AAV-mediated Tyrosinase Gene Transfer Restores Melanogenesis and Retinal Function in a Model of Oculo-cutaneous Albinism Type I (OCA1)

Oculo-cutaneous albinism type 1 (OCA1) is characterized by congenital hypopigmentation and is due to mutations in the TYROSINASE gene (TYR). In this study, we have characterized the morpho-functional consequences of the lack of tyrosinase activity in the spontaneous null mouse model of OCA1 (Tyr^c-2j). Here, we show that adult Tyr^c-2j mice have several retinal functional anomalies associated with photoreceptor loss. To test whether these anomalies are reversible upon TYR complementation, we performed intraocular administration of an adeno-associated virus (AAV)–based vector, encoding the human TYR gene, in adult Tyr^c-2j mice. This resulted in melanosome biogenesis and ex novo synthesis of melanin in both neuroectodermally derived retinal pigment epithelium (RPE) and in neural crest–derived choroid and iris melanocytes. Ocular melanin accumulation prevented progressive photoreceptor degeneration and resulted in restoration of retinal function. Our results reveal novel properties of pigment cells and show that the developmental anomalies of albino mice are associated with defects occurring in postnatal life, adding novel insights on OCA1 disease pathogenesis. In addition, we provide proof-of-principle of an effective gene-based strategy relevant for future application in albino patients.     

Inhibition of Dexamethasone-induced Fatty Liver Development by Reducing miR-17-5p Levels

Steatosis is a pivotal event in the initiation and progression of nonalcoholic fatty liver disease (NAFLD) which can be driven by peroxisome proliferator-activated receptor-α (PPAR-α) dysregulation. Through examining the effect of PPAR-α on fatty liver development, we found that PPAR-α is a target of miR-17-5p. Transgenic mice expressing miR-17 developed fatty liver and produced higher levels of triglyceride and cholesterol but lower levels of PPAR-α. Ectopic expression of miR-17 enhanced cellular steatosis. Gain-of-function and loss-of-function experiments confirmed PPAR-α as a target of miR-17-5p. On the other hand, PPAR-α bound to the promoter of miR-17 and promoted its expression. The feed-back loop between miR-17-5p and PPAR-α played a key role in the induction of steatosis and fatty liver development. Mice with high levels of miR-17-5p were sensitive to Dexamethasone-induced fatty liver formation. Inhibition of miR-17-5p suppressed this process and enhanced PPAR-α expression in mice treated with Dexamethasone. Clofibrate, Ciprofibrate, and WY-14643: three agents used for treatment of metabolic disorders, were found to promote PPAR-α expression while decreasing miR-17-5p levels and inhibiting steatosis. Our studies show that miR-17-5p inhibitor and agents used in metabolic disorders may be applied in combination with Dexamethasone in the treatment of anti-inflammation, immunosuppression, and cancer patients.     

慢性乙型肝炎肝纤维化患者血清Copeptin和PCT表达水平及其临床价值研究

目的 探讨慢性乙型肝炎肝纤维化患者血清和肽素（Copeptin）和降钙素原（procalcitonin,PCT）表达水平,并分析其在肝纤维化诊断中的价值。方法 共纳入2019年2月～2021年12月在广西医科大学第四附属医院就诊的195例慢性肝病单感染HBV患者作为HBV组,并选择同一期间在该院进行健康体检的志愿者作为对照组。其中HBV组中慢性乙型肝炎（CHB）患者117例（CHB组）,CHB相关肝硬化（LC）患者78例（LC组）,对所有参与患者进行临床指标分析,采用ELISA方法检测患者血清Copeptin和PCT表达水平。METAVIR评分系统评估CHB组中接受肝活检的患者的肝纤维化程度;Child-Pugh分级评估LC患者的肝功能障碍程度。比较Copeptin,PCT表达水平与肝纤维化和肝功能的关系。ROC曲线评估肝纤维化诊断生物标志物。结果 HBV组血清Copeptin(2.39±0.73 ng/ml vs 1.19±0.39ng/ml)和PCT(2.11±0.69 ng/ml vs 0.97±0.31 ng/ml)水平明显高于对照组（t=11.912,12.108,均P &l...     

慢性荨麻疹患者外周血IL-17和IL-23的表达及临床意义

目的分析慢性荨麻疹患者外周血IL-17,IL-23的表达及临床意义。方法分析2009年1月～2013年1月在荆门市康复医院接受治疗的慢性荨麻疹患者的临床资料,并列为观察组。另纳入同期健康体检者作为对照组。结果该研究共纳入研究对象91例,其中观察组患者61例,对照组30例。观察组患者外周血IL-17及IL-23水平显著高于对照组,相比较差异具有统计学意义(P＜0.001)。Pearson相关性检验显示:观察组患者外周血IL-23与IL-17水平呈现显著正相关,差异具有统计学意义(r=0.504,P＜0.001)。经4周治疗后,观察组患者外周血IL-17水平显著降低,与治疗前比较差异具有统计学意义(65.31±18.75pg/ml vs 121.53±31.19 pg/ml,t=10.710,P＜0.001)。以治疗后IL-17水平的中位数(66.79 pg/ml)为界,将患者分为A(IL-17＜66.79 pg/ml),B(IL-17≥66.79 pg/ml)两组。Kaplan-Meier分析显示:治疗后1年,A组患者累积复发率显著低于B组,相比较差异具有统计学意义［13.79%,(4/29) vs 37.93%,(11/29)Log-rank χ2=4.344,P=0.037］。结论慢性荨麻疹患者外周血IL-17,IL-23表达升高,且可以一定程度判断预后。     

Development of a Liver-specific Tet-On Inducible System for AAV Vectors and Its Application in the Treatment of Liver Cancer

Recombinant adeno-associated virus (rAAV) are effective gene delivery vehicles that can mediate long-lasting transgene expression. However, tight regulation and tissue-specific transgene expression is required for certain therapeutic applications. For regulatable expression from the liver we designed a hepatospecific bidirectional and autoregulatory tetracycline (Tet)-On system (Tet_bidirAlb) flanked by AAV inverted terminal repeats (ITRs). We characterized the inducible hepatospecific system in comparison with an inducible ubiquitous expression system (Tet_bidirCMV) using luciferase (luc). Although the ubiquitous system led to luc expression throughout the mouse, luc expression derived from the hepatospecific system was restricted to the liver. Interestingly, the induction rate of the Tet_bidirAlb was significantly higher than that of Tet_bidirCMV, whereas leakage of Tet_bidirAlb was significantly lower. To evaluate the therapeutic potential of this vector, an AAV-Tet_bidir-Alb-expressing interleukin-12 (IL-12) was tested in a murine model for hepatic colorectal metastasis. The vector induced dose-dependent levels of IL-12 and interferon-γ (IFN-γ), showing no significant toxicity. AAV-Tet_bidir-Alb-IL-12 was highly efficient in preventing establishment of metastasis in the liver and induced an efficient T-cell memory response to tumor cells. Thus, we have demonstrated persistent, and inducible in vivo expression of a gene from a liver-specific Tet-On inducible construct delivered via an AAV vector and proved to be an efficient tool for treating liver cancer.     

慢加急性肝衰竭患者血清Clusterin 和CCR5 表达水平及其对预后预测价值研究

目的 探讨血清丛生蛋白(Clusterin）及趋化因子受体5（CCR5）在慢加急性肝衰竭（acute-on-chronic liverfailure，ACLF）患者中的表达以及两者联合检测对ACLF 患者预后的预测价值。方法 选取2018 年1 月～ 2020 年12月唐山市传染病医院收治的84 例ACLF 患者作为观察组，同期选择在该院体检的80 例健康体检者作为对照组。采用酶联免疫吸附试验(ELISA）法测定患者血清中Clusterin 和CCR5 水平；根据患者预后28 天情况，将其分为存活组（n=48）和死亡组（n=36）。采用受试者工作特征曲线（receiver operating characteristic curve，ROC）分析血清Clusterin 与CCR5联合检测对ACLF患者预后的预测价值；Spearman 相关性分析血清Clusterin 和CCR5水平与终末期肝病模型(modelfor end-stage liver disease，MELD) 评分、慢性肝衰竭- 序贯器官衰竭评分(chronic liver failure-sequential organ failureassessment，CLIF-SOFA) 的相关性；采用多因素Logistic 回归分析ACLF 患者预后的影响因素。结果 与对照组相比，观察组Clusterin（87.37±9.99μg/ml vs 104.85±15.14μg/ml） 及CCR5（11.55±2.86μg/ml vs 15.68±3.01μg/ml） 水平降低，差异具有统计学意义（t=8.767，9.010，均P<0.05）。与存活组相比，死亡组Clusterin（77.40±9.26μg/ml vs94.85±10.54μg/ml）及CCR5（8.58±1.98μg/ml vs 13.78±3.52μg/ml）水平降低，差异具有统计学意义（t=7.904，7.962，均P<0.05）。ROC 曲线显示，血清Clusterin 与CCR5 联合预测的曲线下面积（area under curve，AUC）（0.927）最大，其敏感度和特异度分别为88.90% 和83.30%。经Spearman 相关性分析Clusterin 与MELD，CLIF-SOFA 评分呈负相关（r=-0.524，-0.457，均P<0.05），CCR5 与MELD，CLIF-SOFA 评分呈负相关（r=-0.611，-0.358，均P<0.05）。多因素Logistic 回归分析显示，血清Clusterin，CCR5 及IL-6 为ACLF 患者预后不良的影响因素（均P ＜ 0.05）。结论 Clusterin 及CCR5 在ACLF 患者血清中表达下调，且联合检测二者在预测ACLF 患者短期预后方面具有良好的参考价值。     

超声诊断脂肪肝与血脂水平及肝功能的关系

目的探讨超声定量分析脂肪肝与血脂、肝功能之间关系。方法对在永新县人民医院经超声诊断为脂肪肝的患者331例(脂肪肝组,其中轻度脂肪肝102例,中度脂肪肝136例,重度脂肪肝93例)及健康体检经超声诊断无脂肪肝的正常者200例(对照组)采集空腹静脉血3 mL,采用罗氏7600生化全自动分析仪检测测定总胆固醇(TC)、三酰甘油(TG)、丙氨酸转氨酶(LT)及氨酰转肽酶(GGT)水平。结果脂肪肝组的TC、TG、GGT、ALT水平及异常检出率明显高于对照组(P<0.05或P<0.01);重度脂肪肝患者TC、TG、GGT及ALT异常率均明显高于轻、中度脂肪肝患者,中度脂肪肝患者高于轻度脂肪肝患者(均P<0.05)。结论超声对脂肪肝分型准确,结合血脂、肝功能的异常情况对脂肪肝诊断及预后具有指导价值。更多还原     

酒精性脂肪肝和肝硬化患者各种血清酶的检测及其临床评价

目的 探讨酒精性脂肪肝血清特异性的鉴别指标，并用其来判断病程，指导临床诊断和治疗。方法 对76例酒精性脂肪肝、62例酒精性肝硬化以及70例非酒精性脂肪肝患者的血清酶和血脂进行测定及分析。结果 与非酒精性脂肪肝相比，酒精性脂肪肝的谷草转氨酶（AST）和γ-谷氨酰转肽酶（GGT）的异常率以及谷草转氨酶／谷丙转氨酶（AST／ALT〉1）的构成比的增高，差异均具有统计学意义；与酒精性脂肪肝相比，酒精性肝硬化的AST／ALT比值，AST和GGT的水平都有明显增高。结论 AST、AST／ALT（〉1）、GGT等指标对鉴别酒精性脂肪肝以及判断其病程，具有一定的临床意义。     

Outcome of Orthotopic Liver Transplantation in the Aetiological and Clinical Variants of Acute Liver Failure

Thirty-three patients with acute liver failure underwent orthotopicliver transplantation, including 16 with fulminant hepatic failure,15 with late–onset hepatic failure and two with severeacute liver failure (coagulopathy without encephalopathy). Twenty–three(70 per cent) survived to leave hospital and 21 of these arecurrently alive and well. Outcome correlated with the serumbilirubin level before transplantation (p<0.05) but no correlationwas found with the variant of acute liver failure, grade ofencephalopathy, cerebral oedema, serum creatinine, white cellcount, prothrombin time or platelet count at the time of transplantation.Severe coagulation factor deficiencies did not constitute aclinical problem. One patient developed a neurological deficitsecondary to cerebral oedema, but otherwise the morbidity reflectedthat observed in the general population after transplantation.Careful monitoring of intracranial pressure and surveillance(with early aggressive therapy) for bacterial and fungal infectionsis very important in achieving a successful outcome after transplantation.     

HBeAg定量与慢性乙肝患者血清FasFasL表达水平及肝损伤的预示价值研究

目的探析慢性乙肝患者肝损伤程度与HBe Ag指标以及血清Fas Fas L表达水平之间的关系。方法以我院收治的32例慢性乙肝患者作为研究观察组展开分析,与健康人群进行对比,比较HBe Ag定量检测结果以及Fas、Fas L表达水平。结果健康人群的HBe Ag定量检测结果为0.56±0.05PEIU/m L,处于正常范围内,中度肝损害以及重度肝损害患者的HBe Ag定量检测结果分别为0.82±0.06PEIU/m L和0.99±0.08PEIU/m L,两两之间的比较有统计学意义(P0.05)。健康人群的Fas检测结果为1.0±0.62%,低于中度以及重度肝损害患者水平(P0.05);Fas L检测结果为17.1±3.4%,高于中度以及重度肝损害患者水平(P0.05),比较有统计学意义。结论慢性乙肝患者的HBe Ag指标以及FAS、Fas L水平与健康人群相比均会发生较大改变,并且其变化情况与其肝损害程度之间存在密切关系,可以此预示患者肝损害的严重程度,对病情进行评估。     

人工肝血浆置换治疗慢性重型肝炎疗效观察与护理

目的观察人工肝血浆置换治疗慢性重型肝炎的临床疗效及总结护理体会。方法92例慢性重型肝炎患者随机分为对照组(n=52)及治疗组(n=40)。对照组给予常规内科治疗,在此基础上对治疗组进行血浆置换治疗。观察治疗前后患者临床症状的变化,测定血液肝肾功、凝血酶原活动度及电解质变化。对比两组存活率。总结护理注意要点。结果经治疗后患者临床症状显著改善(P<0.05);患者胆红素明显降低(P<0.05);白蛋白、胆碱酯酶、凝血酶原活动度明显增加(P<0.05);电解质紊乱明显改善(P<0.05);而对肾功影响不明显。未出现严重并发症。治疗组存活率明显高于对照组(P<0.05);其中早、中期患者存活率高于对照组(P<0.05),而晚期存活率比较无显著性差异。结论在常规内科治疗基础上加用人工肝血浆置换是治疗慢性重型肝炎的一种安全有效的肝脏替代治疗方法,且在病程早期进行效果较好。     

IL-17在乳腺癌中的表达及意义

目的：探讨白细胞介素17（IL-17）在乳腺癌患者血清和T细胞中的表达及与乳腺癌功能指标的相关性。方法采用流式细胞仪分析乳腺癌患者外周血 CD3＋ CD4＋ IL-17＋ T 细胞百分数;ELISA 法测定血清 IL-17水平;化学发光免疫分析法检测 CA153和 CA125的水平,并分析 IL-17水平与各指标的相关性。结果乳腺癌患者外周血中 CD3＋ CD4＋ IL-17＋ T 细胞百分数与健康对照组差异有统计学意义（P ＜0．05）;血清 IL-17水平与健康对照组差异有统计学意义（P ＜0．05）;血清 IL-17水平与CD3＋ CD4＋ IL-17＋ T 细胞百分数及 CA153水平均呈正相关;血清 IL-17水平与 CA125水平无显著相关性。结论IL-17水平与乳腺癌有关。     

解毒化瘀Ⅱ方对急性肝衰竭大鼠肝细胞Bcl-2及细胞色素C表达的影响

目的通过观察解毒化瘀Ⅱ方对急性肝衰竭大鼠肝线粒体Bcl-2、细胞色素C表达的影响，探讨其抗肝衰竭的作用机制。方法以硫代乙酰胺（TAA）皮下注射复制急性肝衰竭大鼠模型，SPF级Wistar大鼠84只，随机分为空白组，模型组，解毒化瘀Ⅱ方低、中、高剂量组，安宫牛黄丸组，乳果糖组；造模前3d开始灌胃给药，共给药5．5d；采用Western Blot法检测各组大鼠肝细胞线粒体Bcl-2表达及细胞色素C在肝线粒体和细胞质中的表达情况。结果与空白组比较，模型组大鼠肝线粒体内Bcl-2表达强度显著降低，细胞色素C在细胞质中的表达增强，在线粒体中的表达减弱；解毒化瘀Ⅱ方能提高急性肝衰竭大鼠肝线粒体内Bcl-2的表达，抑制细胞色素C从肝线粒体释放到细胞质中，并呈现剂量-效应关系。结论解毒化瘀Ⅱ方对暴发性肝衰竭大鼠肝细胞的保护作用机制有可能是通过增强肝细胞线粒体Bcl-2表达，抑制肝线粒体膜通透性转换孔开放，阻止细胞色素C释放到细胞质，抑制肝细胞凋亡的发生。     

乙型肝炎病毒相关慢加急性肝衰竭患者血清miR-328-3p 表达水平及其与疾病严重程度及预后的相关性研究

目的 探究微小核糖核酸（microRNA,miR）-328-3p表达水平与乙型肝炎病毒相关慢加急性肝衰竭（hepatitis B virus-related acute-on-chronic liver failure,HBV-ACLF）严重程度及预后的关系。方法 选取 2018年 12月～2021年 12月陕西省人民医院收治的 HBV-ACLF患者 120例（HBV-ACLF组）、慢性乙型肝炎（chronic hepatitis B,CHB）患者 120例（CHB组）,另选取同期健康体检者 120例为健康组。比较三组一般资料及血清 miR-328-3p表达水平,比较不同分期 HBV-ACLF患者血清 miR-328-3p表达水平;分析 HBV-ACLF患者血清 miR-328-3p表达水平与终末期肝病模型（model for end-stage liver disease,MELD）评分的相关性;比较不同预后 HBV-ACLF患者基线资料及血清 miR-328-3p表达水平;评估血清 miR-328-3p和 MELD评分对 HBV-ACLF患者预后的预测价值; COX回归分析 HBV-ACLF患者预后的影响因素。结果 健康组、 CHB组和 HBV-ACLF组血清 miR-328-3p（1.03±0.34,1.85±0.62和 2.76±0.92）表达水平比较,差异有统计学意义（F=200.241,P＜ 0.05）;早期、中期、晚期组 HBV-ACLF患者血清 miR-328-3p（2.30±0.58,2.88±0.72和 3.63±0.91）表达水平逐渐升高,差异有统计学意义（F=28.023,P＜ 0.05）;HBV-ACLF患者血清 miR-328-3p表达水平与 MELD评分、血清天门冬氨酸氨基转移酶（aspartate aminotranferase,AST）、总胆红素（total serum bilirubin,TBIL）和丙氨酸氨基转移酶（alanine aminotransferase,ALT）呈正相关（r=0.598,0.418,0.375,0.339,均 P＜ 0.05）;死亡组 HBV-ACLF患者 MELD评分（28.62±5.72）和血清 miR-328-3p（3.34±0.67）表达水平均高于生存组（21.14±4.23,2.32±0.46）,差异有统计学意义（t=8.237,9.878,均 P＜ 0.05）;血清 miR-328-3p和 MELD评分预测 HBV-ACLF患者预后的曲线下面积（area under curve,AUC）分别为 0.861（95%CI:0.791～0.932）,0.853（95%CI: 0.781～0.925）,且二者联合预测 HBV-ACLF患者预后的 AUC为 0.945（95%CI:0.902～0.989）,高于 miR-328-3p和 MELD评分单独预测（Z=1.991,2.137,均 P＜ 0.05）,且其敏感度和特异度分别为 96.2%,86.8%;回归分析显示, MELD评分 [OR(95%CI)=2.532(1.635～3.920)]和 miR-328-3p[OR(95%CI)=2.928(1.836～4.668)]均是 HBV-ACLF患者死亡的危险因素（均 P＜ 0.05）。结论 HBV-ACLF患者血清 miR-328-3p表达水平较高,与 HBV-ACLF疾病严重程度和预后显著相关,且血清 miR-328-3p与 MELD评分联合能较为有效地预测 HBV-ACLF患者预后,有一定临床参考价值。     

Adoptive Transfer of Ex Vivo HO-1 Modified Bone Marrow�Cderived Macrophages Prevents Liver Ischemia and Reperfusion Injury

Macrophages play a critical role in the pathophysiology of liver ischemia and reperfusion (IR) injury (IRI). However, macrophages that overexpress antioxidant heme oxygenase-1 (HO-1) may exert profound anti-inflammatory functions. This study explores the cytoprotective effects and mechanisms of ex vivo modified HO-1-expressing bone marrow–derived macrophages (BMDMs) in well-defined mouse model of liver warm ischemia followed by reperfusion. Adoptive transfer of Ad-HO-1-transduced macrophages prevented IR-induced hepatocellular damage, as evidenced by depressed serum glutamic-oxaloacetic transaminase (sGOT) levels and preserved liver histology (Suzuki scores), compared to Ad-β-gal controls. This beneficial effect was reversed following concomitant treatment with HO-1 siRNA. Ad-HO-1-transfected macrophages significantly decreased local neutrophil accumulation, TNF-α/IL-1β, IFN-γ/E-selectin, and IP-10/MCP-1 expression, caspase-3 activity, and the frequency of apoptotic cells, as compared with controls. Unlike in controls, Ad-HO-1-transfected macrophages markedly increased hepatic expression of antiapoptotic Bcl-2/Bcl-xl and depressed caspase-3 activity. These results establish the precedent for a novel investigative tool and provide the rationale for a clinically attractive new strategy in which native macrophages can be transfected ex vivo with cytoprotective HO-1 and then infused, if needed, to prospective recipients exposed to hepatic IR–mediated local inflammation, such as during liver transplantation, resection, or trauma.