外周血单个核细胞内人类免疫缺陷病毒DNA和RNA定量对病毒转录活性的区分

Quantitative results of human immunodeficiency virus total deoxyribonucleic acid and ribonucleic acid in peripheral blood mononuclear cells to distinguish the activity of viral transcription

目的基于人类免疫缺陷病毒Ⅰ型(HIV-1)感染者外周血单个核细胞(PBMCs)中HIV-1总DNA和RNA定量检测结果对感染细胞内病毒的转录活性进行区分。方法采集2017年10月至2018年12月于哈尔滨医科大学附属第四医院感染科就诊的HIV-1感染者血液样本,分离PBMCs细胞,采用PCR荧光探针法对PBMCs细胞内HIV-1总DNA和RNA进行定量检测,并计算两者比值(Ratio)。根据Ratio值筛选出HIV-1转录活跃组样本和相对非活跃组样本,另外选择健康人PBMCs样本作为对照组。对3组样本进行基因转录组表达谱检测以及人口特征差异性检验,并对基因表达谱检测结果进行主成分分析以验证对3组样本病毒转录活性区分的准确性。结果从60例感染HIV-1患者的PBMCs样本中筛选出HIV-1转录活跃组样本(10例)和相对非活跃组样本(11例),另外选择6例健康人PBMCs样本作为对照组。其中转录活跃组样本Ratio值为165.2~738.93,平均为(339.27±189.68);相对非活跃组Ratio值为4.67~42.39,平均为(17.65±11.78)。转录活跃组和相对非活跃组样本间的CD4+T细胞计数(P=0.049)和Ratio值(P<0.001)差异均具有统计学意义;3组样本年龄(P=0.989)和性别(P=0.650)分布差异无统计学意义。对3组样本的PBMCs基因表达谱主成分分析结果显示:对照组与HIV-1感染者(包括转录活跃组和相对非活跃组)间区分明显。转录活跃组和相对非活跃组间有部分样本重合,同时结果也显示当HIV-1感染者的CD4+T淋巴细胞计数与健康人无显著差异时,其细胞内的基因表达与健康人接近。结论基于HIV-1总DNA和RNA定量检测结果及两者间比值可以较好地区分PBMCs内病毒转录活性。HIV-1感染细胞内部病毒的不同转录激活状况可导致其基因表达谱的异质性。

Objective Based on the quantitative results of total deoxyribonucleic acid(DNA)and ribonucleic acid(RNA)of human immunodeficiency virus type 1(HIV-1)in peripheral blood mononuclear cells(PBMCs)of patients with HIV-1 infection,transcriptional activity of HIV was distinguished.Methods Blood samples and separate PBMCs from healthy persons and HIV-1 infectors were collected from October 2017 to December 2018 in the Department of Infectious Diseases,the Fourth Affiliated Hospital of Harbin Medical University,PCR fluorescent probe method was applied to detect quantitative total DNA and RNA of HIV-1 in PBMCs,and the ratio values between them were calculated.According to ratio values,HIV-1 active transcription group and relatively inactive group were screened.In addition,PBMCs samples from healthy people were selected as controls.Gene transcriptome expression profile detection and populational characteristics difference were performed among the three groups,and principal component analysis was performed on the gene expression profiles among the three groups to verify the accuracy of distinguishing intracellular viral transcriptional activity.Results The HIV-1 active group samples(10 cases)and the relatively inactive group samples(11 cases)were screened form PBMCs samples of 60 HIV-1 infectors,and the PBMCs samples from 6 healthy people were selected as control group.The ratio value in active transcription group was 165.2-738.93,the mean and standard deviation was(339.27±189.68).In relatively inactive group,the ratio value was 4.67-42.39,the mean and standard deviation was(17.65±11.78).There were statistically significant differences in CD4+T counts(P=0.049)and ratio values(P<0.001)between the active transcription group and the relatively inactive group;there were no significant differences in age(P=0.989)and gender(P=0.650)distribution among the three groups.The principal component analysis results of gene expression profiles of PBMCs samples from the three groups showed that there was a clear difference between healthy people and HIV-1 infectors(active ranscription and relatively inactive groups),but some samples overlapped between the active ranscription group and the relatively inactive group.When CD4+T lymphocyte count of HIV-1 infectors was the same as healthy people,the state of gene expression within infected cells is similar to that of healthy people.Conclusions The ratio value of quantitative detection results of total DNA and RNA of HIV-1 could better distinguish the intracellular viral transcriptional activity.Differently transcriptional activation status of virus in infected cells could lead to intracellular heterogeneity of gene expression profile.