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Objective@#To establish the immortalized mouse brain microvascular pericytes model and to apply to the cerebrovascular toxicants screening study.@*Methods@#Brain pericytes were isolated from 3 weeks of mice by tissue digestion. Immortalized pericyte cell line was constructed by infecting with LT retrovirus. Monoclone was selected to purify the immortalized pericyte cell line. The pericyte characteristics and purity were explored by immunocytochemistry. Cell proliferation was measured by using the Pomega MTS cell Proliferation Colorimetric Assay Kit. Pericytes were treated with 0, 160, 320, 640, 1 280, 2 560 μmol/L lead acetate, 0, 5, 10, 20, 40, 80 μmol/L cadmium chloride and 0, 5, 10, 20, 40, 80 μmol/L sodium arsenite in 24 hours. Cell toxicity of each group was determined by MTS assay, median lethal dose (LD50) was calculated in linear regression.@*Results@#Mouse brain pericytes were successfully isolated by tissue separation and enzyme digestion method. After immortalized by LT retroviruses, monoclone was selected and expanded to establish pericyte cell line. The brain pericytes exhibited typical long spindle morphology and positive staining for α-SMA and Vimentin. The proliferation of brain pericytes cell lines was very slowly, and the doubling time was about 48 hours. The proliferation of immortalized brain pericytes cell lines was very quickly, and the doubling time was about 24 hours. After lead acetate, cadmium chloride and sodium arsenite treatment for 24 hours respectively, gradual declines in cell viability were observed. The LD50 of lead acetate was 2 025.0 μmol/L, the LD50 of cadmium chloride was 36.6 μmol/L, and the LD50 of sodium arsenite was 33.2 μmol/L.@*Conclusion@#The immortalized mouse brain microvascular pericyte model is established successfully by infecting with LT retrovirus, and can be applied to screen cerebrovascular toxicants. The toxicity of these toxicants to immortalized mouse brain microvascular pericyte is in sequence: sodium arsenite,cadmium chloride, lead acetate.
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Objective@#To investigate the effects of cerebral cavernous malformation 3 (CCM3) gene knockout on the lead exposure-induced blood-brain barrier malfunction in mice brain, and the relationship between CCM3 knockout and the Alzheimer's disease (AD).@*Methods@#Wide type (WT) mice and CCM3+/- mice were divided into 4 groups, control group and lead exposed group in WT as well as CCM3+/- mice. Lead exposed groups were treated with 0.05% lead acetate in drinking water for 12 weeks, while control group drink deionized water freely. Blood lead and brain lead levels in each group were detected by graphite furnace atomic absorption spectrometry. The brain tissue of each group was made into paraffin sections, whose morphology were observed by HE staining. The expression of Aβ1-42 in brain tissue was detected by immunohistochemistry and the brain capillaries were labeled by VRGFR2. The protein expression of Claudin-5, ZO-1, and p-Tau was detected by Western blot. The brain tissue RNA was extracted and the relative expression of LRP-1 mRNA was detected by qRT-PCR.@*Results@#The levels of blood lead WT (216.07±84.16) and CCM3+/- (189.64±101.86) μg/L in lead exposed group were higher than those in control group WT (19.52±11.46) and CCM3+/- (11.79±8.20) μg/L, the difference was statistically significant (t=4.18, P=0.006; t=3.79, P=0.016). The levels of brain lead WT (1.78±0.69) and CCM3+/- (1.74±0.66) μg/L were higher than those in control group WT (1.06±0.87) and CCM3+/- (0.97±0.64) μg/L, the difference was statistically significant (t=3.67, P=0.018; t=3.88, P=0.015). The HE staining showed no obvious lesions in the brain of each group of mice. The results of immunohistochemistry showed that there was no Aβ1-42 deposition in the brain of mice in each group. The numbers of microvessels in the brain of CCM3+/- mice in the lead exposed group were decreased. Compared with the relative expression levels of Claudin-5 (WT: 1.30±0.03, CCM3+/-: 1.07±0.08) in control group mice brain, the relative expression of Claudin-5 (WT: 0.96±0.04, CCM3+/-: 0.59±0.01) was decreased with statistical significance (F=199.27, P<0.001). The relative expression level of LRP-1 gene mRNA in brain of lead exposed group (WT: 0.32±0.10, CCM3+/-: 0.06±0.01) was higher than that of unexposed group (WT:1.00±0.06, CCM3+/-:2.12±0.18), the difference was statistically significant (F=288.29, P<0.001). The relative expression level of LRP-1 gene mRNA in brain of CCM3+/- mice exposed to lead was lower than that of WT mice ((0.06±0.01)vs(0.32±0.10), t=26.90, P<0.001).@*Conclusion@#The mice did not show significant AD-like lesions under low-does lead exposure, but resulted in early damage of brain blood-brain barrier and early changes of AD-like lesions in mice, with CCM3+/- mice being sensitive to lead exposure stronger than that of WT mice, suggesting that deletion of CCM3 gene may be one of the potential risk factors for accelerating the development of AD in mice exposed to lead.
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In the course of using aspirin treatment,the patients with cardiocerebrovascular diseases may still have ischemic events,that is the phenomenon of aspirin resistance.Gene polymorphism and aspirin resistance related research has become a hot spot.This article reviews the gene polymorphisms associated with aspirin resistance.
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Objective To study the clinical effects of methylenetetrahydrofolate reductase(MTHFR) C677T gene mutation on mild vascular cognitive impairment(VCI)in patients with cerebral infarction.Methods The prospective study was conducted in 180 patients with atherosclerotic acute cerebral infarction hospitalized from June 2015 to May 2016.180 patients were divided into normal homocysteine(Hcy)group (Hcy 5.46~16.20 μmol / L,n=130)and high Hcy group(Hcy>16.20 μmol / L,n=50).MTHFR C677T genotypes were detected by polymerase chain reaction-restriction endonuclease fragment length polymorphism.The VCI state and risk factors for VCI were analyzed with Montreal Cognitive Assessment scale(MoCA)and Mini Mental State Examination(MMSE)score in patients with acute cerebral infarction on admission and 14 days,30 days and 90 days after admission.Results MMSE and MoCA scores at 14 days,30 days,90 days after admission were lower in high Hcy group than in normal Hcy group(P<0.05 or P<0.01).MMSE and MoCA scores were lower in T/T genotype than in C/C genotype(P<0.05)at 14 day after admission,and lower in C/T genotype than in C/C genotype at 30 day and 90 day follow up(P<0.05or P<0.01).Logistic regression analysis showed that Hcy may be independent risk factors for mild VCI(OR =1.274,95%CI:1.027~1.264,P=0.018).Conclusions MTHFR C677T gene mutations may cause hyper-homocysteinemia,and promote the emergence of VCI in patients with cerebral infarction.MTHFR C677T gene mutations may be a genetic susceptibility factor for VCL
الملخص
In the course of using aspirin treatment,the patients with cardiocerebrovascular diseases may still have ischemic events,that is the phenomenon of aspirin resistance.Gene polymorphism and aspirin resistance related research has become a hot spot.This article reviews the gene polymorphisms associated with aspirin resistance.
الملخص
Objective To study the value of methionine loading test (MLT) in the mild vascular cognitive impairment (VCI) after acute cerebral infarction.Methods The fasting plasma homocystine (Hcy) level and homocystine level after MLT were measured by high-performance liquid chromatography methods.We chose 240 patients with normal level of fasting plasma Hcy (normal group),159 patients with normal level of Hcy after MLT,81 patients with hyperhomocysteinemia after MLT (hyperhomocysteinemia group),and 112 patients with fasting hyperhomocysteinemia (fasting hyperhomocysteinemia group) in this study.The Montreal Cognitive Assessment (MoCA) and Mini-Mental State Examination (MMSE) were conducted in normal,hyperhomocysteinemia and fasting hyperhomocysteinemia groups on admission,at 7d,14 d,30 d after treatment.Results Logistic regression analysis showed that the increased level of Hcy might be an independent risk factor for VCI [OR:1.285,95%CI:1.038-1.265,P<0.05].The scores of MMSE and MoCA were lower in patients with fasting hyperhomocysteinemia and patients with hyperhomocysteinemia after MLT than in patients with normal fasting plasma Hcy at 7 d,14 d and 30 d after treatment (P<0.01 or 0.05),while the scores had no significant differences among the three group on admission (P>0.05).There were no significant differences in MMSE and MoCA scores between patients with fasting hyperhomocysteinemia and patients with hyperhomocysteinemia after MLT on admission,7 d,14 d and 30 d after treatment (P>0.05).Conclusions Hcy may be an independent risk factor for VCI.The MLT can discover the dormant vascular risk factors for VCI,which offers a valuable detection method for early intervention and prevention in the clinical medicine.
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Objective To evaluate the effect of the plasma homocystine (Hcy) after methionine loading test (MLT) on the recurrence of ischemic vascular event,including cerebral infarction,transient ischemic attack (TIA),acute coronary syndrome,other vascularembolism,in cerebral infarction patients.Methods The fasting plasma homocystine (FHcy) and homocystine after MLT (PHcy) levels were measured by high-performance liquid chromatography.383 cerebral infarction patients with normal Hcy level were selected and divided into hyperhomocysteinemia after MLT (PHcy) group (n=135) and non-hyperhomocysteinemia after MLT (NPHcy) (n=248).Recurrence rates of ischemic vascular events within a 5-years follow-up period was observed.Results The levels of FHcy,PHcy and △Hcy (PHcy level subtracted FHcy level) were higher in males than in females in the two groups (all P<0.05).The recurrence rates of cerebral infarction/TIA,acute coronary syndrome and other vascular embolism events were higher in PHcy group than in NPHcy group within the follow-up period (all P<0.05).By forward stepwise logistic analysis,we found that the increased PHcy and △Hcy levels were the independent risk factors for recurrent ischemic vascular events [odds ratio (OR):0.509,2.107,95% confidence interval (CI):0.286-0.904,1.185-3.745,both P<0.05].Conclusions PHHcy may be an independent risk factor for recurrence ischemic vascular events in cerebral infarction.
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BACKGROUND: Hyperhomocysteinemia is a new independent risk factor for coronary heart disease (CHD), but its association with cerebrovascular diseases is still controversial. The level of fasting plasma total homocysteine (plasma tHcy) alone is not enough to reveal the effect of hyperhomocysteinemia on cerebral infarction (CI).OBJECTIVE: To explore the association of hyperhomocysteinemia with folacin and vitamin B12 and with CI in young and middle-aged people, as well as the role of methionine loading test in the diagnosis of latent hyperhomocysteinemia.DESIGN: A case-control study, Spearman correlation analysis.PARTICIPANTS: A total of 85 patients were hospitalized within 2 days after the onset of CI at the Department of Neurology, the General Navy Hospital of Chinese PLA, from 2000 to 2003. There were 63 males and 22females aged 29-55 years old with the mean age of (48.74±5.05) years.There were 48 cases of lacunar CI and 37 cases of arterial thrombotic CI.Meanwhile, 44 normal controls, 30 males and 14 females aged 29-55 years old with the mean age of (47.75±6.71), were recruited from the department staff and those who came to the hospital for routine health examination.METHODS: Fasting vein blood of 6 mL was collected from the patients on the 1st day of hospitalization, and 2 mL of the blood was used for detecting the level of fasting plasma tHcy using high efficiency liquid chromatography. Methionine of 0.1 g/kg was taken orally by patients immediately after blood sampling; 4 hours later, the level of loading plasma tHcy was also determined. The remaining 4 mL was used for detecting the level of serum folacin and vitamine B12 with bioradiation assay.MAIN OUTCOME MEASURES: ① The level of fasting and loading plasma tHcy in CI group and control group. ② Incidence of hyperhomocysteinemia in CI group and control group [Hyperhomocysteinemia was confirmed if hyperhomocysteine was higher than 95% of the upper limit of fasting plasma tHcy or 4-hour loading plasma tHey of normal control group, that is, fasting plasma tHcy >17.26 μnol/L in males and >14.17 μnol/L in females; and loading plasma tHcy should be > 44.57 μmol/L in males and > 40.02 μmol/L in females. ③ The level of serum folacin and vitamine B12 in CI group and control group. ④ Single factor analysis of fasting plasma tHcy and related risk factors.RESULTS: Totally 85 CI patients and 44 normal controls were recruited in this study and all data were statistically analyzed. ① The level of fasting and loading plasma tHcy in CI group and control group: Both fasting and loading plasma tHcy was significantly higher in CI group than in control group [(22.49±9.80), (13.08±2.33) μmol/L; (48.07±11.20), (37.23±3.48)μmol/L, (t=8.409, 8.187, P < 0.01)]. ② Incidence of hyperhomocysteinemia: The incidence was obviously higher in CI group than in control group [68% (58/85), (9%, 4/44), X2=40.628, P < 0.01]. Totally 35 patients (41%, 35/85) had higher fasting plasma tHcy than that of normal controls, and 23 (27%, 23/85) had higher loading plasma tHcy than that of normal controls. ③ The level of serum folacin and vitamine B12 in CI group and control group: They were [(5.73±2.52), (7.14±2.20) μg/L] in CI group,significantly lower than control group [(473.47±190.19), (576.70±212.05) rng/L,(t =3.151, 2.809, P < 0.01)]. ④ Single factor analysis of plasma tHcy with related risk factors: Fasting and loading plasma tHcy was found obviously associated with sex, and folacin and vitamine B12 level (r = -0.306 to 0.488, P < 0.01), but not with other risk factors and neurological deficit scores (r = 0.021-0.173, P > 0.05). Moreover, only plasma tHcy level was proved to have significant positive correlation with fasting blood glucose (r=0.186, P < 0.05).CONCLUSION: Hyperhomooysteinemia is an independent risk factor for CI in young and middle-aged people. Methionine loading test is an essential means for detecting latent hyperhomocysteinemia. Insufficiency of folacin and vitamine B12, two important nutrition factors, may lead to hyperhomocysteinemia and indirectly result in CI.
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Objective To explore the possible effect of the plasma homocysteine level on the risk of recurrent cerebral infarction patients by follow-up research in hope for finding a new theoretical evidence for the therapy and the prophylaxis of cerebral infarction.Methods We determined the free plasma total homocysteine(tHcy)of 151 patients with acute cerebral infarction and 52 age-and gender-matched healthy controls by high-performance liquid chromatography,then we divided the patients into hyperhomocysteinemia (Hhcy)group and no hyperhomocysteinemia(Nhhcy)group according to the outcome.Within a 5-years follow-up period,we observed the recurrence of cerebral infarction in the 2 groups.Results There was no statistical deference in common information between Hhcy and Nhhcy group.But the recurrence rate of the Hhcy group,being 44.26%,was significantly higher than 12.22%of Nhhcy group(P