中国组织工程研究与临床康复
中國組織工程研究與臨床康複
중국조직공정연구여림상강복
JOURNAL OF CLINICAL REHABILITATIVE TISSUE ENGINEERING RESEARCH
2008年
7期
1393-1396
,共4页
糖尿病,2型%血小板膜糖蛋白类%糖尿病血管病变%流式细胞术%血小板聚集功能%组织构建
糖尿病,2型%血小闆膜糖蛋白類%糖尿病血管病變%流式細胞術%血小闆聚集功能%組織構建
당뇨병,2형%혈소판막당단백류%당뇨병혈관병변%류식세포술%혈소판취집공능%조직구건
背景:研究已证实血小板活化参与了糖尿病血管病变的发生发展.血小板膜糖蛋白Ib/IX/V复合物是血小板膜上主要的糖蛋白分子之一,是血管性血友病因子和凝血酶的受体,在血小板活化中起重要作用.目的:观察2型糖尿病患者血小板膜糖蛋白Ib/IX/V复合物及其组分糖蛋白Ibα表达的变化.设计:病例-对照实验.单位:华中科技大学同济医学院附属协和医院中西医结合科.对象:根据1999年世界卫生组织制定的糖尿病诊断标准选择2005-12/2007-01本院门诊就诊的外周血小板计数＞ 50×109 L-1 的2型糖尿病患者51例,男28例,女23例,纳入对象在检查前两周内未服用影响血小板功能的药物.根据病情控制情况将患者分为控制良好组(n =25)和控制不良组(n =26);根据是否合并血管病变分为合并血管病变组(n =27)和无血管病变组(n =24).选择同期本院健康体检者23名为健康对照组,年龄、性别与病例组匹配.纳入对象或家属对检测项目和试验知情同意.试验经医院伦理委员会批准.方法:患者均在清晨就诊时抽取空腹肘静脉血进行检测.健康体检者于来院体检时进行相同检测.①生化指标检测:由本院检验科完成空腹血糖、糖化血红蛋白和空腹面胰岛素的检测.②糖蛋白Ib/IX/V复合物及其组分糖蛋白Ibα的表达的检测:采集清晨空腹静脉血3 mL,38 g/L枸橼酸钠抗凝后10 g/L多聚甲醛固定45 min.取50 μL固定后全血加至流式检测管,同时分别向不同管中加入20 μL糖蛋白Ib/IX/V 复合物单克隆抗体CD42a-b-c-d和PE标记的糖蛋白Ibα单克隆抗体CD42b,混匀后室温避光孵育30 min,然后CD42a-b-c-d标记的样品中加入20 μL FITC标记的羊抗鼠 IgG,混匀后室温避光孵育30 min.于FACS420型流式细胞仪上检测样本平均荧光强度.③血小板聚集率测定:参照文献检测血小板聚集率.主要观察指标:①生化指标.②糖蛋白Ib/IX/V复合物及糖蛋白Ibα的表达情况.③血小板最大聚集率.结果:实验纳入的51例2型糖尿病患者和23名健康体检者全部进入结果分析.①生化指标检测结果:控制良好组和控制不良组患者空腹胰岛素与健康对照组比较,差异有显著性意义(P ＜ 0.01).控制不良组患者空腹血糖和糖化血红蛋白均明显高于健康对照组和控制良好组,差异有显著性意义(P ＜ 0.01).②糖蛋白Ⅰb/Ⅸ/Ⅴ复合物和糖蛋白Ⅰbα表达情况的比较:控制良好组和控制不良组患者糖蛋白Ⅰb/Ⅸ/Ⅴ复合物和糖蛋白Ⅰbα的表达明显低于健康对照组,控制不良组明显低于控制良好组,差异均有显著性意义(P ＜ 0.05～0.01).无血管病变组和合并血管病变组患者均明显低于健康对照组,差异有显著性意义 (P ＜ 0.01).无血管病变组和合并血管病变组患者糖蛋白Ⅰbα明显低于健康对照组,合并血管病变组明显低于无血管病变组,差异均有显著性意义(P ＜ 0.05).糖蛋白Ⅰb/Ⅸ/Ⅴ复合物平均荧光强度与空腹血糖、糖化血红蛋白和空腹胰岛素呈明显负相关(r =-0.634,-0.573,-0.649,P ＜ 0.05);糖蛋白Ⅰbα平均荧光强度与空腹血糖和糖化血红蛋白呈明显负相关(r =-0.602,-0.543,P ＜0.05).③血小板聚集功能的比较:2型糖尿病患者血小板最大聚集率较正常人明显升高,差异有显著性意义(t =-3.852,P ＜ 0.01),控制不良组明显高于控制良好组,合并血管病变组高于无血管病变组,差异均有显著性意义(P ＜ 0.05).结论:2型糖尿病患者早期无血管病变时即存在血小板活化,发生血管病变后活化更明显,且血小板活化与血糖呈正相关.血小板糖蛋白Ⅰb/Ⅸ/Ⅴ复合物作为凝血酶和血管性血友病因子的受体,可能参与了2型糖尿病血管病变的发生发展.
揹景:研究已證實血小闆活化參與瞭糖尿病血管病變的髮生髮展.血小闆膜糖蛋白Ib/IX/V複閤物是血小闆膜上主要的糖蛋白分子之一,是血管性血友病因子和凝血酶的受體,在血小闆活化中起重要作用.目的:觀察2型糖尿病患者血小闆膜糖蛋白Ib/IX/V複閤物及其組分糖蛋白Ibα錶達的變化.設計:病例-對照實驗.單位:華中科技大學同濟醫學院附屬協和醫院中西醫結閤科.對象:根據1999年世界衛生組織製定的糖尿病診斷標準選擇2005-12/2007-01本院門診就診的外週血小闆計數＞ 50×109 L-1 的2型糖尿病患者51例,男28例,女23例,納入對象在檢查前兩週內未服用影響血小闆功能的藥物.根據病情控製情況將患者分為控製良好組(n =25)和控製不良組(n =26);根據是否閤併血管病變分為閤併血管病變組(n =27)和無血管病變組(n =24).選擇同期本院健康體檢者23名為健康對照組,年齡、性彆與病例組匹配.納入對象或傢屬對檢測項目和試驗知情同意.試驗經醫院倫理委員會批準.方法:患者均在清晨就診時抽取空腹肘靜脈血進行檢測.健康體檢者于來院體檢時進行相同檢測.①生化指標檢測:由本院檢驗科完成空腹血糖、糖化血紅蛋白和空腹麵胰島素的檢測.②糖蛋白Ib/IX/V複閤物及其組分糖蛋白Ibα的錶達的檢測:採集清晨空腹靜脈血3 mL,38 g/L枸櫞痠鈉抗凝後10 g/L多聚甲醛固定45 min.取50 μL固定後全血加至流式檢測管,同時分彆嚮不同管中加入20 μL糖蛋白Ib/IX/V 複閤物單剋隆抗體CD42a-b-c-d和PE標記的糖蛋白Ibα單剋隆抗體CD42b,混勻後室溫避光孵育30 min,然後CD42a-b-c-d標記的樣品中加入20 μL FITC標記的羊抗鼠 IgG,混勻後室溫避光孵育30 min.于FACS420型流式細胞儀上檢測樣本平均熒光彊度.③血小闆聚集率測定:參照文獻檢測血小闆聚集率.主要觀察指標:①生化指標.②糖蛋白Ib/IX/V複閤物及糖蛋白Ibα的錶達情況.③血小闆最大聚集率.結果:實驗納入的51例2型糖尿病患者和23名健康體檢者全部進入結果分析.①生化指標檢測結果:控製良好組和控製不良組患者空腹胰島素與健康對照組比較,差異有顯著性意義(P ＜ 0.01).控製不良組患者空腹血糖和糖化血紅蛋白均明顯高于健康對照組和控製良好組,差異有顯著性意義(P ＜ 0.01).②糖蛋白Ⅰb/Ⅸ/Ⅴ複閤物和糖蛋白Ⅰbα錶達情況的比較:控製良好組和控製不良組患者糖蛋白Ⅰb/Ⅸ/Ⅴ複閤物和糖蛋白Ⅰbα的錶達明顯低于健康對照組,控製不良組明顯低于控製良好組,差異均有顯著性意義(P ＜ 0.05～0.01).無血管病變組和閤併血管病變組患者均明顯低于健康對照組,差異有顯著性意義 (P ＜ 0.01).無血管病變組和閤併血管病變組患者糖蛋白Ⅰbα明顯低于健康對照組,閤併血管病變組明顯低于無血管病變組,差異均有顯著性意義(P ＜ 0.05).糖蛋白Ⅰb/Ⅸ/Ⅴ複閤物平均熒光彊度與空腹血糖、糖化血紅蛋白和空腹胰島素呈明顯負相關(r =-0.634,-0.573,-0.649,P ＜ 0.05);糖蛋白Ⅰbα平均熒光彊度與空腹血糖和糖化血紅蛋白呈明顯負相關(r =-0.602,-0.543,P ＜0.05).③血小闆聚集功能的比較:2型糖尿病患者血小闆最大聚集率較正常人明顯升高,差異有顯著性意義(t =-3.852,P ＜ 0.01),控製不良組明顯高于控製良好組,閤併血管病變組高于無血管病變組,差異均有顯著性意義(P ＜ 0.05).結論:2型糖尿病患者早期無血管病變時即存在血小闆活化,髮生血管病變後活化更明顯,且血小闆活化與血糖呈正相關.血小闆糖蛋白Ⅰb/Ⅸ/Ⅴ複閤物作為凝血酶和血管性血友病因子的受體,可能參與瞭2型糖尿病血管病變的髮生髮展.
배경:연구이증실혈소판활화삼여료당뇨병혈관병변적발생발전.혈소판막당단백Ib/IX/V복합물시혈소판막상주요적당단백분자지일,시혈관성혈우병인자화응혈매적수체,재혈소판활화중기중요작용.목적:관찰2형당뇨병환자혈소판막당단백Ib/IX/V복합물급기조분당단백Ibα표체적변화.설계:병례-대조실험.단위:화중과기대학동제의학원부속협화의원중서의결합과.대상:근거1999년세계위생조직제정적당뇨병진단표준선택2005-12/2007-01본원문진취진적외주혈소판계수＞ 50×109 L-1 적2형당뇨병환자51례,남28례,녀23례,납입대상재검사전량주내미복용영향혈소판공능적약물.근거병정공제정황장환자분위공제량호조(n =25)화공제불량조(n =26);근거시부합병혈관병변분위합병혈관병변조(n =27)화무혈관병변조(n =24).선택동기본원건강체검자23명위건강대조조,년령、성별여병례조필배.납입대상혹가속대검측항목화시험지정동의.시험경의원윤리위원회비준.방법:환자균재청신취진시추취공복주정맥혈진행검측.건강체검자우래원체검시진행상동검측.①생화지표검측:유본원검험과완성공복혈당、당화혈홍단백화공복면이도소적검측.②당단백Ib/IX/V복합물급기조분당단백Ibα적표체적검측:채집청신공복정맥혈3 mL,38 g/L구연산납항응후10 g/L다취갑철고정45 min.취50 μL고정후전혈가지류식검측관,동시분별향불동관중가입20 μL당단백Ib/IX/V 복합물단극륭항체CD42a-b-c-d화PE표기적당단백Ibα단극륭항체CD42b,혼균후실온피광부육30 min,연후CD42a-b-c-d표기적양품중가입20 μL FITC표기적양항서 IgG,혼균후실온피광부육30 min.우FACS420형류식세포의상검측양본평균형광강도.③혈소판취집솔측정:삼조문헌검측혈소판취집솔.주요관찰지표:①생화지표.②당단백Ib/IX/V복합물급당단백Ibα적표체정황.③혈소판최대취집솔.결과:실험납입적51례2형당뇨병환자화23명건강체검자전부진입결과분석.①생화지표검측결과:공제량호조화공제불량조환자공복이도소여건강대조조비교,차이유현저성의의(P ＜ 0.01).공제불량조환자공복혈당화당화혈홍단백균명현고우건강대조조화공제량호조,차이유현저성의의(P ＜ 0.01).②당단백Ⅰb/Ⅸ/Ⅴ복합물화당단백Ⅰbα표체정황적비교:공제량호조화공제불량조환자당단백Ⅰb/Ⅸ/Ⅴ복합물화당단백Ⅰbα적표체명현저우건강대조조,공제불량조명현저우공제량호조,차이균유현저성의의(P ＜ 0.05～0.01).무혈관병변조화합병혈관병변조환자균명현저우건강대조조,차이유현저성의의 (P ＜ 0.01).무혈관병변조화합병혈관병변조환자당단백Ⅰbα명현저우건강대조조,합병혈관병변조명현저우무혈관병변조,차이균유현저성의의(P ＜ 0.05).당단백Ⅰb/Ⅸ/Ⅴ복합물평균형광강도여공복혈당、당화혈홍단백화공복이도소정명현부상관(r =-0.634,-0.573,-0.649,P ＜ 0.05);당단백Ⅰbα평균형광강도여공복혈당화당화혈홍단백정명현부상관(r =-0.602,-0.543,P ＜0.05).③혈소판취집공능적비교:2형당뇨병환자혈소판최대취집솔교정상인명현승고,차이유현저성의의(t =-3.852,P ＜ 0.01),공제불량조명현고우공제량호조,합병혈관병변조고우무혈관병변조,차이균유현저성의의(P ＜ 0.05).결론:2형당뇨병환자조기무혈관병변시즉존재혈소판활화,발생혈관병변후활화경명현,차혈소판활화여혈당정정상관.혈소판당단백Ⅰb/Ⅸ/Ⅴ복합물작위응혈매화혈관성혈우병인자적수체,가능삼여료2형당뇨병혈관병변적발생발전.
BACKGROUND: It has been proved that platelet activation is involved in the development of diabetic angiopathy. Glycoprotein (GP) Ib/IX/V complex is one of the main platelet membrane glycoproteins, and the receptor of both von Willebrand Factor and thrombin. It plays a key role in the process of platelet activation.OBJECTIVE: To observe the expression changes of GP Ib/IX/V complex and its component GP Ibα in patients with type 2 diabetes mellitus. DESIGN: Case-control study. SETTING: Department of Integrated Chinese and Western Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology.PARTICIPANTS: A total of 51 type 2 diabetic outpatients who visited Union Hospital were enrolled from December 2005 to January 2007. The diagnosis was based on the independent criteria from WHO in 1999. Of all the 51 patients, there were 23 females and 28 males, with a peripheral platelet count of over 50×109 L-1. All the subjects had no history of administrating drugs two weeks before the examination, which would potentially influence platelet count. According to disease controlling condition, the patients were assigned to well controlled patient (WCP) group (n = 25) and poorly controlled patient (PCP) group (n =26); and according to whether angiopathy was accompanied, diabetic patients were divided into vascular disease (VD) group (n =27) and non-vascular disease (NVD) group (n =24). Meanwhile 23 healthy subjects were enrolled as normal control group. Informed consents were obtained from subjects and their relatives. The experiments were approved by the ethical committee of Union Hospital.METHODS: Fasting venous blood was harvested from all the subjects' elbow on the early morning of visit day.①Biochemical analysis: Fasting plasma glucose (FPG), glycosylated hemoglobin (HbA1c) and fasting plasma insulin (FINS) was measured by the Clinical Chemistry Department in Union Hospital.② Measurement of platelet membrane GP Ib/IX/V complex and its component GP Ibα: First, 3 mL cubital fasting blood was drawn from each subject and was anti-coagulated with 38 g/L natrium citricum. After that, all samples were fixed with 10 g/L paraform for 45 minutes. Then 50 μL well-fixed blood was added into the polystyrene tube, meanwhile 20 μL monoclonal antibody, such as CD42a-b-c-d and PE-labeled CD42b, was respectively mixed gently with the blood sample and incubated at room temperature in dark for 30 minutes. Next, 20 μL FITC-labeled rat IgG was mixed with the sample containing CD42a-b-c-d and incubated equally. In the end all blood samples were analyzed by FACS420 flow cytometry and the results were expressed as mean fluorescence intensity (MFI). ③ Platelet maximun aggregation rate (MAR) was detected according to reference.MAIN OUTCOME MEASURES: ①Biochemical indicators;②The expressions of GP Ib/IX/V complex and GP Ibα;③Platelet MAR.RESULTS: Fifty-one patients with type 2 diabetes mellitus and twenty-three healthy subjects were all involved in the result analysis.①There were significant differences in FINS in WCP group and PCP group compared with normal controls (P ＜ 0.01). FPG and HbA1c were significantly higher in PCP group compared with normal control group and WCP group (P ＜ 0.01).②Expressions of GP Ib/IX/V complex and GP Ibα were significantly lower in WCP group and PCP group compared with normal control group (P ＜ 0.01), significantly lower in PCP group than in WCP group (P ＜ 0.05), and also significantly lower in VD group and NVD group compared with normal control group (P ＜ 0.01). Moreover the expression of GP Ibα in VD group and NVD group was significantly lower than that of normal control group (P ＜ 0.05), and it also significantly decreased in VD group compared with NVD group (P ＜ 0.05). MFI of GP Ib/IX/V complex had an obvious negative correlation with FBG, HbA1c and FINS (r =-0.634, -0.573, -0.649, P ＜ 0.05), and GP Ibα MFI was obviously negatively correlated with FBG and HbA1c (r =-0.602, -0.543, P ＜ 0.05).③Platelet MAR of diabetic patients were remarkably higher than in healthy subjects (t =-3.852, P ＜ 0.01). Platelet MAR in PCP and VD groups were respectively higher than those in WCP and NVD groups (P ＜ 0.05). CONCLUSION: Platelet activation exists in the early stage of type 2 diabetes mellitus without diabetic angiopathy, and is more obvious after diabetic angiopathy. There is a positive correlation between platelet activation and blood glucose. As a receptor of thrombin and von Willebrand Factor, GP Ib/IX/V complex may be involved in the development of diabetic angiopathy.
