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Alzheimer's disease (Alzheimer's disease, AD) is a multiple degenerative diseases of the central nervous system in old age and the pre-elderly major pathological changes including diffuse brain atrophy and beta-amyloid (β-amyloid protein of Aβ) deposition of senile plaques (senile plaques, SP), neurofibrillary tangles (neurofibrillary tangles, NFTs) and a large number of neurons lost. Accompany the the society aging process, the incidence of AD increased year by year, has become a serious hazard to human health, medical and social problems. The etiology and pathogenesis of AD is not yet clear. Studies have shown that inflammation of the brain caused by a variety of factors led to the activation of microglia reaction plays an important role in the occurrence of the development of AD. Complement system as an important part of the humoral immunity, are also involved in the inflammatory reaction process. In the brain of AD patients, the researchers found that the classic complement pathway early components C1, C2, C4, and complement activation ultimately the formation of membrane attack complex (Membrane attack complex, MAC) upregulation. In addition to the classical pathway, Strohmeyer found that the alternative pathway of complement associated with pathologic lesions of AD Immunohistochemistry showed the existence of alternative pathways critical factor factor B and factor D in the brains of patients with AD. In addition, weak evidence to support the mannan-binding lectin (Mannan binding lectin, MBL) pathway is also involved, Lanzrein found in the serum of AD with the control group, MBL did not have significant difference, but in the cerebrospinal fluid of AD patients with MBL there are significantly reduced. However, complement each component role in AD pathogenesis is still unclear. In recent years, the study of the complement C1q may play multiple roles. Some scholars have found toxic effects: reported that Clq Ap multivalent combination of participation in the the senile plaques nucleation process. The domestic study also found that C1q may be one of the factors that lead to brain neurons oxidative toxicity and neuronal death. Some scholars believe that C1q have neuroprotective effect: Pisalyaput on C1q can enhance microglial phagocytosis of Aβ, and can protect neurons against Aβ-induced neurotoxicity confirmed by in vitro experiments. In addition, studies have found that it can inhibit the neurotoxicity caused by Ap and serum P protein. Recently, Deborah, it is also beneficial to microglial phagocytosis of apoptotic neurons, and regulate the inflammatory reaction and then. In addition, however, complement C1q promote intracranial chronic inflammatory response of microglia led to it? This is possible because the complement C1q may promote the combination of Aβ and microglia. First, complement Clq both with Ap (as described above), but also combined with microglia. Now was confirmed that the microglia C1q receptor present on C1qR. Second, complement Clq, Aβ and microglia three position is very close to the brain tissue of patients with AD. Immunohistochemistry showed that deposition of Ap fiber around the senile plaques surrounding the presence of activated microglia and complement Clq. Complement C1q likely to promote the combination of the latter two combined with Aβ and microglial cells, thereby promoting the activation of microglial cells, and then the inflammatory response. Our study is the concentration of C1q and the same concentration of Ap fiber stimulation of BV-2 cells, and using C1qA (competitive with Aβ binding) blocking test, the final determination of the degree of activation of BV-2 cells-CD45 expression and inflammatory factor TNF-α, IL-6 protein and mRNA expression levels. BV-2 cells CD45 expression by flow cytometry; TNF-α, IL-6 protein concentration using ELISA technology, method reference of TNF-α, IL-6 ELISA test kit instructions attached; TNF-α, IL -6 mRNA was measured by quantitative PCR method with reference to the corresponding quantitative PCR instructions. The test results show that the the complement C1q increase BV-2 cells CD45 expression, increased protein and mRNA expression of TNF-α and IL-6 in the addition of complement C1q was no significant change. [Materials and Methods] 1. Material BV-2 microglial cells were purchased from the Shanghai Institute of Cell Bank; amyloid Aβ1-40 (Sigma, USA); complement C1q (the Israeli the Prospec company); complement C1qA (China Taiwan Abnova Company); CCK-8 reagent (Dojindo Laboratories Co., Ltd. in Japan); IL-6 and TNF-α ELISA kit (Austrian the Bender Company); NP40 (Cayman Chemical, USA); quantitative PCR kit (U.S. complex energy gene Company). Method 2.1 BV-2 cells cultured BV-2 cells in a 37 ° C water bath after 1000rpm centrifuged for 5 minutes to remove the frozen liquid, and then placed in 25cm2 flasks, at 37 ° C, 5? 2 under the conditions, containing 10? S DMEM high glucose medium cultured cells passaged until the cells were grown to 80% -90% confluent. 2.2 aggregation state of Aβ1-40 Preparation and identification with sterile deionized water Aβ1-40 was diluted to lmg / mL at 37 ° C incubated for 7 days, so that it becomes fibrillar aggregation of Aβ. 5μl of the liquid droplets and then take in the carbon support film 200 mesh deionized copper grids, air dried for 1 minute, using negatively stained with 2% uranyl acetate for 1 minute, using TEM. 2.3 Determination of cell viability resuspended culture flasks BV-2 microglia, 5 × 105/ml were seeded in 96-well culture plate, divided into 7 experimental group, a positive control group and a blank group. each containing 3 holes, cultured for 12h after each experimental group were added to the lipopolysaccharide LPS (1μg / L) of the positive control group, the experimental group of Aβ1-40 (100mg / L) the C1q (10nmol / L), C1q (50nmol / L), Aβ1-40 (100mg / L) C1q (10nmol / L), Aβ1-40 (100mg / L) C1q (50nmol / L), Aβ1-40 (100mg / L) C1q (10nmol / L) C1qA (10nmol / L), Aβ1-40 (100mg / L) C1q (50nmol / L) C1qA (50nmol / L), the blank group was not added (which contains both drugs and more than one group before the first dosing plus drug mixing at 37 ℃ incubated 1h), removal of the supernatant after 24 h culture, Add 100μl containing 10? K-8 culture medium after the culture was continued 2h, measuring the absorbance of each well using a microplate reader at 450nm wavelength. calculated according to the formula cell viability of each well. 2.4 in each experimental group BV-2 cells CD45 expression was measured in 6-well plates, repeat the above process, training 24 h after absorbing the supernatant and add 0.25% trypsin digestion of cells to be digested completely sucked trypsin added 1ml precooled PBS solution and the cells were resuspended, and then transferred to a 1.5ml microcentrifuge tube, 2000rpm centrifuge 5min, suction addition 200μl PBS was added to the supernatant and then resuspended cells still 2000rpm centrifugal 5min, and was repeated twice, and then with 100 μl PBS resuspended cells and joined 2μl flow rat anti-mouse CD45 antibody, and mix to incubate at 4 ℃ refrigerator for 30min, and then according to the above process again with PBS, the cells were washed 3 times with flow cytometry expression of CD45 BV- 2 the number of cells. 2.5 in each experimental group supernatant and cell lysate IL-6, TNF-α Determination in 24-well plates Repeat the above process, after 24 h of culture supernatant and stored at -20 ℃, and then use PBS washed hole cells, and containing 0.5% NP40 was added to each well 200μl After washing in PBS cells were lysed, and Finally, the cell lysate was centrifuged and the supernatant was saved at -20 ℃. Detection, all specimens removed from the refrigerator melting after all specimens after 3000rpm centrifuged for 20 minutes, I step the ELLISA kit instructions, and finally with a microplate reader at 450nm wavelength measured absorbance (A) values, the standard curve IL-6, TNF-α the concentration is calculated according to the curve. 2.6 in each experimental group, IL-6, TNF-α mRNA of determination, in a 6-well plates was repeated the above process, gettering supernatant after 24 h culture, total RNA was extracted using TRIZOL method, and RNA concentration was measured, I experiment multiplexed The energy provided by quantitative PCR kit instructions. Statistical analysis Measurement data were expressed as mean ± standard deviation (x ± s) said application SPSS13.0 statistical software for statistical analysis of the results using one-way ANOVA (One-way ANOVA), the homogeneity of variance with LSD method for group multiple comparisons, heterogeneity of variance with Dunnett's T3 multiple comparisons between groups. P lt; 0.05 as statistically significant. [Results] 1. Groups BV-2 cells in cell survival results of the comparison in each experimental group BV-2 microglia cell viability was no significant difference (F = 2.020, P = 0.103). Groups BV-2 cells CD45 expression by flow cytometry of the result of the comparison groups have different number of BV-2 microglial cells express CD45. Each experimental group BV-2 microglial cells express CD45 Quantity significant difference (F = 59.031, P = 0.000). , LPS (1μg / L) after treatment, BV-2 microglia expression of CD45 increase in number compared with the other groups, the difference was statistically significant (P lt; 0.05); separate Aβ1-40 (100mg / L treatment) C1q (50nmol / L) BV-2 microglial cells express CD45 in quantity than the LPS group outside the group, compared with LPS group decreased, the differences were statistically significant (P lt; 0.05). 3 groups supernatant and cell lysate of IL-6, TNF-α concentrations measured value of the IL-6 concentration results in 3.1 groups supernatant and cell lysate: supernatant and cell lysate of each experimental group fluid IL-6 concentration difference statistically significant (supernatant IL-6 concentration comparison F = 77.838, P = 0.000; comparison of the IL-6 concentration in the cell lysate F = 24.038, P = 0.000). After LPS treatment, IL-6 concentrations measured in the BV-2 cell supernatants and cell lysates compared with the rest of the group increased significantly compared, the difference was statistically significant (P lt; 0.05), the rest of the group IL-6 concentration in the supernatant and cell lysate mutual difference was not statistically significant (P gt; 0.05). Measured value of 3.2 groups of TNF-a concentration in the supernatant and cell lysate: TNF-α concentration in the supernatant and cell lysate of each experimental group difference was statistically significant (supernatant concentration of TNF-a comparison F = 157.891, P = 0.000; the cell lysate TNF-α concentrations compare F = 166.897, P = 0.000). The concentration of TNF-a by LPS, BV-2 microglial cell supernatants and its cell lysate and the rest of the group increased compared, the differences were statistically significant (P lt; 0.05). In addition, Aβl-40 (100mg / L) C1q (50nmol / L) treatment, with the exception of LPS outside groups compared to the supernatant was measured TNF-a increased compared with LPS group reduce their differences both have a statistically significant (P lt; 0.05). 4 groups of IL-6, TNF-a mRNA of expression result of 4.1 in each group BV-2 microglial cells of IL-6 mRNA expression levels: IL-6 mRNA expression levels in each experimental group difference was statistically significant (F = 66.743, P = 0.000). Which after LPS treatment, BV-2 microglial cells to IL-6 mRNA expression levels higher than other groups, the difference was statistically significant (P lt; 0.05); while the other group was no significant difference (P gt; 0.05) 4.2 in each group BV-2 microglial cells TNF-αmRNA expression levels: the level of expression of TNF-αmRNA each experimental group difference was statistically significant (F = 65.695, P = 0.000). The LPS-treated BV-2 microglia TNF-αmRNA expression levels increased compared to the other groups, the difference was statistically significant (P lt; 0.05); Aβ1-40 (100mg / L) of C1q (50nmol / L) treated BV-2 microglia TNF-αmRNA expression levels compared with LPS group increased compared to the rest of the group, the differences were statistically significant (P lt; 0.05) [Conclusion] a complement C1q enhanced Aβ activation of microglia. Complement C1q enhanced Aβ induced inflammatory response of microglia microglia that complement Clq and AD intervention significantly increased the secretion of TNF-a levels.
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