高糖及TNF-α对RAW264.7细胞向破骨细胞分化的影响

【摘要】 目的 观察高糖及TNF-α的培养条件对RAW264.7细胞向破骨细胞诱导分化的影响。方法 在正常、高糖(30 mmol/L)及TNF-α(10 μmol/L)条件下培养RAW264.7细胞后,加入浓度为100 ng/mL的细胞核转录因子κB受体激活物的配体(receptor activator of NF-κB ligand, RANKL)为诱导剂,诱导RAW264.7向破骨细胞分化;诱导9天后,抗酒石酸酸性磷酸酶(TRAP)染色,比较各组TRAP+细胞数,RT-PCR及Western Blot检测各组破骨细胞标志基因CTR和MMP-9的表达。结果 不同的培养条件下RANKL均能诱导RAW264.7分化为成熟的破骨细胞,其中TNF-α环境中RAW264.7形成的TRAP+阳性细胞数、CTR和MMP-9的表达最高,而在高糖环境下最低。结论 TNF-α可以促进RAW264.7向破骨细胞分化,而高糖对这个过程可能是抑制作用,这一现象符合Ⅰ型和Ⅱ糖尿病患者骨质破坏的表现;高糖及TNF-α的培养条件下RANKL对RAW264.7的作用可模拟糖尿病足病变微环境中OC的诱导分化的过程。     

基于Kirkpatrick's四级培训评估模型腔镜腹股沟疝培训课程的效果分析

目的探讨腔镜腹股沟疝技术培训班的临床效果。 方法对参加中山大学附属第六医院胃肠、疝和腹壁外科2018年8月20至24日举办的腹股沟疝腔镜技术培训班的64名学员进行回顾性横断面研究。开展腔镜疝修补时间0~6年,平均手术时间（2.1±1.6）年。对学员培训前、培训结束时及培训3~6个月后前后进行问卷调查学员对培训课程的反馈,培训前及培训后3~6个月的手术表现,并基于Kirkpatrick's四级培训评估模型对结果进行分析。腹股沟疝腔镜技术培训班包括腔镜基础技能培训、腹股沟疝手术相关理论授课及手术演示。 结果有64.5%学员对培训课程表示满意。培训后3个月发出调查问卷64份,回收39份,回答率约39/64（60.9%）。回复的学员中,培训前23名有腹腔镜经腹腹膜前疝修补术（TAPP）经验,10名有腹腔镜完全腹膜外疝修补术（TEP）经验,6名是初学者。其中,有TAPP经验的学员中,每一步骤平均自信心得分从培训前的（3.23±0.94）分提高到（3.73±0.68）分,差异有统计学意义（P<0.05）;有TEP经验的10名学员中,每一步骤平均自信心得分从（2.99±0.84）分提高到（3.76±0.58）分,差异有统计学意义（P<0.05）。以初发单侧腹股沟疝为例,TAPP手术时间从培训前的97.6 min减少到培训后76.3 min,TEP手术时间从培训前102.0 min减少到培训后76.5 min（P<0.05）;TAPP手术出血量从培训前13.9 ml减少到培训后7.7 ml,TEP手术出血量从培训前17.0 ml减少到培训后8.4 ml,学员手术表现得到明显提升（P<0.05）。在6名初学者中,有4名已开展TAPP,2名仍未开展TAPP或TEP。 结论开展腹腔镜腹股沟疝手术前参加规范化的腔镜腹股沟疝技术培训课程非常重要,通过参加腹腔镜腹股沟疝技术培训班可以明显提升医师的手术水平。     

壳聚糖作为基因递送载体的MUC1基因疫苗治疗胰腺癌的实验研究

【摘要】 目的 研究以壳聚糖作为基因递送载体的MUC1基因疫苗诱导小鼠产生特异性体液及细胞免疫应答的作用,为壳聚糖联合MUC1基因疫苗用于胰腺癌的免疫治疗提供初步实验依据。方法 将30只小鼠平均分3组,分别接种壳聚糖-MUC1质粒、MUC1质粒及空质粒,通过ELISA法检测小鼠血清MUC1抗体生成情况,通过LDH释放法测定CTL对Capan-2细胞的杀伤活性。结果 接种壳聚糖-MUC1质粒后,小鼠血清抗MUC1抗体水平及CTL对Capan-2细胞的杀伤率均明显升高,与其它2组相比有显著性差异(P<0.05)。 结论 壳聚糖作为一种基因递送载体的MUC1基因疫苗能够有效诱导小鼠产生抗MUC1特异性抗体及产生杀伤MUC1+细胞的CTL,为壳聚糖联合MUC1基因疫苗用于胰腺癌的免疫治疗提供了初步的实验基础。     

止血带的使用方式对膝关节置换术失血量和手术时间的影响

目的探讨不同止血带使用方式对全膝关节置换术中、术后失血量的影响及意义.方法 2006年5月至2009年4月行单侧全膝关节置换术的膝关节骨关节炎患者88例,排除凝血机制异常者,患者根据止血带使用方式不同分为手术开始时开始使用组和安装假体时开始使用组,手术开始时开始使用组46例,安装假体时开始使用组42例,其他操作两组相同,记录手术时间、止血带使用时间,记录术中失血量、术后引流量,计算总失血量和隐形失血量.用两独立样本t检验比较两组间的差异.结果 手术开始时开始使用组：手术时间75～140 min,平均95.9 min;止血带使用时间54～105 min,平均65.2 min.安装假体时开始使用组：手术时间85～150 min,平均98.2 min;止血带使用时间18～30 min,平均24.1 min.术中失血量：手术开始时开始使用组132～640 ml,平均251.8 ml;安装假体时开始使用组320～965 ml,平均570.5 ml,术中失血量有统计学意义（P＜0.01）.术后引流血量：手术开始时开始使用组560～1310 ml,平均630.3 ml;安装假体时开始使用组470～1 190 ml,平均576.9 ml,两组间比较有统计学意义（P＜0.05）.总失血量：手术开始时开始使用组平均1 371.7 ml,安装假体时开始使用组平均1 419.3 ml,两组比较总失血量无统计学意义（P＞0.05）.隐形失血量：手术开始时开始使用组574.7 ml,安装假体时开始使用组437.3 ml,隐形失血量比较有统计学意义（P＜0.05）.手术开始时开始使用组发生肌肉疼痛4例,神经麻痹1例,对照组未发生使用止血带发生的并发症.结论 手术开始时开始使用组可减少术中失血,提供良好的手术视野利于手术操作,但术后失血较多,尤其是隐形失血量较多,对总失血量无明显影响.止血带使用时机对手术时间无影响.     

膝关节周围骨折脱位合并腘动脉损伤

[目的]回顾性分析膝关节周围骨折、膝关节脱位合并胭动脉损伤的早期诊断、治疗。[方法]自1998年3月～2005年5月，共收治膝关节周围骨折、脱位合并有胭动脉损伤患者71例，通过临床检查、血管造影或（和）Doppler检查，及早明确诊断，及时手术探查，修复损伤的动脉、采用简单、快捷、可靠的方法稳定骨折，术后抗凝、解痉、预防感染治疗。[结果]15例伴有严重的小腿软组织毁损初期截肢，其余56例手术成功修复。骨折采用带锁髓内钉固定、钢板螺钉固定或外支架固定。术后吻合口处发生血栓形成2例，经Doppler检查证实后急诊切除血栓部，取自体大隐静脉逆转移植吻合成功。2例发生骨髓炎（均为开放性骨折），经病灶清除庆大霉素骨水泥链珠植入等处理后愈合。2例发生创伤性膝关节炎。其余骨折愈合良好，肢体功能恢复较好。[结论]膝关节周围骨折、脱位应高度怀疑合并有胭血管损伤，通过仔细临床检查，必要时结合Doppler、血管造影尽早明确诊断，及早手术探查修复损伤的血管，稳定骨折。     

小干扰RNA逆转Wnt/β-catenin通路对肝癌耐药细胞株HepG2/ADM的影响研究

目的 了解沉默β-catenin基因对肝癌耐药细胞HepG2的影响。方法 实验分为5组：正常肝细胞（LO2）组、HepG2组、HepG2/ADM组、SiNC-HepG2/ADM阴性转染组和Siβ-catenin-HepG2/ADM转染组；细胞免疫荧光技术检测各组β-catenin的表达情况；设计并筛选出抑制效率最高的Siβ-catenin；Western-blot及RT-PCR技术检测各组β-catenin、P-gp、MRP1的mRNA和蛋白的表达水平；MTT法观察各组对阿霉素（ADM）、氟尿嘧啶（5-FU）、环磷腺苷（VCR）和奥沙利铂（OHP）的敏感性；流式细胞仪检测各组细胞凋亡。结果 50 mol/L的ctnnb1-001在HepG2/ADM中抑制效率最高：78.86%（P<0.05），选其为Si-β-catenin；细胞免疫荧光显示HepG2/ADM中β-catenin荧光最强，转染Si-β-catenin后荧光显著减弱；β-catenin、P-gp和MRP1 mRNA和蛋白在HepG2/ADM组表达较高，mRNA表达分别为：0.92±0.03、7.98±0.43和4.56±0.12（P<0.05），蛋白表达分别为：1.128±0.214、1.678±0.344和1.405±0.212（P<0.05）；转染Siβ-catenin至HepG2/ADM中，β-catenin、P-gp和MRP1在mRNA及蛋白水平均不同程度表达减少，mRNA表达分别为：0.47±0.03、0.66±0.054和0.74±0.03（P<0.05），蛋白表达分别为：0.787±0.032、0.797±0.055和1.390±0.050（P<0.05）；Siβ-catenin-HepG2/ADM转染组较HepG2/ADM组对ADM、5-FU、VCR和OHP的耐药系数（RI）分别为0.61、0.55、0.30、0.55，对化疗药物敏感性显著增强（P<0.05）；Siβ-catenin-HepG2/ADM转染组凋亡率（28.05±0.35）%，较其他组明显增加（P<0.05）。结论 Wnt/β-catenin通路在HepG2中异常激活，其中β-catenin可能正性调控肝癌耐药基因P-gp和MRP1，Si-β-catenin能一定程度阻断Wnt通路，并能一定程度逆转肝癌细胞株HepG2的耐药性和增强化疗敏感性，增加凋亡。     

增生性瘢痕组织中褪黑素受体的表达

目的 检测人增生性瘢痕组织和正常皮肤中褪黑素受体含量的差异,探讨其意义.方法 人增生性瘢痕组织和正常皮肤各10例,免疫组化检测组织中褪黑素受体GPR50的表达,荧光定量PCR(SYBR Green)检测组织中褪黑素受体MT1、MT2 mRNA含量,并将PCR阳性产物基因测序.结果 免疫组化显示褪黑素受体GPR50表达于细胞膜和细胞质,正常皮肤表皮基底层细胞、毛囊和汗腺均有褪黑素受体阳性表达,增生性瘢痕组织表皮、残留的毛囊和汗腺内也存在褪黑素受体表达;同时发现增生性瘢痕真皮成纤维细胞有广泛褪黑素受体阳性表达,而正常皮肤组织中则少见表达.荧光定量PCR显示增生性瘢痕组织中MT1、MT2 mRNA含量分别为(1.16584 4±0.21829)和(0.99550 ±0.14624)拷贝数/μl cDNA,正常皮肤中为(0.99081±0.26485)和(0.77083±0.15927)拷贝数/μl cDNA,增生性瘢痕组织中MT1、MT2 mRNA含量均高于正常皮肤(P相似文献   

西维来司钠对大鼠重症急性胰腺炎肺损伤的作用

目的 探讨西维来司钠(sivelestat sodium)对SD大鼠重症急性胰腺炎(severe acute pancreatitis,SAP)肺损伤的作用及其可能机制.方法 健康成年SD大鼠24只随机分成3组:对照组、模型组、药物组,每组8只.逆行胰胆管注射5%牛磺胆酸钠建立SAP模型.药物组在模型基础上给予西维来司钠干预,对照组和模型组给予等量生理盐水.建模成功6h后处死大鼠,检测各组大鼠血清淀粉酶(AMS)、血清中性粒细胞弹性蛋白酶(NE)活性以及肺脏组织湿干比重(W/D)、髓过氧化物酶(MPO)活性、核因子-κB(NF-κB)活性,光镜下观察肺脏病理改变,并进行病理学评分.结果 与对照组比较,模型组肺组织MPO、NF-κB含量显著升高(P<0.05),W/D比值显著升高(P<0.05),血清AMS、NE显著升高(P<0.05),光镜下可见肺组织出现明显的病理损伤(P<0.05).与模型组相比,药物组的血清NE以及肺组织MPO、W/D、NF-κB含量显著降低(P<0.05),但是AMS却没有显著降低(P>0.05).光镜下肺组织病理损害显著减轻(P<0.05),其损伤程度介于对照组和模型组之间.结论 早期应用西维来司钠对减轻大鼠急性胰腺炎肺损伤程度具有一定的作用.其作用机制可能是:西维来司钠通过抑制NF-κB活性来抑制炎症反应,从而起到减轻重症急性胰腺炎肺损伤的作用.     

基于SEER数据库分析手术在Ⅳ期胆囊癌治疗中的作用

目的 探究手术对Ⅳ期胆囊癌患者预后的影响。方法 从美国SEER数据库筛选2010年1月1日至2015年12月31日间诊断的4 627例Ⅳ期胆囊癌患者,根据是否进行手术治疗分为手术组（3 535例）和非手术组（1 092例）。倾向得分匹配后共得到1 344例,其中手术组和未手术组各672例。采用Cox风险回归模型分析Ⅳ期胆囊癌患者预后的独立风险因素,通过Kaplan-Meier法进行总体和各亚组生存分析和亚组分析,绘制生存曲线并计算中位生存时间（median survival time,MST）。结果 Cox回归模型提示原发灶部位、肿瘤分级、N分期、M分期、放疗、化疗、婚姻状况和手术（HR 0.590,95%CI 0.517~0.672,P<0.001）是Ⅳ期胆囊癌患者预后的独立危险因素。手术组中位（四分位间距）生存时间长于未手术组[7（14）个月 vs 3（8）个月,P<0.001],1年[31.6% vs 16.3%,P<0.001]、3年[9.4% vs 1.4%,P<0.001]生存率均高于未手术组,差异均有统计学意义。亚组分析显示,对于所有原发灶部位、M分期、放疗情况、化疗情况的亚组的Ⅳ期胆囊癌,手术均能改善患者的生存时间和生存结局。结论 手术可以改善Ⅳ期胆囊癌患者的预后情况,且是影响预后的独立危险因素之一。     

关节局部注射对全膝关节置换术后的镇痛效果

目的比较关节局部注射混合局麻药与自控硬膜外镇痛（PCEA）对全膝关节置换（TKA）术后的镇痛效果。方法由同一位医师主刀完成的TKA骨性关节炎（OA）患者58例（63膝）,根据镇痛方式分成两组：局部注射组23例（25膝）,术中关节周围软组织注射2.5g/L布比卡因40ml、酮咯酸30mg和肾上腺素0.25mg的混合液,术后拔除引流管时通过关节腔内放置的细导管注射5g/L布比卡因20ml和酮咯酸30mg。35例（38膝）TKA术后采用PCEA做为对照,比较术后两种镇痛方式的疼痛视觉模拟评分（VAS）、吗啡的使用量、关节被动活动度（PROM）、切口引流量和恶心、呕吐的发生率。结果两组术后的VAS和PROM差异无统计学意义（P〉0.05）,而局部注射组术后吗啡使用量、切口引流量和恶心、呕吐的发生率明显减少（P〈0.05）。结论关节周围软组织和关节内注射混合局麻药对TKA术后的镇痛效果确切,减少了术后出血量、吗啡的使用量和并发症,成本低,且操作简便、实用,是TKA镇痛的有效方法之一。     

Emergence delirium in adults in the post-anaesthesia care unit

Background. Emergence delirium in the post-anaesthesia careunit (PACU) is poorly understood. The goal of this prospectivestudy was to determine frequency and risk factors of emergencedelirium in adults after general anaesthesia. Methods. In this prospective study, 1359 consecutive patientswere included. Contextual risk factors and occurrence of deliriumaccording to the Riker sedation scale were documented. Groupswere defined for the analysis according to the occurrence ornot of agitation, then after exclusion of patients with preoperativeanxiety and neuroleptics, or both, and antidepressants or benzodiazepinestreatments. Results. Sixty-four (4.7%) patients developed delirium in thePACU, which can go from thrashing to violent behaviour and removalof tubes and catheters. Preoperative anxiety was not found tobe a risk factor. Preoperative medication by benzodiazepines(OR=1.910, 95% CI=1.101–3.315, P=0.021), breast surgery(OR=5.190, 95% CI=1.422–18.947, P=0.013), abdominal surgery(OR=3.206, 95% CI=1.262–8.143, P=0.014), and long durationof surgery increased the risk of delirium (OR=1.005, 95% CI=1.002–1.008,P=0.001), while a previous history of illness and long-termtreatment by antidepressants decreased the risk (respectively,OR=0.544, 95% CI=0.315–0.939, P=0.029 and OR=0.245, 95%CI=0.084–0.710, P=0.010). Conclusions. Preoperative benzodiazepines, breast and abdominalsurgery and surgery of long duration are risk factors for emergencedelirium.     

Fibrinogen-thrombin collagen patch reinforcement of highrisk colonic anastomoses in rats

AIM To evaluate the effectiveness of human fibrinogenthrombin collagen patch(TachoSil~?) in the reinforcement of high-risk colon anastomoses.METHODS A quasi-experimental study was conducted in Wistar rats(n = 56) that all underwent high-risk anastomoses(anastomosis with only two sutures) after colectomies. The rats were divided into two randomized groups: Control group(24 rats) and treatment group(24 rats). In the treatment group, high-risk anastomosis was reinforced with TachoSil~? (a piece of Tacho Sil? was applied over this high-risk anastomosis, covering the gap). Leak incidence, overall survival, intra-abdominal adhesions, and histologic healing of anastomoses were analyzed. Survivors were divided into two subgroups and euthanized at 15 and 30 d after intervention in order to analyze the adhesions and histologic changes. RESULTS Overall survival was 71.4% and 57.14% in the TachoSil~? group and control group, respectively(P = 0.29); four rats died from other causes and six rats in the treatment group and 10 in the control group experienced colonic leakage(P 0.05). The intra-abdominal adhesion score was similar in both groups, with no differences between subgroups. We found non-significant differences in the healing process according to the histologic score used in both groups(P = 0.066).CONCLUSION In our study, the use of TachoSil~? was associated with a non-statistically significant reduction in the rate of leakage in high-risk anastomoses. TachoSil~? has been shown to be a safe product because it does not affect the histologic healing process or increase intra-abdominal adhesions.     

Effects of intravenous anesthetics on bacterial elimination in human blood in vitro

Backround : Since anesthetics are widely used in critically ill patients, this study investigates anesthetic effects on neutrophil and monocyte function concerning bacterial elimination in human whole blood. Methods : The effects of thiopental (20 and 200 μg/ml), propofol (5 and 50 μg/ml), midazolam (0.15 and 1.5 μg/ml) and ketamine (3 and 30 μg/ml) on elimination of Escherichia (E.) coli from whole blood were investigated in vitro after incubation for 1 h in both clinical (1) (n=10) and 10-fold higher (h) (n=11) concentrations. These data were compared to neutrophil and monocyte phagocytosis (1; n=6) and burst activity (1; n=10, h; n=11), measured by flow cytometry. To enable quantification of the clearance process, a defined number of 10⁵ colony forming units of E. coli were added to the blood assays and bacterial growth was determined. Results : All anesthetics delayed bacterial clearance from the blood in the 10-fold concentration (P<0.05). Thiopental (1+h) and propofol (h) suppressed neutrophil (59±3% and 38±6%) and monocytic (45±6% and 30±11%) oxidative burst (P<0.01). Phagocytosis was reduced even after propofol (1) in polymorphonuclear leukocytes (PMN) (34±9%; P<0.05) and monocytes (35±11%). Ketamine (h) prolonged bacterial elimination (P<0.01), which did correlate with inhibition of monocytic phagocytosis, by 26±14%. Midazolam application (h) resulted in an inhibition of PMN-respiratory burst by 19±6% (P<0.05) and impaired bacterial clearance (P<0.05). Conclusion : Thiopental, propofol, midazolam and ketamine affect E. coli clearance and neutrophil and monocyte oxidative burst and phagocytosis in vitro only in high concentrations, while thiopental inhibited monocytic burst and propofol impaired PMN phagocytosis even in clinically used concentrations. These data suggest that i.v. anesthetics in concentrations recommended for general anesthesia seem to have minor influence on the investigated host defense mechanisms.     

不同尿钙水平的Gitelman综合征患者临床特点比较

目的观察不同尿钙水平Gitelman综合征(GS)患者的临床特点,探讨尿钙在GS疾病临床分型中的价值。方法收集2016—2018年来自中国国家罕见病注册系统(NRSC)、在北京协和医院行SLC12A3基因检测诊断为GS患者的临床资料,分析其尿钙特点,比较不同尿钙水平患者的临床和实验室检查指标。氢氯噻嗪试验按照标准操作流程进行,测定患者基线和用药后3 h内氯离子排泄分数改变量的最大值(ΔFECl)。结果共有83例GS患者被纳入研究,其中低尿钙患者53例(63.86%)。低尿钙组尿钙/肌酐比明显低于非低尿钙组[(0.085±0.058)mmol/mmol比(0.471±0.284)mmol/mmol,t=7.349,P<0.001]。两组患者在年龄、性别、估算肾小球滤过率、血压、血尿电解质水平、代谢性碱中毒方面差异均无统计学意义。低尿钙组患者乏力(χ2=4.595,P=0.032)及多尿(χ2=5.778,P=0.016)发生比例低于非低尿钙组,两组患者在其他临床症状方面差异无统计学意义。低尿钙和非低尿钙组各有16例患者行氢氯噻嗪试验,中位ΔFECl结果分别为0.539%(0.430%,1.283%)和0.829%(0.119%,1.298%),均提示对氢氯噻嗪无反应,组间差异无统计学意义(U=130.000,P=0.956)。结论GS患者中低尿钙比例为63.86%,尿钙水平与疾病临床表型、NCC功能损伤严重程度之间均无明确相关性。     

Neurolytic thoracic paravertebral block in cancer pain

Background : Paravertebral block has successfully been used in the treatment of acute and chronic pain. The duration of paravertebral block could theoretically be prolonged by using neurolytic agents.
Methods : We retrospectively analyzed the results of neurolytic paravertebral blocks performed in 7 patients suffering from intense cancer-related thoracic pain. Thirty-seven spinal nerve roots were blocked during 20 visits. Nerve roots were identified by eliciting paresthesia radiating to the painful area. Each root was blocked separately. After test block using 0.5% bupivacaine, the paravertebral blocks were performed with 1–4 ml of 7% phenol in aqua.
Results : No technical failures or complications were recorded in the patient files. Pain relief lasted over 2 months after 4 visits (20%), from 1 week to 1 month after 5 visits (25%), and less than 1 week after a single visit (5%). After 9 visits (45%), the results were poor with no significant pain relief.
Conclusion : Neurolytic paravertebral block with phenol doses used in our patients appears to have only limited use. Some patients with pain restricted to a small number of thoracic segments may benefit from its use. Because of complication risks, this technique should be limited to intractable pain in cancer patients with poor prognosis.     

Fluid absorption in endoscopic surgery

Fluid absorption is an unpredictable complication of endoscopicsurgery. Absorption of small amounts of fluid (1–2 litre)occurs in 5–10% of patients undergoing transurethral prostaticresection and results in an easily overlooked mild transurethralresection (TUR) syndrome. Large-scale fluid absorption is rarebut leads to symptoms severe enough to require intensive care.Pathophysiological mechanisms consist of pharmacological effectsof the irrigant solutes, the volume effect of the irrigant water,dilutional hyponatraemia and brain oedema. Other less widelyknown factors include absolute losses of sodium by urinary excretionand morphological changes in the heart muscle, both of whichpromote a hypokinetic circulation. Studies in animals, volunteersand patients show that irrigation with glycine solution shouldbe avoided. Preventive measures, such as low-pressure irrigation,might reduce the extent of fluid absorption but does not eliminatethis complication. Monitoring the extent of absorption duringsurgery allows control of the fluid balance in the individualpatient, but such monitoring is not used widely. However, theanaesthetist must be aware of the symptoms and be able to diagnosethis complication. Treatment should be based on administrationof hypertonic saline rather than on diuretics. New techniques,such as bipolar resectoscopes and vaporizing instead of resectingtissue, result in a continuous change of the prerequisites forfluid absorption and its consequences.     

维生素D受体在糖尿病肾病足细胞损伤及蛋白尿缓解中的作用

目的探讨维生素D受体(VDR)在糖尿病肾病(DKD)足细胞中的表达水平及在足细胞损伤及蛋白尿缓解中的作用。方法(1)本研究纳入了65例诊断患有2型糖尿病(伴或不伴蛋白尿)的患者,并纳入了25例年龄和性别相匹配的健康体检者为对照组。根据白蛋白/肌酐(ACR)的尿排泄比例对2型糖尿病患者进行分组,分别为无蛋白尿(ACR<30 mg/g,n=24)、微量白蛋白尿(ACR 30~300 mg/g,n=18)和临床蛋白尿(ACR>300 mg/g,n=23)。另选择25例经肾活检确诊的DKD患者作为DKD组。正常肾脏组织标本均取自泌尿外科同一时期肾脏肿瘤切除患者10例。将各组检测指标进行对比,同时采用实时定量PCR、ELISA法和免疫组化法检测VDR在各组患者的血液、尿液样本和肾脏组织中的表达情况,以及使用Pearson相关分析分析VDR与尿蛋白的相关性。(2)在2型糖尿病肾病小鼠模型中对上述结果进行验证,将遗传背景均为C57BLKs/J的雄性db/db小鼠及同窝出生的db/m小鼠,随机分为正常对照组(A组)、DKD对照组(B组)、DKD二甲基亚砜处理组(C组)、DKD帕立骨化醇(VDR激动剂)处理组(D组),C、D组连续腹腔注射处理8周,对照组不做任何处理。小鼠10周龄时开始连续干预8周,在小鼠22周龄(开始干预后12周)检测各组小鼠体重、血、尿生化指标对比;Western印迹法检测β⁃catenin、VDR的变化;免疫荧光观察足细胞标志蛋白podocin及足细胞损伤蛋白α⁃SMA的表达变化。结果(1)与正常健康对照组相比,无蛋白尿组、微量白蛋白尿组和临床蛋白尿组的糖尿病患者血浆中VDR的mRNA和蛋白水平均较低(均P<0.05);与无蛋白尿组的糖尿病患者相比,微量白蛋白尿组和临床蛋白尿组的糖尿病患者血浆中VDR的mRNA和蛋白水平均较低(均P<0.05)。(2)与正常健康对照组相比,无蛋白尿糖尿病组和DKD组患者血浆中VDR的mRNA和蛋白水平均较低(均P<0.05);与无蛋白尿糖尿病组患者相比,DKD组患者血浆中VDR的mRNA和蛋白水平亦较低(均P<0.05)。(3)免疫组化结果显示,DKD组肾组织中VDR的表达明显少于正常对照组。(4)DKD患者血浆中VDR mRNA相对水平与ACR呈负相关(r=-0.342,P<0.05)。(5)各组尿液上清液中VDR的水平与血浆中的水平呈相反趋势。(6)Western印迹结果显示,B组、C组肾小球足细胞β⁃catenin蛋白表达高于D组(均P<0.05),VDR蛋白的表达低于D组(均P<0.05);免疫荧光结果显示,B组、C组肾小球足细胞podocin的表达低于D组(均P<0.05),α⁃SMA的表达高于D组(均P<0.05)。结论VDR高表达缓解DKD足细胞损伤及蛋白尿。     

Clinical significance of in vitro donor-specific hyporesponsiveness in renal allograft recipients as demonstrated by the MLR

A longitudinal study was carried out on 19 recipients of cadaveric renal allografts, monitoring their anti-donor and anti-third party responses in the mixed lymphocyte reaction (MLR) at the time of transplantation and at 3, 6, and 12 months post-transplant. Two patterns of responses were identified: in the first (n=11), patients showed, or later developed, donor-specific hyporesponsivenes, and in the second (n=8), patients had persistent antidonor and anti-third party responses. After 1 year, the serum creatinine, number of episodes of acute rejection and biopsy findings were compared in both groups. In the first group, the mean serum creatinine was 136.4 mmol/l, the total number of acute rejection episodes was three and in nine of the ten available biopsies, there were minimal cellular infiltrates and normal appearance of the glomeruli, tubules and blood vessels. In the second group, the mean serum creatinine was 163 mmol/l, the total number of acute rejection episodes was 12 and in five of the seven biopsies available, evidence of ongoing rejection was obtained. The difference in mean serum creatinine was not statistically significant (P>0.05), but the difference in the numbers of acute rejection episodes was (P>0.05). It is concluded that in some renal allograft recipients, a state of donor-specific hyporesponsiveness develops, and this state may be associated with better graft out-come at 1 year. These data may be useful in selecting patients for reduced immunosuppressive therapy.