L-homocysteic acid (HCA) and other amino acids were conjugated to rat brain material (extracted rat brain protein) with glutaraldehyde to form HCA- and amino acids-brain material conjugates. The specificity of monoclo...L-homocysteic acid (HCA) and other amino acids were conjugated to rat brain material (extracted rat brain protein) with glutaraldehyde to form HCA- and amino acids-brain material conjugates. The specificity of monoclonal antibody (McAb) was tested on serial dilution test and absorption test on enzyme-linked immunosorbent assay (ELISA) using these conjugates as antigens instead of amino acids-BSA (bovine serum albumin) conjugates used previously. The characterized McAb was applied for immunohistochemical staining using PAP (peroxidase antiperoxidase) technique in combination with silver enhancement of diamino-benzene (DAB) products. The results indicated that McAb to L-HCA reacted with L-HCA-brain material conjugates, but not with other amino acids-brain material conjugates so far tested. McAb absorbed with L-HCA-brain material abolished or decreased immunoreactivity of L-HCA-brain material with McAb. The antibody selectively stained subpopulation of cells and processes in the hippocampus fixed with glutaradehyde. Absorption of McAb with L-HCA-brain material abolished immunohistochemical staining. These results suggested that McAb was specific for L-HCA-brain materials and could be used for imunohistocytochemistry. This would provide a new tool for immunohistochemical visualization and localization of L-HCA in the nervous system.展开更多
BACKGROUND: Recent researches demonstrate that onset of cerebral infarction always accompanies with obvious changes of function of thyroid axis; while, high-homocysteic acidemia has been proved as one of risk factors ...BACKGROUND: Recent researches demonstrate that onset of cerebral infarction always accompanies with obvious changes of function of thyroid axis; while, high-homocysteic acidemia has been proved as one of risk factors of vascular dementia and Alzheimer disease. Meanwhile, it is found that level of plasma homocysteic acid is positive correlation with injured degrees of cognitive function and brain damage. OBJECTIVE: To observe the changes of function of thyroid and level of homocysteic acid among patients with vascular dementia and compare with those patients without dementia cerebral infarction. DESIGN: Randomized grouping and contrast observation. SETTING: Department of Neurology, People’s Hospital Affiliated to Yunyang Medical College, South China Hospital of Wuhan University. PARTICIPANTS: A total of 38 patients with vascular dementia were hospitalized in the Department of Neurology, People’s Hospital Affiliated to Yunyang Medical College from February 2004 to December 2005. All patients met the diagnostic criteria of the Fourth Edition of Diagnostic and Statistical Manual of Mental Disorder (DSM-IV) established by American Psychiatric Association. Based on educational degrees, Mini-mental Status Examination (MMSE) was classified into illiteracy (≤ 17 points), education of primary school (educational duration ≤ 6 years, ≤ 24 points) and education of middle school or above (educational duration > 6 years, ≤ 24 points). Forty patients with non-dementia cerebral infarction were regarded as the control group and checked with CT examination. There were no significant differences of sex and age between the two groups. All patients and relatives were provided the consent. METHODS: Within 24 hours after hospitalization, patients with vascular dementia received MMSE scores, and the degrees were classified based on the scoring results: mild (20-24 points), moderate (10-19 points) and severe (below 10 points). Levels of thyroxine were measured with radioimmunodetection and content of homocysteic acid was measured with high performance liquid chromatogram (HPLC) electrochemical detection. MAIN OUTCOME MEASURES: Levels of homocysteic acid and thyroxine among patients with vascular dementia and non-dementia cerebral infarction. RESULTS: A total of 38 patients with vascular dementia and 40 patients with non-dementia cerebral infarction were involved in the final analysis. ① Levels of triiodothyronine (T3), thyroxine (T4) and free T3 (FT3) were (0.9±0.4) μg/L, (92.9±26.4) μg/L and (3.9±1.8) pmol/L in vascular dementia group respectively, which were higher than those in control group [(1.3±0.3) μg/L, (110.2±28.7) μg/L, (7.2±2.1) pmol/L, t =2.766 6-7.433 6, P < 0.01]; while, level of homocysteic acid was (29.57±7.12) μmol/L in vascular dementia group, which was higher than that in control group [(24.53±4.98) μmol/L, t =3.637 7, P < 0.01]. There were no significant differences of free T4 (FT4) and thyrotropic-stimulating hormone (TSH) between the two groups (P > 0.05). ② Levels of FT3 of patients with mild, moderate and severe vascular dementia were (1.0±0.2), (0.9±0.1) and (0.8±0.1) μg/L, respectively; levels of homocysteic acid were (26.52±4.84), (29.59±5.56) and (32.71±6.17) μmol/L, respectively. There were significant differences among patients at the three degrees of vascular dementia (F =3.59-32.4, P < 0.01). However, there were no significant differences of T4, FT4 and TSH among the three kinds of patients (P > 0.05). CONCLUSION: Levels of thyroxine of patients with vascular dementia decrease; however, levels of homocysteic acid increase. Therefore, the results can indirectly reflect severities of vascular dementia.展开更多
目的研究补阳还五汤抗同型半胱氨酸(Hcy)诱导的脐静脉内皮细胞(HUVEC)损伤的作用及其机制。方法采用Hcy造模,将内皮细胞随机分为10组,即空白组(10%空白血清),模型组(Hcy+10%空白血清),补阳还五汤低剂量组(5%含药血清+Hcy)、中剂量组(10...目的研究补阳还五汤抗同型半胱氨酸(Hcy)诱导的脐静脉内皮细胞(HUVEC)损伤的作用及其机制。方法采用Hcy造模,将内皮细胞随机分为10组,即空白组(10%空白血清),模型组(Hcy+10%空白血清),补阳还五汤低剂量组(5%含药血清+Hcy)、中剂量组(10%含药血清+Hcy)、高剂量组(20%含药血清+Hcy),10%FBS+Hcy对照组,Rho激酶(ROCK)阻断剂Y27632+Hcy组,靶分子肌球蛋白轻链激酶(MLCK)阻断剂ML-7+Hcy组,丝裂原活化蛋白激酶(MAPK)抑制剂SB203580+Hcy组,细胞外信号调节激酶(ERK)抑制剂PD98059+Hcy组,24 h后,用IP裂解液(含PMSF)裂解细胞。用蛋白质印迹法(Western blot)测定ROCK、MLCK蛋白水平,用RT-PCR技术测定ROCK、MLCK m RNA的表达情况,并用激光共聚焦测定细胞骨架结构蛋白纤维肌动蛋白(F-actin)的变化。结果 Western blot与RT-PCR结果显示:模型组与空白组比较,HUVEC细胞ROCK、MLCK蛋白以及其m RNA水平明显上升,而补阳还五汤含药血清组和抑制剂组对其有明显的下调作用。激光共聚焦检测骨架蛋白F-actin实验中,与空白组比较,模型组细胞应力纤维明显增多;而补阳还五汤各剂量组相对于模型组,应力纤维的形成明显降低。结论补阳还五汤可能通过调节内皮细胞Rho/Rho激酶信号通路,阻止细胞骨架的改变,减轻内皮细胞损伤而发挥其抗动脉粥样硬化的作用。展开更多
文摘L-homocysteic acid (HCA) and other amino acids were conjugated to rat brain material (extracted rat brain protein) with glutaraldehyde to form HCA- and amino acids-brain material conjugates. The specificity of monoclonal antibody (McAb) was tested on serial dilution test and absorption test on enzyme-linked immunosorbent assay (ELISA) using these conjugates as antigens instead of amino acids-BSA (bovine serum albumin) conjugates used previously. The characterized McAb was applied for immunohistochemical staining using PAP (peroxidase antiperoxidase) technique in combination with silver enhancement of diamino-benzene (DAB) products. The results indicated that McAb to L-HCA reacted with L-HCA-brain material conjugates, but not with other amino acids-brain material conjugates so far tested. McAb absorbed with L-HCA-brain material abolished or decreased immunoreactivity of L-HCA-brain material with McAb. The antibody selectively stained subpopulation of cells and processes in the hippocampus fixed with glutaradehyde. Absorption of McAb with L-HCA-brain material abolished immunohistochemical staining. These results suggested that McAb was specific for L-HCA-brain materials and could be used for imunohistocytochemistry. This would provide a new tool for immunohistochemical visualization and localization of L-HCA in the nervous system.
文摘BACKGROUND: Recent researches demonstrate that onset of cerebral infarction always accompanies with obvious changes of function of thyroid axis; while, high-homocysteic acidemia has been proved as one of risk factors of vascular dementia and Alzheimer disease. Meanwhile, it is found that level of plasma homocysteic acid is positive correlation with injured degrees of cognitive function and brain damage. OBJECTIVE: To observe the changes of function of thyroid and level of homocysteic acid among patients with vascular dementia and compare with those patients without dementia cerebral infarction. DESIGN: Randomized grouping and contrast observation. SETTING: Department of Neurology, People’s Hospital Affiliated to Yunyang Medical College, South China Hospital of Wuhan University. PARTICIPANTS: A total of 38 patients with vascular dementia were hospitalized in the Department of Neurology, People’s Hospital Affiliated to Yunyang Medical College from February 2004 to December 2005. All patients met the diagnostic criteria of the Fourth Edition of Diagnostic and Statistical Manual of Mental Disorder (DSM-IV) established by American Psychiatric Association. Based on educational degrees, Mini-mental Status Examination (MMSE) was classified into illiteracy (≤ 17 points), education of primary school (educational duration ≤ 6 years, ≤ 24 points) and education of middle school or above (educational duration > 6 years, ≤ 24 points). Forty patients with non-dementia cerebral infarction were regarded as the control group and checked with CT examination. There were no significant differences of sex and age between the two groups. All patients and relatives were provided the consent. METHODS: Within 24 hours after hospitalization, patients with vascular dementia received MMSE scores, and the degrees were classified based on the scoring results: mild (20-24 points), moderate (10-19 points) and severe (below 10 points). Levels of thyroxine were measured with radioimmunodetection and content of homocysteic acid was measured with high performance liquid chromatogram (HPLC) electrochemical detection. MAIN OUTCOME MEASURES: Levels of homocysteic acid and thyroxine among patients with vascular dementia and non-dementia cerebral infarction. RESULTS: A total of 38 patients with vascular dementia and 40 patients with non-dementia cerebral infarction were involved in the final analysis. ① Levels of triiodothyronine (T3), thyroxine (T4) and free T3 (FT3) were (0.9±0.4) μg/L, (92.9±26.4) μg/L and (3.9±1.8) pmol/L in vascular dementia group respectively, which were higher than those in control group [(1.3±0.3) μg/L, (110.2±28.7) μg/L, (7.2±2.1) pmol/L, t =2.766 6-7.433 6, P < 0.01]; while, level of homocysteic acid was (29.57±7.12) μmol/L in vascular dementia group, which was higher than that in control group [(24.53±4.98) μmol/L, t =3.637 7, P < 0.01]. There were no significant differences of free T4 (FT4) and thyrotropic-stimulating hormone (TSH) between the two groups (P > 0.05). ② Levels of FT3 of patients with mild, moderate and severe vascular dementia were (1.0±0.2), (0.9±0.1) and (0.8±0.1) μg/L, respectively; levels of homocysteic acid were (26.52±4.84), (29.59±5.56) and (32.71±6.17) μmol/L, respectively. There were significant differences among patients at the three degrees of vascular dementia (F =3.59-32.4, P < 0.01). However, there were no significant differences of T4, FT4 and TSH among the three kinds of patients (P > 0.05). CONCLUSION: Levels of thyroxine of patients with vascular dementia decrease; however, levels of homocysteic acid increase. Therefore, the results can indirectly reflect severities of vascular dementia.
文摘目的研究补阳还五汤抗同型半胱氨酸(Hcy)诱导的脐静脉内皮细胞(HUVEC)损伤的作用及其机制。方法采用Hcy造模,将内皮细胞随机分为10组,即空白组(10%空白血清),模型组(Hcy+10%空白血清),补阳还五汤低剂量组(5%含药血清+Hcy)、中剂量组(10%含药血清+Hcy)、高剂量组(20%含药血清+Hcy),10%FBS+Hcy对照组,Rho激酶(ROCK)阻断剂Y27632+Hcy组,靶分子肌球蛋白轻链激酶(MLCK)阻断剂ML-7+Hcy组,丝裂原活化蛋白激酶(MAPK)抑制剂SB203580+Hcy组,细胞外信号调节激酶(ERK)抑制剂PD98059+Hcy组,24 h后,用IP裂解液(含PMSF)裂解细胞。用蛋白质印迹法(Western blot)测定ROCK、MLCK蛋白水平,用RT-PCR技术测定ROCK、MLCK m RNA的表达情况,并用激光共聚焦测定细胞骨架结构蛋白纤维肌动蛋白(F-actin)的变化。结果 Western blot与RT-PCR结果显示:模型组与空白组比较,HUVEC细胞ROCK、MLCK蛋白以及其m RNA水平明显上升,而补阳还五汤含药血清组和抑制剂组对其有明显的下调作用。激光共聚焦检测骨架蛋白F-actin实验中,与空白组比较,模型组细胞应力纤维明显增多;而补阳还五汤各剂量组相对于模型组,应力纤维的形成明显降低。结论补阳还五汤可能通过调节内皮细胞Rho/Rho激酶信号通路,阻止细胞骨架的改变,减轻内皮细胞损伤而发挥其抗动脉粥样硬化的作用。
