Separation of adipose-derived stromal vascular fraction cells by a variety of physical methods: A comparative study / 中国组织工程研究

BACKGROUND: Increasing attention has been paid to vascular components of the adipose-derived matrix and adipose-derived stem cells in tissue engineering. Existing methods for separating the vascular components of the adipose-derived matrix mainly include enzymatic and bolus injection, both of which have fatal disadvantages. OBJECTIVE: To search for a method for preparing adipose-derived stromal vascular fractions with high efficiency, safety, and simplicity. METHODS: The group without any treatment was used as the negative control, and the enzymatic hydrolysis method served as the positive control. The enzymatic hydrolysis method, traditional bolus method, modified bolus method, glass beads method and built-in ultrasonic waves method were compared through cell volume, survival rate, cell fragments, cell viability, increment rate and detection of microbial infection. The enzymatic hydrolysis method and the common bolus injection method were commonly used in the separation of vascular component cells of the fat source matrix; the improved bolus method was a method obtained by improving on the basis of the ordinary bolus method; the glass bead method was to use the glass bead to oscillate. The shear force generated was obtained by adding glass beads to the fat granules and shaking at 2 500 r/min for 9 minutes to prepare stromal vascular fraction cells. Using the built-in ultrasonic method, adipose tissue was treated at 25 W for 36 seconds to obtain stromal vascular fraction cells through a cavitation effect. RESULTS AND CONCLUSION: (1) The size of stromal vascular fraction cells isolated by five methods showed no significant difference (P > 0.05). (2)The cell viability was lowest in the negative control group, and highest in the enzymatic hydrolysis group. The cell viability in the enzymatic hydrolysis, glass bead, and built-in ultrasonic wave groups was significantly higher than that in the modified and traditional bolus groups (P < 0.05). (3) The cell survival rate and cell proliferation rate in the enzymatic hydrolysis, glass bead, and built-in ultrasonic wave groups were significantly higher than those in the modified and traditional bolus groups (P < 0.05). (4) The cell fragmentation rate and cell apoptosis rate in the enzymatic hydrolysis, glass bead, and built-in ultrasonic wave groups were significantly lower than those in the modified and traditional bolus groups (P < 0.05). (5) These results indicate that the built-in ultrasonic method and the glass bead method are better in enriching vascular components of the adipose-derived matrix. But glass bead method adds exogenous products, so it increases the risk of pollution. Built-in ultrasonic method inserts the ultrasound probe into the adipose tissue, but as long as the ultrasound probe is thoroughly sterilized, the risk of contamination is minimized. In general, the built-in ultrasonic method and the glass bead method are superior to modified and traditional bolus methods.

T. thermophilus Rhamnolipids Induce Cytogenetic Damage on Human Lymphocytes and Bind DNA in vitro.

Aims: Bacteria including Pseudomonas and T. thermophilus secretes rhamnose–containing glycolipid biosurfactants called rhamnolipids (RLs), known as bacterial virulence factors. The aim of this investigation was the evaluation of DNA damage induced on human lymphocytes by both RLs itself, secreted in a host organism by pathogens during a bacterial attack or symbiosis and in combination with the camptothecin (CPT), and on calf thymus DNA. Study Design: Human lymphocytes and calf thymus DNA were treated with isolated T. thermophilus RLs for studying DNA damage in vitro. Methodology: RLs DNA damaging action was evaluated by the Sister Chromatid Exchanges (SCEs) methodology, a method for estimating genotoxicity of human exposure to different chemicals or other mutagenic agents and by DNA electrophoretic mobility experiments. Results: RLs at concentrations of 100 and 150 μg/mL reveal significant toxicity. The highest concentration of 200 μg/mL reveals higher genotoxicity. The frequency of SCEs/cell was increased two times over the control level. When CPT, an antineoplastic drug with DNA damaging action, was tested together with RLs the genotoxic activity was reduced significantly (P<0.01) compared to the action caused by CPT itself. Sequential increase in the concentration of RLs results in the proportional reduction of Proliferation Rate Index (PRI) which is a cytostatic index. Also, Mitotic Index (MI), a cytotoxic index, was also significantly decreased at concentration of 200 μg/mL RLs. Addition of RLs in the same concentration together with CPT doesn’t affect the MI so much. Moreover, RLs are obviously capable for strong binding to plasmid or calf thymus DNA in vitro. Conclusion: RLs exert genotoxicity, cytotoxicity and cytostaticity in human lymphocytes and play probably a protective role for cells against CPT due to RLs’ detergent capability to enrobe CPT and DNA, providing a significant property that might support its possible involvement in DNA horizontal transfer phenomena.

Inhibited effects of veliparib combined doxorubicin for BEL-7404 proliferation of human liver cancer cell line

OBJECTIVE@#To explore inhibition effects of veliparib as PARP inhibitor combined doxorubicin for BEL-7404 proliferation of human liver cancer cell line.@*METHODS@#BEL-7404 was taken as the object of study and conventional culture was performed. It was treated by doxorubicin and (or) veliparib after 24 h. Cell proliferation rate was detected by four methyl thiazolyl tetrazolium (MTT) assay, cell apoptosis was measured with annexin V-FITC/PI double staining method by flow cytometry, DNA damage degree evaluation by single cell gel electrophoresis assay, and cytosolic C levels of the mitochondrial and cytosol by polyacrylamide gel electrophoresis (Western blotting).@*RESULTS@#Cell proliferation rate of doxorubicin combined veliparib group was lower than that of the control group and doxorubicin alone treated group significantly (P<0.01), the apoptosis rate was significantly higher than that of the control group and doxorubicin alone treated group (P<0.05). At the same time, DNA damage level of doxorubicin combined with veliparib group was significantly higher than doxorubicin alone treatment group and the control group (P<0.01), and cytochrome C in the cytosol was significantly higher than that of control group and doxorubicin alone treated group (P<0.01).@*CONCLUSIONS@#Veliparib, PARP inhibitor could inhibit PARP activity, block tumor cell DNA repair, and have significant sensitizing effect for hepatocellular carcinoma cell line BEL-7404 treated with doxorubicin. This might provide a new target for clinical treatment of hepatic carcinoma.

ROS1 Expression in Invasive Ductal Carcinoma of the Breast Related to Proliferation Activity

PURPOSE: ROS1 is an oncogene, expressed primarily in glioblastomas of the brain that has been hypothesized to mediate the effects of early stage tumor progression. In addition, it was reported that ROS1 expression was observed in diverse cancer tissue or cell lines and ROS1 is associated with the development of several tumors. However, ROS1 expression has not been studied in breast cancer to date. Therefore, we investigated ROS1 expression at the protein and gene level to compare expression patterns and to verify the association with prognostic factors in invasive ductal carcinoma (IDC) of the breast. MATERIALS AND METHODS: Tissue samples from 203 patients were used. Forty-six cases were available for fresh tissue. We performed immunohistochemical staining and real-time polymerase chain reaction (PCR). RESULTS: ROS1 expression was significantly lower in proportion to higher histologic grade, higher mitotic counts, lower estrogen receptor expression, and a higher Ki-67 proliferation index, although ROS1 expression was not significantly associated with the survival rate. The result of real-time PCR revealed similar trends, however not statistically significant. CONCLUSION: Higher ROS1 expression may be associated with favorable prognostic factors of IDC and its expression in IDC is related to the proliferation of tumor cells.

Quantification of AgNOR expression in exfoliated oral mucosal cells of tobacco chewers with and without lesion.

Background: Nucleolar organizer regions (NOR) are associated with proliferative activity and represent a diagnostic and prognostic marker. Materials and Methods: Smears were taken from smokers, tobacco chewers, oral squamous cell carcinoma patients, and normal subjects and evaluated by 2 silver-staining nucleolar organizer region (AgNOR) counting methods: (1) mean number of AgNORs per nucleus (mAgNOR); and (2) percentage of nuclei with >3 and >5 AgNORs (pAgNOR). Results: A statistically significant difference was observed between normal subjects, smokers, tobacco chewers, and oral cancer patients and between tobacco chewers with and without lesion. No significant difference was observed between tobacco chewers and smokers except in the percentage of >5 criteria. Conclusions: AgNOR enumeration using noninvasive methods, such as the cytobrush appears to be useful technique in distinguishing between normal mucosa, mucosa with and without lesions exposed to carcinogens, such as tobacco and frank oral carcinoma.

HDAC1 Expression in Invasive Ductal Carcinoma of the Breast and Its Value as a Good Prognostic Factor

BACKGROUND: Histone deacetylase 1 (HDAC1) is associated with the expression and function of estrogen receptors and the proliferation of tumor cells, and has been considered a very important factor in breast tumor progression and prognosis. Several studies have reported an association between HDAC1 expression and poorer prognosis in cancers including breast cancer, with a few exceptions. However, because of the dearth of studies on HDAC1 expression in breast cancer, its significance for breast cancer prognosis has not been well defined. Therefore, we examined HDAC1 expression in invasive ductal carcinoma (IDC), the most common breast cancer, and investigated its potential prognostic significance. METHODS: We used 203 IDC tissue samples. Immunohistochemical stains for HDAC1 and real-time polymerase chain reaction for HDAC1 mRNA were performed and the results were compared to generally well-established prognostic factors in breast cancer and patient survival rates. RESULTS: HDAC1 expression was significantly reduced in proportion to higher histologic grade, higher nuclear pleomorphism score, and higher mitotic counts, and with lower estrogen receptor expression. Furthermore, it was significantly associated with the survival rate. CONCLUSIONS: HDAC1 expression is a good prognostic indicator in IDC.

DC loaded with CPP-Id increase the efficiency of CTL proliferation and enhance its cytotoxicity in vitro / 中国癌症杂志

Background and purpose:Dendritic cells(DCs) possess specialized feature such as pathogen recognition,antigen capturing and processing machinery,and stimulating naive T lymphocyte to have antitumor ability that allow them to act like professional APCs.This paper is aimed to confirm the impacts on the proliferation and secretion of INF-? of tumor specific CTL and its cytotoxocity induced by DC loaded with different antigen.Methods:After the stimulation of DC loaded with different antigen,the proliferative rate of allolymphocytes was measured by MTT and the cytotoxocity of CTL was evaluated with LDH method.The INF-? secreted by activated T lymphocytes was detected by ELISPOT.Results:The DC loaded with CPP-Id(320%?15%) had significantly induce T lymphocyte proliferating when comparing with the induction by DC loaded with Id(57%?10%)(P

The Effect of RGP Lens and Reverse Geometry Lens on Corneal Epithelial Proliferation Rate in Rabbit

PURPOSE: To investigate proliferation rate of the corneal epithelium with rigid gas permeable lens(RGP) or reverse geometry lens(RGL) wearing. METHODS: Twenty-four rabbits were fitted with either a RGP or RGL on right eye, with left eye serving as a control. They were sacrificed at 1, 3, 7, and 14 days after lens fitting. after immunohistochemistry using 5-bromo-2-deotyuridine, BrdU-labeled cells were counted in 10-medium power fields (X200) in each sample using light microscope by 1.0 mm intervals between superior limbus and the center. RESULTS: The number of BrdU-labeled cells was highest in peripheral cornea, and lowest in limbus(p<0.05). The BrdU-labeled cells of limbus increased by 127% but labeled-cells of the center decreased by 24% in RGP group after 1-day fitting. Increase of the labeled cells was observed in the mid-peripheral and central cornea by 12% and 121% each but the decrease of labelled cells was found at the limbus by 16% in RGL fitting group at 1 day. After 14days, Both RGP and RGL group showed decrease of BrdU-labeled cells in limbus by 8% each, and in the center by 32% and 63%, respectively. CONCLUSIONS: Normal rabbit cornea was characterized by different epithelial proliferation rates according to location. However these pattern was significantly altered with both RGL and RGP fitting group. The change was greater in RGL group than in RGP group. This suggested wearing the RGL might be less physiologic than RGP fitting.