Isolation, culture and homing to the liver of mouse adipose-derived stem cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the method for the isolation, cultivation, and purification of adipose-derived stem cells (ADSCs) and examine the oncogenesis and homing of ADSCs to the liver in vivo.</p><p><b>METHODS</b>ADSCs were isolated from female mice by digestion with 0.075% collagenase I and the morphology of the isolated cells was observed with examination of the cell surface markers and cell cycle. BALB/c mice were injected with 1×10(6) ADSCs on the back to evaluate the oncogenesis of ADSCs or with 1×10(6) ADSCs stained with 5, 6-carboxyfluorescein diacetate-succinimidyl ester (CFSE) via the tail vein to examine the cell homing to the liver.</p><p><b>RESULTS</b>The isolated ADSCs highly expressed CD29 and CD44 and were negative for CD34, CD45, CD11b and CD14. Cell cycle distribution analysis showed cell percentages in G0/G1, S, and G2/M phases of 80.1%, 7.9%, and 12%, respectively. The ADSCs had a low immunogenicity and did not express CD40, CD80, CD86, MHCI, MHCII or PDL-1. After stimulation with IFN-γ, the expression of CD40, CD80 and PDL-1 were up-regulated slightly in the cells. Dorsal injection of the ADSCs did not result in any tumor formation within 1 month, and ADSCs injected via the tail vein showed cell homing to the liver.</p><p><b>CONCLUSION</b>Murine ADSCs can be isolated and expanded effectively by collagenase digestion and adherent culture. The isolated ADSCs can successfully reside in the liver after implantation, and thus may serve as a promising candidate cell in stem cell therapy of liver diseases.</p>

Activated hepatic stellate cells promote expression of regulatory T cells / 中华肝胆外科杂志

Objective Accumulating reports have suggested that hepatic stellate cells (HSCs) exhibit immunosuppressive ability and may be responsible for the occurrence and development of hepatocellular carcinoma (HCC).The mechanisms through which HSCs affect T-cell-induced adaptive immune responses and the relationship with the regulatory T cells (Treg cells) were studied.Methods We isolated HSCs from wildtype mice to demonstrate the influence of HSCs on T-cell proliferation and explored their effect on Treg cells through mixed leukocyte reactions (MLRs) in vitro.Results We found that activated HSCs could induce T-cell hyporesponsiveness in adaptive immune response by inhibiting the proliferation of T cells andincreasing the quantity of Treg cells.Conclusion Activated HSCs may lead to hypoergia of T cells in adaptive immune reaction and up-regulate the expression of Treg cells,thus facilitating immunotolarance.

Experimental study of human adipose derived mesenchymal stem cells differentiated into hepatocyte-like cells in vivo and vitro / 中华肝胆外科杂志

ObjectiveTo isolate and culture human adipose derived mesenchynal stem cells (hADSCs) and study the potential of hADSCs to differentiate into hepatocyte-like cells. MethodsTo ensure the removal of contaminating hematopoietic cells, hADSCs were selected based on plastic adherence. Cell surface antigen was confirmed by flow cytometry; ultramicrostructure was detected by transmission electron microscopy; adipogenic and osteogenic differentiation of hADSCs was analyzed by oil Red O staining and yon Kossa staining. hADSCs were exposed to differentiation medium containing EGF,FGF,OSM, HGF,TSA in vitro. 10% CCl4 (100 μ1/20 g body weight )was injected into immune-deficient BALB/c-nu mice and hADSCs (5 × 105 cells) were simultaneously administrated from the tail vein. Blood samples were collected and concentration of aminotransferase and direct bilirubin were detected. Administration without hADSCs was used as a control. One month later, we sacrificed the mice and liver sections were examined by histochemical immunofluorescence with human ALB specific antibodies. ResultshADSCs exhibited fibroblast-like morphology, and expressed CD73, CD90,CD105, and were lacking of CD34 and CD45. Adipogenic and osteogenic differentiation showed that hADSCs have the capacity of multidifferentiation. The differentiated cells showed hepatocyte-like cell morphologies and hepatocyte-specific markers including albumin (alb) and α-fetoprotein (AFP). The bioactivity assays revealed that these hepatocyte-like cells could uptake low-density lipoprotein (LDL).Histochemical immunofluorescence showed that hADSCs were incorporated into injured livers. Some human alb-positive cells were found in liver sections after implantation of undifferentiated hADSCs. Transaminase activity in the experimental group was lower than in the control group. Conclution hADSCs can differentiated into functional hepatocyte-like cells and can relieve CCl4 induced BALB/c-nu acute liver injury.

Immune modulatory activity of activated hepatic stellate cells in the tumor microenvironment / 中华肝胆外科杂志

Objective To determine immune modulatory activity of activated hepatic stellate cells( HSCs) in hepatocellular carcinoma and immune response in tumor microenvironment. Methods Cell proliferation was measured by BrdU incorporation with a microtiter plate reader at 450 nm. The effect of HSCs on T cell proliferation was measured by MLR. Mouse hepatic cancer cell line H22 were implanted on the backs of BALB/c mice to establish the subcutaneous transplanted tumor model. Then the mice were sacrificed after 20 days for anatomical and size determination. Furthermore, Paraffin-embedded tissue was removed by serial tissue sectioning and immunohistochemically examined for expression of T lymphocyte subsets. T lymphocyte subsets in splenocytes were detected by FCM. Apoptotic mononuclear cells were evaluated by FITC-labeled Tunel assay. Results We determined that HSCsCM promoted hepatocellular carcinoma(HCC) cell line proliferation and HSCs inhibit T cell proliferation by MLR in vitro. We also examined normal immune mice to assess the immunosuppression of HSCs in the development of HCC. In the co-transplantation with HSCs group, T cells and their subtypes decreased obviously in the tumors and the spleen. The results showed that co-transplanted HSCs can induce more PD-L1 expression and more mononuclear cell apoptosis in tumor tissue. Conclusion Our results demonstrated that HSCs promote HCC progression partially because of their immune regulatory activity. Our data supply new information to support an integral role for HSCs in promoting HCC progression and suggest that HSCs may serve as a therapeutic target for HCC.

Fetal liver stem cells for the treatment of murine liver injury / 中华普通外科杂志

Objective To isolate and indentify fetal hepatic progenitor/stem cells, and study the feasibility and effectiveness of their transplantation on acute liver injury in nude mice. Methods The primitive cells isolated from 13.5dpc pregnant mouse fetal hver by way of enzyme digesting were cultured in vitro and liver specific markers as AFP, CK19, Albumin, c-Met, were identified by immunofluorescence and RT-PCR on colonies. 4 × 105 cells were transplanted into nude mice with carbon tetrachloride induced acute liver injury. Hapatic functions were measured pre- and -post transplantation on days 1,2,4, 7. Meanwhile, hepatic pathology was studied. Results Compared to control group the hepatic functions gradually recovered in transplant group, on days 1,2,4 after fetal hepatic stem cell transplantation. The hepatic pathology significantly improved in stem cells transplantation group. Conclution Fetal hepatic progenitor/stem cell were successfully yielded by enzyme digest. Stem cells transplantation improved the hepatic function and pathology in acute hapatic injury model of nude mice.

Application of carbon nanotubes in modified oxidase biosensors / 中国组织工程研究

Carbon nanotubes are a novel kind of nano-materials, and contribute to modify electrodes using their good capacity of electric transmission. Furthermore, carbon nanotubes may reduce the overpotential caused by the oxidation-reduction reaction of chemical materials, and ameliorate the oxidation-reduction reversibility of the biomolecules; Their wide specific surface area leads to enzyme immobilization and promote the electric transmission between enzyme active center and electrode surface; Functionalized carbon nanotubes bonding with various biomolecules will benefit to fix these biomolecules on the electrode surface. With the development of preparation and purification techniques, carbon nanotubes may widen the prospect of biosensors applied. The aim of this paper is to summarize and analyze the application, research status and development tendency of carbon nanotubes with regard to biosensors. Moreover, this paper reviews various carbon nanotubes modified oxidase biosensors, such as adsorption, paste, array and covalent bond, together with their application on DNA biosensor. The preparation, characteristics and research status of various sensors are also summarized.

Study of Subcelluar-localization of Serum Inhibited Gene Si1 Protein / 生物化学与生物物理进展

A serum inhibited gene Si1(GenBank acession number:AY050169) was previously cloned and identified by differential expression of genes in U251 cells.For the further study of biological function of Si1, prediction procedure was performed to predict its subcelluar-localization.Relative experiments were carried out at the same time.The expression of EGFP/Si1 recombinant in HeLa cells showed Si1 protein located in nuclear which corroborated the prediction results of PsortⅡ, Proloc, Cello version2, Subnuclear compartments prediction system, NUCLEO and NUCPRED.According to the PredictNLS prediction, twelve different fragments of EGFP/Si1 recombinants were constructed to identify precise NLS regulation sequence.Findings proved that the real NLS regulation sequence was not the same as the software predicted(1 206 bp～1 239 bp on Si1 ORF), but located on 1 395 bp～1 594 bp of Si1.A tumor relatived mutation/EGFP recombinant localization result showed though the mutation site(1 639bp on Si1 ORF) does not located in NLS regulation sequence, it did affect wildtype Si1 protein divert to nuclear and may affect its natural function in cell, perhaps it is the main reason for highly mutation rate of Si1 in tumor.

Contents of trace and constant elements and therapeutic effects of Chinese patent medicine on cardio-cerebral vascular disease: fuzzy classified principle / 中国组织工程研究

BACKGROUND: It is discovered that trace elements in Chinese patent medicine play an important role in pharmaceutical efficacy and structural analysis. A new topic has been formed gradually in a further research on pharmaceutical efficacy and effective components of Chinese patent medicine in the view of inorganic trace elements. The fuzzy classified study on the interrelationship between trace and constant elements of Chinese herbs and therapeutic effects is to discuss the trace-element action of drugs in the holistic aspect of trace elenent and research rather scientifically the holistic coordination among trace elements in Chinese herbs.OBJECTIVE: To determine the contents of 21 kinds of trace and constant elements in Chinese patent medicines in the treatment of cardio-cerebral vascular disease. To study with fuzzy classified method the interdependence between trace, constant elements of drugs and therapeutic effects so as to provide a new approach for the quality evaluation of Chinese patent medicine.DESIGN: Testified observed study based on various Chinese patent medicines.SETTING: Chemistry teaching-research room in a university and department of cardiology in a general hospital of a chemical industrial group corporation.MATERIALS: Twenty-one kinds of common Chinese patent medicines for the treatment of cardio-cerebral vascular diseases in clinic, namely di' ao xinxuekang capsule(DXK), xueshuan xinmaining capsule(XMN), shan haidan capsule(SHD), buchang naoxintong capsule(NXT), linaoxin capsule(LNX), xinnaosu capsule(XNS), lixin wan(LXW), suxiao jiuxin wan (SJX), xinbao wan(XBW), yinxingye pian(YXY), fufang danshen pian (DSP), niuhuang jiangya wan(NHJY), naosaitong pian(NST), qiangli tianma duzhong wan ( TD W ) , tongsaimai pian ( TSM ) , tongmai ningxin infusion(TMNX), maitong capsule(MTC), fufang yinxing wan ( YXW ) , da huoluo dan(DHL), guanxin suhe capsule(GXSH) and xiaoshuan tongluo capsule (XSTL).METHODS: Wet digestion was used to manage samples, atomic absorption spectrophotometric method(AAS) method was used to determine the contents of 10 kinds of trace and constant elements in 21 Chinese patent medicines,namely Na, Ca, Mg, K, Fe, Cr, Ni, Cu, Zn and Mn. Data range method was applied for the standardized management of statistical indexes of various element contents in relevant drugs, dotprod(U, V) method was used to measure correlation coefficient(r) among the classified objects so as to determine fuzzy matrixes in universe. The interrelationship was analyzed between the contents of trace elements of various Chinese medicines and pharmaceutical efficacy. By the analysis of fuzzy matrixes in universe, it was to evaluate firmly on the efficacy similarity of various kinds of Chinese patent medicines with quantitative indexes.MAIN OUTCOME MEASURES: ① Results of element contents; ② Results of fuzzy cluster analysis on elements.RESULTS: The standardized management of statistical indexes was carried on the contents of Na, Ca, Mg, K, Fe, Cr, Ni, Cu, Zn and Mn in various drugs to determine correlation coefficient(r) among the classified objects so as to determine fuzzy matrixes in universe. When(r) = 0.55, there were 9 kinds of drugs similar in cluster, namely xueshuan xinmaining capsule, buchang naoxintong capsule, lixin wan, xin bao wan, qiangli tianmai duzhong wan,tongsaimai pian, maitong capsule, da h uoluo dan and xiaoshuan tongluo capsu le. When(r) =0. 65, there were 6 kinds of drugs similar in cluster,namely buchang naoxintong capsule, xin bao wan, qiangli tianmai duzhong wan, tongsaimai pian, maitong capsule and xiaoshuan tongluo capsule. When(r) =0. 87, there were 2 kinds of drugs similar in cluster, namely buchang naoxintong capsule and xin bao wan.CONCLUSION: It is indicated in the research that the drugs are in rich of trace and constant elements. The larger the correlation coefficient of drugs is,the more similar the pharmaceutical efficacy is. It is discovered the close interrelationship of the trace and constant elements in drugs with their pharmaceutical efficacy. In this research, fuzzy classified method is used to discuss, in the view of trace elements, the similarity of quantitative indexes in efficacy of Chinese patent medicine commonly administrated in the treatment of cardio-cerebral vascular disease, which has provided a new approach for the quality evaluation of Chinese patent medicine.

Inhibitory Effect of K_6[P_2W_(18)O_(62)]?14H_2O on Human Breast Tumor Cell MCF-7 Growth in Vitro / 中国药房

OBJECTIVE: To study the in vitro anti-tumor activity of tungstophosphate compound(K6[P2W18O62]?14H2O) with Dawson structure.METHODS: MCF-7 cells were treated with different concentration of K6[P2W18O62]?14H2O,then its IC50 was measured by MTT assay;flow cytometry was applied to analyze cell cycle and apoptosis by computing the ratio of MCF-7 cells in different phase and its apoptotic ratio.Cell growth and viability were measured by trypan blue staining and cell counting by computing the ratio of living cells.The synergetic anti-tumor activity of tungstophosphate compound(K6[P2W18O62]?14H2O) and pharmorubicin was studied.RESULTS: K6[P2W18O62]?14H2O inhibited MCF-7 cell proliferation with IC50 of(33.7?3.2) ?mol?L-1,it reduced the proportions of MCF-7 cells at S phase and G1 phase,with the apoptotic rate at 3.7%～29.2% within 48 h.The percentage of living cells was 95.37%～76.78% at 48 h.As compared with pharmorubicin alone,the addition of K6[P2W18O62]?14H2O to pharmorubicin showed a significantly higher inhibitory action on proliferation of MCF-7 cells(P