【作者】 廉馨； 王绿娅； 蔺洁； 陈立伟； 潘晓冬； 杜兰平； 秦彦文； 刘舒； 薛红； 陈保生；

【Author】 Lian Xin WANG Lu-ya , LIN Jie , Chen Li- wei Pan Xiao-dong , DU Lang-Ping,Qing Yan-wen, LIU Shu , Xue Hong Chen Bao-sheng, Beijing Institute of Heart Lung and Blood Vessel Diseases, Beijing 100029, China

【机构】 南华大学基础医学院生物化学分子生物学教研室； 北京市心肺血管疾病研究所动脉粥样硬化研究室； 中国医学科学院基础医学研究所国家医学分子生物学重点实验室；

【摘要】 目的利用流式细胞技术,检测家族性高胆固醇血症(FH)患者淋巴细胞低密度脂蛋白受体(LDL-R)活性,试图建立适合临床应用的LDL—R活性检测方法。方法(1)分离1例临床确诊为纯合FH先证者及其双亲淋巴细胞,以血脂正常者为对照,(2)采用免疫细胞组化法检测细胞表面LDL-R数量,RT—PCR法检测LDL—R的mRNA水平,灰度扫描分析;(3)流式细胞技术检验细胞结合和摄取荧光标记的低密度脂蛋白(LDL)的能力,激光共聚焦显微镜观察验证。结果(1)FH先证者及其父母亲淋巴细胞表面LDL—R数量明显降低,FH先证者、其父、其母和正常对照的免疫组化灰度扫描光密度值分别为0.08、0.12、0.15和0.42;(2)RT-PCR法检测结果显示先证者及其父母亲淋巴细胞LDL-R的mRNA水平明显降低,分别相当于正常对照的17.2%、41.4%和51.7%;(3)流式细胞仪检测结果显示FH先证者及其父母亲淋巴细胞表面LDL-R对LDL的结合与摄取能力均明显低于正常对照,分别相当于正常对照的13.6%、33.0%、32.2%,和47.8%、47.8%、40.8%;激光共聚焦显微镜观察验证。结论(1)纯合FH先证者淋巴细胞表面的LDL-R蛋白数量、mRNA水平与LDL-R结合能力均明显低于其杂合父母,其父母明显低于正常对照;(2)FH先证者及其父母的淋巴细胞表摄取LDL的能力明显低于正常对照,而先证者与其父母之间的差异不明显;(3)本文建立了流式细胞技术检测淋巴细胞LDL-R活性的方法,FH纯合子、杂合子及正常人的测定值不重叠,可用于评价患者LDL-R活性。更多还原

【Abstract】 Obejective To establish detection method to evaluate LDL - receptor function of lymphocyte cells of family hypercholesterolemia by means of flow cytometry. Methods 1. Isolated the lymphocyte cell of a FH proband, and his parents, using a blood with normal lipid level as control. 2. We analysis the expression of LDL - R at both transcription and translation level using RT - PCR and immunohistochemistry staining, gray scanning to semi - quantitate. 3. The binding and internalization of LDL to LDL receptor were detected by flow cytometer ( FCM) and co - focus microscope. Result 1. The density scan analysis showed that the proband’s and his parents’LDL - R on the cell membrane is significant low compared with normal control. The gray scale is about 0. 08 (proband) 0. 12 (father) , 0. 15 (mother) and 0. 42 (control). 2 The expression of LDL - R at both transcription and translation level decreases notably. Comparing with the control the relative expression level is 17. 2% (proband) , 41. 4% (father) , 51. 7% (mother). 3 The FCM analysis results showed that the LDL - R binding and internalization activity on the lymphocytes of the FH proband and his parents are lower than the normal significantly, only about 13.6% (proband), 33.0% (father), 32.2% ( mother); 7. 8% (proband) , 47. 8% (father) , 40. 8% (mother). Conclusion 1. The proband’s LDL - R transcription level, translation level, and LDL binding level is notably lower than his parents whom are also significant lower than control. 2. The internalization LDL level of proband and his parent are lower than control. But there was no significant difference in the family. 3 We established a method to detect LDL - R function on lymphocyte cell. The analysis results of FH homozygote, heterozygote, and normal control are not overlapped. The technique can be used as a method to evaluate LDL - R function.更多还原