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Background Lead is ubiquitous in the environment pollutants, long-term accumulation in the human body, arising from systemic multi-system damage, especially the more serious harm to children [1]. Studies have shown that lead has a strong affinity nerve, can accumulate in the nerve tissue, causing long-term irreversible damage of the function of the nervous system [2]. The kidney as lead distribution and excretion of the major organs, and lead poisoning one of the target organs [3]. In recent years found no safety threshold, the toxic effects of lead, the body lead toxic [4]. While many countries have taken a number of measures to reduce environmental lead contamination, but chronic lead poisoning is still one of the major health problems faced by the modern urban residents [5]. Blood - brain barrier (Blood brain barrier, BBB) is an important structure of the central nervous system, its morphological basis, including a series of anatomical structures: the brain capillary endothelial cells and endothelial cells in the blood and between neurons close connection, the base film, the projections, as well as glial cells (glial membrane). BBB ensures a high degree of stability of the central nervous system environment within the required normal central nervous system function. Nephron (Renal Unit), mainly by glomerular and tubular kidney structure and function of the basic unit of the glomerular capillary plexus, organizational support during mesangial, mesangial cells play an important role; kidney The small tube is a single layer of epithelial tubule reabsorption of some of the ingredients in the original urine and excretion of waste. Visible BBB or nephron structure and / or function changes are caused by the core of many diseases pathophysiological changes. At present, the lead-induced brain, kidney injury studies in animal models and epidemiological investigation based [6]. Research on cells mainly to the hippocampus, cerebral cortex neuron cells and human renal tubular epithelial cells (HK-2) study the astrocytes (C8), brain capillary endothelial cells and human glomerular systematic study of mesangial cells (HMC) is relatively small. This study is on us early animal experiments basis, C8, people shares artery endothelial cells (HFAEC) and HMC, HK-2 as the research object, observe the lead acetate in vitro culture C8, HFAEC and HMC, HK-2 impact, to explore lead acetate to the cellular and molecular mechanisms of brain damage and kidney damage. Purpose of the first part of lead acetate on astrocytes and human femoral artery endothelial cells by observing the lead acetate explore lead acetate C8 C8 and HFAEC cell morphology, biochemical markers of apoptosis-related protein expression and the number of apoptotic cells, and HFAEC mechanism of injury and provide a theoretical basis, in order to lead to the damage of the BBB. Method 1 with different concentrations (5, 10, 20, 40 mol / L) the role of lead acetate (exposure) the logarithmic phase C8 and HFAEC, 6,12,24,48 h, at the same time without lead acetate group as the control group, the MTT method The detection of lead acetate on cell growth inhibition. (2) selection of 5, 10, 20 micromol / L lead acetate exposed to C8 and HFAEC 24h, the same without lead acetate group as the control group, using Giemsa HE staining and morphological changes were observed by transmission electron microscopy. 5, 10, 20 micromol / L lead acetate the C8 and HFAEC 24h after exposure, the spectrophotometer exposure group and control group C8 and HFAEC culture supernatant of lactate dehydrogenase (LDH) and malondialdehyde (MDA) content indirect understanding of cellular injury. 4. 5,10,20 μmol / L lead acetate exposed to C8 and HFAEC 24h, DNA Ladder method the observed exposure group and the control group the C8 and HFAEC genome DNA damage. 5, 10, 20 micromol / L lead acetate exposed to C8 and HFAEC 24h, the immunohistochemical assay exposure group and the control group C8 and the HFAEC c-Jun, p53, Bax, Bcl-2, caspase-3 protein expression. 6. 5,10,20 μmol / L lead acetate exposed to C8 and HFAEC 24h RT-PCR method to detect exposure group and control group C8 and HFAEC Caspase-3 mRNA transcription. 5, 10, 20 micromol / L lead acetate exposed to C8 and HFAEC 24h after exposure group and the control group of the Western blotting method detection C8 and HFAEC Caspase-3 protein expression. 8. 5,10,20 μmol / L lead acetate exposed to C8 and HFAEC 24h after PI-the by Hoechst33342 double the the staining the flow cytometry exposure group and control group the C8 and HFAEC apoptosis rate. 1 different concentrations of lead acetate (5, 10, 20, 40 mol / L) exposed to C8 different time of HFAEC (6, 12, 24, 48 h) after each exposure group cell growth inhibition rate than the control group were significantly liters (P lt; 0.01), and there is concentration and time dependent manner. Lead acetate (5, 10, 20 micromol / L) exposed to C8, HFAEC 24 hours after the inverted phase contrast microscopy revealed cell density lower than control group, smaller cell bodies and astrocytes synaptic shorten thinner cell reduce connection; Giemsa, HE staining observed in exposed groups than the control group cytoplasmic concentration, a large number of nuclear stain, shrinkage, the nucleolus cracking, part of the nucleus into the renal type horseshoe, electron microscopy also found that exposure group nucleus pyknosis, side shift, nuclear quality gap increases, and increase with the concentration of lead acetate formation of apoptotic bodies. 3 spectrophotometer culture supernatant LDH, MDA content found 5,10,20 μmol / L lead acetate exposure group C8 the culture supernatant LDH and MDA content in HFAEC were significantly higher than the control group (P lt; 0.01) . DNA ladder method to detect C8, 5,10,20 μmol / L lead acetate exposure group cells HFAEC found that the more obvious phenomenon of DNA damage compared to the control group cell genome. Immunohistochemistry test results showed 5,10,20 μmol / L lead acetate exposure group C8, HFAEC cells c-jun, p53, Bax, caspase-3 protein expression compared with the control group cells expressing increased significantly, while Bcl -2 protein expression level of lead acetate exposure group than the control group cells reduced cell. RT-PCR assay results show 5,10,20 μmol / L lead acetate treated group C8 HFAEC caspase-3 mRNA transcription the cell transcription level compared with the control group was significantly higher. 7 Western blotting assay 5,10,20 μmol / L lead acetate exposure group C8 HFAEC expression of Caspase-3 protein expression levels compared to the control cells was significantly increased, and was positively correlated with the dose of lead acetate. PI-by Hoechst33342 double staining and flow cytometry results display, 5, 10, 20, mol / L lead acetate exposure group C8 HFAEC apoptosis rate of apoptosis rate compared with the control group was significantly higher (P lt; 0.01 ), and a dose relationship. Conclusion lead acetate caused the C8 and HFAEC genomic DNA damage by generating lipid peroxides, further affecting the change in the amount of Apoptosis Related Proteins Bax/Bcl-2 ratio increased expression of activated caspase-3 apoptotic genes, eventually procure the C8 and HFAEC apoptosis. And may be provided by to apoptotic destruction to procure C8 and HFAEC, the structure and function of the blood-brain barrier. Objective of the second part of lead acetate on human mesangial cells and human renal tubular epithelial cells by observing the lead acetate HMC and HK-2 cells morphology, biochemical markers, apoptosis-related protein expression and the number of apoptotic cells of lead acetate on the HMC and HK-2 mechanism of injury, provide a theoretical basis for the lead-induced kidney injury. The same way as the first part of the results. Exposure to different concentrations of lead acetate (5, 10, 20, 40 mol / L) the HMC and HK-2 at different times (6, 12, 24, 48 h) after each exposure group cell growth inhibition compared the control group was significantly higher (P lt; 0.01), and there is concentration and time dependent manner. 5, 10, 20 micromol / L lead acetate exposure the HMC and HK-2 after 24 h, inverted phase contrast microscopy revealed cell density lower than control group, small rounded cell body; Giemsa, HE staining observed in the exposed group than the control group cytoplasmic concentration, a large number of nuclear stain, condensation, nucleolar cleavage, part of the nucleus into kidney type, horseshoe-shaped and so on, the same electron microscopic studies revealed nuclear pyknosis exposure group, side shift, the nucleoplasm gap increases, and with the concentration of lead acetate to increase the formation of apoptotic bodies and increased. Spectrophotometer culture supernatant LDH, MDA content, 5, 10, 20, mol / L lead acetate exposure group HMC and HK-2 culture supernatant LDH and MDA content than the control group were significantly higher (P lt; 0.01). DNA ladder detection the HMC and HK-2 gene, 5, 10, 20, mol / L lead acetate exposure group HMC and HK-2 gene compared with the control group cell genome appears more obvious phenomenon of DNA damage. The immunohistochemistry assay HMC and HK-2 apoptosis-related protein expression, the results show 5,10,20 μmol / L lead acetate exposure group cells in c-Jun, p53, Bax, caspase-3 expression than control cells expression was significantly increased, while the expression of Bcl-2 protein expression levels of lead acetate exposure group compared with the control group was significantly lower. RT-PCR method to detect 5,10,20 μmol / L lead acetate HMC and HK-2 cells exposed to 24 hours after Caspase-3 mRNA transcription was significantly higher compared to the control cells. 7 Western blotting assay 5,10,20 μmol / L lead acetate exposure HMC and HK-2 cells after 24 h Caspase-3 protein expression levels significantly higher than the control group. 8 PI-by Hoechst33342 double staining and flow cytometry, 5, 10, 20, mol / L lead acetate exposure group HMC and HK-2 apoptosis rate significantly increased apoptosis rate compared with the control group (P lt ; 0.01), and was positively correlated with lead acetate dose. The conclusions lead acetate HMC and HK-2 genomic DNA damage caused by generating lipid peroxides further affect the change in the amount of expression of apoptosis-related factors, the Bax/Bcl-2 ratio increased activation of Caspase-3 apoptotic genes high expression, leading to the HMC and HK-2 apoptosis. By promoting HMC and HK-2 apoptosis and may undermine the structure and function of the kidneys.
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