Both Wnt/ß-catenin and ERK5 signaling pathways are involved in BDNF-induced differentiation of pluripotent stem cells into neural stem cells.

Although brain-derived neurotrophic factor (BDNF) induces the differentiation of induced pluripotent stem cells (iPSCs) into neural stem cells (NSCs), its exact mechanism remains unelucidated. Wnt/ß-catenin and ERK5 are two important signalling pathways of the Wnt and MAPK signalling cascades and are speculated to be closely related to the differentiation of cells. In this study, we reported the role of the Wnt/ß-catenin and ERK5 signalling pathways on the BDNF-induced differentiation of iPSCs into NSCs. We examined the expression of ß-catenin and p-ERK5 using small interfering RNA (siRNA)-induced silencing of ß-catenin and ERK genes. We found that BDNF significantly improved the efficiency of iPSC differentiation and that the expression of ß-catenin and p-ERK5 in the BDNF culture medium was significantly upregulated. Furthermore, we found that the expression of the ß-catenin component was downregulated by siRNA-ß-catenin, and the expression of the p-ERK5 component was downregulated by siRNA-ERK5. Flow cytometry showed that the differentiation rate of iPSCs was also significantly decreased by RNA interference. The results suggested that the Wnt/ß-catenin and ERK5 signalling pathways are activated in the process of BDNF-induced iPSC differentiation. Interestingly, our study showed that siRNA-ERK5 not only inhibits the activity of the ERK5 signalling pathway but also partially controls the activity of the Wnt/ß-catenin signalling pathway. The results suggested that the Wnt/ß-catenin and ERK5 signalling pathways are not independently involved in the process of BDNF-induced iPSC differentiation. Our study showed that BDNF promotes the differentiation of iPSCs into NSCs by activating the Wnt/ß-catenin and ERK5 signalling pathways, and an interconnected relationship may exist between the Wnt/ß-catenin and ERK5 signalling pathways.

7.0 tesla high resolution MRI study on intracerebral migration of magnet-labeled neural stem cells in a mouse model of Alzheimer's disease.

OBJECTIVE: To observe the migration characteristics of neural stem cells (NSCs) labeled with the MRI contrast agent superparamagnetic iron oxide (SPIO) in the brain of APP/PS1 transgenic mice with Alzheimer's disease (AD) by 7.0 T high resolution MRI. METHODS: C57BL/6 mouse NSCs were cultured, amplified, labeled with Feridex and Poly-l-lysine (FE-PLL) and evaluated by transmission electron microscopy (TEM). Using the random number table method, 24 APP/PS1 transgenic AD mice aged 12 months were equally assigned to two groups: animals in group A were transplanted with FE-PLL labeled NSCs and those in group B were transplanted with non-labeled NSCs in the right hippocampus. Twelve wild-type mice of the same age and born from the same litter were used as the control group (group C) and transplanted with FE-PLL labeled NSCs. Using the 7.0 T high resolution MR scanner, the transplanted NSCs were traced in vivo at 1 day, 1 and 2 weeks after cell transplantation. The MRI findings were compared with the histopathological findings. RESULTS: C57BL/6 mouse NSCs were cultured and amplified successfully. TEM showed large amounts of iron-containing particles in the cytoplasm of transplanted cells. MRI in group A showed the presence of spheroid low signals at the injection point of the hippocampus on T2*WI one day after transplantation; one weeks later, the low signals were seen diffusing to the surroundings along the injection point, and covering almost the whole hippocampal area but the intensity of the low signals became weaker gradually; two weeks after transplantation, almost all low signals disappeared. In group B, no significant change in low signals was observed in the transplantation area at all designated time points. Although low signals were also observed in the hippocampus after transplantation of FE-PLL labeled NSCs in group C, their size and location remained almost unchanged. Prussian blue staining showed that migration of the FE-PLL labeled NSCs in the hippocampus of the AD mice was consistent with the MRI findings at all designated time points. CONCLUSION: NSCs underwent diffuse and non-directional migration to the surroundings after they were transplanted to the hippocampus of APP/PS1 transgenic AD mice, and this migration pattern could be traced in vivo by MRI when they were labeled with magnet.

Survivin-targeted nanoparticles for pancreatic tumor imaging in mouse model.

We investigated the potential of targeting survivin, an inhibitor of apoptosis, in visualize pancreatic tumor in mouse model using targeted magnetic nanoparticles (MNPs) and magnetic resonance imaging (MRI). Chitosan-coated MNPS and survivin antisense oligonucleotide(ASON) were conjugated to give Sur-MNPs. Accumulations of targeted, non-targeted nanoparticles or nonsense oligonucleotide-MNPs (NSON-MNPs) in the liver, spleen, kidney and tumors were determined. Targeted nanoparticles were highly accumulated in BxPC-3 cells but not in non-cancer cells. In vivo MRI showed a significant T2 signal reduction in tumors of mice injected with targeted nanoparticles but slight signal change in tumors of mice injected with non-targeted nanoparticles or NSON-MNPs. Prussian blue staining demonstrated highly accumulated Sur-MNPs in tumor mass compared with normal pancreatic, kidney and liver tissues. Our data show that the MNPs functionalized with ASON lead to the targeted localization in pancreatic tumors. Survivin targeted nanoparticles could be used for detection of pancreatic tumors.

VEGF-C ShRNA inhibits pancreatic cancer growth and lymphangiogenesis in an orthotopic fluorescent nude mouse model.

The aim of this study was to assess the inhibitory efficacy of short hairpin RNA (ShRNA) targeting vascular endothelial growth factor C (VEGF-C) in an orthotopic pancreatic cancer mouse model. BxPC-3 human pancreatic cancer cells expressing green fluorescent protein (GFP) were orthotopically implanted onto the pancreas of nude mice. All mice were randomly divided into four groups when the average tumor size had reached 100 mm(3) and were treated with either vehicle or gemcitabine at 150 mg/kg; or intravenous VEGF-C ShRNA at 150 mg/kg; or intratumoral VEGF-C ShRNA at 150 µg/kg. In vivo fluorescence imaging was performed to monitor tumor growth and metastasis during the study. Real-time quantitative polymerase chain reaction (RT-qPCR) and an enzyme-linked immunosorbent assay (ELISA) were performed to determine the mRNA and protein level of VEGF-C in tumor tissues. Lymphatic vessel marker D2-40, blood vessel marker CD31 and proliferation marker Ki67 expression of the tumor tissues were analyzed by immunohistochemistry staining. Intravenous and intratumoral VEGF-C ShRNA treatment significantly inhibited tumor growth, downregulated the expression of VEGF-C mRNA, reduced tumor microlymphatic vessel density (MLVD), and inhibited cancer cell proliferation. Gemcitabine, as the standard treatment for pancreatic cancer, demonstrated a stronger inhibitory effect on tumor growth, with less inhibition of MLVD and more inhibition of microvessel density (MVD) and proliferation than VEGF-C ShRNA. These results indicate that different mechanisms are associated with the efficacy of gemcitabine and VEGF-C ShRNA.

In vitro study of SPIO-labeled human pancreatic cancer cell line BxPC-3.

The survivin gene is highly expressed in pancreatic cancer. The purpose of this study was to design and synthesize functionalized magnetic iron oxide nanoparticles (MNPs) targeting survivin gene for the detection of pancreatic cancer. The pancreatic cancer cell line BxPC-3 with survivin gene expression was selected in this study. The healthy lung fibroblast cell was used as a control. Chitosan-coated MNPs (CS@MNPs) and antisense oligodeoxynucleotide of survivin gene were conjugated to MNPs to give Sur-MNPs. Fourier transform infrared spectroscopy was performed to confirm the conjunction of chitosan. The interactions of MNPs, CS@MNPs, and Sur-MNPs in BxPC-3 cells were observed, recorded and analyzed. The size, morphology, cell uptake, cytotoxicity and stability of those particles were assessed by transmission electron microscope, Prussian blue staining, MTT assay and agarose gel electrophoresis. The magnetic resonance signal intensities of pancreatic cells labeled with CS@MNPs and MNPs, and Sur-MNPs, were compared on T2-weighted images. The results demonstrated that the level of cellular uptake of CS@MNPs was higher than that of naked MNPs. The Sur-MNPs had a suitable size (12 nm sized core), high stability, no cytotoxicity and good water dispersion. Sur-MNPs did not accumulate in healthy lung fibroblast cells, while being taken up by BxPC-3 cells. The Sur-MNPs in BxPC-3 cells could be visualized on T2-weighted images, which suggested that Sur-MNPs could be used to detect the expression of survivin gene. Thus, Sur-MNPs may be a potential molecular imaging probe targeting survivin gene for early detection of pancreatic cancer cells.

Lymphangiogenesis and biological behavior in pancreatic carcinoma and other pancreatic tumors.

Lymphatic vessels in primary tumor tissue play an important role in lymphatic metastasis. Lymphatic metastasis of malignant neoplasms is significantly related to prognosis, influencing both recurrence and survival. The aim of this study was to investigate the correlation of intra-tumoral lymphatic vessel density (iLVD) and peri-tumoral lymphatic vessel density (pLVD) with biological behavior and prognostic parameters in pancreatic carcinoma (PC) and other pancreatic tumors. Lymphangiogenesis was examined using the D2-40 monoclonal antibody in 33 cases of PC, 7 neuroendocrine tumors of the pancreas (NETP), 7 solid pseudopapillary tumors of the pancreas (SPTP) and 3 cystadenomas of the pancreas (CP). Positively-stained microvessels were counted at magnification x400 in dense lymphatic vascular foci (hotspots). The LVD of PC was compared to 3 other pancreatic tumors. The relationships among the LVD, the extent of differentiation, lymphatic invasion, lymph node metastasis and other clinicopathological parameters of PC were analyzed. There was no difference in the iLVD among PC, NETP, SPTP and CP. The pLVD of NETP was markedly higher than that of PC, SPTP and CP. The pLVD of PC was significantly higher than that of SPTP and CP, but there was no difference between SPTP and CP. The pLVD of PC was significantly associated with the extent of differentiation, lymphatic invasion and lymph node metastasis, whereas it was not associated with age, gender, tumor size, tumor location and peri-pancreatic invasion. The iLVD of PC was not correlated with these clinicopathological parameters. There was no difference in iLVD and no marked difference in pLVD among the pancreatic tumors. Detection of pLVD is of greater importance than detecting iLVD in these pancreatic tumors, as pLVD can be utilized for the prediction of lymph node metastasis, thus aiding in the evaluation of patient prognosis.

[3.0 T magnetic resonance imaging of pancreatic cancer in an orthotopic nude murine model].

OBJECTIVE: To assess the methods of establishing an orthotopic nude murine model of human pancreatic cancer and explore the manifestations of MRI (magnetic resonance imaging) and its pathological fundamentals so as to provide research rationales for human pancreatic cancer. METHODS: The BXPC-3 cell of human pancreatic cancer was orthotopically planted in nude mice. And the animals were examined by a clinical 3.0 T magnetic resonance imager. The MRI examinations were analyzed along with their pathological findings. RESULTS: Among 18 mice, 15 of them grew tumors with a success ratio of 83.3%. The pathological findings conformed to those of high differentiation pancreatic parenchyma cancer. Comparing with the neighbor muscles, the tumors were homogeneous (66.7%, 10/15) or heterogeneous (33.3%, 5/15) of hypointense on T1-weighted images while homogeneous (26.7%, 4/15) or heterogeneous (73.3%, 11/15) hyperintense on T2-weighted images with heterogeneous enhancement. The border became obviously enhanced and there was mild central enhancement while the necrotic part showed no enhancement. CONCLUSION: 3.0T MRI can detect pancreatic neoplasms ≥ 2 mm and visualize clearly their locations, shapes, dimensions and internal structures in an orthotopic nude murine model. Thus it provides a visible framework for further studies of human pancreatic cancer.