A sensitive electrochemiluminescence cytosensor for quantitative evaluation of epidermal growth factor receptor expressed on cell surfaces.

A sensitive electrochemiluminescence (ECL) strategy for evaluating the epidermal growth factor receptor (EGFR) expression level on cell surfaces was designed by integrating the specific recognition of EGFR expressed on MCF-7 cell surfaces with an epidermal growth factor (EGF)-funtionalized CdS quantum dots (CdSQDs)-capped magnetic bead (MB) probe. The high sensitivity of ECL probe of EGF-funtionalized CdSQD-capped-MB was used for competitive recognition with EGFR expressed on cell surfaces with recombinant EGFR protein. The changes of ECL intensity depended on both the cell number and the expression level of EGFR receptor on cell surfaces. A wide linear response to cells ranging from 80 to 4×10(6)cellsmL(-1) with a detection limit of 40cellsmL(-1) was obtained. The EGF-cytosensor was used to evaluate EGFR expression levels on MCF-7 cells, and the average number of EGFR receptor on single MCF-7 cells was 1.35×10(5) with the relative standard deviation of 4.3%. This strategy was further used for in-situ and real-time evaluating EGFR receptor expressed on cell surfaces in response to drugs stimulation at different concentration and incubation time. The proposed method provided potential applications in the detection of receptors on cancer cells and anticancer drugs screening.

Coexpression of hyperactivated AKT1 with additional genes activated in leukemia drives hematopoietic progenitor cells to cell cycle block and apoptosis.

The phosphatidylinositol 3-kinase/AKT pathway is an integral component of signaling involved in the development of many cancers, including myeloid leukemias such as chronic myeloid leukemia and acute myeloid leukemia (AML). Increased AKT1 activity is frequently seen in AML patients, providing leukemic cells with growth and survival promoting signals. An important aspect of AKT1 function is its involvement in cellular metabolism and energy production. Under some circumstances, strong activation of AKT1 increases oxidative stress, which can cause apoptosis when cells progressively build up excess free radicals. This has been described in hematopoietic cells overexpressing activated AKT1; however, whether this is true in cells coexpressing other genetic events involved in leukemia is not known. This prompted us to investigate the effect of constitutively active AKT1 (myristoylated AKT1) in hematopoietic progenitor cells expressing constitutively active signal transducer and activator of transcription 5, Fms-related tyrosine kinase 3-internal tandem duplication, or antiapoptotic B-cell lymphoma 2. Surprisingly, myristoylated AKT1 was incompatible with proliferation driven by both signal transducer and activator of transcription 5 and Fms-related tyrosine kinase 3-internal tandem duplication, which triggered cell cycle block and apoptosis. Moreover, transplantable cells of B-cell lymphoma 2-transgenic mice were impaired in their engraftment ability to recipient mice when expressing hyperactivated AKT1. This was linked to AKT1-mediated proapoptotic functions and not to impairment in homing to the bone marrow. Although cells expressing hyperactivated AKT1 displayed higher levels of reactive oxygen species both in vitro and in vivo, the addition of the antioxidant N-acetyl-L-cysteine significantly reduced apoptosis. Taken together, the results indicate that constitutive AKT1 activity is incompatible with growth- and survival-promoting ability of other activated genes in AML.

A novel electrochemical biosensor for monitoring protein nitration damage affected by NaNO2/hemin/H2O2.

A sensitive and facile electrochemical biosensor has been developed for monitoring the protein nitration damage affected by the nitro free radicals(NO2). The NO2 radicals is generated from hemin-catalyzed oxidation of nitrite (NO(-)2) in the presence of hydrogen peroxide (H2O2). In this work, nanocomposite films of graphene-gold nanoparticles (EG-AuNPs) were modified on the glassy carbon electrode by electropolymerization. Bovine serum albumin (BSA) was then further assembled on EG-AuNPs film through Au-S bond. The damage of BSA molecule was caused by the NO2 radicals which was generated from the NaNO2/hemin/H2O2 nitration reagent. The differential pulse voltammetry was used to detect the damage of BSA molecule. Fluorescence spectra and circular dichroism spectrum further confirmed the nitration damage of BSA. Moreover, the lowest concentration at which the BSA damage was detected is 28.9 µM NO(-)2 or H2O2, and the volume ratio of NO(-)2 and H2O2 was 1:1 in the hemin/NO(-)2/H2O2 nitration reagent. The results demonstrated that the proposed electrochemical method can be used to detect protein damage affected by nitration reagent. The developed electrochemical biosensor is envisioned to have promising applications in protein damage studies.

Interleukin-6 primarily produced by non-hematopoietic cells mediates the lipopolysaccharide-induced febrile response.

Interleukin-6 (IL-6) is critical for the lipopolysaccharide (LPS)-induced febrile response. However, the exact source(s) of IL-6 involved in regulating the LPS-elicited fever is still to be identified. One known source of IL-6 is hematopoietic cells, such as monocytes. To clarify the contribution of hematopoietically derived IL-6 to fever, we created chimeric mice expressing IL-6 selectively either in cells of hematopoietic or, conversely, in cells of non-hematopoietic origin. This was performed by extinguishing hematopoietic cells in wild-type (WT) or IL-6 knockout (IL-6 KO) mice by whole-body irradiation and transplanting them with new stem cells. Mice on a WT background but lacking IL-6 in hematopoietic cells displayed normal fever to LPS and were found to have similar levels of IL-6 protein in the cerebrospinal fluid (CSF) and in plasma and of IL-6 mRNA in the brain as WT mice. In contrast, mice on an IL-6 KO background, but with intact IL-6 production in cells of hematopoietic origin, only showed a minor elevation of the body temperature after peripheral LPS injection. While they displayed significantly elevated levels of IL-6 both in plasma and CSF compared with control mice, the increase was modest compared with that seen in LPS injected mice on a WT background, the latter being approximately 20 times larger in magnitude. These results suggest that IL-6 of non-hematopoietic origin is the main source of IL-6 in LPS-induced fever, and that IL-6 produced by hematopoietic cells only plays a minor role.

Inflammation- and tumor-induced anorexia and weight loss require MyD88 in hematopoietic/myeloid cells but not in brain endothelial or neural cells.

Loss of appetite is a hallmark of inflammatory diseases. The underlying mechanisms remain undefined, but it is known that myeloid differentiation primary response gene 88 (MyD88), an adaptor protein critical for Toll-like and IL-1 receptor family signaling, is involved. Here we addressed the question of determining in which cells the MyD88 signaling that results in anorexia development occurs by using chimeric mice and animals with cell-specific deletions. We found that MyD88-knockout mice, which are resistant to bacterial lipopolysaccharide (LPS)-induced anorexia, displayed anorexia when transplanted with wild-type bone marrow cells. Furthermore, mice with a targeted deletion of MyD88 in hematopoietic or myeloid cells were largely protected against LPS-induced anorexia and displayed attenuated weight loss, whereas mice with MyD88 deletion in hepatocytes or in neural cells or the cerebrovascular endothelium developed anorexia and weight loss of similar magnitude as wild-type mice. Furthermore, in a model for cancer-induced anorexia-cachexia, deletion of MyD88 in hematopoietic cells attenuated the anorexia and protected against body weight loss. These findings demonstrate that MyD88-dependent signaling within the brain is not required for eliciting inflammation-induced anorexia. Instead, we identify MyD88 signaling in hematopoietic/myeloid cells as a critical component for acute inflammatory-driven anorexia, as well as for chronic anorexia and weight loss associated with malignant disease.

Emergence of NK-cell progenitors and functionally competent NK-cell lineage subsets in the early mouse embryo.

The earliest stages of natural killer (NK)-cell development are not well characterized. In this study, we investigated in different fetal hematopoietic tissues how NK-cell progenitors and their mature NK-cell progeny emerge and expand during fetal development. Here we demonstrate, for the first time, that the counterpart of adult BM Lin(-)CD122(+)NK1.1(-)DX5(-) NK-cell progenitor (NKP) emerges in the fetal liver at E13.5. After NKP expansion, immature NK cells emerge at E14.5 in the liver and E15.5 in the spleen. Thymic NK cells arise at E15.5, whereas functionally competent cytotoxic NK cells were present in the liver and spleen at E16.5 and E17.5, respectively. Fetal NKPs failed to produce B and myeloid cells but sustained combined NK- and T-lineage potential at the single-cell level. NKPs were also found in the fetal blood, spleen, and thymus. These findings show the emergence and expansion of bipotent NK/T-cell progenitor during fetal and adult lymphopoiesis, further supporting that NK/T-lineage restriction is taking place prethymically. Uncovering the earliest NK-cell developmental stages will provide important clues, helping to understand the origin of diverse NK-cell subsets, their progenitors, and key regulators.

Identification of an NK/T cell-restricted progenitor in adult bone marrow contributing to bone marrow- and thymic-dependent NK cells.

Although bone marrow (BM) is the main site of natural killer (NK)-cell development in adult mice, recent studies have identified a distinct thymic-dependent NK pathway, implicating a possible close link between NK- and T-cell development in adult hematopoiesis. To investigate whether a potential NK-/T-lineage restriction of multipotent progenitors might take place already in the BM, we tested the full lineage potentials of NK-cell progenitors in adult BM. Notably, although Lin(-)CD122(+)NK1.1(-)DX5(-) NK-cell progenitors failed to commit to the B and myeloid lineages, they sustained a combined NK- and T-cell potential in vivo and in vitro at the single-cell level. Whereas T-cell development from NK/T progenitors is Notch-dependent, their contribution to thymic and BM NK cells remains Notch-independent. These findings demonstrate the existence of bipotent NK-/T-cell progenitors in adult BM.

Distinct and overlapping patterns of cytokine regulation of thymic and bone marrow-derived NK cell development.

Although bone marrow (BM) represents the main site for postnatal NK cell development, recently a distinct thymic-dependent NK cell pathway was identified. These studies were designed to investigate the role of cytokines in regulation of thymic NK cells and to compare with established regulatory pathways of BM-dependent NK cell compartment. The common cytokine receptor gamma-chain (Il2rg) essential for IL-15-induced signaling, and FMS-like tyrosine kinase 3 (FLT3) receptor ligand (Flt3l) were previously identified as important regulatory pathways of the BM NK cell compartment based on lack of function studies in mice, however their complementary action remains unknown. By investigating mice double-deficient in Il2rg and Flt3l (Flt3l(-/-) Il2rg(-/-)), we demonstrate that FLT3L is important for IL2Rg-independent maintenance of both immature BM as well as peripheral NK cells. In contrast to IL-7, which is dispensable for BM but important for thymic NK cells, IL-15 has a direct and important role in both thymic and BM NK cell compartments. Although thymic NK cells were not affected in Flt3l(-/-) mice, Flt3l(-/-)Il2rg(-/-) mice lacked detectable thymic NK cells, suggesting that FLT3L is also important for IL-2Rg-independent maintenance of thymic NK cells. Thus, IL-2Rg cytokines and FLT3L play complementary roles and are indispensable for homeostasis of both BM and thymic dependent NK cell development, suggesting that the cytokine pathways crucial for these two distinct NK cell pathways are largely overlapping.

[Bone marrow stromal cells co-modified with BMP-2 and bFGF gene].

AIM: To establish modified bone marrow stromal cells(BMSCs) which can express BMP-2 and bFGF stably. METHODS: BMP-2 and bFGF gene were amplified by RT-PCR, and then cloned into the expression vector pcDNA3.0. After being confirmed by DNA sequencing, pcDNA3.0-BMP-2 and pcDNA3.0-bFGF were co-transfected into rat BMSCs with Lipofectamine 2000 reagent. The expression of BMP-2 and bFGF gene in rat BMSCs was detected by RT-PCR, Western blot, immunohistochemical staining and ELISA. RESULTS: BMP-2 and bFGF gene were cloned, and their sequences were identical with those in GenBank. The expression plasmids, pcDNA3.0-BMP-2 and pcDNA3.0-bFGF, were constructed and co-transfected into rMSCs successfully. RT-PCR showed the mass transcription of BMP-2 and bFGF mRNA in transfected BMSCs. Western blot, immunohistochemical staining and ELISA confirmed the expression of BMP-2 and bFGF genes in transfected cells and in the supernatant. CONCLUSION: We have constructed the optimal rat BMSCs which can be used in bone tissue engineering.

[Growth and osteogenesis characteristics of cultured canine mesenchymal stem cells under osteogenic induction].

To investigate the growth and osteogenesis characteristics of cultured canine mesenchymal stem cells (cMSCs) under osteogenic induction. We found the cMSCs were isolated from adult canine using density gradient separation method. The cMSCs attachment formed soon after seeding and grew into colonies with the appearance of fibroblastic cells. The osteogenic induction compound of Dexamethasone (Dex), beta-sodium glycerphosphate (beta-GP), ascorbic acid (AA) was added to passaged cMSCs and the proliferation and osteogenic differentiation of them was studied. The morphology of cells was observed by light micrograph and transmission electron microscope. The proliferation and growth characteristics of cMSCs were observed during primary and passage cultures through MTT. The differentiation were assayed by alkaline phophatase (ALP) and osteocalcin (OCN). We found the cMSCs have an active proliferative ability in primary and passage culture, and cMSCs under osteogenic induction have the typical characteristic of a secretory cell; the osteogenic induction compound may induce cMSCs to differentiate to osteoblasts. There are higher expression of ALP and OCN in passage 3 cMSCs under osteogenic induction than that of the osteoblasts osteogenic induction condition. Our research suggest the cMSCs in our culture system are mainly undifferentiated osteoprogenitors and can differentiate to osteoblast under osteogenic induction.

[The study of osteogenous differentiation of MSCs transfected with different gene].

This experiment is sought to study the contribution of different gene in osteogenous differentiation of bone marrow stromal cells (MSCs) and optimize the seed cells of bone tissue engineering. Firstly, we obtained the full length gene of BMP-2, VEGF165 and bFGF by RT-PCR, and cloned into the expression vector pcDNA3. 0. After to be transfected, the MSCs cell lines which could express each of the target protein were selected out by G418. RT-PCR and immunohistochemistry were used to confirm the exogenous gene expression in MSCs. MTT showed that almost all of the MSCs which have been transfected with exogenous gene had more strong proliferative potential than the untransfected group did. There was a notable increase of ALP activity in transfected cell compared with the control group. The concentration of OCN in cell culture medium had a increase in some degree except of VEGF group. The outstanding osteogenous differentiation could be observed in BMP-2 and bFGF transfected MSCs. The results showed that the MSCs modified by BMP-2 and bFGF would be more effective in bone tissue engineering field.

[Research of osteoblast-induced rat mesenchymal stem cells cocultured with beta-TCP/PLLA composite of different ratio].

There are three key factors in tissue engineering: seeding cells, scaffold and their interaction. Although mesenchymal stem cells (MSCs) are potential seeding cells, the problem of what phase MSCs should be used is not yet solved. On the other hand, degradable porous scaffolds which have good mechanics and good biocompatibility are preferred. To choose the optimum seeding cells and the suitable ratio of beta-TCP/PLLA porous scaffold, we observed the phenotype of the male SD rat's osteoblastic MSCs and detected the amount of alkaline phosphatase, osteocalcin and type I collagen secreted by the osteoblastic rMSCs in different phase. About 10, 14 and 20 days after induction, the induced cells came into proliferative phase, matrix synthesis phase and mineralization phase, respectively. Then we chose the suitable cells and seeded them on beta-TCP/PLLA composite scaffolds with different ratios (beta-TCP/PLLA = 1:1; beta-TCP/PLLA = 1:2; and beta-TCP/PLLA = 2:1). Fluorescence microscope, scanning electron microscope and MTT assay were used to observe and to detect the biocompatibility of the scaffolds. The results indicated that all of these materials have biocompatibility to some extent. Cells can grow well on all of the scaffolds. However, scaffold beta-TCP/PLLA = 2:1 seems to be a more suitable tissue engineering scaffold on account of its minimal influence on cell growth and differentiation.

[Dynamic alteration of blood lipid and hemodynamics parameters and vascular intima in the process of atherosclerosis].

To establish and observe rabbit hyperlipemia and atherosclerosis model, we combined the method of high-lipid diet and immuoreactive injure. We divided 45 New Zealand rabbit into control group and high-lipid group. According to the time (4, 8, 12 weeks) of established model, the control group and high-lipid group were divided into 3 groups respectively. The blood-lipid, the hemodynamics parameter and vascular intima were determined and observed. The results showed: (1) After feeding 4, 8, 12 weeks, TC and LDL-C of the high-lipid group in the serum increased obviously. After feeding 8 week, TG of the high-lipid group began to increase obviously. HDL-C of the high-lipid group was higher than control group, but with a descendent trend. (2) Blood viscosity of the high-lipid group increased obviously under the 0.512 S(-1) and 5.96 S(-1) at 12 week. Blood flow increased obviously at 8 and 12 week. SBP increased evidently at 8 and 12 week. Alteration of the plasma viscosity and vascular diameter were not obvious. (3) By observing with optical microscope the intima of the control group is smooth. The intima of the high-lipid group has a light incrassation at 4 week. The intima of the high-lipid group has a obvious incrassation at 8 and 12 week. By observing with through transmission electron microscopy (TEM), the lipid vacuole is under the endothelium cell. (4) By adopting the immunohistochemistry, the foam cell that derived from smooth muscle cell were determined. We concluded that the blood lipid can have a prefigurative role in atherosclerosis; blood flow and blood pressure and blood viscosity increase at low shear rate in the course of the atherosclerosis; vascular intima is incrassate and the composition of the AS plaque is variational continually.