两种原代树鼩肝细胞的分离和培养方法的研究

目的 探讨体外原代培养树嗣肝细胞的分离方法.方法 以成年树鼩和新生树鼦做为肝供体,分别采用体外两步灌流法和Percoll梯度液离心方法获取肝细胞并进行体外培养;以台盼蓝染色法测细胞存活率,在相差倒置显微镜下观察细胞形态变化,MTT法测培养细胞活性,并采用PAS染色法鉴定.结果 分离收获成年树鼩肝细胞较新生树鼩肝细胞存活率高;培养过程中,新生树胸肝细胞较成年树鼩肝细胞生长快,增殖能力强,具有统计学意义;PAS染色观察,新生树鼩和成年树鼩的肝细胞中充满大量糖原颗粒,两者差异无显著性.结论 两种方法均可用于原代树鼩肝细胞的体外培养.     

乙型肝炎病毒假病毒体外感染树鼩原代肝细胞模型的建立

目的：建立乙型肝炎病毒假病毒（HBVpp）体外感染树鼩原代肝细胞（PTH）模型。方法：通过肝脏原位两步灌注法分离PTH并对其冻存方法进行优化,通过共转染293T细胞生产基于慢病毒包装系统的HBVpp并考察其感染的种属和组织特异性。结果：通过肝脏原位两步灌注法分离了PTH,并优化了PTH冻存液的配方;包装的HBVpp具有与HBV真病毒类似的感染种属和组织特异性。结论：该模型的建立对深化HBV进入肝细胞机制的研究具有重要意义。     

树鼩原代皮肤成纤维细胞的分离培养技术

建立稳定的树鼩(Tupaiabelangeri)皮肤成纤维细胞的体外培养体系,可为有关此类细胞的实验和疾病树鼩细胞模型提供技术支持。取树鼩大腿内侧皮肤用组织块贴壁法和胶原酶Ⅰ消化法分离皮肤细胞,胰蛋白酶差别消化法纯化细胞;用MEM(10%FBS)完全培养基和含低血清生长添加物(LSGS)的培养基培养细胞;免疫荧光和蛋白印迹法鉴定细胞,并测定细胞的生长、冻存和复苏特性。经树鼩皮肤细胞分离效果比较,胶原酶消化法比组织块贴壁法更适合用于树鼩原代皮肤细胞分离;对分离及冻存复苏后细胞生长状况观察比较发现,添加了LSGS的MEM培养基更利于细胞存活、生长;细胞形态观察、免疫荧光和蛋白印迹检测鉴定所分离的细胞为树鼩皮肤成纤维细胞。成功建立了树鼩原代皮肤细胞的分离、纯化方法,并优化了该细胞的培养条件。     

人轮状病毒G1P[8]型感染树鼩原代小肠上皮细胞模型的建立

目的探究树鼩原代小肠上皮细胞的增殖特性,建立人轮状G1P[8]型病毒体外感染树鼩原代小肠上皮的细胞模型。方法采用胶原酶XI和中性蛋白酶I联合消化法获取树鼩原代小肠上皮细胞,经纯化和鉴定,用人轮状G1P[8]型病毒感染细胞,测定培养上清的病毒滴度和载量,并用Western blot和间接免疫荧光检测法检测人轮状病毒G1P[8]型VP6蛋白的表达情况,评价人轮状G1P[8]型病毒体外对树鼩原代小肠上皮细胞的感染性。结果分离的树鼩原代小肠上皮细胞经传代培养纯化,获得纯度达90%树鼩原代小肠上皮细胞。对树鼩原代小肠上皮细胞、原代肾细胞、HCT116细胞和MA104细胞进行轮状病毒易感性比较,确定树鼩原代小肠上皮细胞可以被人轮状病毒G1P[8]感染,培养72 h时病毒滴度可达到2.0×105TCID_(50)/m L。经Western blot和间接免疫荧光发现在人轮状G1P[8]型病毒感染树鼩原代小肠上皮细胞1~5 d均能检测到人轮状病毒VP6蛋白的表达和分布。结论确立了树鼩原代小肠上皮细胞的分离、纯化与培养方法,并建立了人轮状G1P[8]型病毒感染树鼩原代小肠上皮细胞的体外模型。     

表皮生长因子对肝细胞醋氨酚急性损伤的保护作用

本工作采用无血清培养的小鼠原代肝细胞制备了醋氨急性肝损伤模型,然后利用该模型观察了表皮生长因子（ＥＧＦ）对肝细胞的保护作用。结果如下：（１）小鼠原代肝细胞无血清培养液中加入终深度度为２０ｍｍｏｌ／Ｌ的醋氨酚培养１２－１４ｈ后,培养液中ＧＰＴ和ＧＰＴ的活性明显升高,可作为一种适当的肝细胞损伤。（２）提前１ｈ加入不同剂量（５０,１００,５００,１０００ｎｇ／ｍｌ）的ＥＧＦ可减轻醋氨酚相起的肝细胞损伤。     

乙型肝炎病毒进入肝细胞机制研究进展

乙型肝炎病毒(hepatitis B virus,HBV)感染早期进入肝细胞机制研究一直是HBV研究领域的热点和难点．简单易得的HBV体外感染细胞模型是HBV感染进入机制研究无法逾越的主要障碍．近年来,随着新型HBV体外感染细胞模型的建立和应用(HepRG细胞和树鼩原代肝细胞),HBV的进入机制研究取得了一系列重大发现．综述了近几年HBV进入肝细胞机制的最新研究进展,主要包括HBV表面蛋白进入相关结构域的鉴定,已发现的候选HBV进入相关分子和尚待解决的问题．     

荧光定量PCR观察鸭胚肝细胞培养上清DHBV载量的动态变化

通过实时荧光定量PCR(FQ-PCR)观察后天感染和先天感染鸭乙型肝炎病毒(DHBV)的鸭胚肝细胞培养上清中DHBV载量的动态变化,探讨适宜于抗HBV药物的体外实验、HBV生物学特性及乙型肝炎发病机制研究的鸭胚肝细胞模型.筛选先天感染DHBV的阳性鸭胚并进行肝细胞原代培养,对DHBV阴性鸭胚肝细胞原代培养,并以不同浓度的DHBV强阳性血清感染细胞.于细胞接种后不同时间点收集培养上清,FQ-PCR检测分析培养上清DHBV DNA的含量.结果显示:先天感染的鸭胚肝细胞接种第2 d上清中DHBV载量最高,第3 d大幅下降,然后缓慢上升,第8 d达到高峰,至第15 d时仍然与高峰时接近,DHBV载量的数量级都在10<'8>copies/mL没有变化;后天感染不同浓度的DHBV阳性血清的鸭胚肝细胞培养上清中的DHBV载量动态变化趋势基本一致,DHBV感染后第3 d大幅下降,以后逐渐上升,至第11 d达高峰,第15 d又略有下降.结论:先天感染DHBV的鸭胚肝细胞的培养上清病毒载量上升至峰值时间短,病毒载量稳定,更适合于抗HBV药物的体外实验研究.     

EV71病毒对树鼩原代肾上皮细胞感染模型的建立

目的建立EV71对树鼩原代肾细胞的感染模型。方法胰蛋白酶消化法获得树鼩的原代肾细胞,用EV71感染树鼩肾细胞,测定1、2、4、6和8 d培养上清病毒滴度,分别用Western blot和间接免疫荧光法检测细胞中EV71病毒VP1蛋白的表达,以确定EV71病毒对树鼩原代肾细胞的感染性。结果对分离得到的树鼩原代肾细胞进行传代纯化和形态鉴别,建立以树鼩原代肾细胞为主的细胞培养。用EV71病毒感染树鼩原代肾细胞,感染后48~96 h病毒滴度可达到1.3×10~6TCID_(50)/m L,说明EV71病毒可有效感染树鼩原代肾细胞并有效增殖。Western blot检测发现,EV71病毒VP1蛋白可在感染后2~8 d的树鼩原代肾细胞中有效检出,间接免疫荧光法则在感染后2~6 d细胞的细胞质中检测到病毒VP1蛋白的分布。结论在成功建立树鼩原代肾细胞培养的基础上,确定了EV71病毒对树鼩原代肾细胞的感染性和病毒增殖特性,初步建立了EV71树鼩原代肾细胞感染模型。     

Rb蛋白功能缺陷降低肝细胞对TGF-β的敏感性

目的：探讨肝细胞内视网膜母细胞瘤（Rb）基因缺陷对转化生长因子-β（TGF-β）诱导的细胞周期静止的影响。方法：分离并培养原代肝细胞,特异性siRNA腺病毒封闭细胞内Rb基因表达后加入TGF-β诱导,然后MTT法检测细胞生长变化,流式细胞仪检测细胞周期变化,并用Western blot和荧光定量逆转录聚合酶反应（FQ-RT-PCR）法检测细胞中pRb、E2F、c-MYC和p16的表达变化。结果：原代培养肝细胞感染Rb-siRNA重组腺病毒后,正常对照肝细胞抑制率高于Rb基因封闭的肝细胞（69.76%,p<0.01）,流式细胞检测可见对照组TGF-β诱导后处于G1期的肝细胞数明显增多（99.80%,p<0.05）,而在Rb基因封闭组中,TGF-β诱导后各细胞周期分布与未诱导的正常肝细胞基本一致。Western blotting检测可见48h时对照组TGF-β诱导后,E2F和c-MYC蛋白表达减少而p16高表达,Rb基因封闭组TGF-β诱导后c-MYC表达减少而p16表达增高。结论：TGF-β可诱导肝细胞静止于G1期,而Rb基因功能缺陷的肝细胞对TGF-β的诱导敏感性显著降低。     

兔肝细胞体外分离及培养方法的实验研究

目的：研究体外兔肝细胞分离及培养方法,比较不同培养基条件下兔肝细胞培养过程。方法：采用非灌注胶原酶消化法分离兔肝细胞,分别采用RPIM1640培养液（含10％新生牛血清）,DMEM培养液（含10％新生牛血清）,DMEM培养液（含10％胎牛血清）培养,计数法观察原代细胞增殖变化,MTF法观察传代细胞增殖情况。培养细膨采用PAS染色法鉴定,电镜观察细胞超微结构。结果：分离的肝细胞细胞活率大于85％;DMEM培养液（含10％胎牛血清）培养肝细胞生长状态较另两种培养液中的肝细胞强,DMEM培养液（含10％新生牛血清）中的细胞增殖能力较RPIM1640培养液（含10％新生牛血清）高,具有统计学意义。PAS染色和透射电镜观察培养细胞胞质中有大量糖原颗粒。结论：本实验采用的分离方法可获得较纯的肝细胞,而且操作简便实用。DMEM培养液较RPIM1640培养液更加适宜原代培养肝细胞生长,胎牛血清对培养肝细胞的生长促进作用明显高于新生牛血清。     

Analysis of factors related to ELISA-HBsAg negative and HBV DNA positive blood donors

This report analyzed factors relating to ELISA-HBsAg and Hepatitis B virus (HBV) DNA in blood donors. To provide a reference for an accurate screening model for HBV infection in donated blood, we collected rel-evant information from 124 blood donors testing ELISA-HBsAg negative and HBV DNA positive in 2017, including ELISA-HBsAg Max s/co, gender, age, residence, education level, blood donation record and alanine aminotransferase(ALT) value. Meanwhile, 99 blood donors with the results ELISA-HBsAg negative and HBV DNA negative were randomly selected as control. Univariate logistic analysis was conducted for possible correlation factors, then multivariate logistic regression analysis was performed for statistically significant observation indicators. The results showed that the Ct value of HBV DNA mixed test in the observation group donor was higher than that of HBV DNA single test (P<0.05). If one of the two ELISA-HBsAg s/co values was within the range of 0.259 to 0.304, the chance of HBV DNA positive was increased. Univariate logistic regression analysis showed that ELISA-HBsAg Max s/co, gender, age, blood donation history and ALT value were all risk factors in the observation group. Multivariate logistic regression analysis showed that ELISA-HBsAg Max s/co, (OR=5352.448, P<0.05), age (OR= 4.527, P<0.05) and blood donation history (OR=0.441) were risk factors. The study concluded that ELISA-HBsAg Max s/co, age and number of blood donations are risk factors for ELISA-HBsAg negative and HBV DNA positive blood donors, women or donors under 26 years of age had the lowest risk.     

Influence of backward bifurcation in a model of hepatitis B and C viruses

In the 1990s, liver transplantation for hepatitis B and C virus (HBV and HCV) related-liver diseases was a very controversial issue since recurrent infection of the graft was inevitable. Significant progress has been made in the prophylaxis and treatment of recurrent hepatitis B/C (or HBV/HCV infection) after liver transplantation. In this paper, we propose a mathematical model of ordinary differential equations describing the dynamics of the HBV/HCV and its interaction with both liver and blood cells. A single model is used to describe infection of either virus since the dynamics in-host (infected of the liver) are similar. Analyzing the model, we observe that the system shows either a transcritical or a backward bifurcation. Explicit conditions on the model parameters are given for the backward bifurcation to be present. Consequently, we investigate possible factors that are responsible for HBV/HCV infection and assess control strategies to reduce HBV/HCV reinfection and improve graft survival after liver transplantation.     

Serum progranulin levels are elevated in patients with chronic hepatitis B virus infection,reflecting viral load

Progranulin (PGRN) is implicated in infection, immunity and host defense, but its role in the pathogenesis of HBV infection remains unknown. Here we investigated whether there is dysregulated production and the clinical significance of circulating PGRN in patients with chronic HBV infection. Serum concentrations of PGRN were analyzed by enzyme-linked immunosorbent assay. Serum PGRN levels were significantly higher in patients with chronic HBV infection than healthy subjects. PGRN levels were significantly associated with HBV-DNA levels, but did not correlate with the concentrations of alanine aminotransferase and aspartate aminotransferase. This study demonstrates increased circulating PGRN production and association between PGRN levels and viral loads in patients with chronic HBV infection, suggesting a functional role of PGRN in the pathogenesis of HBV infection.     

应用微环DNA技术体外诱导和制备重组的乙型肝炎病毒共价闭合环状DNA

乙型肝炎病毒(hepatitis B virus,HBV)共价闭合环状DNA(covalently closed circular DNA,cccDNA)是病毒慢性感染的分子基础。本课题组前期研究通过Cre/loxP介导的位点特异性DNA重组策略,在细胞核内由前体质粒诱导重组cccDNA(rcccDNA~(loxP))产生,首次建立了HBV cccDNA的体外培养细胞和小鼠实验模型。本研究基于大肠埃希菌ZYCY10P3S2TPhiC31重组酶诱导表达系统,建立了一种体外诱导HBV rcccDNA(rcccDNAattR)微环产生和纯化的策略。纯化的rcccDNA~(attR)微环具有超螺旋结构,细胞培养实验证实其能支持功能性的HBV复制和抗原表达。与普通的线性HBV复制子编码质粒相比,rcccDNA~(attR)尾静脉高压注射小鼠模型能诱导显著延长的病毒抗原血症。因此,本研究在原核表达系统和实验小鼠水平提供了一种更为简化的HBV cccDNA实验模型系统,并再次显示rcccDNA具有显著的稳定性,能作为一种基本策略在小鼠模型中诱导病毒持续感染。     

用重组8型腺相关病毒载体介导的乙型肝炎病毒持续感染小鼠模型评价核苷类似物的抗病毒效果

探索用重组8型腺相关病毒载体携带1.3拷贝乙型肝炎病毒(rAAV8-1.3HBV)介导的HBV持续感染小鼠模型评价核苷酸类似物抗病毒药物的抗病毒效果.首先,通过将rAAV8-1.3HBV经尾静脉注射到30只C57BL/6小鼠体内,建立HBV持续感染模型并对模型成功率进行检测,将建模成功的27只小鼠随机分成6组.然后采取灌胃的方式给予不同剂量的抗病毒药物恩替卡韦(ETV)及拉米夫定(LAM),每日1次,连续l0d,后停药15d,同时设置生理盐水及空白对照组.其中ETV分为高剂量(1.0 mg/(kg·d))和低剂量(0.1 mg/(kg·d) 两组;LAM分为高剂量(500 mg/(kg·d)) 和低剂量(100 mg/(kg·d)) 两组.检测给药前后和停药前后小鼠模型血清中HBV DNA、HBeAg和HBsAg表达水平并比较变化情况.结果发现连续给药10d后,各给药组与生理盐水组相比,血清中HBV DNA水平均显著下降,具有统计学差异(P＜0.05).停药15d后,低剂量的ETV与LAM两组血清HBV DNA水平出现反弹,差异存在统计学意义(P＜0.05).在整个实验过程中,各组小鼠血清中HBeAg和HBsAg表达水平均未出现明显变化.上述结果表明,ETV和LAM能有效抑制模型小鼠中HBV病毒的复制,而对HBeAg和HBsAg表达水平无明显影响;提示AAV8-1.3HBV介导的HBV持续感染小鼠模型制备简单,成模率高,可有效体现出ETV和LAM抗HBV的作用效果,从而用于核苷酸类似物抗HBV药物的筛查.     

Affibody-displaying bio-nanocapsules effective in EGFR,typical biomarker,expressed in various cancer cells

The expression of epidermal growth factor receptor (EGFR) across a wide range of tumor cells has attracted attention for use as a tumor marker in drug delivery systems. Therefore, binding molecules with the ability to target EGFR have been developed. Among them, we focused on affibodies that are binding proteins derived from staphylococcal protein A. By displaying affibody (Z_EGFR) binding to EGFR on the surface of a bio-nanocapsule (BNC) derived from a hepatitis B virus (HBV), we developed an altered BNC (Z_EGFR-BNC) with a high specificity to EGFR-expressing cells. We considered two different types of Z_EGFR (Z955 and Z1907), and found that the Z1907 dimer-displaying BNC ([Z1907]₂-BNC) could effectively bind to EGFR-expressing cells and deliver drugs to the cytosol. Since this study showed that [Z1907]₂-BNC could target EGFR-expressing cells, we would use this particle as a drug delivery carrier for various cancer cells expressing EGFR.     

重组8型腺相关病毒介导HBV急性感染树鼩模型建立

目的利用重组8型腺相关病毒介导1.3拷贝HBV基因组（1.3HBV,ayw亚型）在树鼩肝脏表达,建立HBV急性感染树鼩模型。方法通过大腿内侧静脉注射将携带有1.3 HBV的重组8型腺相关病毒（recombi-nant adeno-associated virus 8,rAAV8-1.3HBV）导入树鼩肝脏,通过ELISA检测树鼩血清中HBsAg、HBeAg、HBsAb、HBeAb、HBcAb,荧光定量PCR检测树鼩肝脏和血清中HBV DNA,全自动生化分析仪检测血清中ALT水平,并观察感染后肝脏的病变情况。结果 HBV感染主要血清标志物1～2周内均检测阳性;30 d后肝组织仍可检测到病毒抗原阳性细胞;55 d时肝组织HBV DNA拷贝数仍可达到104～105;树鼩血清中HBV DNA拷贝数持续一个月高于正常组;肝组织炎细胞略增多,血清ALT水平持续升高。结论 rAAV8所携带的HBV基因组高效专一导入树鼩肝细胞并复制表达,成功建立HBV急性感染树鼩模型,为进一步探索rAAV8树鼩慢性感染模型打下一定的基础。