MRGPRX2 is critical for clozapine induced pseudo-allergic reactions.

BACKGROUND: Clozapine is one of the most widely used second-generation antipsychotics in clinic. However, allergy-like symptoms such as rash and angioedema have been reported frequently, and the mechanism is still not clear. Mas-related G protein-coupled receptor X2 (MRGPRX2) expressed on mast cells is a crucial receptor for drug induced pseudo-allergic reactions. Therefore, we explored whether the symptoms induced by clozapine were associated with allergic reaction through MRGPRX2. METHODS: The effects of clozapine on pseudo-allergic reactions were evaluated by mast cells degranulation and calcium mobilization assay in vitro, and mice hindpaw swelling, serum histamine detection, avidin and H&E staining assay in vivo. The overexpressed MRGPRX2 cells membrane chromatography (MRGPRX2-HEK293/CMC), MRGPRX2-HEK293 cells calcium mobilization assay and molecular docking were applied to research the correlation between clozapine and MRGPRX2. RESULTS: The study showed that clozapine induced the release of ß-hexosaminidase, histamine and monocyte chemoattractant protein-1 (MCP-1), and trigged calcium mobilization in mast cells. In vivo, clozapine induced paw swelling, degranulation and vasodilation. Furthermore, clozapine could activate the calcium mobilization obviously in MRGPRX2-HEK293 cells, not in NC-HEK293 cells. Clozapine also had a good retention characteristic on MRGPRX2-HEK293/CMC column and the K D value is (2.33 ± 0.21)×10-01M. CONCLUSIONS: Our findings demonstrated that clozapine could induce pseudo-allergic reactions and MRGPRX2 might be the critical receptor for it.

[Effects of interplanting on soil aggregate distribution and stability in economic orchard at hilly slope red soil of southern China]. / 套种对南方红壤坡耕地经济果园土壤团聚体分布及稳定性的影响.

We investigated the effects of interplanting on soil aggregate distribution and stability of red soil in economic orchard in a new-constructed slope land, based on 12 kiwifruit planting experi-mental plots with a slope of about 12°, a length of 18 m, and a width of 1.5 m. Three types of interplanting patterns were implemented by interplanting purple sweet potato (PSP), hairy vetch (HV), and weeds (W) for three years, respectively, taking the bare land with no vegetation as control (CK) to determine the aggregate indicators at 0-15 cm soil layer. The results showed that the quantity and size of soil water stabilized aggregates (WR>0.25) all tended to increase which ranked in the order of PSP>HV>W>CK. The order of soil aggregate structure damage rate (PAD) and fractal dimension (D) were CK>W>HV>PSP, indicating that soil aggregate in PSP was the most stable, followed by HV treatment. Along the downslope, the content of WR>0.25, mean weight diameter (MWD), and geometric mean diameter (GMD) all tended to decrease, while the PAD and D increased, indicating that soil structure turned to be poorer in downslope of the new-constructed slope land. D was negatively correlated with MWD, GMD, and the content of >0.25 mm aggregates. It was concluded that interplanting could increase the quantity and size of soil aggregates, improve soil structure and soil quality of economic orchard in hilly slope land.

Maxillary sinus floor elevation with a tissue-engineered bone composite of deciduous tooth stem cells and calcium phosphate cement in goats.

The study aimed to assess the effect of maxillary sinus floor elevation with tissue-engineered bone constructed from deciduous tooth stem cells (DTSCs) and calcium phosphate cement (CPC). The stem cells from goat deciduous teeth (SGDs) were isolated and transfected by means of the adenovirus with an enhanced green fluorescent protein gene (AdEGFP). As many as 18 bilateral maxillary sinuses of nine goats were randomly allocated into three groups (n = 6/group): group A (SGDs-CPC compound), group B (CPC alone) and group C (autogenous bone obtained from an iliac crest). All the samples were evaluated by computed tomography (CT), histology and histomorphometric analysis. Furthermore, the fate of implanted SGDs was traced using an immunohistochemical staining method in the decalcified samples. SGDs might be differentiated into osteoblasts in an osteogenic medium. In the present study, three-dimensional CT analysis showed that the volume of newly formed bone in group A was greater than that in the other two groups. After a healing period of 3 months, sequential analyses of triad-colour fluorescence labelling, histology and histomorphology indicated that the SGDs-CPC compound primarily promoted bone formation and mineralization at 2 and 3 months after the operation. Moreover, the areas of new bone formation in elevated sinuses were 41.82 ± 6.24% in the SGDs-CPC group, which was significantly higher than the 30.11 ± 8.05% in the CPC-alone group or the 23.07 ± 10.21% in the autogenous bone group. Immunohistochemical staining revealed that GFP and OCN were both expressed in the new bone tissue for the samples with eGFP, which suggested that the implanted SGDs might have contributed to new bone formation on the elevated sinus floor. SGDs can promote new bone formation and maturation in the goat maxillary sinus, and the tissue-engineered bone composite of SGDs and CPC might be a potential substitute for existing maxillary sinus floor elevation methods. Copyright © 2014 John Wiley & Sons, Ltd.

Induction of Osteogenic Differentiation of Human Adipose-Derived Stem Cells by a Novel Self-Supporting Graphene Hydrogel Film and the Possible Underlying Mechanism.

Graphene and its derivatives have received increasing attention from scientists in the field of biomedical sciences because of their unique physical properties, which are responsible for their interesting biological functions. With a range of extraordinary properties such as high surface area, high mechanical strength, and ease of functionalization, graphene is considered highly promising for application in bone tissue engineering. Here, we examined the effect of using a self-supporting graphene hydrogel (SGH) film to induce the osteogenic differentiation of human adipose-derived stem cells (hADSCs). In comparison to conventional graphene and carbon fiber films, the SGH film had higher mechanical strength and flexibility. Moreover, we found that the SGH film was nontoxic and biocompatible. Of particular interest is the fact that the film alone could stimulate the osteogenic differentiation of hADSCs, independent of additional chemical inducers. Such effects are stronger for the SGH film than for graphene or carbon fiber films, although the induction capacity of the SGH film is not as high as that of the osteogenic-induced medium. The excellent osteoinductivity of the SGH film is closely related to its remarkable physical properties that include specific nanostructures, surface morphology, strong cell adherence, reasonable surface hydrophilicity, and high protein absorption.

[Preliminary study of akermanite's effect on stem cells from goat deciduous teeth in proliferation and osteogenic differentiation].

PURPOSE: To explore the characters of attachment, proliferation and osteogenic differentiation of stem cells from goat deciduous teeth (SGDs) cultured on akermanite ceramic. METHODS: SGDs were collected with enzyme digestion, and cultured. Under culture condition in growth medium, actin filament labeling and CCK-8 analysis were used to reveal attachment and proliferation. Expression level of alkaline phosphatase (ALP) and osteogenic differentiation genes (ALP, COL I, OPN and OCN) were examined by pNPP method and real-time PCR. The data was analyzed using SPSS 13.0 software package. RESULTS: Compared with ß-TCP, SGDs seeded on akermanite ceramic not only spread out better, but also grew faster since day 4. In osteogenic medium, cells of akermanite group had stronger ALP activity at day 4 and 7. The gene expression of akermanite was more than the ß-TCP group. The gene OCN expression level increased significantly at day 7 (P<0.01). CONCLUSIONS: SGDs can attach and grow well on akermanite ceramic. Compared with ß-TCP, akermanite ceramic shows an advantage in osteogenic differentiation of SGDs.

[Ectopic osteogenesis of SGDs in goat's muscle pouch].

PURPOSE: To investigate ectopic osteogenesis of composites SGDs with porous calcium phosphate cement(pCPC) in goat's muscle pouch. METHODS: SGDs were cultivated with modified tissue culture techniques, then were induced into osteoblasts in the third passage, the osteogenic-induced SGDs were combined with pCPC and transplanted into the goat left dorsal muscle pouch, the pCPC without cells was transplanted into the right dorsal muscle pouch as negative controls. The transplants were harvested at 2-,4-,6-,8- week and prepared for histological examination. The morphologic quantitative analysis was made by SPSS 16.0 software package. RESULTS: Bone formation was not detected in pCPC without cells by histological examination. 2,4,6,8 weeks after transplantation in SGDs-pCPC group, the percentages of bone formation were (1.24±0.25)%,(1.59±0.23)%,(4.12±0.39)% and (5.68±0.58)%,respectively.There was no significant difference in bone formation at 2 and 4 weeks after transplantation (P>0.05). At 8- week, the percentages of bone formation were higher than that at 6- week in SGDs-pCPC group, and both significantly higher than that at 2- and 4- week(P<0.05). CONCLUSION: SGDs combined with pCPC have the ability of ectopic osteogenesis.

[Experimental study on stem cells from goat deciduous teeth compounded with porous calcium phosphate cement for ectopic osteogenesis].

PURPOSE: To observe the biocompatibility and ectopic bone-like tissue formation of stem cells from goat deciduous teeth (SGDs) with porous calcium phosphate cement (pCPC). METHODS: The expression of STRO-1 on SGDs was measured with flow cytometry (FCM); the 4th passage SGDs were cultured in induced-mineralization medium in vitro for 7 days. Combined with pCPC, the adhesion and growth of the compounds were observed with scanning electron microscopy (SEM); the ectopic bone-like tissue formation was observed 8 weeks after the compounds implanted subcutaneously into the nude mice. RESULTS: On the third day of SGDs compounded with pCPC, SEM verified that the cells adhered closely and tightly with pCPC, protruded pseudopods and secreted matrix. 8 weeks after the compounds implanted in ectopic sites, HE staining confirmed the formation of bone-like tissue; Immunohistochemistry showed the strongly positive expression of OCN protein in the implanted materials. CONCLUSIONS: SGDs may differentiate into osteoblast and are potential to induce bone matrix formation; combined with pCPC, the compounds may generate bone-like tissue.

[Isolation, cultivation and induced-mineralization of dental pulp cells from goat deciduous teeth in vitro].

PURPOSE: To isolate and cultivate dental cells from goat deciduous teeth ,and explore changes of its biological characters before and after induced-mineralization. METHODS: Pulp cells were cultivated with modified tissue block enzymolytic method, cell lineage in the second passage with SAB methods was checked out. Induced-mineralized cultivation was adopted in the fourth passage, some examinations were used to compare with normal cultivated cells: cell proliferative capality, mineralized ability test, cell morphology change, protein(OCN) expression level, related osteogenic genes(ALP,COL-I,OCN,OPN) expression. RESULTS: Modified tissue block enzymolytic method could culture better pulp cells derived from goat deciduous teeth. Immunohistochemical staining showed that pulp cells were from mesenchyma. MTT method showed that induced-mineralization pulp cells proliferated more slowly than un-induced cells. Compared with uninduced-mineralization cells, induced-mineralization cells had stronger ALP activity and Alizarin red staining rate, its proteins(OCN) and mineralized genes(ALP,OCN) expression were significantly upregulated. CONCLUSIONS: Pulp cells can be cultivated derived from goat exfoliated deciduous teeth with modified tissue block enzymolytic method. Fourteen days after continuous induced-mineralization culture , pulp cells derived from the goat deciduous teeth might own the potential in differentiating to osteoblast and form bone-like tissue.

Norcantharidin potentialize the chemosensitivity of adriamycin through the NF-κB/IκBα signaling pathway / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the synergetic effect of norcantharidin (NCTD) and adriamycin (ADR) on the proliferation and apoptosis of multiple myeloma (MM) cells.</p><p><b>METHODS</b>Human MM cell line U266 cells were treated with NCTD alone (10 µmol/L) or in combination with ADR (0.25 µmol/L). MTT and Annexin V/PI staining were used to determine cell viability and apoptosis. The protein expression of nuclear factor-κB P65 (NF-κB P65), phosphorylated NF-κB p65 (p-NF-κB p65), NF-κB P65 inhibitor IκBα, phosphorylated IκBα (p-IκBα), survivin, Bcl-2 and Bax were determined by Western blot. Immunohistochemistry was used to determine the expression of vascular endothelial growth factor (VEGF).</p><p><b>RESULTS</b>(1) NCTD potentiated the cytotoxicity and pro-apoptotic effects induced by ADR. The combination of NCTD and ADR had synergistic anti-proliferation effect. (2) Combination of ADR and NCTD downregulated the expression of nuclear NF-κB P65 and cytoplasm p-IκBα induced by ADR. The expression of nuclear NF-κB P65 and cytoplasm p-IκBα decreased from 2.08 ± 0.29 and 0.39 ± 0.07 to 0.48 ± 0.08 and 0.02 ± 0.01 respectively, while the expression of the cytoplasm NF-κB P65 and IκBα were unchanged in the ADR alone group and the combined group. (3) The expression of survivin and bcl-2 decreased from 0.31 ± 0.05 and 0.23 ± 0.05 to 0.03 ± 0.02 and 0.05 ± 0.02, while the expression of Bax increased from 0.46 ± 0.06 to 0.62 ± 0.08 respectively in ADR alone group and combined group. (4) The positive rate of VEGF in ADR group and combination group were (44.6 ± 4.4)% and (27.0 ± 2.1)% respectively, indicating that NCTD could potentiate the inhibition effect on VEGF induced by ADR.</p><p><b>CONCLUSIONS</b>The results suggest that NCTD can potentialize the chemosensitivity of multiple myeloma cells to ADR through regulating NF-κB/IκBα signaling pathway and NF-κB-regulated gene products including survivin, Bcl-2, Bax and VEGF.</p>

[Culture and green fluorescent protein gene transfection of pulp cells from goat deciduous teeth in vitro].

PURPOSE: To isolate and culture pulp cells from goat deciduous teeth, and transfect green fluorescent protein gene. METHODS: Pulp cells from goat deciduous teeth were obtained by tissue culture. Cell growth curve was measured by counting the number of cells, HE and alkaline phophatase(AKP) stain, as well as immunhistochemical stain of vimentin and keratin were performed. Virus supernatant was used to infect cell green fluorescent protein gene. RESULTS: For the pulp cells, the cell group double time was 43.79 hours. AKP stain and immunhistochemical stain of vimentin were both positive, while immunhistochemical stain of keratin was negative. The infected cells expressed green fluorescent. CONCLUSIONS: Pulp cells could be cultured from goat deciduous teeth, and express green fluorescent successfully.

[A study of osteogenic induction of dental pulp stem cells from deciduous teeth in vitro].

UNLABELLED: URPOSE: To isolate and culture dental pulp stem cells from human deciduous teeth, and induce them to osteoblasts. METHODS: Dental pulp stem cells were separated from deciduous teeth with enzyme digestionìthen isolated and purified by limited dilution. The cells clone form rate was carried out, and cell growth curve was measured by counting the number of cells. HE stain and immunhistochemistry stain of VimentinìCD44 and STRO-1 were tested. Then the cells were induced to osteoblasts, and HE stain,AKP stain, Von Kossa stain,Van Gieson stain,and immunhistochemistry stain of osteocalcin were used for evaluation. RESULTS: Dental pulp stem cells were obtained from deciduous teeth by limited dilution. The cells were induced to osteoblasts, which elicited a biological and morphologic characteristics similar to those of osteoblasts. CONCLUSIONS: The results show that dental pulp stem cells are obtained from deciduous teeth and induced to osteoblasts successfully.