关键词：丙型肝炎病毒 丙型肝炎病人 外周血单个核细胞 细胞免疫
摘 要 为探讨丙型肝炎(HC)病人细胞免疫功能和丙型肝炎病毒(HCV)的致病机及机体对其免疫保护作用，收集24例HC病人(急性3例，慢性21例)，用3 H-TdR掺入法研究病人外周血单个核细胞(PBMC)对不同HCV抗原增殖反应，并用流式细胞仪(FACS)检测了PBMC中CD4+ 、CD8+ 淋巴细胞亚群在HCV抗原刺激后的变化。结果：HC病人PBMC对HCV合成肽CP9 ，NS4 和基因重组抗原C，E1 ，E2 ，NS3 刺激后出现不同程度增殖反应，刺激指数(SI)分别为1.69±0.51，1.61±0.54，1.68±0.58，1.49±0.44，1.44±0.44和1.33±0.33。3例急性HC中2例病人的PBMC对HCV抗原呈有效增殖反应(SI≥2.1)，且血清HCVRNA阴转伴ALT正常。细胞表型分析显示：增殖的细胞表型是CD4+ 淋巴细胞，而CD8+ 淋巴细胞增殖反应较弱。结论：HC病人PBMC确实存在对HCV抗原的增殖反应；CD4+ 淋巴细胞比CD8+ 淋巴细胞增殖反应要强，急性HC病人PBMC对HCV抗原有效的增殖反应预示可能有良好的临床愈合。
中图号 R374.21
Abstract To explore the cellularimmune function in patients with hepatitis C and study the HCV pathogenic mechanism andimmune protection effect against HCV infection in host. PBMC proliferative responses invitro to HCV synthetic peptides (Cp9 ， Ns4 ) and recombinant HCVproteins (C，E1 ，E2 ，NS3 ) were evaluated in 21patients with chronic hepatitis C and 3 patients with acute hepatitis C. Phenotypiccharacteristics of proliferating cells in PBMC were detected by FACS after PBMC werestimulated with HCV antigens. Results showed that lymphoproliferative responses of PBMCfrom patients with hepatitis C to synthetic peptides (Cp9 ， NS4 )and recombinant proteins (C，E1 ，E2 and NS3 ) weredetected and its stimulation index(SI) were 1.69±0.51， 1.61±0.54， 1.68±0.58，1.49±0.44， 1.44±0.44 and 1.33±0.33 respectively. Two out of threepatients with acute hepatitis C had efficient lymphoproliferative response to HCV antigens(SI
2.1)and their serum HCV-RNA were seroconversion and serum ALT values were returned to normalwhen studied. Phenotype analysis showed that phenotypic characteristics of proliferatingcells was CD4+ lymphocytes and CD8+ lymphocytes proliferativeresponses was less than that of CD4+ lymphocytes. The results indicate thatPBMC from patients with hepatitis C exist proliferative responses to different HCVantigens. CD4+ lymphocytes proliferative response is greater than that ofCD8+ lymphocytes. The efficient lymphoproliferative responses to HCVantigens in patients with acute hepatitis C suggests that the patients possibly have agood clinical prognosis.
Key words Hepatitis C virus，Patients with hepatitis C，Peripheralblood mononuclear cell，Cellular immune
Hepatitis C virus (HCV) has been identified as a major etiological agent of parentallyand community-acquired NANB hepatitis and most patients with hepatitis C are not able toeliminate the virus[1～2] . However， it is still not known that HCVpathogenic mechanism and immune protecting effect against HCV infection in host at present.The presence of antibodies to most HCV antigens in patients with chronic hepatitis Csuggests that the humoral immune responses to the virus is unable to stop the progressionof disease[3～8] . In order to explore the cellular immune function inpatients with hepatitis C peripheral blood mononuclear cell (PBMC) proliferativeres-ponses in vitro to HCV synthetic peptides (Cp9 ，NS4 ) andgene recombinant HCV proteins (C，E1 ，E2 ，NS3 )were evaluated in 21 patients with chronic hepatitis C and 3 patients with acute hepatitisC. We are try to analysis whether there are a relationship between PBMC immune responsesin patients with hepatitis C to various HCV antigens and clinical prognosis of HCVinfection and to select HCV predominant antigens which are to be thought of having a goodimmunogenicity and provide the basis for studing the function of PBMC from patients withhepatitis C and HCV vaccine development in future.
MATERIAL AND METHODS
HCV-seropositive individuals For thepresent study we selected 24 individuals with serum HCV-RNA positive detected by reversetranscription-nested polymerase chain reaction (RT-nested-PCR). The assay wasperformed as previously described[9] ， Briefly， RNA was extracted from50μl of serum by a modification of a method described elsewhere[10] .After reverse transcription at 42°C for 90 minutes， complementary DNA was amplified bytwo round PCR by using primers from the core region of HCV genome. (positive sense：5′G G T C G C A A C C T C G T G G A A G G 3′，negative sense：5′A T C G A T G A C C TT A C C C A A A T 3′). The amplification profile was as follows：denaturation at 95°Cfor 1 minute， annealing at 55°C for 1 minute and extension at 72°C for 45 seconds，total of 30 cycles. PCR products (209pb) were subjected to electrophoresis in 2%agarose gels， stained with ethidium bremide， and visualized under ultravioletillumination.
Patients with chronic hepatitis C This group consisted of 21patients [17 male and 4 female； median age： 44 years， (range， 27～65)]whohad elevated serum levels(>40IU/L)of alanine amino transferase(ALT) for 1 yearand liver histology compatible with the diagnosis of chronic hepatitis by the diagnosiscriterion of viral hepatitis prevention and treatment in the fifth national symposium oninfectious diseases and parastie diseases， May， 1995， Beijing， P.R.China.
Patients with acute hepatitis C This group consisted of 3individuals [1 male and 2 female，median age： 30 years (range， 25～37)] witha history of blood transfusion， whose serum levels of ALT>40 IU/L for no more than 3months and whose signs and symptoms were compatible with the dia-gnosis of acute hepatitisby the diagnosis criterion of viral hepatitis prevention and treatment in the fifthnational symposium on infectious diseases and parasite diseases， May， 1995， Beijing，P.R.China.
HCV-seronegative individuals As controls for the specificity ofPBMC responses，we selected 8 healthy individuals without a clinical history of hepatitisand without symptoms or signs of liver disease and serum HCV RNA negative [6 male and 2female， median age： 29 years (range， 21～41)].
HCV antigens HCV recombinant proteins： The HCV proteins core，envelope 1(E1 )， envelope 2 (E2 ) nonstructural 3(NS3 )were supplied and gifted by prof. Zhuang Hui (Dept. of Microbiology， BeijingMedical University， P.R.China) and Dr.wang Xingtai， Dr. Zhang Shuming (Dept.of hepatitis， national institute for the control of pharmaceutical and biologicalproducts. Beijing， P.R.China).
HCV synthetic peptides： The HCV synthetic peptides Cp9 ，NS4 weresyntheted by Institute of Biochemistry Chinese Academy of Medicine， Shanghai， P.R.China).
Cell Culture-medium The medium used of all cell cultures consistedof RPMI 1640 supplemented with 25 mmol/L HEPES， 2 mmol/L L-glutamine (all fromGibcoBRL，Scotland)， Penicillin (100u/ml)， streptomycin (100μg/ml) and5% (voL/voL) pooled human serum(RPMI-HS). The AB serum was obtained fromhealthy blood donors with blood group AB+ and was only used when antibodiesto HCV were absent.
PBMC proliferation assay Heparinized venous blood drawn frompatients with hepatitis C were diluted in Hank’s liquid. PBMC were separated byFicoll-Hypaque (The second agent factory， Shanghai， P.R.China)density gradientcentrifugation. Proliferation assays were performed as described [4-5] .Briefly， PBMC(2×105 cells) in 0.2 ml of RPMI-HS were cultured in96-well flat-bottom microplates in the presence or absence of HCV synthetic peptides orHCV recombinant proteins at various final concentration for 6 days at 37°C in anatmosphere of 5% CO2 in air. 0.5 μCi of [3 H] thymidine(3 H-TdR，Purchased from Institute of radionuelide， Chinese Academy ofMedicine， Shanghai， P.R.China) was added in each well and DNA incorporatedradioactivity was measured afte an additional 16 to 18 hours by liquid scintillationcounting. All proliferation assays were performed in triplicate.The data represent themean counts per minute (cpm). Results were expressed as stimulation index (SI：after cpm incorporated in response to antigens minus cpm incorporated in the absence ofantigens divided by cpm incorporated in the absence of antigens).SI2.1 ispositive (or efficient) proliferative responses.
Determination of the cellular phenotype PBMC， which werestimulated with HCV antigens or not， were analyzed by immunofluorescence on afluorescence activated cell sorter (FACS) after staining with the fluorescein isothiocyanate-conjugated monoclonal antibodies (anti-CD4 ，anti-CD8 ，Purchased from Dept. of Immunology Beijing Medical University， P.R.China) andspecific fluorescence intensity was determined on a FACS ta flow cytometer (Becton ＆Dickinson).
Statistical analysis Results are expressed as mean ±S.Statisticalanalysis was performed using the student’s test. A level of P<0.05 was consideredto be statistically significant.
RESULTS
Proliferation response of PBMC to differentdoses HCV synthetic peptides To select appropriate optimum antigens doses wefirst adopt 1μg， 4μg， 8μg and 12μg/ml HCV synthetic peptides and detectedproliferation response of their PBMC to synthetic peptides CP9 and NS4 .Fig 1 showed the proliferation response of their PBMC to different do-ses HCV syntheticpeptides in vitro. PBMC from 8 individual with hepatitis C appear to be have diffe-rentproliferative responses to CP9 and NS4 in diffe-rent doses. As thesynthetic peptides doses became higher the proliferative response of their PBMC to HCVsynthetic peptides CP9 and NS4 were stronger gradually. However，proliferative response had no significant difference between 8μg dose group and 12μgdose group after the date was analyzed by student’s test. None of the HCV-RNA negativecontrol individual responded to synthetic peptides CP9 and NS4 .
Fig 1 Dose-response curves of PBMCstimulated with HCV synthetic peptides CP9 and NS4
(PBMC (2×105 cells) from 8 individual with hepatitis C and 8 individualwith HCV-seronegative were incubated for 6 days respectively with 1μg， 4μg， 8μg and12μg/ml CP9 and NS4 .Each point was a mean value of 8 samples.
Proliferative response of PBMC from patients withhepatits C to different HCV antigens The proliferative responses to HCV antigensin 24 patients with hepatitis C showed as table 1. PBMC from 25% (6/24) of thesepatients proliferated in response to at least one HCV antigens. According to the ratioof positive proliferative response(SI2.1) PBMC from patients with hepatitis C proliferativeresponses to recombinant HCV C，E1 ，E2 and NS3 proteinswere 26.7%(4/15)， 18.2%(2/11)，18.2%(2/11)and 9.1%(1/11)respectively and proliferative responses to HCV synthetic peptides CP9 ， NS4 were 25% (6/24) and 16.7%(4/24) in order. The most frequent responseswere to antigens of HCV C region.HCV-RNA column in Fig.1 showed that the two out ofthree patients with acute hepatitis C had efficient proliferative response of their PBMCto HCV antigens (SI2.1) and their serum HCV-RNA converted negative and hadnormal level of ALT as well.
Tab 1 PBMC proliferative response to differentHCV antigens in patients with hepatitis C
| Patient no. | Age (Yr) | Sex | Anti- HCV | HCV- RNA | Path | PBMC proliferation in response to | |||||
| Synthetic peptides | Recombinent proteins | ||||||||||
| CP9 | NS4 | C | E1 | E2 | NS3 | ||||||
| 1 | 65 | M | + | + | CHR | 1.10 | 1.05 | ||||
| 2 | 37 | F | + | - | ACUΔ | 2.27 | 2.13 | ||||
| 3 | 28 | M | + | + | CHR | 1.46 | 1.55 | ||||
| 4 | 48 | M | + | + | CHR | 1.81 | 1.85 | ||||
| 5 | 54 | F | + | + | CHR | 2.79 | 1.13 | ||||
| 6 | 28 | M | + | + | CHR | 1.80 | 2.03 | ||||
| 7 | 61 | M | + | - | CHR | 1.08 | 2.14 | ||||
| 8 | 29 | M | + | + | CHR | 1.96 | 1.77 | ||||
| 9 | 27 | F | - | + | CHR | 1.26 | 1.98 | ||||
| 10 | 28 | M | + | + | CHR | 1.91 | 1.74 | 2.68 | 2.68 | ||
| 11 | 34 | M | + | - | CHR | 2.16 | 3.50 | 1.96 | |||
| 12 | 48 | M | + | + | CHR | 1.80 | 1.47 | 1.48 | |||
| 13 | 27 | F | + | + | ACUΔ | 1.60 | 1.32 | 1.67 | |||
| 14 | 25 | M | + | - | ACUΔ | 3.46 | 1.58 | 2.34 | 2.16 | 2.21 | 1.65 |
| 15 | 27 | F | + | + | CHR | 1.01 | 1.32 | 1.01 | 1.45 | 1.40 | 0.97 |
| 16 | 41 | F | + | + | CHR | 1.20 | 1.15 | 1.00 | 0.99 | 1.04 | 1.12 |
| 17 | 40 | M | + | + | CHR | 2.21 | 1.65 | 2.40 | 1.57 | 1.65 | 2.47 |
| 18 | 37 | M | NT | + | CHR | 1.10 | 1.03 | 1.05 | 1.32 | 1.15 | 1.02 |
| 19 | 58 | M | + | + | CHR | 2.18 | 2.10 | 2.40 | 1.62 | 1.59 | 1.50 |
| 20 | 49 | M | NT | + | CHR | 1.28 | 1.16 | 1.09 | 1.28 | 1.10 | 1.03 |
| 21 | 60 | M | + | + | CHR | 1.24 | 1.38 | 2.08 | 2.34 | 2.23 | 1.17 |
| 22 | 32 | M | + | + | CHR | 1.18 | 1.07 | 1.61 | 1.55 | 1.44 | 1.25 |
| 23 | 54 | M | + | + | CHR | 1.45 | 1.22 | 1.34 | 1.03 | 1.05 | 1.23 |
| 24 | 30 | M | + | + | CHR | 1.18 | 1.04 | 1.02 | 1.03 | 1.24 | 1.17 |
 ±s | 1.69 | 1.61 | 1.68 | 1.49 | 1.44 | 1.33 | |||||
| ±0.63 | ±0.55 | ±0.58 | ±0.44 | 0.44± | ±0.43 | ||||||
| Ratio of positive response | 6/24 | 4/24 | 4/15 | 2/11 | 2/11 | 1/11 | |||||
Δ ACU， acute hepatitis C；CHR，chronic hepatitis C；A，abnormal； N， normal； NT， no test；
* Serum ALT values measured on the same day as when PBMCproliferation was assessed.
—， Positive proliferation responses was SI2.1.
Phenotype analysis To determine which typeof cells proliferated in response to HCV antigens in vitro phenotypic characteristics ofproliferating cells in PBMC cultivation for 6 days with HCV antigens were detected byimmunofluorescence on FACS. Table 2 showed that the PBMC from patients with chronic Cappeared to be proliferated after stimulated with HCV antigens and phenotypiccharacteristics of proliferating cells was CD4 + lymphocyte andphenotypic characteristics of CD8 + lymphocyte was less than thatof CD+ 4 lymphocyte after stimulated with HCV antigons， of whichCD+ 4
Tab 2 Phenotypic characteristics of proliferatingcells sti-
mulated with HCV antigens
| Cells Phenotype | no stimulated with HCV antigens | Stimulated with HCV antigens | ||
| CP9 | NS4 | C | ||
| CD+ 4 | 40.05±3.48 | 44.34±1.69Δ | 43.48±1.92 | 44.28±4.46Δ |
| CD+ 8 | 20.27±1.27 | 21.19±2.34 | 21.56±2.33 | 22.5±2.59 |
| CD+ 4 /CD+ 8 | 1.97±0.19 | 2.09±0.24 | 2.02±0.18 | 1.99±0.21 |
*each value was a mean of 5 samples ±s
Δ compared with no HCV antigens stimulating，P<0.05
PBMC were cultured for 6 days in the presence of HCV synthetic peptides CP9 ，NS4 and HCV recombinant C protein or not.
lymphocyte proliferative responses to HCV synthetic peptide CP9 and recombinantantigen C had significant increased in comparison with HCV antigens stimulating before.
DISCUSSION
Persistence of HCV in the infected host is a very common event that occurs inmore than 60% of infected patients. The complex interplay between virus and immunecells and the possible viral interference with an efficient development of the protectiveimmure response are still to be elucida-ted[6] . In particular，information on the role played by T and B cells with selective specificity for differentHCV antigens in the neutralization of circulating and intracellular virus is still poorlyunderstood.
To gain insight into the immune function responsible for clearing viral infection wedetected the lymphoproliferative response to HCV antigens in 24 patients with hepatitis C.In preliminary experiment 8 patients with chronic hepatitis C and 8 healthy individualswere studied for PBMC proliferative response to different concentrations (1μg， 4μg，8μg and 12 μg/ml) of HCV synthetic peptide CP9 and NS4 . Theresult showed that lymphoproliferative response was more vigorous in presence of higherdose peptide antigens and that proliferative response of their PBMC was stronger in 12μg/mldose group than that of 8 mg/ml dose group. However， there was no statisticalsignificance between the two dose groups. In order to show the nature of proliferationresponse of PBMC from patients with hepatitis C and save expensive HCV antigens 8μg/mlantigens dose was taken in later experiment.
The study shows that the majority of HCV-infected patients exhibit CD+ 4 T-cell responses to viral antigens. This was established by assessing the HCV-specificPBMC proliferative response in vitro， which depeneds on the presence of T cells thathave been exposed to HCV antigens and have therefore undergone clonal expansion in vivo.However， the proliferative response intensity of PBMC is less than that of other’sreport[3～6] . These are possible correlated with lower titer of HCV ininfected body， inferior HCV immunogen， weak immune responses to HCV antigens ininfec-ted host and not much purity of HCV antigens used in our experiment. From thepoint of view of selecting HCV predominant antigens core antigens (recombinant HCV Cprotein and synthetic peptide CP9 ) were a good immunogen because the PBMCproliferative responses to core antigens were the most vigorous in comparison with PBMCproliferative responses stimulated with any other HCV antigens (recombinant HCV E1 ，E2 ，NS3 and synthetic peptide NS4 ). This investigation result canprovide a feasible method from cellular immune prevention for selecting HCV predominantantigens used in development HCV vaccine in future.
The phenotypic characteristics of proliferating cells indicated that HCV-specific CD+ 4 T lymphocytes proliferated more vigorous than that of CD+ 8 Tlymphocytes measured by FACS. The result can suggest the PBMC stimulated with solubleexogenous antigens is expected to induce a preferential activation of CD4 -positiveHLA class Ⅱ-restricted T cells and not of CD8 -positive HLA class Ⅰ-res-trictedcytotoxic T lymphocytes which are believed to be the main effectors for the elimination ofvirus-infected cells[11，12] . Because exogenous antigens arephagocytized and dealed with by macrophange and present antigens to the HLA-calss Ⅱmolecules to form heteromolecular complexes that are recognized by CD+ 4 T lymphocytes and induce CD+ 4 T lymphocytes to expand andproliferate.
The relationship between the immune res-ponse of PBMC from patients with hepatitis Cto HCV antigens and outcome of infection is now difficult to determine. There are a fewdifferent opinions in some researchers([3～7] . Our study showed thatthe efficient proliferation response (SI2.1) of PBMC from patients with acutehepatitis C likely indicated that the lymphoproliferative response to HCV antigens areassociated with clinical prognosis of HCV infection. Indeed， in the patients withacute hepatitis C， whose PBMC proliferative response to HCV antigens are stronger，their serum HCV-RNA were negative and serum ALT were normal when studied. In the otherpatients with acute and chronic hepatitis C， whose PBMC did not demonstrate efficientproliferative responses to HCV antigens， their serum HCV-RNA were detectable and serumALT were elevated. Because the cases studied is not enough further studies in a largegroup of patients with hepatitis C and using more various antigens in PBMC proliferationassay are required. The present findings may show that PBMC from patients with hepatitisC exist proliferative responses to HCV proteins/peptides， of which core antigens werea good immunogen than any other HCV antigens used in our studies and suggest that thesignificant lympho-proliferative responses to HCV antigens are more frequently observed inacute infective patients who were able to eradicate the virus than in chronic HCVinfective patients.
*This study was supported by the science foundation of public healih bureau ofZhejiang province in China.
作者单位：Dou Jun(窦骏) Liu Kezhou(刘克洲) Chen Zhi(陈智)Wo Jian’er(沃建尔)He Nanxiang(何南祥) Xu Chenhuai(徐陈槐) Zhang Mingtai(章明太) Wang Xinzi(王信子) Institue of Infectius Disease，Zhejiang Medical University ，Hangzhou 310003(Dou Jun now at：Department ofMicrobiology Nanjing Railway Medical College，Nanjing 210009)
第一作者：男，43岁，博士，副教授
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(1998-04-03收稿；1998-07-13修回) (Dou Jun(窦骏)， Liu Kezhou(刘克洲)，Chen Zhi(陈智)1， Wo Jian’er(沃建尔)，He Nanxiang(何)