【作者】 王冬菊；

【导师】 肖小敏；

【作者基本信息】 暨南大学 ， 妇产科学， 2010， 博士

【摘要】 目的：1.建立从人足月胎盘中大量分离纯化滋养细胞的实验方法；2.分离纯化乙肝携带产妇胎盘滋养细胞,观察HBV抗原和DNA在滋养细胞中的表达,了解乙肝携带产妇胎盘滋养细胞中是否存在HBV的复制；3.以HBV重组质粒转染滋养细胞来源细胞株,构建HBV感染滋养细胞的体外模型,以肝癌细胞作为对照,观察模型中HBV相关核酸和蛋白质的表达水平；4.设计构建针对HBV SmRNA的shRNA (short hairpin RNA)表达载体,与HBV重组质粒共转染滋养细胞,观察不同shRNA质粒的基因敲减效果,筛选出具有显著干扰作用的siRNA序列。方法：1.收集20例正常足月妊娠胎盘,剪取50g绒毛组织,胰酶联合DNase I消化组织,简化的密度梯度离心法联合反复贴壁法分离纯化滋养细胞,细胞免疫荧光标记法、激光扫描共聚焦显微镜技术(Laser scanning confocal microscopy, LSCM)检测滋养细胞内CK-7蛋白的表达,计算滋养细胞纯度。2.收集9例血清HBsAg. HBeAg双阳性产妇胎盘,分离纯化滋养细胞,细胞免疫荧光标记法、LSCM技术检测滋养细胞纯度；微粒子酶免分析法(Microparticle enzyme immunoassay, MEIA)、荧光定量PCR (Fluorescencequantitative PCR, FQ-PCR)检测体外培养24、48、72h滋养细胞培养上清HBsAg、HBeAg和HBV DNA的表达量,确定滋养细胞是否感染HBV; FQ-PCR检测感染HBV滋养细胞中是否表达HBV复制的指标共价闭合坏DNA (covalently closed circular DNA, cccDNA)3.通过脂质体介导法,以HBV重组质粒pcDNA3-3HBV转染滋养细胞来源细胞株JEG-3、JAR和肝癌细胞HepG2; PCR检测胞内HBV两种DNA分子形态rcDNA (relaxed circular DNA)和cccDNA的表达,FQ-PCR检测培养上清HBV DNA的滴度；RT-PCR (reverse transcription PCR)检测HBsAg编码mRNA的表达：细胞免疫荧光标记法、结合LSCM技术检测细胞内HBsAg和HBcAg的表达；MEIA检测培养上清HBsAg、HBeAg的分泌量,从DNA、RNA、蛋白质三个层面对细胞模型进行观察。G418筛选培养基对转染后JAR和JEG-3细胞进行筛选。4.设计构建4种针对HBV SmRNA的shRNA表达载体,分别命名为pS-603、pS-475、pS-66、pS294,设置错义链载体pS-scramble和空白载体pS-0为干扰特异性对照和空白对照。脂质体介导法与HBV重组质粒共转染JAR细胞,转染后48、72、96h收集细胞及培养上清。细胞免疫荧光标记结合LSCM技术、Western blotting定量检测胞内HBsAg的表达；MEIA定量检测培养上清HBsAg的分泌量；实时荧光定量逆转录PCR (Fluorescence quantitative reverse transcription PCR, FQ-RT-PCR)检测胞内SmRNA表达量；FQ-PCR检测培养上清HBV DNA滴度,分析比较不同shRNA的干扰效果。结果：1.经胰酶、DNase I联合消化胎盘组织,简化的密度梯度离心法联合反复贴壁法,每50 g胎盘组织可获得约108、纯度约85%,活力约88%的滋养细胞。2.9例乙肝携带产妇胎盘滋养细胞体外培养24、48、72h,培养上清的HBsAg表达量(S/N值)分别为3.72±1.41、1.99±0.87和1.75±0.99,HBeAg的表达量(S/CO值)分别为2.26±1.31、0.62±0.36和0.49±0.40,提示9例血清HBsAg、HBeAg双阳性产妇胎盘组织皆感染了HBV；其中,HBsAg和HBeAg 24h表达量皆高于48h、72h(p<0.05)。上清HBV DNA的滴度分别为(2.39±1.87)×103 copies/mL、(1.04±0.94)×103 copies/mL和(0.78±0.68)×103 copies/mL,3个时间点之间无明显差异。9例乙肝胎盘滋养细胞样本中2例样本检测到少量HBV cccDNA的表达,分别为6.75×103 copies/mL、2.22×103 copies/mL。3.转染pcDNA3-3HBV的JEG-3、JAR、HepG2细胞胞内表达rcDNA、cccDNA和SmRNA.培养上清可检测到HBV DNA的表达,JEG-3与JAR细胞上清HBV DNA的表达量无显著性差异,HepG2培养上清HBV DNA滴度明显高于2种绒癌细胞的表达(p<0.05)转染细胞内表达HBsAg和HBcAg;培养上清HBsAg和HBeAg的表达量比较,JEG-3与JAR细胞无显著性差异,HepG2细胞则明显高于2种绒癌细胞(p<0.05)。经G418筛选,转化的JAR细胞形成持续生长的阳性克隆系,而JEG-3在筛选过程中逐渐死亡4. shRNA与HBV重组质粒共转染JAR细胞后,pS-603、pS-475、pS-66组细胞内、培养上清HBsAg,胞内SmRNA和培养上清HBV DNA的表达量,随转染后时间推移逐渐降低(P<0.05)pS-294、pS-scramble和pS-0组细胞HBsAg编码mRNA和蛋白质的表达、培养上清HBV DNA的滴度无显著性差别,并且不随时间推移而改变。结论：1.简化的密度梯度离心法联合反复贴壁法,可获得大量纯化的足月胎盘滋养细胞。2.HBV感染的胎盘中可能存在低水平的HBV复制,但滋养细胞中HBsAg、HBeAg和HBV DNA的表达可能不是HBV持续复制的结果。3.HBV重组质粒构建的HBV感染滋养细胞体外模型中,存在HBV的复制和病毒蛋白的表达。4.RNA干扰可抑制HBV感染滋养细胞体外模型中病毒蛋白的表达。4种shRN表达质粒中,pS-603、pS-475、pS-66干扰效果明显,而pS-294干扰效果不明显。更多还原

【Abstract】 Objective1. To establish a practical protocol for the harvest of large amount of purified term human placental trophoblast.2. To observe the expression of hepatitis B virus (HBV) antigens and DNA in trophoblasts isolated from gravidae with HBV surface antigen (HBsAg) and e antigen (HBeAg) seropositivity and, to investigate wheather there is HBV replication in HBV affected placental trophoblasts.3. To establish an in vitro model of HBV-affected-trophoblast by transfecting trophoblast-derived cell strains with recombinant HBV expressing plasmid. Comparing with transfected hepatocarcinoma cell strain HepG2, we study the expression of viral nucleic acid and protein in the model.4. Design and construct different short hairpin RNA (shRNA) expressing vectors targeting at HBsAg coding mRNA (SmRNA), as to analyze their gene knock-down efficiency after being co-transfected with recombinant HBV expressing plasmid into the host cells, and to select the most capable siRNA (small interfering RNA) sequence. Methods1.50g of chorion tissue from 20 normal term placentas were isolated respectively, digested by trypsin and DNase I, purified by the combination of modified gradient gravity centrifugation and repeatedly adherence to culture vask. Immunofluo-rescence labeling of intracellular cytokeratin-7 (CK-7) was measured by laser scanning confocal microscopy (LSCM) for the calculation of the purity of trophoblasts.2. Placental trophoblasts from 9 gravidae with HBsAg and HBeAg seropositivity were isolated, purified and cultured in vitro for 72 hours. The culture supernatant were collected at 24h,48h,72h and subjected to HBsAg/HBeAg detection by Microparticle Enzyme Immunoassay (MEIA) as well as to HBV DNA measurement by fluorescence quantitative PCR (FQ-PCR), as to confirm wheather the sample was affected by HBV. HBV covalently closed circular DNA (cccDNA) was detected by FQ-PCR as to study if there was HBV replication in HBV affected trophoblasts.3. Trophoblast derived cell strains JEG-3 and JAR, as well as hepatocarcinoma cell strain HepG2, were used for liposome mediated transfection of recombinant HBV expressing plasmid. After transfection, cell samples and culture supernatant were collected for laboratory examination at 24h,48h and 72h. The intracellular rcDNA (relaxed circular DNA) and cccDNA of HBV were detected by PCR, SmRNA was detected by RT-PCR (reverse transcription PCR). HBV DNA in culture supernatant was determined by FQ-PCR. Intracellular HBsAg/HBcAg were examined by immunofluorescence labeling, HBsAg/HBeAg in culture supernatant were measured by MEIA. Meanwhile, G418 containing culture medium was applied for the selection of transformed choriocarcinoma cell line.4. Design and construct 4 kinds of shRNA expressing vectors targeting at SmRNA, named pS-603、pS-475、pS-66、pS294 respectively. pS-scramble and empty vector pS-0 were used as transfection specificity control and negative control respectively. ALL shRNA expressing plasmids were co-transfected into the host cells with recombinant HBV expressing plasmid by LipofectamineTM 2000 respectively. After transfection, cell samples and culture supernatant were collected for laboratory examination at 48h,72h and 96h. Intracellular HBsAg were measured quantitatively by immunofluorescence staining and Western blotting, HBsAg in culture supernatant were measured by MEIA. The expression of SmRNA was determined by FQ-RT-PCR (fluorescence quantitative reverse transcription PCR). Supernatant HBV DNA titer was measured by FQ-PCR.Results1. After being digested by trypsin and DNase I, purified by the combination of gradient gravity centrifugation and repeatedly adherence to culture vask, the harvest of trophoblast yeilded to about 108 cells per 50g chorion tissue with 85% purity and 88% vitality on average.2.9 trohpoblast samples from the gravidae with HBsAg/HBeAg seropositivity were prooved to be HBV-affected. HBsAg expression (S/N ratio) in the culture supernatant were 3.72±1.41 at 24h,1.99±0.87 at 48h and 1.75±0.99 at 72h respectively. HBeAg (S/CO ratio) were 2.26±1.31 at 24h,0.62±0.36 at 48h and 0.49±0.40 at 72h, respectively. At 24h, the expression of HBsAg and HBeAg were higher than those at 48h and 72h (p<0.05). Supernatant HBV DNA titer were (2.39±1.87)×103 copies/mL at 24h, (1.04±0.94)×103copies/mL at 48h, (0.78±0.68)×103 copies/mL at 72h respectively, but there was no significant differences about the DNA titers among the 3 timepoints. A low expression of HBV cccDNA was detected in 2 samples among the 9 samples, which were 6.75×103 copies/mL and 2.22×103 copies/mL.3. RcDNA, cccDNA and SmRNA were detected in the transfected JEG-3, JAR and HepG2 cells. HBV DNA was detected in the culture supernatant. There was no significant differences about supernatant HBV DNA titer between JEG-3 and JAR, yet HepG2 had higher titer than the two choriocarcinoma cells did (p< 0.05). Intracellular HBsAg/HBcAg were detected by immunofluorescence labeling. There was no significant differences in supernatant HBsAg/HBeAg expression between JEG-3 and JAR, yet HepG2 had higher expression of the two viral antigens than the two choriocarcinoma cells did (p< 0.05). Positive clones of transformed JAR were selected by G418 medium successfully, but JEG-3 extincted gradually during the selection procedure.4. After co-transfection of shRNA expressing vectors and recombinant HBV plasmid, the expression of intracellular and supernatant HBsAg, SmRNA, as well as the supernatant HBV DNA, declined gradually day after day in pS-603, pS-475 and pS-66 groups. There were no significant differences in the expression of HBsAg, SmRNA and HBV DNA among pS-294, pS-scramble and pS-0 groups.Conclusion1. The combination of modified gradient gravity centrifugation and repeatedly adherence to culture vask might be a pratical protocol for the harvest of large amount of purified term human placental trophoblast.2. There might be a low level of viral replication in HBV affected placenta. But the expression of HBsAg/HBeAg and HBV DNA might not result from continuos replication of HBV.3. There were viral replication and protein expression in the in vitro model of HBV-affected-trophoblast constructed by transfecting recombinant HBV expressing plasmid into the host cell.4. pS-603, pS-475 and pS-66 were able to knock down the expression of HBV SmRNA but pS-294 was not.更多还原