乙肝肝移植受体术后骨髓CD34^＋细胞和PBMCs内乙肝病毒DNA的同步检测

Quantitative detection of hepatitis B virus DNA in bone marrow hematopoietic stem and progenitor cells as well as peripheral blood mononuclear cells of recipients after liver transplantation

目的肝移植术后乙肝复发严重影响预后，HBV感染的病毒来源值得研究。采用PCR技术可检测到术后病人外周血单个核细胞（PBMCs）中HBV DNA的存在，然而PBMCs的寿命有限，其中的病毒来源仍不清楚。该研究对此问题做了初步的探索。方法采集13例乙肝肝移植术后受体外周血和骨髓标本，运用Ficoll密度梯度离心法结合免疫磁性分离法（MACS）分别从外周血和骨髓标本中分离出PBMCs和骨髓来源CD34^-细胞即造血干祖细胞，利用实时荧光定量PCR法检测细胞中的HBV DNA，同时检测血清HBV标志物和血清HBV DNA。结果13例病人术前血清HBV DNA阳性8例，阴性5例。术后平均37个月的随访期内（16～77个月）血清中HBV DNA，HBsAg和HBeAg检测结果全为阴性；13例PBMCs中均检测到HBV DNA的存在，阳性率为100％。PBMCs中DNA含量对数平均值为3．40±0．85；13例骨髓来源CD34^＋细胞中都检测到HBV DNA的存在，阳性率也是100％。CD34^＋细胞中DNA含量对数平均值为3．30±0．58。病人PBMCs和CD34^＋细胞内DNA含量对数值均数差异无统计学意义（P〉0．05）。同一病人PBMCs与CD34^＋细胞HBV DNA同时为阳性。术前血清HBV DNA检测阳性和阴性病人的PBMC内DNA含量对数值均数差异无统计学意义（P〉O．05）；术前血清HBV DNA检测阳性和阴性病人的CD34^＋细胞内DNA含量对数值均数差异也无统计学意义（P〉0．05）。结论现有预防措施下，乙肝肝移植受体虽然外周血清中不能检测到HBV DNA和HBV抗原成分的存在，但术后骨髓CD34^＋细胞和PBMCs中皆长期存在HBV DNA。含有HBV DNA的骨髓来源CD34^＋细胞可能是含有HBV DNA的PBMCs的来源，也可能是肝移植术后PMBCs内HBV DNA长期存在的原因，及以后导致HBV复发的潜在危险因素。然而，现在尚无有效方法可以完全清除这些残存病毒，术后长期的抗病毒药物预防措施是必要的。

Objective Hepatitis B virus （HBV） reinfection after liver transplantation is a major problem. Though HBsAg and HBV DNA can not be detected in some patients＇ serum after transplantation, PCR has confirmed the existence of HBV DNA in these patients＇ peripheral blood mononuclear cells （PBMCs）. In order to discuss the potential mechanism of persistent HBV infection of PBMCs, the following research has been done. Methods HBV DNA in bone marrow hematopoietic stem and progenitor cells （CD34^＋ cells） as well as PBMCs of HBV-related recipients after liver transplantation were detected by Fluorescence quantitative polymerase chain reaction （FQ PCR）. Blood and bone marrow specimens were taken simultaneously from 13 recipients who have been receiving lamivudine combined with hepatitis B immune globulins （HBIG） prophylaxis after liver transplantation. HBV DNA in serum, PBMCs and CD34^＋ cells was measured by FQ-PCR. Meanwhile, HBV markers in serum were tested by ELISA. In addition, CD34^＋ cells were isolated from bone marrow specimens using Magnetic cell sorting and separation technique （MACS）. Results FQ PCR confirmed the existence of HBV DNA in both the PBMCs and the CD34^＋ cells from bone marrow in 13 of the 13 recipients, while HBV DNA, HBsAg, HBeAg in serum can not be detected during a mean follow up period of 37 months（16 77 months） after liver transplantation. In all PBMCs samples, HBV DNA was positive. HBV DNA level in PBMCs was 1. 638 × 10^2- 1. 332 × 10^3 copies/10^6 （HBV DNA copies in every 10^6 PBMCs）, the logarithmic average was 3.40±0.85. The difference of HBV DNA levels in PBMCs between HBV DNA positive and negative patients prior to OLT was not statistically significant. In all CD34^＋ cells samples, HBV DNA was positive. HBV DNA level in CD34^＋ cells was 1. 428× 10^2- 1. 266× 10^3 copies/10^6（HBV DNA copies in every 10a CD34^- cells）, the logarithmic average was 3. 30±0. 58. The difference of HBV DNA levels in CD34^＋ cells between HBV DNA positive and negative patients prior to OLT was not statistically significant. The difference of HBV DNA levels in PBMCs and CD34^＋ cells was not statistically significant （P〉0.05）. HBV DNA detection revealed an apparent association between PBMCs and CD34 cells. That is to say, when PBMCs were HBV DNA positive, CD34^＋ cells were uniformly HBV DNA positive. Conclusion There is long-term persistence of HBV DNA in both bone marrow CD34^＋ cells and PBMCs after liver transplantation despite the use of currently available prophylactic approaches. It seems that PBMCs harboring HBV DNA may originate from the bone marrow CD34^＋ cells containing HBV DNA, which may account for one of the reasons of hepatitis B （HB） recurrence post transplantation. Up to now, complete clearance of HBV DNA is still not possible. The long-term prophylactic use of anti-viral drugs to suppress HBV replication seems to be necessary.