Separation and Identification of Microplastics in Digestive System of Bivalves / 分析化学

As a new type of pollutants,microplastics (small pieces of plastic with the longest dimension less than 5 mm) are bringing wide concerns by policy makers and researchers. The impact of microplastics on marine life is closely related to human health. Bivalves, as filter feeding creatures, can easily ingest microplastics in their feeding process. The microplastics in these bivalves can easily enter the human body through the food chain. In this study,a bivalve sample pretreatment method was established for separating the microplastics in their digestive system. Qualitative and quantitative analysis of microplastics was carried out by micro-Fourier transformed infrared spectroscope (μ-FT-IR) and Stereo microscope. By comparing the digestion systems of using 10% KOH and 30% H2O2, respectively, it was found that the digestion system using 10% KOH had better digestion efficiency. The recoveries of polypropylene (PP),polyethylene (PE), polystyrene (PS) and polyvinyl chloride (PVC) ranged from 96.7% to 98.6%, with RSD of less than 3.2%. Therefore, 10% KOH was used to digest organic tissue for separating the microplastics from the bivalves'digestive system. With this digestion system,the microplastics in Chlamys farreri from local markets and Mytilus galloprovincialis from both local markets and wild environments in Qingdao were separated and analyzed. The results showed that microplastics were found in over 80% of the individuals purchased from the market and 40% in the collected wild individuals. The average abundance of microplastics in Chlamys farreri purchased from different markets varied from 5.2 to 19.4 items/individual and 3.2-7.1 items/g, while in Mytilus galloprovincialis, the numbers varied from 1.9 to 9.6 items/individual and 2.0-12.8 items/g. Farmed mussels contained more microplastics (average 1. 9 items/individual, 3. 17 items/g) than wild mussels (average 0.53 items/individual, 2.0 items/g). There were various colors of microplastics. The detected microplastics came from three shapes: fibers, fragments and granules. Fibrous microplastics, being the most dominant ones,accounted for 84.11%. The average size of fibrous microplastics(0.66±0.70 mm) was larger than that of the other two shapes of microplastics. The number of microplastics decreased with increasing microplastic sizes. Microplastics of less than 500 μm coming from different markets were in the range of 26% to 84%. There was great spatial difference in the size of microplastics. The polymer composition of the microplastic was identified by μ-FT-IR. And it was found that the most common polymer component sin the samples were cellophane(CP),followed by polypropylene(PP). In the farmed mussels,PP was the most abundant component. The results showed that the abundance and types of microplastics in cultured or wild bivalves were closely related to their growing environment.

Killing effect of coexpressing cytosine deaminase and thymidine kinase on rat vascular smooth muscle cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Vascular smooth muscle cell (VSMC) proliferation following arterial injury plays a critical role in a variety of vascular proliferative disorders, such as atherosclerosis and restenosis after balloon angioplasty. Herpes simplex virus-thymidine kinase (HSV-TK)/ganciclovir (GCV) and E. coli cytosine deaminase (CD)/5-fluorocytosine (5-Fc) suicide gene systems have been successfully employed in cardiovascular gene therapy, respectively. We reasoned that coexpression of both HSV-TK with CD suicide genes would lead to increased cell killing. To test this imagine, the adenoviral vectors expressing TK and/or CD genes were developed and tested on vascular smooth muscle cells.</p><p><b>METHODS</b>Adenoviral vectors, including Ad-EF1alpha-CD-cytomegalovirus (CMV)-TK coexpressing both CD and TK double suicide genes, Ad-EF1alpha-CD and Ad-CMV-TK expressing CD and TK respectively, and control vector Ad-CMV-LacZ, were constructed and prepared with homologous recombination in RecA + E. coli cells. Integration and expression of CD and/or TK gene were identified by PCR and Western blot. Primary cultured VSMCs were infected at a multiplicity of infection (MOI) of 20 with exposure to their matching prodrugs 5-Fc and GCV. Cell mortality was measured by methyl thiazolyl tetrazolium (MTT) assays. Flow cytometry analysis was used to detect cell death. Apoptotic cells were analyzed using Hoechst 33342 fluorescence dye as a DNA probe. Genomic DNA cleavage of apoptotic VSMCs was tested by agarose gel electrophoresis.</p><p><b>RESULTS</b>Recombinant adenovirus expressing CD and/or TK suicide genes were successfully constructed. Both single and double suicide genes could be integrated into adenoviral genome and expressed. Cytotoxic effects of Ad-EF1alpha-CD-CMV-TK double suicide genes combined with 5-Fc and GCV were higher than those of Ad-CMV-TK and Ad-EF1alpha-CD single gene groups. The rate of cell survival was only (9 +/- 3)% in the Ad-EF1alpha-CD-CMV-TK group, but (37 +/- 3)% in the Ad-CMV-TK and (46 +/- 4)% in the Ad-EF1alpha-CD groups (P < 0.05). Flow cytometry analysis indicated that the killing mechanisms of the groups were different. Necrosis and apoptosis were involved in the mechanism of the double gene group. Based on the DNA stainability with Hoechst 33342, the apoptotic rates of VSMCs in the Ad-EF1alpha-CD-CMV-TK [(11.0 +/- 2.1)%] and Ad-CMV-TK [(12.0 +/- 2.2)%] groups were higher than those in Ad-CMV-LacZ [(1.2 +/- 0.11)%] and Ad-EF1alpha-CD [(5.0 +/- 1.8)%] groups (P < 0.05, respectively). DNA smear could be observed in both Ad-CMV-TK and Ad-EF1alpha-CD-CMV-TK groups after administration of prodrugs.</p><p><b>CONCLUSIONS</b>The killing effect on rat VSMCs mediated by adenoviral CD/TK double suicide genes is superior to that of single suicide gene. The killing mechanism of recombinant adenovirus coexpressing CD/TK double suicide genes is mainly through cytotoxic effect and apoptosis.</p>

Effect of antisense thrombin receptor and p21 double gene co-expression system on the proliferation and apoptosis in human aortic smooth muscle cells / 中国医学科学院学报

<p><b>OBJECTIVE</b>To focus on the study of the effect on proliferation and apoptosis of human aortic smooth muscle cells (ASMC) by adeno-associated virus (AAV) vector carrying antisense thrombin receptor (ATR) and p21 double gene co-expression system.</p><p><b>METHODS</b>Cultured human AMSC was infected with recombinant AAV containing ATR, p21 single gene and AP double gene respectively. The integration and expression of genes were confirmed by semi-quantitative RT-PCR. The anti-proliferation effect was determined by MTT assay. Cell cycle and apoptotic cell counts were measured through Flow Cytometry. The rate of apoptotic cells was examined with acridine orange/ethidium bromide(AO/EB) stain.</p><p><b>RESULTS</b>RT-PCR indicated that the exogenous genes had been integrated into ASMC. The rates of cell survival were decreased by 16.67%, 21.60%, and 29.4% and the cell counts of G0/G1 phase were (61.8 +/- 2.9)%, (82.5 +/- 4.0)%, (80.4 +/- 6.1)% in ATR, p21 and AP group respectively after rAAV infected 4 days. The level and area of apoptotic peak were greater in AP double gene than ATR and p21 single gene. Cell stain indicated that apoptotic cells were (7.2 +/- 3.3)%, (10.7 +/- 5.6)%, and (18.3 +/- 2.7)% in each transgene group compared with (1.5 +/- 0.8)% in control group.</p><p><b>CONCLUSION</b>AP double gene co-expression system has powerful effect for inhibiting proliferation and inducing apoptosis ASMC than ATR and p21 single gene and that is a superior way for gene therapy to restenosis.</p>