Role of uterine natural killer cells in angiogenesis of human decidua of the first-trimester pregnancy

Decidualization is accompanied by extensive angiogenesis, which is an essential step in the maturation of new blood vessels in mammalian pregnancy. The purpose of this study was to determine a distribution of uNK cells (CD56 uNK or CD56bright cells) in human decidua of the first-trimester pregnancy, and investigate whether uNK cells in human decidua could express vascular endothelial growth factor (VEGF-A) and/or angiopoietin2 (Ang2). Our immunohistochemical staining results demonstrated that a great amount of uNK (CD56 ) cells scattered throughout the decidual stroma and near endometrial gland and spiral vessels in human decidua. The protein expression of VEGF-A and Ang2 was detected in decidual stroma cells, capillary endothelial cells and glandular cells in tissue specimens. There was a positive correlation between microvessel density (MVD) and the number of the CD56-positive uNK cells in decidual stroma, and also between the number of the CD56-positive uNK cells and VEGF-A protein expression in the tissue. In addition, we found that uNK cells in human decidua could express VEGF-A mRNA, but not Ang2 mRNA, in isolated uNK cells in human decidua of the first-trimester gestation by combination of LCM and Nested-PCR. Our study indicated that uNK cells, through expressing VEGF-A, may play an important role in the angiogenic response at the time of human decidualization and early placenta development.     

β-EP和胃泌素在大鼠舌下腺的免疫组织化学定位

In order to study the distribution of β-EP and gastrin immunoreactive cells in the sublingual gland, we used the Advidin Biptin-Peroxidase Complex (ABC) immunohistochemical technique to detect the expression of β-EP and gastrin in rat sublingual glands. The results show that the epithelial cells of striated ducts and interlobular ducts appeared β-EP and gastrin positive in immunoreaction and the mucosus and serous acinar cells were immunoreactive negative. The poei-tive substances were distributed in the cytoplasm with negative nuclei. These results suggest that β-EP and gastrin are ex-pressed in the epithelial cells of ducts in the rat sublingual gland, which may play an important role in the secretion and functional regulation of the sublingual gland.     

Establishment of a novel corneal endothelial cell line from domestic rabbit, Oryctolagus curiculus

To develop a rabbit corneal endothelial (RCE) cell line, in vitro culture of RCE cells was initiated from Oryctolagus curiculus corneas and a novel RCE cell line was established in this study. To initiate the primary culture of RCE cells, corneas from rabbit eyes were sliced and attached into glutin-coated wells with endothelial cell surface down. After being cultured at a time-gradient interval from 48 to 6 h, the corneal slices were detached and reattached into new wells, respectively. Cells in the wells containing only a pure population of RCE cells were collected and cultured in 20% FBS-DMEM/F12 medium con- taining chondroitin sulfate, ocular extract, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), carboxymethyl-chitosan, N-acetylglucosamine hydrochloride, glucosamine hydrochloride, culture medium of rabbit corneal stromal cells and oxidation-degradation products of chondroitin sul- fate at 37℃, 5% CO2. The cultured RCE cells, in quadrangle and polygonal shapes, proliferated to con- fluence 3 weeks later. During the subsequent subculture, the shape of RCE cells changed gradually from polygonal to more fibroblastic. A novel RCE cell line, growing at a steady rate, with a population doubling time of 53.8 h, has been established and subcultured to passage 67. Chromosome analysis showed that the RCE cells exhibited chromosomal aneuploidy with the modal chromosome number of 44. The results of immuno-cytochemical staining with neuron specific enolase (NSE) confirmed that the RCE cells were in neuroectodermal origin. Combined with the results of vascular endothelial growth factor (VEGF) treatment and endothelial cell morphology recovery, it can be concluded that the cell line established here is an RCE cell line. This RCE cell line may serve as a useful tool in theoretical re- searches of mammalian corneal endothelial cells, and may also have potential application in artificial corneal endothelium development.     

Effects of adeno-associated virus (AAV) of transforming growth factors β1 and β3 (TGFβ1,3) on promoting synthesis of glycosaminoglycan and collagen type II of de-differentiated nucleus pulposus (NP) cells

The effects of AAV-TGFβ1 and AAV-TGFβ3 on promoting synthesis of glycosaminoglycan and collagen type Ⅱ of dedifferentiated rabbit lumbar disc NP cells were studied in this work. The rabbit lumbar disc NP cells were isolated and cultured. The earlier and later dedifferentiated NP cells were established by subculture. The AAV transfection efficiency to dedifferentiated NP cells was analyzed with AAV-EGFP in vitro. After dedifferentiated NP cells were transfected by AAV-TGFβ1 or AAV-TGFβ3, their biological effects on promoting synthesis of glycosaminoglycan or collagen type Ⅱ were detected and compared by the methods of 35S incorporation or immunoblotting. The experimental results showed that AAV could transfect efficiently the earlier dedifferentiated NP cells, but its transfection rate was shown to be at a low level to the later dedifferentiated NP cells. Both AAV-TGFβ1 and AAV-TGFβ3 could promote the earlier dedifferentiated NP cells to synthesize glycosaminoglycan and collagen type Ⅱ, and the effect of AAV-TGFβ1 was better than that of AAV-TGFβ3. For the later dedifferentiated NP cells, the AAV-TGFβ3 could promote their synthesis, but AAV-TGFβ1 could slightly inhibit their synthesis. Therefore, AAV-TGFβ1 and AAV-TGFβ3 could be used for the earlier dedifferentiated NP cells, and the TGFβ3 could be used as the objective gene for the later dedifferentiated NP cells.     

Comparison of two in vitro angiogenesis assays for evaluating the effects of netrin-1 on tube formation

Netrin-1 is a neural guidance cue that also regulates vascu- lar development. Controversial results, however, have been obtained concerning the roles of netrin-1 in vascular devel- opment both in vivo and in vitro. In the present study, two in vitro angiogenesis assays were compared to evaluate the effects of netrin-1 secreted by retrovirally transduced mel- anoma cells （Mel2a-netrinl） on tube formation. The results showed that there was no obvious difference in tube forma- tion induced by conditioned media （CM） from the control, Mel2a-netrinl and Mel2a cells in a matrigel assay. The results of another in vitro assay, in which endothelial cells were co-cultured with human fibroblasts, however, showed that Mel2a-netrinl CM inhibited the tube formation, sup- posedly through blocking the elongation and coalescence of human umbilical vein endothelial cells （HUVECs）. These results confirmed that the matrigel assay is not able to demonstrate the anti-angiogenic roles of netrin-1.     

Collagen scaffolds modified with collagen-binding bFGF promotes the neural regeneration in a rat hemisected spinal cord injury model

Nerve conduit is one of strategies for spine cord injury(SCI)treatment.Recently,studies showed that biomaterials could guide the neurite growth and promote axon regeneration at the injury site.However,the scaffold by itself was difficult to meet the need of SCI functional recovery.The basic fibroblast growth factor(bFGF)administration significantly promotes functional recovery after organ injuries.Here,using a rat model of T9 hemisected SCI,we aimed at assessing the repair capacity of implantation of collagen scaffold(CS)modified by collagen binding bFGF(CBD-bFGF).The results showed that CS combined with CBD-bFGF treatment improved survival rates after the lateral hemisection SCI.The CS/CBD-bFGF group showed more significant improvements in motor than the simply CS-implanted and untreated control group,when evaluated by the 21-point Basso-Beattie-Bresnahan(BBB)score and footprint analysis.Both hematoxylin and eosin(H&E)and immunohistochemical staining of neurofilament(NF)and glial fibrillary acidic protein(GFAP)demonstrated that fibers were guided to grow through the implants.These findings indicated that administration of CS modified with CBD-bFGF could promote spinal cord regeneration and functional recovery.     

Dihydrotanshinone I inhibits angiogenesis both in vitro and in vivo

Dihydrotanshinone I （DI）, a naturally occurring compound extracted from Salvia miltiorrhiza Bunge, has been reported to have cytotoxicity to a variety of tumor cells. In this study, we investigated its anti-angiogenic capacity in human umbilical vein endothelial cells. DI induced a potent cytotoxicity to human umbilical vein endothelial cells, with an IC50 value of approximately 1.28 μg/ml.At 0.25-1μg/ml, DI dose-dependently suppressed human umbilical vein endothelial cell migration, invasion, and tube formation detected by wound healing, Transwell invasion and Matrigel tube formation assays, respectively. Moreover, DI showed significant in vivo anti-angiogenic activity in chick embryo chorioailantoic membrane assay. DI induced a 61.1% inhibitory rate of microvessel density at 0.2 μg/egg. Taken together, our results showed that DI could inhibit angio-genesis through suppressing endothelial cell proliferation, migration, invasion and tube formation, indicating that DI has a potential to be developed as a novel anti-angiogenic agent.     

Endothelial progenitor cells in debate

Adult stem/progenitor cells play important roles in tissue homeostasis and have important implications for regenerative medicine. It was once thought that formation of new blood vessels in adult only occurs through angiogenesis, a process whereby new vessels are formed from existing mature endothelial cells; while vasculogenesis, where new vessels are derived from differentiation of endothelial progenitor cells （EPCs）, was thought to occur exclusively in embryos. The discovery of adult EPCs a few years ago has changed this old paradigm; and subsequent studies showed that EPCs may be a promising tool for the treatment of vascular disorders. However, there have been conflicting reports on subtypes, surface markers, and functions of EPCs; and thus the exact origin and identity of EPCs remain to be defined. A common approach to obtain EPCs is to isolate and culture mononuclear cells from peripheral blood and to select adherent cells for further analyses. In the June issue of Cell Research, Zhang et al. attempted to isolate putative ＂EPCs＂ using this approach,     

Application of the sodium hyaluronate-CNTF scaffolds in repairing adult rat spinal cord injury and facilitating neural network formation

The present study aimed to explore the potential of the sodium hyaluronate-CNTF (ciliary neurotrophic factor) scaffold in activating endogenous neurogenesis and facilitating neural network re-formation after the adult rat spinal cord injury (SCI). After completely cutting and removing a 5-mm adult rat T8 segment, a sodium hyaluronate-CNTF scaffold was implanted into the lesion area. Dil tracing and immunofluorescence staining were used to observe the proliferation, differentiation and integration of neural stem cells (NSCs) after SCI. A planar multielectrode dish system (MED64) was used to test the electrophysiological characteristics of the regenerated neural network in the lesioned area. Electrophysiology and behavior evaluation were used to evaluate functional recovery of paraplegic rat hindlimbs. The Dil tracing and immunofluorescence results suggest that the sodium hyaluronate-CNTF scaffold could activate the NSCs originating from the spinal cord ependymal, and facilitate their migration to the lesion area and differentiation into mature neurons, which were capable of forming synaptic contact and receiving glutamatergic excitatory synaptic input. The MED64 results suggest that functional synapsis could be established among regenerated neurons as well as between regenerated neurons and the host tissue, which has been evidenced to be glutamatergic excitatory synapsis. The electrophysiology and behavior evaluation results indicate that the paraplegic rats’ sensory and motor functions were recovered in some degree. Collectively, this study may shed light on paraplegia treatment in clinics.     

Polycomb chromobox 4 enhances migration and pulmonary metastasis of hepatocellular carcinoma cell line MHCC97L

We recently report that the expression of polycomb chromobox 4(Cbx4)is significantly correlated with the overall survival of a great cohort of hepatocellular carcinoma(HCC)patients and it enhances hypoxia-induced vascular endothelial growth factor(VEGF)expression and angiogenesis in HCC cells through enhancing sumoylation of hypoxia inducible factor-1alpha(HIF-1α).Here we continue to investigate the potential effects of Cbx4 on the migration and metastasis of the metastatic HCC cell line MHCC97L.Our results show that Cbx4 overexpression in the cell line increases the in vitro vessel formation of vascular endothelial cells in its SUMO interaction motifs-dependent manner,and promotes the in vitro migration of the cancer cell,which can be effectively abrogated by anti-VEGF antibody.Although Cbx4 expression does not impact the in vitro growth of MHCC97L cells,it still promotes the progression and metastasis of orthotopically transplanted tumors in nude mice.These results further support the role of Cbx4 as a SUMO E3 ligase in the progression and metastasis of HCC.     

Identification of microRNA transcriptome reveals that miR-100 is involved in the renewal of porcine intestinal epithelial cells

MicroRNAs play important roles in various cellular processes, including differentiation, proliferation and survival. Using a pig model, this study sought to identify the miRNAs responsible for crypt-villus axis renewal of the small intestinal epithelium.Compared to the villus upper cells, there were 15 up-regulated and 41 down-regulated miRNAs in the crypt cells of the jejunum.Notably, we found that miR-100 was expressed more in the villus upper cells than in the crypt cells, suggesting an effect on intestinal epithelium differentiation. Overexpression of miR-100 increased the activity of alkaline phosphatase, confirming that miR-100 promoted IPEC-J2 cell differentiation. MiR-100 can inhibit cell proliferation as evidenced by CCK-8 and cell cycle assay results. We also showed that miR-100 significantly inhibited the migration of IPEC-J2 cells and promoted cell apoptosis through caspase-3-dependent cleavage of Bcl-2. Furthermore, FGFR3 was identified as a potential target of miR-100 by bioinformatics analysis. We confirmed that overexpression of miR-100 suppressed FGFR3 expression in IPEC-J2 cells by directly targeting the FGFR3 3′-UTR. This is the first report of miRNAs acting on the renewal of the intestinal crypt-villus axis.Our results also showed that miR-100 promotes the differentiation and apoptosis, and inhibits the proliferation and migration of enterocytes of pigs.     

Retinal ganglion cells of high cytochrome oxidase activity in the rat

Retinal ganglion cells in the rat were studied using the heavy metal intensified cytochrome oxidase and horseradish peroxidase histochemical methods.The results show that a population of large retinal ganglion cells was consistently observed with the cytochrome oxidase staining method in retinas of normal rats or rats which received unilateral thalamotomy at birth.These cytochrome oxidase rich ganglion cells appeared to have large somata,3-6 primary dendrites and extensive dendritic arbors,and are comparable to ganglion cells labeled by the wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP).However,the morphological details of some of the cells revealed by the cytochrome oxidase staining method are frequently better than those shown by the HRP histochemical method.These results suggest that the mitochondrial enzyme cytochrome oxidase can be used as a simple but reliable marker for identifying and studying a population of retinal genglion cells with high metabolic rate in the rat.     

Substance P enhances the proliferation of rat anterior pituitary cells in vitro

The undecapeptide substanceP(SP) was shown to be intimately involved in both the structural and functional aspects of the anterior pituitary.Yet,in addition to its influences on hormonal secretion,SP may well possess more actions in this master gland.The present study was ftherefore aimed to investigate the effect of SP on the proliferation of rat anterior pituitary cells in primary culture,It was found that SP could dose-dependently increase the incorporation of tritiated thymidine(3H-TdR) into cultured anterior pituitary cells.Other mammalian tachykinins such as neurokinin A and neurokinin B had similar effect but to varying degrees.The equipotent analogue of SP,Norleucine^11-SP(Nle^11-SP),also acted likewise.with its action antagonizable by spantide,a SP receptor blocker.To further characterize the nature of cells responsive to the challenge of SP,immunocytochemical staining against S-100 protein and some adenohypophyseal hormones was performed alone or plus autoradiography.The results showed that the percentage of S-100 proteinimmunorective cells was apparently elevated by the addtion of Nle^11-Sp for 48h,which indicates a preferential proliferation of folliculo-stellate cells under the regime .This was confirmed by increases in immunocytochemical or autoradiographical labelling indices of anterior pituitary Substance P and anterior pituitary cell proliferation.Cells treated similarly.Taken together,These results reveal that the trophic action of SP observed previously in other tissues is also present at least in cultured rat anterior pituitary cells.with responding cells being predominantly folliculo-stellate cells as typified by S-100 proteinimmunoreactivity.Therefore,an intra-pituitary trophicaction of SP in vivo could be anticipated.     

A Preliminary Study of the Noninvasive Remote Control System for Rat Bio-Robot

<正> A system is described here that can noninvasively control the navigation of freely behaving rat via ultrasonic,epidermaland LED photic stimulators on the back.The system receives commands from a remote host computer to deliver specifiedelectrical stimulations to the hearing,pain and visual senses of the rat respectively.The results demonstrate that the three stimuliwork in groups for the rat navigation.We can control the rat to proceed and make right and left turns with great efficiency.Thisexperiment verified that the rat was able to reach a setting destination in the way of cable with the help of a person through theappropriate coordination of the three stimulators.The telemetry video camera mounted on the head of the rat also achieveddistant image acquisition and helped to adjust its navigation path over a distance of 300 m.In a word,the non-invasive motioncontrol navigation system is a good,stable and reliable bio-robot.