Netrin-1 promotes the vasculogenic capacity of human adipose-derived stem cells.

Adipose derived stem cells (ADSCs) have been increasingly explored for use in cell-based therapy against ischemic diseases. However, unsatisfactory angiogenesis limits the therapeutic efficacy. Netrin-1, a known axon guidance molecule, improves neovascularization in the ischemic region. Thus, our study was performed to evaluate the potential effect of Netrin-1 on the angiogenic behaviors of human ADSCs (hADSCs). hADSCs acquired from human abdominal adipose tissue were modified by liposome transfection of Netrin-1 plasmid, and the proliferation of hADSCs was determined by Cell Counting Kit-8 (CCK-8) assay. The transcript levels of pro-invasive proteins such as matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP-9), were measured to test migratory and invasive capabilities, and the levels of vascular endothelial growth factors were assayed to monitor angiogenic activity. Our results showed that Netrin-1 overexpression enhanced the proliferation of hADSCs, and promoted the migration and invasion of hADSCs, as indicated by increased levels of MMP-2 and MMP-9. Furthermore, Netrin-1 overexpression increased the expression of vascular endothelial growth factor and placental growth factor in hADSCs. Our results highlighted the possibility that genetic modification of hADSCs by Netrin-1 overexpression might be beneficial for cell transplantation therapy against ischemic diseases.

Decline of three farmland pest species in rapidly urbanizing landscapes.

Urbanization is a pressing challenge for earth's humans because it is changing not only natural environments but also agricultural lands. Yet, the consequences of cropland loss on pest insect populations that largely depend on these habitats remain largely unclear. We used a 17-year data set to investigate the dynamics of three moth pest species (i.e., striped stem borer, yellow stem borer, and pink stem borer) and their driving forces across the largest mega-urban region of China. Total abundance of three pest species is declined by about 80%, which was strongly associated with cropland loss during rapid urbanization. Our findings indicate that not only the increasing conversion of natural areas to human-dominated landscapes but also that of agricultural lands to urban landscapes can be critical to insect populations. It is therefore essential to monitor and understand the insect dynamics in rapidly urbanizing regions, which are currently found in many developing countries worldwide.

Challenges for Obstetricians and the Countermeasures of COVID-19 Epidemic.

Since the outbreak of the novel coronavirus in Wuhan, China, as obstetricians, we also face great challenges. We need to identify pregnant patients with 2019 coronavirus disease infection timely, and give them appropriate treatment in order to obtain a good maternal and infant prognosis. Here, we would like to share a case and provide some suggestions on how to screen, diagnose and treat pregnant women with 2019 coronavirus disease infection during the outbreak.

Conditioned Medium from Human Umbilical Vein Endothelial Cells Promotes Proliferation, Migration, Invasion and Angiogenesis of Adipose Derived Stem Cells.

Preeclampsia (PE) is a pregnancy-specific hypertensive complication, closely related to endothelial dysfunction. Adipose derived stem cells (ADSCs) have the capacity to differentiate into endothelial cells for vascular repair. Therefore, we hypothesized that induced endothelial differentiation of ADSCs might hold great potential for the treatment of PE. In this study, the primary ADSCs and human umbilical vein endothelial cells (HUVECs) were isolated by the collagenase digestion method. The supernatant of HUVECs was collected from the first generation of cells. Then, ADSCs were divided into two groups: ADSCs alone group and induced ADSCs (iADSCs) group. In iADSCs group, ADSCs were induced by HUVECs conditioned medium and ADSCs special culture medium at a ratio of 1:1 over a two-week period. In order to identify the endothelial characteristics of iADSCs, CD31 and CD34 were examined by flow cytometry. The proliferation, migration, invasion and angiogenesis assays were employed to compare the bioactivity of iADSCs and ADSCs. Furthermore, The levels of angiogenic related factors including vascular endothelial growth factor (VEGF) and placenta growth factor (P1GF) were detected by RT-PCR and Western blotting. Results showed conditioned medium from HUVECs promoted ADSCs proliferation, migration, invasion and angiogenesis. In addition, the levels of VEGF and P1GF were significantly enhanced in iADSCs group. This study uncovered the iADSCs application potential in the therapy and intervention of PE.

[In vivo and in vitro research study of the influence on placenta angiogenesis by gene silencing of netrin-1].

OBJECTIVE: To study influence on angiogenesis of placenta by gene silencing of netrin-1. METHODS: Netrin-1 gene in human umbilical vein endothelial cells (HUVEC) and placenta of pregnant rats were silenced by RNA interference. The following methods were used in this study, including the phenytetrazoliumromide (MTT) for viability, clone formation for proliferation, transwell for migration, and tube formation for angiogenesis in vitro. The change of fetal growth was recorded. Placental microvessel density in pregnant rats was measured by immunohistochemical CD(34) staining in vivo. RESULTS: (1) HUVEC: viability and proliferation of HUVEC were remarkably inhibited by gene silencing of netrin-1, which number of clone formation, migration cell, tube formation were from (69 ± 6)%, 86 ± 17, 37 ± 9 decreased to (46 ± 5)%, 46 ± 13 and 17 ± 5 (P < 0.05) respectively. (2) Placenta of pregnant rats: after netrin-1 gene silenced, fetal weight were decreased from (2.39 ± 0.17) g to (2.12 ± 0.10) g (P < 0.05). Placental microvessel density was decreased from (258 ± 38)/mm(2) to (197 ± 32)/mm(2) in vivo (P < 0.05). CONCLUSIONS: Gene silencing of netrin-1 could inhibit viability, proliferation, migration, tubal formation of HUVEC and angiogenesis of placenta. Netrin-1 plays an important role in regulating angiogenesis in placenta.

[Correlation of netrin-1 expression with invasion of extra villous trophoblasts].

OBJECTIVE: To investigate mechanism of netrin-1 regulating invasion of extra villous trophoblasts. METHODS: RT-PCR was used to detect six receptors expression including UNC5A, UNC5B, UNC5C, UNC5D, DCC and neogenin in extra villous trophoblast cell line TEV-1. The TEV-1 cells were cultured and devided into seven groups according to the concentration of netrin-1 adding into the medium, which include 10 microg/L, 50 microg/L, 100 microg/L, 500 microg/L, 1000 microg/L, 5000 microg/L and the control (the concentration of netrin-1 was 0 microg/L)groups. The proliferation and invasion of TEV-1 induced by netrin-1 were determined by CCK-8 assay and transwell invasion assay respectively. RESULTS: (1) Only neogenin and UNC5B were found to be expressed on TEV-1 by RT-PCR method. (2) In CCK-8 proliferation assay, after 72 hours culture, the proliferation of TEV-1 were 1.55 +/- 0.29 in 10 microg/L, 1.72 +/- 0.31 in 50 microg/L, 2.15 +/- 0.35 in 100 microg/L, 1.42 +/- 0.25 in 500 microg/L, 1.50 +/- 0.27 in 1000 microg/L, and 1.38 +/- 0.23 in 5000 microg/L group, which were all higher than 1.00 +/- 0.16 in control group significantly (P < 0.05). (3) In matrigel invasion assay, after 6 hours culture, the number of the trans-membrane cells in various netrin-1 group, including 41 +/- 4 in 10 microg/L, 47 +/- 5 in 50 microg/L, 55 +/- 6 in 100 microg/L, 44 +/- 5 in 500 microg/L, 43 +/- 5 in 1000 microg/L and 42 +/- 5 in 5000 microg/L group, were all higher than 30 +/- 4 in control group with statistical significance (P < 0.05). (4) The fold changes of neogenin were 1.50 +/- 0.16 in 10 microg/L, 1.83 +/- 0.19 in 50 microg/L, 2.24 +/- 0.25 in 100 microg/L, 2.12 +/- 0.24 in 500 microg/L, 2.12 +/- 0.23 in 1000 microg/L and 2.13 +/- 0.23 in 5000 microg/L group, which were all higher than 1.00 +/- 0.11 in control group significantly (P < 0.05). There were significant difference between group 10 microg/L and 50 microg/L, group 50 microg/L and 100 microg/L (P < 0.05). There were no significant difference between group 100 microg/L and 500 microg/L, group 1000 microg/L and 5000 microg/L (P > 0.05). (5) The fold changes of UNC5B 1.09 +/- 0.11 in 10 microg/L, 1.47 +/- 0.14 in 50 microg/L, 1.61 +/- 0.16 in 100 microg/L, 1.85 +/- 0.19 in 500 microg/L, 2.21 +/- 0.21 in 1000 microg/L and 2.42 +/- 0.23 in 5000 microg/L group, were all higher significantly when compared with 1.00 +/- 0.07 in control group (P < 0.05). There were significant difference between all groups (P < 0.05). CONCLUSION: Netrin-1 can promote the potential of proliferation and invasion of extravillous trophoblasts in vitro through its receptors including neogenin and UNC5B.

Effects of Smac gene over-expression on the radiotherapeutic sensitivities of cervical cancer cell line HeLa.

BACKGROUND: The second mitochondria-derived activator of caspases (Smac) is a novel proapoptotic gene, which plays an important role in the apoptosis-inducing effects of irradiation on tumor cells. The purpose of this study was to investigate the effects of extrinsic Smac gene transfer and its over-expression in radiotherapeutic sensitivities of cervical cancer cells. METHODS: After the Smac gene was transferred into the cervical cancer cell line HeLa, subcloned cells were obtained by persistent G418 selection. Cellular Smac gene expression was detected by RT-PCR and Western blot, while in vitro cell viabilities were detected by trypan blue staining assay. After treatment with X-ray irradiation, cellular radiotherapeutic sensitivities were investigated by tetrazolium bromide colorimetry. Cellular apoptosis and its rate were determined by electronic microscopy, annexin V-FITC and propidium iodide staining flow cytometry. The expression and activities of cellular caspase-3 were assayed by Western blot and colorimetry. RESULTS: Smac mRNA and protein levels in HeLa/Smac cells and the selected subclone cell line of cervical cancer were significantly higher than those of HeLa (P < 0.01). There was no significant difference in cellular viabilities between them (P > 0.05). However, after irradiation with 8 Gy X-ray, growth activities of HeLa/Smac were reduced by 22.42% (P < 0.01). When compared with those of HeLa, partial HeLa/Smac cells presented characteristic morphological changes of apoptosis under electronic microscope, with higher apoptosis rates (16.4% vs. 6.2%, P < 0.01); the caspase-3 expression levels in HeLa/Smac cells were improved significantly (P < 0.01), while its activities were increased by 3.42 times (P < 0.01). CONCLUSIONS: Stable transfer of the extrinsic Smac gene and its over-expression in cervical cancer cell line could significantly enhance the expression and activities of cellular caspase-3 and ameliorate apoptosis-inducing effects of irradiation on cancer cells, which was a novel strategy to improve radiotherapeutic effects on cervical cancer.