【作者】 黎彦君；

【导师】 玉艳红；

【作者基本信息】 广西医科大学 ， 传染科， 2012， 硕士

【摘要】 目的：通过研究隐球菌性脑膜炎(Cryptococcal meningitis, CM)外周血和脑脊液(Cerebrospinal fluid, CSF)中hFgl2(hunman Fibrinogen-like protein2,人Fgl2凝血酶原酶)凝血酶原酶阳性表达的细胞种类及其表达与病情严重程度的关系,了解隐球菌性脑膜炎患者hFgl2凝血酶原酶的表达与临床意义,探讨hFgl2凝血酶原酶的作用机制。方法：将观察对象分为隐球菌性脑膜炎组(A组)和健康对照组(B组),分别取A组20例和B组20例外周血提取白细胞行hFgl2凝血酶原酶蛋白(简称hFgl2蛋白)行免疫组化制片,其中A组所取外周血分别来自患者住院确诊后第一次检测脑脊液隐球菌数(第一期)、隐球菌数目下降一半(第二期)、隐球菌数目为零或隐球菌培养阴性(第三期)的三个不同时期；并收集A组配对患者脑脊液提取白细胞行hFgl2蛋白免疫组化制片。观察采集到的hFgl2蛋白表达阳性白细胞图像中阳性表达部位及强度,并使用MIAS医学图像分析软件测定反映hFgl2蛋白表达情况的灰度值(hFgl2蛋白表达越高,平均灰度值数值越低。即：hFgl2蛋白表达情况与所测定的灰度值呈负向关系)。以SPSS13.0统计软件分析比较两组中hFgl2蛋白的表达差异和A组在三个不同时期表达的差异,了解hFgl2在A组中的表达与脑脊液隐球菌数、脑脊液生化检查和隐球菌荚膜抗原定量之间的相关关系。结果：1.hFgl2蛋白在A、B两组外周血单核细胞和中性粒细胞中均有表达,两组单核细胞中hFg12表达的平均灰度值分别为(114.49±11.82)、(137.56±10.64),经比较,差异有统计学意义(P<0.05)。两组中性粒细胞hFgl2表达的平均灰度值分别为(111.71±8.62)、(143.56±12.57),经比较,差异有统计学意义(P<0.05)。再行组内单核细胞与中性粒细胞hFgl2蛋白表达的平均灰度值比较,差异无统计学意义(P>0.05)。2.hFgl2蛋白在隐球菌性脑膜炎脑脊液的单核细胞和中性粒细胞中均有表达,阳性组与阴性对照组单核细胞hFgl2表达的平均灰度值为(113.33±8.07)、(147.25±9.72),经比较,差异有统计学意义(P<0.05)；阳性组与阴性对照组中性粒细胞hFgl2表达的平均灰度值分别为(115.44±8.67)(149.25±8.57),经比较,差异有统计学意义(P<0.05)行阳性组内单核细胞与中性粒细胞hFgl2蛋白表达的平均灰度值比较,差异无统计学意义(P>0.05)。3.hFgl2在A组三个不同时期外周血的单核细胞和中性粒细胞中的表达均为阳性,单核细胞hFgl2表达的平均灰度值分别为(101.87±12.11)、(106.63±8.38)、(118.37±11.26),三组比较,差异有统计学意义(P<0.05)。再行组内两两比较差异均有统计学意义(P<0.05),中性粒细胞hFgl2表达的平均灰度值分别为(103.44±13.51)、(107.31±8.87)、(120.334±9.92),三组比较,差异有统计学意义(P<0.05)。再行组内两两比较差异均有统计学意义(P<0.05)。单核细胞和中性粒细胞hFgl2表达的平均灰度值同期进行比较,差异无统计学意义(P>0.05)。4.hFgl2在A组三个不同时期脑脊液的单核细胞和中性粒细胞中的表达均为阳性,单核细胞hFgl2表达的平均灰度值分别为(101.314±8.19)、(106.284±7.62)、(120.264±12.62)。三组比较,差异有统计学意义(P<0.05)。再行组内两两比较差异均有统计学意义(P<0.05)。中性粒细胞hFgl2表达的平均灰度值分别为(103.23±9.08)、(110.38±8.75)、(121.06±10.43),三组比较,差异有统计学意义(P<0.05)。再行组内两两比较差异均有统计学意义(P<0.05)。单核细胞和中性粒细胞hFgl2表达的平均灰度值同期进行比较,差异无统计学意义(P>0.05)。5.A组外周血单核细胞和中性粒细胞hFgl2蛋白表达的平均灰度值与脑脊液隐球菌数均呈负相关(单核细胞r。(隐球菌数)=-0.36,P=0.033,中性粒细胞rs(隐球菌数)=-0.374,P=0.011)(hFg12蛋白的平均灰度值与隐球菌性脑膜炎组的严重程度呈负相关,即隐球菌性脑膜炎组外周血单核细胞和中性粒细胞中hFgl2蛋白表达强度与脑脊液隐球菌数成正相关)。hFgl2蛋白的平均灰度值与脑脊液生化检查和隐球菌荚膜抗原定量的相关性经检验差异无统计学意义(P>0.05)。6.A组脑脊液单核细胞和中性粒细胞,hFgl2蛋白表达的平均灰度值与脑脊液隐球菌数均呈负相关(单核细胞rs(隐球菌数)=-0.323,P=0.008,中性粒细胞rs(隐球菌数)=-0.354,P=0.007)。hFgl2蛋白的平均灰度值与脑脊液生化检查和隐球菌荚膜抗原定量的相关性经检验差异无统计学意义(P>0.05)。结论：1.hFg12蛋白能够表达于隐球菌性脑膜炎患者及健康对照组外周血单核细胞和中性粒细胞中,且表达程度相当。2.hFgl2蛋白能够表达于隐球菌性脑膜炎脑脊液单核细胞和中性粒细胞中,且表达程度相当。3.hFg12蛋白在隐球菌性脑膜炎患者外周血中单核细胞和中性粒细胞的表达与在脑脊液单核细胞和中性粒细胞中的表达一致,其表达强度均随着病情的减轻而逐渐下降.4.隐球菌性脑膜炎患者hFg12蛋白表达的强度与脑脊液隐球菌数成正相关。更多还原

【Abstract】 Objective:To detect the protein expressions of hfg12 (human fibrinogen like protein 2) from peripheral blood monocytes as well as Cerebrospinal fluid in patients with Cryptococcal meningitis and try to make clear the correlation and possible involvement between these diseases and the expression of hFgl2.Research hFgl2 prothrombin enzyme positive expression of mononuclear cells and Cerebrospinal fluid cryptococcal number、CSF protein and Cryptococcal capsular antigen quantitative to determine the correlation between the degree of The severity of meningitis, and understand the sensitivity of the reflection hFgl2 prothrombin enzyme expression determination of the clinical significance.Methods:1.Acquisition with complete clinical data of 20 cases with Cryptococcal meningitis(A) peripheral blood smear as experimental,20 cases of the healthy check-up peripheral blood smear as control group (B).2. Patients in group B were detected in the hospital diagnosed the first time the number of cerebrospinal fluid cryptococcal, cryptococcal fall by half the number of cryptococcal number of zero or cryptococcal collection of three peripheral blood in three different periods of culture-negative; And collection of matching patients with cryptococcal meningitis lumbar puncture the cerebrospinal fluid extract cells3.Application immunohistochemistry methods were used in the treatment of Cryptococcal meningitis group and the normal group peripheral blood monocytes.And using the two-step immunohistochemistry methods detected to Fgl2 protein expression in the collected in the Cerebrospinal fluid.4.The statistical data of expression was analysed by SPSS13.0.Results:1.Fgl2 proteins was detected in two groups of peripheral blood monocytes.Its expression of average gray level respectively was(Group A): (114.49±11.82), (Group B):(137.56±10.64), The two groups by statistical analysis, a statistically significant difference(P<0.05), Two sets of neutrophil cell expression hFgl2 average gray value (111.71±8.62), (143.56±12.57), by comparison, the difference was statistically significant (P<0.05).Prompt hFgl2 protein highly expressed in peripheral blood mononuclear cells and neutrophil cell of the cryptococcal meningitis group·2.hFgl2 protein expression in cryptococcal meningitis in cerebrospinal fluid mononuclear cells.. Average gray value of its expression with the negative control group was (103.33±8.07)、(147.25±9.72), The two groups by statistical analysis, a statistically significant difference(P<0.05), Positive and negative control group, the average gray value of neutrophils hFgl2 expression, respectively (115.44±8.67) (149.25±8.57), by comparison, the difference was statistically significant (P<0.05) single-core line-positive groupcells compared to the average gray value and to neutrophils hFgl2 protein expression was no significant difference (P> 0.05).Prompt hFgl2 protein highly expressed in the cryptococcal meningitis group cerebrospinal fluid mononuclear cells and neutrophil cell.3.the hFgl2 expression in Peripheral blood mononuclear cells and neutrophil cell.in three different periods of cryptococcal meningitis were positive, which Cerebrospinal fluid cryptococcal number of hospital diagnosed the first time, cryptococcosis decreased by half the number, cryptococcal number is zero or cryptococcal culture negative. Average gray value of its expression with the negative control group was (101.87±12.11) (106.63±8.38)、(118.37±11.26) respectively. There was significant difference in three groups (P<0.05). The average gray value of neutrophils hFgl2 expression, respectively (103.44±13.51) (107.31±8.87), (120.33±9.92), three groups, the difference was statistically significant (P<0.05). Further pairwise comparisons within the group differences were statistically significant (P<0.05). Expression by monocytes and neutrophils hFgl2 average gray value over the same period were compared, no statistically significant difference (P> 0.05).4. the hFgl2 expression in Cerebrospinal fluid mononuclear cells in three different periods of cryptococcal meningitis were positive, which Cerebrospinal fluid cryptococcal number of hospital diagnosed the first time, cryptococcosis decreased by half the number, cryptococcal number is zero or cryptococcal culture negative. Average gray value of its expression with the negative control group was (101.31±8.19)、(106.28±7.62)、(120.26±12.62) respectively. There was significant difference in three groups (P<0.05). The average gray value of neutrophils hFgl2 expression, respectively (103.23±9.08), (110.38±8.75), (121.06±10.43), three groups, the difference was statistically significant (P <0.05). Further pairwise comparisons within the group differences were statistically significant (P<0.05). Expression by monocytes and neutrophils hFgl2 average gray value over the same period were compared, no statistically significant difference (P> 0.05).Prompt the expression of hFgl2 in cryptococcal meningitis Cerebrospinal fluid’s positive values and disease severity of a positive correlation.5. In Peripheral blood mononuclear cells and neutrophil cell, the average gray value of hFg12 protein in Cryptococcal meningitis group and cerebrospinal fluid cryptococcal number was negatively correlated (P<0.05),it means the average gray value of hFgl2 protein with the cryptococcal meningitis group, the severity was negatively correlated, Cryptococcal meningitis in peripheral blood mononuclear cells of hFgl2 protein expression intensity of cerebrospinal fluid cryptococcal number into a positive correlation. There was no significant difference in the correlation of the average gray value of hFgl2 protein and CSF protein and cryptococcal capsular antigen quantitative(P>0.05).6. In cerebrospinal fluid mononuclear cells and neutrophil cell, the average gray value of hFgl2 protein in Cryptococcal meningitis group and cerebrospinal fluid cryptococcal number was negatively correlated (P<0.05), it means the average gray value of hFgl2 protein with the cryptococcal meningitis group, the severity was negatively correlated, Cryptococcal meningitis in Cerebrospinal fluid mononuclear cells of hFgl2 protein expression intensity of cerebrospinal fluid cryptococcal number into a positive correlation. There was no significant difference in the correlation of the average gray value of hFgl2 protein and CSF protein and cryptococcal capsular antigen quantitative(P>0.05).Conclusions:1. Fg12 protein can be in the human peripheral blood monocytes.2. hFgl2 protein can be expressed in cryptococcal meningitis in cerebrospinal fluid mononuclear cells· 3 the espression of hFgl2 protein reduced with the cryptococcal meningitis severity of the disease mitigate,and the espression of hFgl2 protein in peripheral blood and cerebrospinal fluid mononuclear cells showing the synchronization. prothrombinase hFgl2 is expected to become another evaluation of the degree of damage in the nervous system of patients with cryptococcal meningitis·更多还原