Although serum creatinine and/or serum cystatin C have been widely used in clinical practice to estimate glomerular filtration rate (GFR), they cannot replace nuclide labeled GFR measurement when measuring independent renal function and reflecting the true level of GFR. Nuclide labeled GFR methods include nuclide renal dynamic imaging method and nuclide plasma clearance method. These two methods have their own advantages and disadvantages, thus, we expect to objectively and comprehensively introduce their characteristics for rational use by clinicians.

肾脏衰老有哪些形态、结构和功能的变化 衰老是一个自发的渐变过程,使人体产生一系列变化,其中肾脏是易于发生衰老的器官之一.肾脏衰老时外观上会有如下变化:体积缩小,质量减轻,肾皮质萎缩;肾脏表面粗糙化;更多单纯性肾囊肿形成.微观结构上也有改变,包括:动脉硬化,肾小球动脉弯曲缩短,管壁内膜增厚,肾血流量减少;肾小球硬化,肾小管萎缩,肾单位减少;肾脏衰老时"老废物质"蓄积导致肾小管间质纤维化和硬化.

Objective:To study gene therapy for Wilson′s disease (WD) with adeno-associated virus (AAV)-clustered regularly interspaced short palindromic repeats (CRISPR) system in vitro. Methods:Three small-guide RNAs (sgRNAs) were designed and AAV particles containing CRISPR-sgRNA1, 2, 3 or W/O homologous template (HT) were transduced into Toxic milk (TX) mouse hepatocytes, respectively. Gene editing efficiency and HT repair efficiency were evaluated by sequencing, and repair template-specific primers were used for PCR to verify HT incorporation.Results:Sanger sequencing data showed that the editing efficiency of sgRNA1 reached (20.2±2.3)%, which was significantly higher than others ( P<0.05). When AAV-HT treatment was combined, the corrected ATP7B gene band could be amplified by PCR, and NGS showed that repair efficiency was (7.9±2.7)%. Conclusion:AAV transduction can achieve pretty high editing efficiency. We selected sgRNA1 and verified its editing efficiency and the repair efficiency of HT, providing a theoretical and experimental basis for further in vivo experiments.

Objective:To study gene therapy for Wilson’s disease with Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system in vitro. Methods:Three crRNAs, W/O homologous template, the single-stranded oligo DNA (ssODN), were transfected into Toxic milk (TX) mouse hepatocytes using CRISPR/Cas9 ribonucleoprotein (RNP) method, respectively. Gene editing efficiency and ssODN repair efficiency were verified by sequencing, and repair template-specific primers were used for PCR to verify ssODN incorporation. Data are expressed as mean±standard deviation of independent samples. Comparisons between two groups were performed by unpaired Student’s t-test. Results:Sanger sequencing showed that the editing efficiency of crRNA001 reached (57.8±5.1)%. The corrected ATP7B gene band can be amplified by PCR using specific primers, and NGS sequencing results showed that the ATP7B gene repair efficiency reached (7.45±2.54)%.Conclusion:Pretty high editing efficiency can be achieved by RNP transfection. We selected crRNA001 and verified its editing efficiency and the repair efficiency of ssODN, providing a theoretical and experimental basis for further in vivo experiments.

目的:探讨内质网应激对肝星状细胞分泌肝细胞生长因子(hypatocyte growth factor,HGF)的影响及其可能机制.方法:通过在培养的大鼠肝星状细胞T6中分别加入5.0μg/mL衣霉素或0.2μmol/L毒胡萝卜素建立肝星状细胞内质网应激模型,4.0 mmol/L 4-苯基丁酸钠(4-phenylbutyrate,4-PBA)和200.0 μmol/L salubrinal作为内质网应激抑制剂对T6细胞进行预处理,重组慢病毒LV-eIf2α-shRNA-GFP敲减T6细胞中的eIf2α mRNA表达,并提取mRNA和全蛋白进行RT-PCR和Western blot实验检测HGF、分子伴侣重链结合蛋白(glucose-regulated protein 78,GRP78)、真核翻译起始因子2α (eukaryotic translation initiation factor 2α,eIf2α)、磷酸化eIf2α、激活转录因子4(activating transcription factor 4,ATF4)以及凋亡信号分子C/EBP同源蛋白(C/EBP homologous protein,CHOP).结果:衣霉素、毒胡萝卜素可诱导T6细胞GRP78升高,激活内质网应激状态的同时抑制HGF表达,4-PBA和salubrinal可阻止内质网应激引起的HGF降低,但对ATF4和CHOP的表达作用不同.慢病毒转染T6降低细胞eIf2α表达的同时成比例降低HGF表达.结论:ERS激活后可通过影响eIf2α表达从而抑制HGF表达.

目的:检测M2-型丙酮酸激酶(pyruvate kinase M2,PKM2)在胆管癌(cholangiocarcinoma,CCA)组织中的表达情况,探讨PKM2下调对胆管癌细胞迁移、侵袭及增殖的影响.方法:实时定量逆转录聚合酶链反应(qRT-PCR)和免疫组织化学(immunohistochemistry,IHC)染色分别检测胆管癌及对应癌旁组织标本中PKM2的mRNA和蛋白表达水平.利用慢病毒表达载体系统在胆管癌细胞株HuCCT-1 、HCCC-9810中下调PKM2,分别用划痕实验、Transwell细胞侵袭实验和CCK-8比色法检测细胞迁移、侵袭及增殖能力.结果:胆管癌组织中PKM2的mRNA和蛋白表达水平明显高于对应癌旁组织.经qRT-PCR和Western blot方法证实稳定转染PKM2 shRNA的胆管癌细胞中PKM2的mRNA和蛋白水平较对照组均明显下降(P＜0.05),与空载对照组(PKM2-NC)和正常对照组(PKM2)相比,实验组细胞(PKM2-shRNA)的迁移、侵袭及增殖能力明显减弱,差异有统计学意义(P＜0.05).结论:胆管癌组织中PKM2表达明显高于癌旁组织,PKM2 shRNA能有效地降低胆管癌细胞中PKM2基因的表达,PKM2基因沉默可以抑制HuCCT-1、HCCC-9810细胞的迁移、侵袭及增殖.

目的:研究胰岛素样生长因子2 mRNA结合蛋白3(IMP3)与胰岛素样生长因子2(IGF2)在乙肝相关性肝癌中的表达,并探讨二者的相关性及与乙肝相关性肝癌临床病理特征的关系.方法:采用免疫组织化学法,检测82例乙肝相关性肝癌组织及对应癌旁组织中IMP3及IGF2的表达;采用实时定量聚合酶链式反应法检测32例冰冻肝癌组织中IMP3及IGF2 mRNA含量;采用蛋白质印迹法检测4例IMP3强阳性染色及4例IMP3阴性染色对应的新鲜冰冻肝癌组织中IMP3及IGF2蛋白含量.结果:IMP3在62.2％肝癌组织中表达,在癌旁组织中不表达.IGF2在59.8％肝癌组织及14.6％癌旁组织中表达.同时,IMP3与IGF2在乙肝相关性肝癌组织中表达密切相关(rn=0.335,P=0.002).另外,IMP3高表达与Edmondson-Steiner 3～4级(P=0.001)、TNM 3～4期(P=0.001)、α-甲胎蛋白(AFP)＞ 200 ng/ml (P=0.012)及伴有门脉癌栓的肿瘤(P=0.018)均密切相关;与IMP3表达相似,IGF2高表达亦与Edmondson-Steiner 3～4级(P=0.001)、TNM 3～4期(P＜ 0.001)及伴有门脉癌栓的肿瘤(P＜0.001)密切相关.结论:IMP3与IGF2在乙肝相关性肝癌组织中表达密切相关,同时,IMP3与IGF2蛋白分子的检测有助于判断肿瘤恶性分化程度及进展.

Objective:To observe the homing capacity of bone mesenchymal stem cells(BMSCs) to rat with acute hepatic failure(AHF). Methods:BMSCs were isolated from rat bone marrow with the method of density gradient centrifugation and stick-to-wall screening,and the surface markers were identified with flow cytometry after amplification in vitro. BMSCs were labeled with Bromodeoxyuridine(BrdU). AHF animals were induced by D-galactosamine(D-gal). Transplantation of labeled BMSCs(1～1.5×106) were performed on the AHF rats(group A)/normal rats(group C),another AHF group(group B) were injected with sodium chloride without BMSCs as a control to group A. Then the mortality of the rats and liver function recovery status were observed. Two weeks after cells transplantation, BMSCs traced with BrdU were detected in the livers by immunohistochemistry. Results:The mortality and status of liver function were not significantly different between A and B group. Both A and C group could find BrdU+ cells, and the number and the disposition of BrdU+ cells in A group was much more than C group's,showing significant difference. Conclusion:BMSCs have capacity of homing to the liver of rat in normal or in injury . But the number of BMSCs homing to liver might be related to the extent of liver injury.

Objective To express and purify PTD-p53 fusion protein and investigate its transduction efficiency.Methods The gene encoding wide-type p53 was isolated,using RT-PCR from A549 cell line and cloned into pTATHA and pET32a prokaryotic expression vectors.Recombinant plasmids were E.coli BL21(DE3)LysS,then the transformed cells were induced with IPTG.The expression and purification of the PTD-p53 and p53 were analyzed by SDS-PAGE.BALB/c mice were immunized with purified p53 protein.The serum was isolated and the antibody specific to p53 was measured by ELISA.The transduction efficiency of PTD-p53 was detected using indirect immunoflure scence assay.Results Prokaryotic expression vectors of PTD-p53 and p53 were constructed correctly.PTD-p53 fusion protein and p53 protein were successfully expressed and purified.p53 specific mouse antiserum was obtained.IFA result indicated that PTD-p53 fusion protein transduced into HepG2 cells efficiently.Conclusion The obtained Tat-p53 fusion protein provides a theretical foundation for the basic research on PTD-p53 treating hepatocellular carcinoma.

目的:建立同时表达4-1BBL、B7.1及B7.2三个共刺激分子基因小鼠原发性肝细胞癌(hepatocellular carcinoma,HCC)细胞系.方法:将B7.1和B7.2全长cDNA的质粒酶切,构建pcDNA3.1-B7.1-IRES-B7.2重组子,酶切法鉴定.用阳离子脂质体(Lipofectamine Reagent)将重组子转染H22,经均霉素(Hygromycin,300μg/ml)筛选,阳性克隆命名为H22-CD80/CD86+细胞.逆转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)检测目的基因在H22-CD80/CD86+变异株的表达.采用同样方法将pCI-neo-4-1BBL质粒转入H22-CD80/CD86+细胞,G418筛选,阳性克隆命名为H22-CD80/CD86/CD137L+细胞.流式细胞仪检测三种基因在细胞克隆中的表达.结果:pcDNA3.1-B7.1-IRES-B7.2重组子经酶切鉴定,同时获得B7.1(862bp)和B7.2(984bp)目的基因片段和5.6kbp线性化pcDNA3.1载体片段;重组子测序结果与Genebank中B7.1和B7.2序列相符,证实构建成功.RT-PCR及FCM检测结果显示B7.1、B7.2及4-1BBL基因分别在H22-CD80/CD86+细胞及H22-CD80/CD86/CD137L+细胞中获得稳定、高效联合表达.结论:pcDNA3.1-B7.1-IRES-B7.2重组子构建正确,H22-CD80/CD86/CD137L+变异株可同时稳定表达B7.1、B7.2和4-1BBL三个共刺激分子基因.

PurposeTo clone and express recombinant human tumstatin in E.coli.MethodsThe human tumstain gene was isolated with RT-PCR from human renal tissue total RNA and cloned into PGEM-T vector for sequencing.Then,the tumstatin gene was subcloned into the pQE-30 expression vector and was transformed into E.coli.The recombinant tumstatin was purified with Ni-NTA His-Bind resin.ResultsThe DNA sequence of cloned tumstatin gene was similar to that reported previously.The construct was expressed in E.coli with high level,accounting for 10% of the total bacterial proteins.This gene product was found with a molecular mass of 28 000 on SDS-PAGE and Western blot,which was identical to that of native tumstatin.The purity of recombinant tumstatin purified by affinity chromatography reached more than 92%.ConclusionThe cloning, expression and purification of human tumstatin has laid a foundation for its antiangiogenesis theraphy of tumor.

ObjectiveTo observe the killing effect on hepatocellular carcinoma(HCC) cell lines by growth response-1(Egr-1) promoter activated herps simplex virus thymidine kinase (tk). MethodsPlasmid pET was constructed by fusing of Egr-1 promoter to the upstream of tk gene and transfect human hepatocellular carcinoma cell lines SMMC-7721(SMMC/ET) with lipofectamine as a delivery system. The cloned cells, after selected with G418, and exposure to γ-radiation by a 60Co source, were added with prodrug GCV. The viability of cell lines was observed. ResultsAfter irradiation, transfected cell lines (0.07±0.03) was killed by prodrug GCV at higher percentage compared with control group(0.88±0.12)(P0.001).The viability was 8% of control group.ConclusionsRadio-sensitized suicide gene activated by γ-ray significantly enhanced killing on HCC.