Conversion of human adipose-derived stem cells into functional and expandable endothelial-like cells for cell-based therapies.

BACKGROUND: Ischemic vascular diseases are the major cause of death worldwide. In recent years, endothelial cell (EC)-based approaches to vascular regeneration are increasingly viable strategies for treating ischemic diseases, but their applications are challenged by the difficulties in their efficient generation and stable maintenance. Here, we show an alternative protocol that facilitates the generation of functional and expandable ETS variant 2 (ETV2)-induced endothelial-like cells (EiECs) from human adipose-derived stem cells (hADSCs), providing a potential source of cells for autologous ECs to treat ischemic vascular diseases. METHODS: hADSCs were obtained from fresh human adipose tissue. Passage 3 hADSCs were transduced with doxycycline (DOX)-inducible ETV2 transcription factor; purified ETV2-hADSCs were induced into endothelial-like cells using a two-stage induction culture system composed of small molecule compounds and cell factors. EiECs were evaluated for their surface markers, proliferation, gene expression, secretory capacity, and effects on vascular regeneration in vivo. RESULTS: We found that short-term ETV2 expression combined with TGF-ß inhibition is sufficient for the generation of kinase insert domain receptor (KDR)+ cells from hADSCs within 10 days. KDR+ cells showed immature endothelial characteristics, and they can gradually mature in a chemically defined induction medium at the second stage of induction. Futher studies showed that KDR+ cells deriving EC-like cells could stably self-renew and expand about 106-fold in 1 month, and they exhibited expected genome-wide molecular features of mature ECs. Functionally, these EC-like cells significantly promoted revascularization in a hind limb ischemic model. CONCLUSIONS: We isolated highly purified hADSCs and effectively converted them into functional and expandable endothelial-like cells. Thus, the study may provide an alternative strategy to obtain functional EC-like cells with potential for biomedical and pharmaceutical applications.

[DNA barcoding and its utility in commonly-used medicinal snakes].

Identification accuracy of traditional Chinese medicine is crucial for the traditional Chinese medicine research, production and application. DNA barcoding based on the mitochondrial gene coding for cytochrome c oxidase subunit I (COI), are more and more used for identification of traditional Chinese medicine. Using universal barcoding primers to sequence, we discussed the feasibility of DNA barcoding method for identification commonly-used medicinal snakes (a total of 109 samples belonging to 19 species 15 genera 6 families). The phylogenetic trees using Neighbor-joining were constructed. The results indicated that the mean content of G + C(46.5%) was lower than that of A + T (53.5%). As calculated by Kimera-2-parameter model, the mean intraspecies genetic distance of Trimeresurus albolabris, Ptyas dhumnades and Lycodon rufozonatus was greater than 2%. Further phylogenetic relationship results suggested that identification of one sample of T. albolabris was erroneous. The identification of some samples of P. dhumnades was also not correct, namely originally P. korros was identified as P. dhumnades. Factors influence on intraspecific genetic distance difference of L. rufozonatus need to be studied further. Therefore, DNA barcoding for identification of medicinal snakes is feasible, and greatly complements the morphological classification method. It is necessary to further study in identification of traditional Chinese medicine.

[DNA barcoding of COI gene in some medicinal animals of Lacertilia].

To identify some medicinal animals of Lacertilia, in total 59 individuals belonging to 12 species 7 genera 3 families, we used the universal barcoding primers to sequence these species, compared with other homologous sequences (564 bp) obtaining from the GenBank and finally constructed phylogenetic trees using Neighbor-joining, Maximum parsimony and Bayesian inference, respectively. As a result, the mean content of G + C (46.5%) was lower than that of A + T (53.5%). As calculated by Kimera-2-parameter model, the whole individuals mean distance for interspecies and intraspecies was 35. 5% and 1.7%, respectively. The mean distance for interspecies was 21 times as much as that for intraspecies. The mean distance for intraspecies of Gekko swinhonis, Hemidactylus frenatus and G. gecko was greater than 2%, respectively. Further analyses suggested that geographical groups of the three species might be of different subSpecies, even species. Of course, incorporating morphological characters and other unlinked genetic markers in future studies will offer further insights into the divergence. On the basis of phylogenetic trees constructed by COI, our results indicated that the taxonomy of the category (family, genus, and species) by DNA barcoding is consistent with morphological characters. Therefore, DNA barcoding is a useful tool for both identification and phylogeny of medicinal animals of Lacertilia, particularly for nonprofessor identifying authentication of Chinese crude drugs of these species.

[Prime study of Gekko gecko in HPLC fingerprint].

OBJECTIVE: To establish HPLC fingerprint of Gekko gecko. METHODS: The relative retention time and relative peak area of exteacts of Gekko gecko were determine by HPLC to confirm proper chromatographic condition and obtain the data. RESULTS: Better distribution of relative retention time and relative peak area were shown under the chromatographic condition and the HPLC fingerprint was established. CONCLUSION: The established HPLC fingerprints of Gekko gecko can be used to identify Gekko gecko and its quality control.

Observation of clinical curative effect in the patients with internal endometriosis by interventional therapy / 中国医师进修杂志

ObjectiveTo explore the curative effect in the patients with internal endometriosis by interventional therapy.MethodsUsing Seldinger technique,34 cases with internal endometriosis wereperformed bilateral uterine artery embolization.Observed postoperative menstrual quantity,dysmenrrhea degree,anemia and the change of the volume of uterine lesions.ResultsAll the patients were followed up for 1-3 years,menstrual quantity average decreasd of 59.1％P ＜ 0.05 ),the symptoms of dysmenorrhea was significantly eased in 28 cases (82.4％,28/34).All the patients of anemia haemoglobin were back to normal,volume of uterus average reduced 43.8％P ＜ 0.05 ),lesion was obviously smaller or disappear.Ultrasonography showed myometrium and blood flow signal of lesion was was obviously reduced.Conclusion Internalendometriosis by interventional therapy can get good results,symptoms improve significantly.

[The molecular mechanism of extravasation in mouse melanoma lung metastasis model].

OBJECTIVE: To study molecular mechanisms underlying the extravasation of mice melanoma cells during lung metastasis. METHODS: B16-RED melanoma cell line was established which stably express the red fluorescent protein. B16-RED cells were compared with B16 cells in ability of proliferation and lung metastasis. A mouse lung metastasis model was established with B16-RED melanoma cells. FITC-dextran was injected i.v. and CD31 indirect immunoflourescence (IIF) staining was made to identify the location of the tumor cells and the time of tumor cell extravasation. Finally, at 48 hours post cell injection, the lung and a normal lung were removed and used for 32K mice microarray analysis. RESULTS: B16-RED was consistent with B16 in cell shape and ability of proliferation and lung metastasis. 52.7% of B16-RED melanoma cells completed the extravasation within 48 hours in mouse lung metastasis model. Many important signal pathways were involved during lung metastasis, including leukocyte transendothelial migration, MAPK signaling pathway, neuroactive ligand-receptor interaction, focal adhesion, cytokine-cytokine receptor interaction, regulation of actin cytoskeleton, axon guidance, calcium signaling pathway, tight junction, etc. CONCLUSION: The extravasation during metastasis is a complex and multiple-steps process, in which many important signal pathways in host tissues were involved.

Hyperthermia increases the therapeutic efficacy of survivinT34A in mouse tumor models.

The use of survivinT34A mutant targeted disruption of survivin, the strongest inhibitor of apoptosis protein overexpressed in tumors, has proved a promising strategy for advanced cancers. However, hyperthermia, as a cytotoxic enhancer, regularly activates the expression of survivin to counteract the heat-induced antitumor activity. Here, we investigated the combinational antitumor effect by using liposome-encapsulated mouse survivinT34A and hyperthermia in mouse models. We observed that the combination treatment of surivinT34A and hyperthermia significantly increased the growth inhibition and apoptosis of tumor cells in vitro compared with single treatment or other controls, which was similar to the effect of survivin silencing in combination with hyperthermia. Moreover, the inhibition of tumor growth in vivo was also remarkably enhanced by combination of surivinT34A and hyperthermia when compared with other treatments. Naturally, the tumor tissues in combination treatment presented the larger necrosis-like areas, more apoptotic cells and less microvessel density. Our findings suggest that the antitumor efficacy of survivin disruption can be enhanced by hyperthermia, which might be a new feasible approach for cancer therapy.

Enhanced antitumor efficacy by blocking activation of the phosphatidylinositol 3-kinase/Akt pathway during anti-angiogenesis therapy.

Anti-angiogenesis has been a promising strategy for cancer therapy. However, many signal pathways are activated during anti-angiogenic treatment to counteract the therapeutic efficacy. Among these pathways, evidence has directly pointed to the phosphatidylinositol 3-kinase/Akt (PI3K/Akt) pathway, whose activation resulted in tolerance to the absence of nutrients and oxygen when tumor angiogenesis has been inhibited. In the present study, we investigated the effects of blocking activation of the PI3K/Akt pathway on cell survival in vitro and tumor growth in vivo during anti-angiogenesis therapy. In modeled microenvironments in vitro, we observed that the phosphorylation of Akt in tumor cells was increased gradually in the absence of serum and oxygen in a time-dependent manner. The specific inhibitors of PI3K inhibited the proliferation of tumor cells in a dose-dependent manner in vitro. Moreover, inhibition was enhanced gradually with increased serum deprivation and/or hypoxia. In a mouse tumor model, we found the phosphorylation of Akt obviously increased following anti-angiogenic therapy using plasmids encoding soluble vascular endothelial growth factor receptor-2, but significantly reduced after treatment with LY294002. Consequently, the combinational treatment exhibited better antitumor effects compared with single treatments, presenting larger necrosis-like areas, more apoptotic cells, less microvessel density and less phosphorylated Akt in tumors. These results suggest that blocking activation of the PI3K/Akt pathway during anti-angiogenesis therapy could enhance antitumor efficacy. Thus, targeting the PI3K/Akt pathway might be a promising strategy to reverse tumor resistance to anti-angiogenesis therapy.

[Anti-tumor effects induced by the DNA vaccine coding human and mouse soluble VEGFR2].

OBJECTIVE: To develop a novel anti-angiogenesis strategy based on a DNA vaccine coding both human and mouse soluble VEGFR2. METHODS: The gene fragments coding human and mouse sVEGFR2 were amplified with PCR and cloned into pVITRO2 to generate pVITRO2-hm-sVEGFR2 recombinant. The in vitro VEGF blocking effect of the pVITRO2-hm-sVEGFR2 expression products on HUVEC cells were evaluated. The anti-tumor effect of pVITRO2-hm-sVEGFR2 was studied in mouse B16 model. The microvessels were stained by using CD31 antibody. RESULTS: The co-expressing vector pVITRO2-hm-sVEGFR2 was constructed successfully, confirmed by the restriction endonuclease digestion and sequencing. The expressing products of pVITRO2-hm-sVEGFR2 could obviously block the function of VEGF on promoting the proliferation of HUVEC in vitro. The tumor growth in mice was also significantly inhibited by pVITRO2-hm-sVEGFR2 expression. CD31 staining demonstrated that the microvessel density obviously decreased in tumor tissues treated with pVITRO2-hm-sVEGFR2. Both anti-tumor and anti-angiogenesis effects of pVITRO2-hm-sVEGFR2 were stronger than that of plasmids which coding only human or mouse sVEGFR2. CONCLUSION: pVITRO2-hm-sVEGFR2 could be a novel DNA vaccine for the anti-tumor therapy by inhibiting angiogenesis.

[IL-15 enhance the therapeutic effects of whole cell vaccine in mouse CT26 colon carcinoma model].

OBJECTIVE: To determine the enhancement effects of IL-15 to the tumor whole cell vaccine in tumor immunotherapy. METHODS: CT26 colon carcinoma model was established with BALB/c mice. Thirty two mice with CT26 colon carcinoma were divided randomly into four groups, which were subcutaneously injected at several spots with PBS (200 microL), CT26 whole cell vaccine (2 x 10(6) cells in 200 microL PBS), mIL-15 (20 microg, encapsulated with liposome in 200 microL 5% glucose solution) and CT26 whole cell vaccine (2 x 10(6) cells in 100 microL PBS) plus mIL-15 (20 microg, encapsulated with liposome in 100 microL 5% glucose solution) respectively every three days for six doses, the plasmid was injected beside the vaccine injecting spots. The size of tumors was measured every four days. All mice were sacrificed 30 days after tumor implantation. The pathologic observation and apoptotic analysis of tumors were preceded. RESULTS: CT26 whole cell vaccine combined with mIL-15 inhibited tumor growth by 45% compared with that of control group, the differences between CT26 + mIL-15 group and the other three groups were significant (P < 0. 05). The HE staining showed that the necrosis areas in tumors of the CT26 + mIL-15 group were larger than those of other three groups. The apoptotic index of tumors from the CT26 + mIL-15 group was (46.7 +/- 7.2)%, higher than that of the other three groups obviously (P < 0.05). CONCLUSION: IL-15 could enhance the therapeutic effects of CT26 whole cell vaccine in tumor immunotherapy.

Vascular endothelial cell antigen (VECA), a novel secreted protein: overexpression in Escherichia coli of the hexahistidine- tagged protein and production of polyclonal antibodies.

A novel VECA (vascular endothelial cell antigen) was previously identified by using an antibody pool against antigens in HUVECs (human umbilical-vein endothelial cells). VECA has been evolutionarily conserved in vertebrate species ranging from frog and fish to mouse and human. Bioinformatics analysis indicated that VECA was 10.1 kDa in size, with a predicted signal sequence and transmembrane domain, indicating that VECA may have important biological functions. The present paper describes a procedure for obtaining and purifying human recombinant VECA expressed in Escherichia coli as a fusion protein, via a human VECA cDNA linked pQE30 expression vector to DNA coding for hexahistidine. The purified protein was used to raise anti-(human VECA) polyclonal antibodies, which were suitable for detecting the presence of VECA in cells, cell-culture supernatant and tissues by immunoblotting and immunohistochemistry. To our knowledge, this is the first study on the protein expression and polyclonal-antibody production for human VECA. In addition, we report for the first time the positive identification of VECA in humans at the protein and subcellular level and provide the first experimental verification that VECA was indeed a secreted protein. The anti-(human VECA) polyclonal antibodies prepared may serve as a useful tool for future biological function studies on VECA.

Selection of root-zone media for higher plant cultivation in space.

OBJECTIVE: To investigate the cultivating effects of several mineral matters used as root-zone media for higher plant growth in space. METHOD: Four kinds of artificial and natural mineral matters were used as plant root-zone media based on lots of investigation and analysis. Nutrient liquid was delivered into the media by a long capillary material, and roots of plants obtained nutrition and water from the media. The related parameters such as plant height and photosynthetic efficiency were measured and analyzed. RESULT: The growing effect in a mixture of coarse and fine ceramic particles with equal quantity proportion was the best, that in fine ceramic particles was the second best, that in clinoptilolite particles was the third and that in diorite particles was the last. CONCLUSION: The mixture of coarse and fine ceramic particles with equal quantity possesses not only fine capillary action, but also good aerating ability, and therefore is capable of being utilized as an effective root-zone media for higher plants intended to be grown in space.

[Selection of light source used for plant cultivation in controlled ecological life support system].

OBJECTIVE: To select suitable light source for higher plant cultivation in the controlled ecological life support system of the future space station. METHOD: The experiment was carried out in the Space Higher Plant Cultivation Ground-based Experimental Facility (SHPCGEF); four combinations of two red and blue light-emitting diode (LED) were utilized as light sources; soilless cultivation technique with porous ceramic tubes and porous ceramic particles was utilized in the growth system. RESULT: The plants grown under the shelf of pure red LED showed a lying-down state in early stage, and stood erect in later period with slender and long stems; the plants under various combinations of red and blue LED grew with nearly normal state, but the plants under the combination of 90% red and 10% blue LED possessed the best comprehensive indexes. CONCLUSION: The normal growth and development of plantlets needs two light sources of red and blue LED, and the combination of 90% red and 10% blue LED is the optimum one among those tested combinations.