<0.01, respectively). The CD45+CD86+ percentage was around 7.5% in virgin CBA/J mice, similar to the 10.6% in CBA/J×DBA/2 mice on day 5.5 of gestation, but increased dramatically to 23.9% by day 9.5 (P<0.01 vs virgin mice and P<0.05 vs CBA/J×DBA/2 mice on day 5.5), and remained at a higher level (27.5%) until day 13.5. However, this trend was not observed in group F during pregnancy. CONCLUSIONS: The increased CD45+CD86+ percentage on day 9.5, when resorption begins, may support the assumption that CD45+CD86+ cells play a role in the course of embryo resorption. Lymphocyte phenotypic analysis in the lymph nodes that drain the pregnant uterus may be helpful to assess the status of local immunity at the feto-maternal interface.     

Survival and neuronal differentiation of transplanted stem cells derived from embryonic hippocampus in adult rats with stroke lesions

AIM: To study whether exogenous neural stem cells (NSCs) derived from embryonic hippocampus differentiates into the neurons after transplanted into the infarct periphery of the brain in a stroke model and to further investigate the behavioral improvement in the rats.METHODS: The NSCs were prepared after isolated from the embryonic hippocampus of green fluorescent protein (GFP)-transgenic rats and cultured. The NSCs were identified using nestin and doublecortin(DCX) as markers. The cortical infarction in rats was induced using photochemical method, named photothrombotic cortical injury (PCI). Twenty adult rats were randomly divided into NSC transplantation group (NSC group) and control group. The cultured NSCs were transplanted into the infarct periphery of the rats in NSC group, and nothing in control group at first day was applied after PCI. The locomotor behavior of animals was checked using the rotarod test at 1st, 7th, 14th and 21st d after PCI. The survival and differentiation of transplanted NSCs were evaluated by immunocytochemical method at 12th week after PCI.RESULTS: The cells derived from the embryonic hippocampus significantly expressed the markers of NSCs. The grafted cells survived at least 12 weeks in the infarct periphery of adult rats and differentiated into mature glial cells and neurons. The density of NeuN+/GFP+ and volume of grafts at 12th week were less than those at 3rd week after transplantation (P<0.05). The time standing on the rod was longer in NSC group than that in control group at 7th, 14th and 21st d after PCI (P<0.01).CONCLUSION: The NSCs derived from embryonic hippocampus survive in the infarct periphery of adult rats up to 12 weeks and differentiate into mature neurons, which might be associated with the improvement of locomotor behavior of stroke animals. The neuronal replacement of neurons differentiated from NSCs may be the underlying mechanism.     

Cell transplantation combined with transmuscle laser revascularization augments neovascularization in rat ischemia hindlimb

AIM: To determine the combined effect of transmuscle laser revascularization (TMR) and endothelial progenitor cells(EPCs) treatment on ischemic hindlimb of nude rats.METHODS: Mononuclear cells (MNCs) isolated from human umbilical cord-blood (HUCB) by density gradient centrifugation were expanded in vitro. Immunocytochemistry and flow cytometry studies were performed. EPCs were labeled with 1, 1- dioctadecyl-1 to 3, 3, 3, 3- tetramethyl-indocarbocyanine perchlorate (DiI) before injected into the laser induced channels or ischemic region. Acute ischemic limb was created in 4 groups of nude rats by ligating right external iliac artery. All animals were divided randomly into the following four groups: TMR+EPCs group: local transplantation of EPCs into laser channels; TMR group: transmuscular channels were created without EPCs; EPCs group: EPCs were injected into ischemic hindlimb; control group: ischemic model without TMR or EPCs. All rats underwent femoral artery ultrasonic blood flow measurements of the ischemic and nonischemic limbs to obtained a flow ratio ［femoral artery flow index (FAFI): right femoral artery flow /left femoral artery flow］ at baseline (after ligating artery immediately) and 28 days postoperation, and then the samples of ischemic limb muscle underwent histochemical and immunohistologic analysis. RESULTS: The attached cells expressed endothelial cell (ECs) markers (KDR, CD34, CD31, AC133 and von Willebrand factor) and exhibited function similar to that of ECs judged by Ac-LDL incorporation. Flow cytometric analysis disclosed that AT cells were positive for CD34 (62%±7%) and AC133 (57.2%±9.8%) at day 7 of culture. 28 days after therapy, FAFI was significantly higher in the TMR +EPCs (0.66±0.09, P<0.01) and EPCs group (0.59±0.09, P<0.05) compared to control group (0.47±0.05). It was significantly higher in TMR +EPCs-，EPCs- and TMR group compared to baseline (TMR+EPCs group: 0.66±0.09 vs 0.39±0.07, P<0.01; TMR group: 0.54±0.12 vs 0.40±0.09, P<0.05; EPCs group: 0.59±0.09 vs 0.38±0.08, P<0.01; control group: 0.47 ±0.05 vs 0.39±0.08, P>0.05). FAFI in the control group was unchanged and no difference was found between TMR group and control group. TMR+EPCs (5.66±0.77), TMR (4.96±0.31) as well as EPCs (4.68±0.44) treatment resulted in an increased number of capillaries in the treated regional area compared with control group (2.60±0.31, P<0.01).CONCLUSION: Nd: YAG-laser revascularization combined with the application of EPCs transplantation significantly ameliorates perfusion and augments neovascularization in this ischemic hindlimb model.     

Premature response to luteinizing hormone of granulosa cells from women with polycystic ovary syndrome

AIM:To demonstrate the relationship between hormones in follicular fluid and the expression of LH receptor in granulosa cells (GC) in anovulatory women with polycystic ovary syndrome (PCOS). METHODS:Follicles were obtained from 12 women with PCOS and 15 women with normal menstrual period through surgery at time between day 7 and day 10 of menstrual cycle. The accumulations of estrogen (E2), progesterone (P), luteinizing hormone (LH), follicle stimulating hormone (FSH) and insulin in follicular fluid were determined by a automatism chemiluminescent microparticle immunoassay (CMIA) for the quantitative determination. The accumulation of androstenediol (A) was determined by ELISA. The amounts of the mRNA expressions of LH receptors from GC and theca cells (TC) respectively were measured by RT-PCR using β-actin as intra-control simultaneously. RESULTS:The levels of LH ［(3.8±2.1 vs 1.7±0.8)IU/L, P<0.01］, A ［(600.0±373.4 vs 212.4±205.4)μg/L, P<0.05］ and expressions of LH receptor mRNA of GC (0.29±0.16 vs 0.12±0.13, P<0.01) and TC (0.46±0.14 vs 0.34±0.09，P<0.05) in the women of PCOS group were statically higher than those in control group. The expression of LH receptor mRNA was not detected by RT-PCR in control group when the diameter of an follicle was less than 7 mm, while it was detected in women with PCOS when it remained as small as 4 mm. Expression of LH receptor mRNA in granulose cells was positive related to the concentration of LH (r=0.67, P<0.01) and insulin (r=0.51, P<0.05) in follicular fluid, and that in theca cells (r=0.60, P<0.01). CONCLUSION:The high level of LH in follicular fluid occurs and GC responds to LH prematurely and more intensively in anovulatory women with PCOS. Larger amount of A and P was produced as a result. All of above may contribute to the mechanism of anovulatory.     

Murine CD200+CK7+ trophoblasts in poly (I∶C)-induced embryo resorption model

AIM: To investigate the relationship between CD200+CK7+ trophoblasts and the resorption of embryos in a poly (I∶C)-induced abortion model. METHODS: The status of CD200 expression was investigated in Balb/c×C57BL/6 and Balb/c×Balb/c mice as induced model of embryo-resorption by an i.p. injection of poly (I∶C). CD200 expression on CK7+ cells from placentas was detected with flow cytometry. CD200+ cells in placenta were observed with immunocytochemical staining. RESULTS: Both the percentage and absolute number of CD200+CK7+ cells were dramatically decreased by injection of poly (I∶C) in Balb/c×C57BL/6 (6.3%±6.2% vs 36.1%±9.3%, P<0.01; and 140±111 vs 1 941±809, P<0.01), as well as in Balb/c×Balb/c mice (8.5%±4.8% vs 26.1%±8.0%, P<0.01; and 701±499 vs 1 886±1 112, P<0.05), with PBS-treated mice as controls. In addition, the lower cell number was statistically correlated with the elevated embryo-resorption detected on day 13.5: from 9.1% to 37.0% in Balb/c×C57BL/6 mice and from 5.8% to 29.0% in Balb/c×Balb/c mice, respectively (P<0.01). In immunocytochemical analysis, CD200+ cells mainly scattered in placenta tissue adjacent to the interface of placenta and uterine. CONCLUSION: Sufficient expression of CD200 on CK7+ cells at the feto-maternal interface may be necessary for the maintenance of embryos.     

Changes of mitochondrial membrane potential and mitochondrial mass in camptothecin-induced Jurkat cells

AIM: To study the changes of mitochondrial membrane potential (△ψm) and mitochondrial mass in apoptosis of Jurkat cells induced by camptothecin (CPT). METHODS: Jurkat cells were treated with CPT. Annexin V-FITC/propidium iodine (PI) double stainig was used to detected early stage of apoptosis and PI staining for analyzing the cell cycle. Jurkat cells were stained by annexin V-PE/DiOC6(3) to detect changes of △ψm. The mitochondrial mass was measured by cytometry with NAO staining. RESULTS: 6 h after treated with 10 μmol/L CPT, the rate of early apoptotic cells (22.59±1.04)% had significantly difference compared with control group (3.93±0.73)% (P<0.01). The necrotic rate (2.48±0.53)% had no significant difference to that in control group (2.78±0.63)% (P>0.05). Apoptotic peak appeared obviously after treated with CPT, the percentage of late apoptotic cells (13.58±0.97)% had distinctly difference compared with control group (3.18±0.51)% (P<0.01). The cells in G0/G1 phase (48.14±0.96)% were much higher than that in control group (44.09±0.43)% (P<0.01). Mitochondrial depolarization was very obviously in CPT group. The percentage of annexin V+DiOC6 (3)- cells was (19.47±0.69)%, while in control group, was (4.21±0.40)% (P<0.01). Mitochondrial mass in CPT group was significantly lower than that in control group, the percentage of NAO+ cells (74.77±1.66) % had significantly difference compared with control group (92.24±1.41)% (P<0.01). CONCLUSION: During the process of CPT-induced apoptosis in Jurkat cells, mitochondrial depolarization was very obviously and mitochondrial mass decreased, indicating that the process of apoptosis is nearly related to the mitochondrial pathway.     

Differentiation of neural stem cells after transplanted into vitreous and the effects on the regeneration of retina ganglion cells

AIM: To investigate the differentiation of neural stem cells (NSCs) after transplanted into vitreous and the effects on the regeneration of retina ganglion cells (RGCs) after optic nerve microcrushed.METHODS: After optic nerve microcrushed in adult rat,2×10⁴/2 μL NSCs or 2 μL 0.1 mol/L PBS was injected into vitreous.Animals were divided into control group (MC group,MC+PBS group) and experiment group (MC+NSCs).Animals in each group were allowed to survive for 3,4,5 weeks,respectively.The regenerating RGCs were labeled retrogradely with granular blue,and the numbers of regenerating RGCs in each retina were observed under fluorescent microscope.In addition after 5 animals in MC+NSCs group survived for 4 weeks,rat eyeballs were removed and prepared as freezing microtome sections for observing the migration of NSCs and NF,GFAP,CNP immumodetection.RESULTS: Compared the mean numbers of regenerating RGCs between experiment group and control group at 3,4,5 weeks,the difference was significant (P<0.01).NSCs expressed NF,GFAP and CNP at 4 weeks and were not found to incorporate into retina.CONCLUSION: It suggests that NSCs enhance the RGCs regeneration after ON microcrushed and differentiate into neurons,astrocytes and oligodendrocytes.     

Isolation of endothelial progenitor cells from cord blood with CD133 immunomagnetic sorting

AIM: To isolate, purify and differentiate endothelial progenitor cells from cord blood in vitro and to study their biological characteristics. METHODS: CD133+ cells were selected from fresh cord blood mononuclear cells (MNC) by magnetic activated cell-sorting system (MACS). EPC was studied by flow cytometry, immunocytochemistry and immunofluorescence staining. Isolated cells were cultured in IMDM medium supplemented with or without VEGF, bFGF, SCF. RESULTS: The percentage of CD133+ cells of cord blood MNC was (1.41±1.14)%, and purity was 75%-85% (FACS method). CD133+ cells were grown on fibronectin-coated chamber slides in the presence of VEGF, bFGF, SCF. Within 1-2 hours of culture cells became adherent. On day 7-10, the adherent cells displayed a typical “cobblestone” morphology. After 14 days of culture, the adherent cells revealed a heterogeneous cell population, comprising small-sized round cells, spindle-like cells and formed tube-like structure. Weibel-Palade bodies were shown on the transmission electron microscopy photomicrographs. Compared with the original, cell markers CD133 and CD34 decreased significantly (77.0%±3.3% to 1.6%±2.2% and 93.1%±4.7% to 37.4%±4.9%, P<0.05), while Flk-1 increased significantly (from 22.3%±3.3% to 94.3%±4.1%, P<0.05) after 14 days of culture with VEGF, bFGF, SCF. The vWF was strongly expressed (77.9%±3.3%) on the 14th day later. CONCLUSION: Vascular endothelial progenitor cells were isolated from cord blood with specific expression of CD133/CD34/Flk-1. With the stimulation of the growth factors, seven-ten days after culture EPCs could be turned to endothelial cells.     

Protective effect of deferoxamine on hypoxic-ischemic encephalopathy in neonatal rat

AIM:To investigate the possible mechanism of deferoxamine on angiogenesis in rat hypoxic-ischemic encephalopathy (HIE). METHODS:SD rats (7 days of age) were used to make HIE model. Model group and treatment group were injected with deferoxamine or normal saline alone 24 hours before hypoxic-ischemic insult. Rats were sacrificed at 1,3,7 or 14 days after hypoxic-ischemic insult. Brain capillary density index (BCDI),the number of proliferating capillary,brain water content and extent of brain atrophy were determined. The expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α(HIF-1α) mRNA was measured. RESULTS:Early water content and late atrophic ratio of the left brain were significantly improved in the treatment group compared to model group (P<0.01). The number of proliferating capillary in the treatment group was significantly higher than that in the model group ［(2.01±0.31)/HPF vs　(0.90±0.25)/HPF,P<0.01］. Deferoxamine markedly up-regulated the expression of VEGF and HIF-1α mRNA in the brain ［VEGF at 12 h: (1.41±0.07) vs (1.10±.15),P<0.05; HIF-1α at 12 h: (1.49±0.12) vs (1.11±0.16),P<0.05］.CONCLUSION:Deferoxamine may promote angiogenesis and attenuate hypoxic-ischemic induced brain injury via up-regulation of HIF-1α and VEGF expression.     

Effect of proliferating cell nuclear antigen specific antisense oligonucleotide on differentiation of cord blood CD34+ cells

AIM:To study the effect of proliferating cell nucler antigen antisense oligonucleotide on ex vivo expansion of cord blood CD34+ hematopoietic stem/progenitor cells. METHODS:CD34+ cells were purified from fresh cord blood by immunomagnetic beads. CD34+ cells were incubated in liquid culture system with different concentrations of PCNA-ASODN. Using flow cytometry, the number of different kinds of stem/progenitor cells and PCNA expression were measured after CD34+ cell incubation. RESULTS:PCNA was lowly expressed in low experiential group, with a positive rate of (27.2±3.6)% and (19.0±1.5)%, the positive rate of control group was (53.8±8.3)% (P<0.01), a high significant difference was observed. Just in low concentration of PCNA-ASODN, the percentage of CD34+cells were increased to (33.4±3.2)%, CD34+CD38-cells expanded (57.8±9.9) folds, and the percentage of CD34+cells were (25.2±2.6)% (P<0.01), the CD34+CD38-cells expanded (43.5±7.4) folds (P<0.05), it has significant difference compared with the control group. CONCLUSION:Low concentration of PCNA-ASODN decreases PCNA expression effectively, slows down differentiation of CD34+cells during ex vivo expansion procedure, and improves the expansion efficiency.     

Phosphatidylinositol 3-kinase regulates hypoxia-inducible factor 1α in pulmonary arteries of rats with hypoxia-induced pulmonary hypertension

AIM:To investigate relationship among phosphatidylinositol 3-kinase (PI3K) and hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) in lung of rats with hypoxia-inducible pulmonary hypertension. METHODS:Forty male adult Wistar rats were randomly divided into five groups (eight rats in each group):control group (C group) and groups with hypoxia for 3, 7, 14 and 21 days (H3, H7, H14 and H21 group). Mean pulmonary arterial pressure (mPAP), right ventric hypertrophy index (RVHI) and vessel morphometry were measured. The levels of HIF-1α mRNA expression in lung tissue was measured by in siteu hybridization (ISH). The protein expression of HIF-1α，VEGF and phosphorylated protein kinase β (P-AKT) were observed by immunohistochemistry or Western blotting. RESULTS:mPAP increased significantly 7 days after hypoxia ［(23.53±1.78) mmHg］, peaked 14 days after hypoxia, then remained on the high level. Pulmonary artery remodeling index (extern diameter 100 μm) and RVIH became evident 14 days after hypoxia. Expression of P-AKT protein in control group was poorly positive, but was up-regulated in pulmonary arterial tunica intima and tunica media in all hypoxia rats. HIF-1α mRNA staining was poorly positive in control，hypoxia for 3 days and hypoxia for 7 days, but began to increase significantly 14 days after hypoxia (0.305±0.104, P<0.05), then remained stable. Expression of HIF-1α protein in control group was poorly positive, but was up-regulated in pulmonary arterial tunica intima in all hypoxic rats. In pulmonary arterial tunica media, the levels of HIF-1α protein was markedly up-regulated after 3 days (0.029±0.019, P<0.05 ), reached its peak 7 days after hypoxia (0.232±0.008, P<0.05), then tended to decline 14 days and 21 days after hypoxia. Expression of VEGF protein began to increase 7 days after hypoxia (0.188±0.018, P<0.05), reached its peak 14 days after hypoxia (0.238±0.017, P<0.05), then remained on the high level in pulmonary arterial tunica intima. Linear correlation analysis showed that P-AKT, HIF-1α mRNA, VEGF and mPAP were correlated with vessel the morphometry and RVHI (P<0.01). P-AKT was positively correlated with HIF-1α and VEGF (tunica intima). CONCLUSION:P-AKT, HIF-1α and VEGF are all involved in the pathogenesis of hypoxia-induced pulmonary hypertension in rats.     

Effects of hypothalamic supraoptic ADH neurons at different times after hypophysectomy in Wistar rats

AIM:To observe the effects of supraoptic ADH neurons and central diabetes insipidus (CDI) in Wistar rats at different times after hypophysectomy.METHODS:Hypophysectomy was undergone by stereotaxic instrument.Water intake,urine output and urine specific gravity (SG) were observed each day.The survival rate of supraoptic ADH neurons was determined by immunofluorescence after hypophysectomy at different times.RESULTS:The rats manifested triphasic CDI after hypophysectomy.The average water intake in experiment group was 73.9 mL vs 30.9 mL in control group (P<0.01).Urine output was 59.7 mL vs 14.7 mL (P<0.01) and SG was 1.015 vs 1.036 (P<0.01).There was minor decrease in ADH neurons at the 3rd day after hypophysectomy (survival 94%,P>0.05),but the cellular body hypertrophy appeared.The survival rate at 10th day was 72％(P<0.01),31% at 20th day and 29% at 30th day.There was no difference between the 20th and 30th day (P>0.05),but they were all less than the 10th day (P<0.01).The ADH immunoreactivity enhanced in median eminence (ME) at the 20th day.CONCLUSION:The triphasic CDI appeard and supraoptic ADH neurons degenerated but the function of those survival neurons had compensatory enhancement after hypophysectomy.     

Effects of interleukin-10 on the calcineurin activity in cultured cardiac fibroblasts induced by arginine vasopressin

AIM: The purpose of the present study was to investigate the effect of interleukin-10 (IL-10) on the proliferation and calcineurin (CaN) activity in cultured cardiac fibroblasts (CFs) induced by arginine vasopressin (AVP).METHODS: The CFs of left ventricle in neonatal Sprague-Dawley rats were isolated and cultured by trypsin digestion and selective plating technique. Then the proliferation rates of cells were determined by using the MTT ［3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide］ assay (A490 value). Cell cycle distribution was determined with flowcytometry technique. The CaN activity was measured by ultra-violet spectrophotography.RESULTS: (1) MTT colorimetry showed that 10-7 mol/L AVP significantly increased A490 value of CFs in comparison with control group (P<0.01). IL-10 attenuated the A490 value of AVP group in a concentration dependent manner. The A490 value of the 10-8, 10-7, 10-6, 10-5 g/L IL-10+10-7 mol/L AVP groups was 0.201±0.007, 0.187±0.006, 0.173±0.010 and 0.157±0.029 respectively, all data significantly lower than those in the presence of AVP alone (P<0.05 or P<0.01). (2) The percentage of the cells in S stage and proliferation index were markedly increased in 10-7mol/L AVP group compared with the control (P<0.01, respectively). In the 10-6 g/L IL-10+10-7 mol/L AVP group, the percentage cells in S stage and proliferation index were significantly lower than those in AVP group (P<0.01, respectively). IL-10 itself had no effect on fibroblast proliferation, but reduced AVP-induced fibroblast proliferation. (3) There was a significantly increase in CaN activity in AVP group compared with control (P<0.01). In the 10-8, 10-7, 10-6 and 10-5 g/L IL-10＋10-7 mol/L AVP groups, the CaN activity was 3.22±0.04, 3.06±0.06, 2.53±0.04 and 2.22±0.04, respectively. IL-10 dose-dependently down-regulated the CaN activity induced by AVP (P<0.01, respectively). However, the CaN activity was still higher in IL-10+AVP group than that in control group (P<0.05 or P<0.01).CONCLUSION: Our data indicate that IL-10 regulates the CaN activity of CFs in the cell proliferation induced by AVP, suggesting that IL-10 plays a role in the regression of cardiac remodeling.     

Effect of microcapsulated catechin on expression of vascular endothelial growth factor in rats with adriamycin induced-nephrotic syndrome

AIM: To study the effect of microcapsulated catechin on vascular endothelial grower factor (VEGF) expression in rats with adriamycin induced-nephrotic syndrome.METHODS: 120 female SD rats were randomly distributed in control group,nephrotic group,dexamethason group,vitamin E group,catechin group and microcapsule group.Rat with nephrotic syndrome were induced by injection of adriblastine (5 mg/kg BW).VEGF concentrations in serum and urine were detected by ELISA assay.VEGF expression in kidney was measured by immunohistochemistry assay.RESULTS: At the end of 4th week and 6th week,VEGF concentration in other groups in kidney,serum and urine were higher than that in control (all P<0.01),and were lower than that in nephritic group (exclude Vit E group,all P<0.01).Serum and urine VEGF concentrations in microcapsule group were lower than those in catechin group (P<0.01,P<0.05,respectively).VEGF expression in microcapsule group in kidney was lower than that in catechin group,but it was not significant different.Urinary protein excretion at 24 h in microcapsule group was lower than that in catechin group (P<0.05),there was positive correlation among urine protein,serum VEGF level,urine VEGF concentration and renal VEGF expression at 24 h (all P<0.01).CONCLUSION: Catechin decreases urinary protein excretion in the rats with nephrotic syndrome through reducing the expression and secretion of VEGF.Microcapusulated catechin is benefit in decreasing the expression and secretion of VEGF.