CN104777309A - 检测外周血单核细胞中HBcAg的表达的方法 - Google Patents
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Abstract
本发明公开了一种检测外周血单核细胞中HBcAg的表达的方法,其使用共聚焦显微镜进行检测。激光共聚焦显微镜可以检测外周血PBMC中HBcAg表达及分布,从而评估HBV DNA复制水平,能够应用在慢性乙型肝炎抗病毒治疗疗效及患者免疫状态的研究上。
Description
技术领域
本申请涉及一种检测外周血单核细胞中HBcAg表达及分布的方法。
背景技术
乙型肝炎病毒(HBV)核心抗原(HBcAg)存在于完整的Dane颗粒的核心内,该核心是HBV的结构蛋白即病毒核壳蛋白,具有重要的生物学特征和临床病理意义。既往研究证明血HBcAg阳性可以提供肝内HBV合成的信息,是病毒存在的直接标志。HBcAg感染的肝细胞是细胞免疫效应攻击的靶细胞,肝细胞溶解后HBcAg可以直接释放入血,故能直接反映肝细胞损害和病情进展的阶段。
发明内容
本发明提供一种新的检测HBcAg表达及分布的方法。
本发明提供一种检测外周血单核细胞中HBcAg表达及分布的方法,其使用共聚焦显微镜进行检测。
所述方法包括如下步骤:a)提取外周血单核细胞;b)将外周血单核细胞进行免疫荧光抗体包被;c)利用激光共聚焦显微镜检测。
所述步骤c中,建立二条通道,分别为一条用于寻找细胞的DAPI通道和一条用于观察HBcAg绿色荧光的GFP通道。
一种使用共聚焦显微镜检测外周血单核细胞中HBcAg分布的方法。
本发明的有益效果是:激光共聚焦显微镜可以动态监测外周血单核细胞(PBMC)中HBcAg表达水平及分布的变化,从而评估HBV DNA复制水平,能够应用在慢性乙型肝炎抗病毒治疗疗效及患者免疫状态的研究上。
附图说明
图1是本实施方式利用Spearman’s分析第一组所有血和PBMC标本中HBVDNA表达水平相关性的结果图;
图2是在63×3放大倍数下,PBMC中的HBcAg阳性表达图,其中A为抗乙型肝炎核心抗原的抗体;B为DEPI,C为前两图叠加效果;
图3是在63×3放大倍数下,PBMC中的HBcAg阴性表达图,其中A为抗乙型肝炎核心抗原的抗体;B为DEPI,C为前两图叠加效果;
图4是在63×放大倍数下计数HBcAg表达阳性的PBMC图,其中A为抗乙型肝炎核心抗原的抗体;B为DEPI,C为前两图叠加效果;
图5是利用Spearman’s分析阳性PBMC百分比与PBMC中HBV DNA水平相关性的结果图。
具体实施方式
下面通过具体实施方式结合附图对本发明作进一步详细说明。
一、患者与分组
该实验纳入的患者为北京大学深圳医院感染科门诊及住院部确诊的慢性乙型病毒性肝炎患者。健康对照组成员为该院医务工作者,血清中HBsAg与HBcAb均阴性。所有受试者血中HIV、HCV、HDV抗体阴性。
第一组:25个未抗病毒治疗的慢乙肝患者(包括免疫清除、免疫耐受及HBV或HBsAg携带等所有免疫阶段的患者,见表1);
第二组:12个干扰素治疗疗程1年后血病毒转阴的慢乙肝患者;
第三组:3个健康患者。
二、实验方法:
1:外周血单核细胞(PBMC)提取:抽取受试者外周血4毫升,在4小时内通过梯度离心法获得细胞,使用PBS洗涤3次,随后悬浮于500ulPBS中存于4℃冰箱。
2:外周血单核细胞免疫荧光方法
1)涂片:将提取的细胞打匀,用枪转移适量至PLL处理好的载玻片上,用枪头平着推开液滴,铺平。自然风干(时间约15~20分钟)。
2)多聚甲醛固定30min;
3)PBS(PH7.4)浸洗3次,每次3min;
4)0.3%TritonX-100PBS,室温孵育30min(配方:10xPBS稀释成1xPBS,加入0.3%TritonX-100);
5)PBS(PH7.4)浸洗3次,每次3min;
6)去电荷试剂4滴孵育30分钟,PBS浸洗1次5min;
7)每块标本滴加50μl稀释后的一抗体(购买于Millipore公司,稀释倍数1:100),4℃孵育16h;
8)PBST浸洗5次,每次5min;
9)每张片滴加50μl稀释后的荧光二抗(购买于Invitrogen公司,按照1:200与PBST进行稀释,PBST配制:0.2%Tween-20加入到PBST中)避光孵育40min,PBS洗2min;
10)PBST浸洗3次,每次10min;
11)每张片滴加50μl DAPI,室温放置3min;
12)PBST浸洗5次,每次5min;
13)使用滤纸吸除明显水分,使用抗荧光淬灭剂(20μl,避免过多)进行封片,避光4℃保存。
3.激光共聚焦
将激光输出功率调节到30%,打开系列扫描通道,建立2条通道,分别为1通道DAPI,颜色较亮,便于寻找细胞;2通道GFP,用于观察HBcAg绿色荧光,图像叠加20层。拍片步骤:将载玻片倒置固定于显微镜上,从低倍镜开始用DAPI寻找细胞,将细胞调至视野中央,使用63x前降低镜头调至63x,取下载玻片在镜头上涂专用油,再固定载玻片,调焦。
4.统计学:秩和检验,Spearman rank correlation.P<0.05为具有统计学意义。所有的统计结果使用SPSS13.0统计软件。
5.结果:
1)检测第一组血与PBMC中HBV DNA表达量
第一组中20个患者血HBV DNA阳性,其中17个患者PBMC标本HBVDNA阳性(85%)。3个PBMCHBVDNA阴性标本来自于血HBeAg阴性患者(表1)。PBMC HBV DNA阳性患者中,对于HBV DNA表达水平,血3.26×106IU/ml(范围1.06×103到1.27×108IU/ml),PBMC3.38×103IU/μg(范围1.52×102到1.47×105IU/μg)。血HBeAg阳性患者血HBV DNA表达量(n=9,中位数4.61×107IU/ml,范围1.36×106到1.27×108IU/ml)明显高于血HBeAg阴性患者(n=8,中位数3.88×104,范围1.06×103到5.32×106IU/ml;P<0.001)。相对于HBeAg阳性患者(中位数2.10×104,范围1.88×103到1.47×105),HBeAg阴性患者PBMC HBV DNA表达量显著下降(中位数1.13×103,范围1.52×102到4.66×103;P<0.001)。
表1.第一组患者情况
缩写:F,女性;M,男性;ALT,谷丙转氨酶;HBsAg,,乙型肝炎病毒表面抗原;HBeAg,乙型肝炎病毒e抗原;
患者第1至第10例为免疫清除阶段;第11至15例为免疫耐受阶段;第16至20例为HBV携带阶段;第21至25为HBsAg携带阶段(即血HBV阴性,血HBsAg阳性);
*为该患者的PBMC标本中HBV DNA为阴性;
中位数(范围):HBeAg阳性患者(性别:4个男性和5个女性;年龄:平均41.3岁,范围20-59岁;HBeAg阴性患者(性别:8个男性和8个女性;年龄:平均40.6岁,范围29-54岁)。
2)血和PBMC中HBV DNA表达相关性
血和PBMC中HBV DNA表达量在血HBeAg阳性患者中存在明显相关性(r=0.78;P<0.05)。在HBeAg阴性患者中,PBMC中HBV DNA表达与血中HBVDNA表达也明显相关(r=0.74;P<0.05)。Spearman’s相关性分析第一组所有血和PBMC标本中HBV DNA水平相关性为0.88(P<0.001;如图1所示)。
3)检测干扰素治疗后血HBV DNA转阴患者PBMC中HBV DNA表达情况
第二组(n=12):5个患者为HBeAg阳性,2个为HBsAg阴性。尽管所有的患者血中HBV DNA均检测不出,但PBMC中的HBV DNA在5/12标本(41.7%)中为阳性。4/5(80%)HBV DNA阳性的PBMC标本在HBeAg阳性患者中,1/5(20%)在HBeAg阴性患者中。2个HBsAg阳性患者血与PBMC中均未检出HBVDNA。检查结果如表2所示。
表2 干扰素治疗后血HBV DNA转阴患者PBMC中HBV DNA与HBcAg表达情况
ND代表未检测出;M代表男性;F代表女性
4)检测HBcAg阳性PBMC
在阳性标本中,胞浆和/或核中可见HBcAg绿色信号,如图2所示。阴性标本中无相应绿色信号,仅有核中DEPI蓝色信号,如图3所示。在63×倍放大倍数(如图4所示)下随机选择区域对HBcAg表达阳性的PBMC细胞进行计数,并计算出阳性百分比,细胞计数总数>1000个。
5)HBcAg阳性PBMC百分比与PBMC中HBV DNA表达量的相关性
HBV DNA阳性PBMC标本中HBcAg阳性PBMC百分比0.2%到39%。在HBV DNA阴性PBMC标本中及对照组不能检测出HBcAg。对于HBcAg阳性PBMC百分比与PBMC中HBV DNA水平,Spearman’s相关性为0.833(P<0.001),如图5所示。
6)分析各个组间核或胞浆HBcAg阳性PBMC百分比
本发明发现HBsAg携带者中仅发现核HBcAg阳性PBMC。HBV携带者中核HBcAg阳性PBMC为主,如表3所示。干扰素治疗后患者中以胞浆HBcAg表达的PBMC为主(P<0.05)。胞浆表达HBcAg的PBMC在免疫耐受者中所占百分比较低,在免疫清除患者中所占百分比较高,(P<0.001)。
表3.PBMC样本中的HBcAg表达量
63×倍放大倍数下每个视野中HBcAg阳性表达的PBMC数
本发明中,PBMC中的HBcAg表达水平与PBMC中HBV载量存在明显相关性。HBcAg在PBMC中分布的位置提示不同的免疫状态。本发明提示利用激光共聚焦显微镜检测PBMC中的HBcAg水平和分布情况可以作为监测HBV复制和感染免疫状态的一种替代方法。本发明使用激光共聚焦显微镜可以检测外周血PBMC中HBcAg表达及分布,从而评估HBVDNA复制水平,能够应用在慢性乙型肝炎抗病毒治疗疗效及患者免疫状态的研究上。
以上内容是结合具体的实施方式对本发明所作的进一步详细说明,不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干简单推演或替换。
Claims (4)
1.一种检测外周血单核细胞中HBcAg的表达的方法,其特征在于,使用共聚焦显微镜进行检测。
2.如权利要求1所述的方法,其特征在于,包括如下步骤:
a)提取外周血单核细胞;
b)将外周血单核细胞免疫荧光;
c)利用激光共聚焦显微镜检测。
3.如权利要求2所述的方法,其特征在于,所述步骤c中,建立两条通道,分别为一条用于寻找细胞的DAPI通道和一条用于观察HBcAg绿色荧光的GFP通道。
4.一种使用共聚焦显微镜检测外周血单核细胞中HBcAg的分布的方法。
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