Screening And Identification Of Differentially Expressed Genes Of PBMC Between CHB And ASCs Subjects

Part I Construction and screening of subtracted cDNA libraries between CHB and ASCs subjectsHeptatitis B virus (HBV) is epidemic all over the world, our country belong to the high epidemic area, the average positive rate of HBsAg is 9.09%. Chronic HBV infection always develops into asymptomatic HBV carriage (ASC), chronic hepatitis B (CHB), liver cirrhosis (LC) and hepatocellular carcinoma (HCC). The mechanisms involving in the different disease states are not clear, but host immunity to HBV plays important roles in the development of different disease states. Host immunity to HBV is regulated by numerous genes, it was speculated that the difference of gene regulation determining the outcomes of HBV infection. Analysis of differentially expressed genes between different infection states might provide information for understanding the mechanisms, and provide clues in search of early diagnostic marks and designing new therapeutic targets.Suppression subtractive hybridization (SSH) is one method for screening the differentially expressed genes and has been widely used in search for gene related todiseases. There are two principles of SSH, one is subtracted hybridization, which excluding the common sequences between two samples, and enriching the differential sequences. At the same time, the comparative concentration of cDNA of different abundance reaches identicalness. So SSH increases the probability of obtaining the low abundance differential genes. Second is suppression PCR, which selectively inhibit the amplification of the non-target sequences and amplify the target genes. Although it is reported the positive rate of subtracted library constructed by SSH was high, there will be false positive. To exclude the false positive, we adopted dot blot hybridization to screening the subtracted libraries.The clinical material for construction of subtracted library is PBMC (Peripheral blood mononuclear cell. PBMC), PBMC including lymphocytes, monocytes and NK cells, so the subtracted library from mRNA represents the differential genes associated with immunity.Although ASC and CHB belong to the spectrum of chronic HBV infection, they represent different immunological states, so we isolated the PBMC from patients to construct subtracted libraries, and to analyze the differential genes related to immunity of different disease states.In our country, most of ASCs subjects acquired HBV infection from either perinatal or horizontal transmission, and more than 90 % developed chronic infection. But in adults suffering from primary HBV infection, 90%-95 % of the subjects can successfully clarify the virus through self-limiting hepatitis and only 5%-10 % of adults become chronic HBV carriers. The analysis of the differentially expressed genes of PBMC between ASCs and normal control might provide clues in elucidating the mechanisms of chronic HBV infection 1. Material and methodsThe participants were 5 ASC subjects, 5 active CHB patients and 5 healthy adults (normal control) from the first Affiliated Hospital, School of Medicine, Zhejiang University. The diagnosis of CHB or ASCs was according to the diagnostic standard from the National Program for Prevention and Treatment of Chronic Viral Hepatitis B. The following patientswere excluded from the study: those on hepatotoxic drug treatment, those consuming alcohol regularly or excessively, those who were anti-hepatitis C virus positive, or anti-HIV positive. For each subject, 3-10 ml venous blood was collected for isolation of PBMC and extraction of RNA. To normalize for individual differences, RNA was pooled from the five individuals in each group to synthesize cDNA. Forward and backward subtraction was performed between CHB and ASCs, ASCs and the normal control. After two rounds of hybridization and PCR amplification of cDNA, subtracted libraries were constructed by T/A cloning from the cDNA. The libraries were rescreened by PCR amplification and dot blot hybridization, a part of the positive clones were sequenced and homologous search to the genebank. 2. ResultscDNA libraries were constructed by SSH between CHB and ASCs, ASCs and the normal control. After screening, besides genes of unknown function or unknown sequences, most of the up-regulated genes in CHB compared to ASCs were related to inflammatory conditions, such as defensin a 3, lactoferrin, proteasome subunit 2 and beta 2-microglobulin, et a). Of the up-expressed genes in CHB compared to ASCs, genes with unknown function were dominating, such as HBxAg transactivated protein 2 (XTP2) . Of the up-expressed genes in ASCs compared to the normal control, checkpoint suppressor 1 was associated with cell cycle arrest induced by DNA damage. Perforin was related inflammation and immune regulation. Most of the up expressed genes in the normal controlcompared to ASCs were associated with inflammation, such as interleukin-1 andinterleukin-8.3. Conclusions(1) Subtracted libraries were constructed successively by SSH between CHB and ASCs, ASCs and the normal control. The results of nucleic sequences analysis indicated that some genes were associated with inflammation and immune, and some genes with unknown function and unknown sequences.(2) SSH was confirmed as a sensitive, effective method in screening differential genes by analysis of subtraction efficacy, library screening and sequencing.Part II: Identification of differential genes by real time RT-PCR and the analysis of plasma levels of defensin a in patients withchronic HBV infectionWe obtained 34 differentially expressed genes in the four libraries by construction and screening the libraries with dot blot hybridization. Due to the limited manpower and time, we selected 3 up expressed genes in CHB for advanced study, the 3 genes were defensin a 3, lactoferrin, protesome subunit 2. These genes were associated with inflammation and have reported related to CHB previously.The genes identified by dot blot hybridization indicated the differential expression existed indeed in the original samples, but the number of original samples was limited, individual difference may result in false positive. To exclude the false positive from individual difference, we identified the differential expression of the 3 genes in the corresponding crowds. Peripheral blood of 23 CHB and 21 ASCs participants was collected, RNA was extracted from, freshly isolated PBMC, real time RT-PCR was used to identify thedifferential expression between the two groups.Defensin a 3 was one of the differentially expressed genes identified by real time RT-PCR, defensin is small antimicrobial peptide, it exhibits a wide variety of microbicidal activities against Gram-positive and -negative bacteria, mycobacteria, fungi and certain enveloped viruses. Defensin is divided into two groups: defensin a and defensin (3, based on their structural features, amino acid composition and number of disulfide bonds. Up to now,six kinds of a-defensin (HNP-1 ~4, HD-5> HD-6) and four kinds of (3-defensin have been found, of which a-defensin 1 ~4 are mainly isolated from neutrophil cells, so they are also called human neutrophils peptidesl~4 (HNP1~4), but they were also found in NK, y8 T lymphocytes, B cells and monocytes/macrophages. Defensin |3 is secreted by the epithelia of skin and mucosa and involves in the defense and immune response of skin and mucosa.A study reported that a-defensinl~3 has activity of anti-HIV, high concentration of a-defensinl~3 (HNP 1-3 ) was found in long-term nonprogressors with HIV-1 infection. That indicated that defensin a take the responsibility of anti-HIV in the patients with HIV. HBV is also a kind of enveloped virus, but the study about interaction of defensin on HBV has not been reported. We first reported the differential expression of defensin a between CHB and ASCs, defensin a 3 was less active than the other defensins against most microbes, and was less active potent chemoattractants for human T cells than defensin a 1 and defensin a 2. Up-regulation of defensin a 3 in CHB might contribute to the development of chronic inflammation.To further investigated the level of protein of defensin a in patients with chronic HBV infection, the plasma level of defensin a 1~3 in the patients with chronic HBV infection was assayed by ELISA. 1. Material and methods(1) Sample collection Sample for real time RT-PCR: The participants were 21 ASC subjects and 23 active CHB patients, For each subject, 2-5 ml venous blood was collected and PBMC was isolated freshly, RNA extracted from the PBMC was reverse transcripted tocDNA for real time RT-PCR.Sample for ELISA: 68 samples were collected in all, including 12 normal subjects, 10 ASCs subjects, 46 CHB subjects The diagnosis of all subjects was according to the diagnostic standard from the National Program for Prevention and Treatment of Chronic Viral Hepatitis B. For each subject, 2-5 ml heparinized venous blood was collected, and plasma was isolated by centrifugation, the plasma level of defensin a was assayed by ELISA.(2) MethodsThe difference on mRNA level of the 3 selected genes was assayed by real time RT-PCR. The plasma level of defensin a 1 ~3 in the patients with chronic HBV infection was assayed by Human HNP 1-3 ELISA Kit from Hycult biotechnology b.v (Netherland).(3) Statistic analysisThe results were expressed as mean ± standard deviation and analyzed by analysis of variance, statistic analysis was performed by SPSS 11.0 statistical program. 2. ResultsThe differential expression of defensin a 3, lactoferrin and protesome subunit 2 was up expressed by 5.28,3.58 and 2.81 fold in CHB subjects compared to ASCs subjects. The plasma concentration of defensin a 1~3 in the normal control, ASCs subjects, CHB subjects was (ug/ml)194.33±l 12.24, 156.00±57.26, 231.00±75.697, respectively, the level of defensin a 1~3 in the CHB subjects was higher significantly than that in the ASCssubjects, but there was no significance of difference between other groups. 3. Conclusion(1) The differential expression of 3 genes, defensin a 3, lactoferrin and protesome subunit 2 was first confirmed as up expressed in CHB compared to ASCs by real time RT-PCR. Up expression of defensin a 3 in patients with chronic hepatitis B might contribute to the development of chronic inflammation.(2) The plasma level of defensin a 1~3 in the subjects with chronic HBV infection was first assayed, and it is significantly higher in CHB subjects than that in ASCs subjects, but there was no significant difference between the nomal control and CHB subjects, the normal control and ASCs subjects.