尿巨噬细胞移动抑制因子与IgA肾病的相关性

目的：探讨尿巨噬细胞移动抑制因子（MIF）水平与IgA肾病（IgAN）患者病情进展之间的关系。方法：用酶免疫方法（EIA）测定35例IgAN患者尿MIF浓度,并与肾脏病理分级、24h尿蛋白（TUP）、内生肌酐清除率（Ccr）、血尿等进行分组分析,以10例健康体检者作对照组。结果：IgAN患者尿MIF浓度较健康人明显增高,差异有统计学意义（P〈0．01）,且随着病理分级增加而逐渐增高,各组间差异有统计学意义（均P〈0．01）,尿MIF水平与24h蛋白尿水平显著相关（r=0．787,P〈0．01）,与Ccr、血尿无显著相关;随着病情控制,治疗后尿MIF较治疗前显著下降,差异有统计学意义（P〈0．01）。结论：IgAN尿MIF浓度明显增高,与病情严重程度相关,对于患者病情的判断有一定价值。     

Coadministration of Adenoviral Vascular Endothelial Growth Factor and Angiopoietin-1 Enhances Vascularization and Reduces Ventricular Remodeling in the Infarcted Myocardium of Type 1 Diabetic Rats

OBJECTIVE

Hyperglycemia impairs angiogenesis in response to ischemia, leading to ventricular remodeling. Although the effects of overexpressing angiogenic growth factors have been studied in inducing angiogenesis, the formation of functional vessels remains a challenge. The present study evaluates the reversal of diabetes-mediated impairment of angiogenesis in the infarcted diabetic rat myocardium by proangiogenic gene therapy.

RESEARCH DESIGN AND METHODS

Ad.VEGF and Ad.Ang1 were intramyocardially administered in combination immediately after myocardial infarction to nondiabetic and diabetic rats. Ad.LacZ was similarly administered to the respective control groups. The hearts were excised for molecular and immunohistochemical analysis at predetermined time points. The myocardial function was measured by echocardiography 30 days after the intervention.

RESULTS

We observed reduced fibrosis and increased capillary/arteriolar density along with reduced ventricular remodeling, as assessed by echocardiography in the treated diabetic animals compared with the nontreated diabetic controls. We also observed increased phosphorylated mitogen-activated protein kinase–activated protein kinase-2, 2 days after the treatment and increased expression of vascular endothelial growth factor (VEGF), Flk-1, angiopoietin-1 (Ang-1), Tie-2, and survivin, 4 days after treatment in the diabetic animals. Gel shift analysis revealed that the combination gene therapy stimulated the DNA binding activity of nuclear factor-κB in the diabetic animals.

CONCLUSIONS

Our preclinical data demonstrate the efficacy of coadministration of adenoviral VEGF and Ang-1 in increasing angiogenesis and reducing ventricular remodeling in the infarcted diabetic myocardium. These unique results call for the initiation of a clinical trial to assess the efficacy of this therapeutic strategy in the treatment of diabetes-related human heart failure.Diabetic individuals who develop an ischemic heart disease (IHD) sustain an unfavorable prognosis for survival compared with other IHD subjects without diabetes (1). This condition may be attributed to impaired coronary collateral vessel development and reduced myocardial vascular perfusion in response to ischemia, leading to profound ventricular remodeling and subsequent heart failure (2). Various studies have linked diabetes-mediated impaired myocardial angiogenesis to alterations in the delicate balance of angiogenic growth factors and cytokines regulating vascular stability (2–4) and compromised signal transduction (4). Several studies have reported the possible role of decreased vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) in the pathogenesis of diabetes-mediated impairment of angiogenesis in the myocardium (5–7).There have been several attempts at preclinical and clinical levels to induce angiogenesis by overexpressing angiogenic factors in the peri-infarct zone after myocardial infarction (MI). Most of the studies have approached this issue using a single gene as the therapeutic agent. Delivery of vectors encoding VEGF₁₆₅ (VEGF) and VEGF-2 was shown to improve collateral vascular perfusion and nourish the oxygen-depleted myocardium, thereby reducing angina and improving heart function in human clinical trials (8–10). However, investigations into the long-term effects of sustained expression of VEGF in mice models revealed deleterious effects due to the formation of leaky immature vessels/hemangiomas and subsequent death of the experimental animal (11,12). Furthermore, transgenic mice overexpressing VEGF revealed lengthy and leaky dermal vessels with evident inflammation (13,14).On the other hand, the Ang-1 system is known to play a critical role in vascular maturation and stabilization, thereby supporting VEGF-induced neovascularization in a complementary manner (6,14,15). Recently, Ang-1 gene therapy has been shown to support the maturation of the immature vasculature in db/db mice (16).In the recent past, work has been done to elucidate the synergistic effect of coadministration of VEGF and Ang-1 in ischemic rat myocardium (17–19). Zhou et al. (18) reported that combined gene therapy using VEGF and Ang-1 significantly reduced myocardial infarct size through the induction of the phosphatidylinositol 3-kinase and Bcl-2 survival pathways and nuclear factor-κB (NFκB) activation.The prospect of a gene therapy using a combination of VEGF and Ang-1 encoding vectors to activate the angiogenic signaling cascade has not yet been explored in the diabetic ischemic myocardium. Diabetes reflects a far more challenging condition, where the VEGF and Ang-1 system is significantly downregulated, hampering the ability of the myocardium to respond to an ischemic stress (2,6), and where the usual revascularization techniques such as coronary artery bypass graft and percutaneous transluminal coronary angioplasty tend to fail, thereby leaving many of the diabetic IHD subjects with no option. Therefore, in this study we aimed at using a combination gene therapy approach involving in vivo adenoviral gene delivery of VEGF and Ang-1, to enhance neoangiogenesis by repairing the impaired angiogenic signaling cascade and thereby reducing ventricular remodeling in streptozotocin (STZ)-induced type 1 diabetic rats. Our findings emphasize the efficacy of coadministration of adenoviral vectors encoding VEGF and Ang-1 in inducing and stabilizing the process of angiogenesis that is impaired in the diabetic myocardium and in reducing ventricular remodeling in the infarcted myocardium in a diabetic milieu, thereby supporting the development of a combination gene therapy for therapeutic myocardial angiogenesis.     

腰痛患者下腰椎MRI上Modic改变与高信号区的发生情况及意义

目的:探讨腰痛患者下腰椎MRI上Modic改变与腰椎间盘局限性高信号区(high-intensity zone,HIZ)的发生情况及意义。方法:对511例腰痛患者(男263例,女248例;年龄20~70岁,平均48岁)腰椎MRI上L4/5和L5/S1节段的Modic改变和HIZ进行评估,统计两者及两者共存于同一节段的发生率。将有Modic改变和/或HIZ的椎间盘分为Modic组、Modic-HIZ组、HIZ组,比较3组的年龄、椎间盘高度、椎间盘退变程度、腰痛VAS和ODI评分。结果:511例患者中,190例(37.18%)209个节段有Modic改变,127例(24.85%)142个椎间盘有HIZ,18例(3.52%)18个节段出现Modic改变和HIZ共存的现象。HIZ组、Modic-HIZ组和Modic组分别为89例(124个节段)、18例(18个节段)、152例(191个节段),患者平均年龄分别为46.0±11.0岁、49.2±9.2岁和53.5±10.6岁,仅HIZ组和Modic组差异有统计学意义(P<0.05);椎间盘平均高度分别为9.93±2.46mm、8.73±2.45mm和7.57±2.21mm,组间两两比较差异有统计学意义(P<0.05);3组椎间盘退变分级均≥Ⅲ级,其中Ⅳ级+Ⅴ级退变率分别为48.39%、72.22%和75.92%,仅HIZ组与Modic组、Modic-HIZ组差异有统计学意义(P<0.05);腰痛VAS分别为8.39±0.32分、8.45±0.30分、8.61±0.54分,ODI评分分别为38.22±4.23分、38.45±4.16分、39.18±3.53分,3组间无统计学差异(P>0.05)。结论:腰痛患者下腰椎Modic改变和HIZ的发生率较高,但两者共存于同一节段的发生率低,当两者共存于同一节段时腰痛并不会明显加重。     

Ⅰ型心肾综合征伴利尿剂抵抗患者炎性细胞因子的变化

目的:探讨炎症反应在Ⅰ型心肾综合征患者并发利尿剂抵抗中的影响。方法:收集2012年8月~2014年8月温州市人民医院收治的Ⅰ型心肾综合征患者,根据是否发生利尿剂抵抗,分为心肾综合征组和利尿剂抵抗组,统一检测两组患者血清TNF-α、IL-1β、IL-6、CRP等炎症因子水平,同时同正常健康人群进行对比,了解3组人群血清中炎性细胞因子的不同。结果:58例Ⅰ型心肾综合征患者,分为心肾综合征组(30例)和利尿剂抵抗组(28例),同时随机抽选健康体检人群(27例),统计分析显示3组间患者炎性细胞因子比较差异有统计学意义(P0.05)。结论:炎症反应不仅参与了Ⅰ型心肾综合征患者的病理生理过程,而且在其严重并发症利尿剂抵抗的发生发展过程中也发挥了重要作用。     

Changes in Mac-1 and CD14 expression on monocytes and serum soluble CD14 level during push/pull hemodiafiltration

BACKGROUND/AIM: Employment of treated dialysate as replacement fluid raises concerns about exposure of patients to pyrogenic substances. This study was undertaken to evaluate the safety of treated dialysate as the replacement fluid for push/pull hemodiafiltration. METHODS: In the present study, changes in the expressions of Mac-1 and CD14 on monocytes, which are upregulated by monocyte activation, were analyzed by flow cytometry, and the serum level of sCD14 which elevates by monocyte activation was measured by enzyme-linked immunosorbent assay (ELISA) during treatment in 7 patients on hemodialysis with regenerated cellulose (RC) membrane, polysulfone (PS) membranes and by push/pull hemodiafiltration (HDF) with PS membranes in a cross-over fashion. RESULTS: During hemodialysis with RC, hemodialysis with PS or push/pull hemodiafiltration with PS, both Mac-1 and CD14 expressions on monocytes significantly increased by passing through the artificial kidneys, and, accordingly, the respective values downstream of the artificial kidneys were significantly higher than the predialysis values, even when the lipopolysaccharide level in dialysate was not detectable by Limulus assay. There was no significant variation in serum sCD14 levels during any of the hemodialysis with RC, hemodialysis with PS or push/pull hemodiafiltration. However, during hemodialysis with PS or push/pull hemodiafiltration with PS, changes in Mac-1 and CD14 expression on monocytes were significantly smaller than those during hemodialysis with RC. CONCLUSION: Monocytes are activated to a greater extent during hemodialysis with RC membranes than during push/pull HDF with PS membranes. We consider that push/pull HDF may be safer than hemodialysis with RC membrane and that it is as safe as hemodialysis with the PS membrane in terms of monocyte activation, when pyrogen-free dialysate is employed.     

Changes in Gene Expression of Selected Genes in Patients with Type 1 Diabetes and Pancreas Transplant in Peripheral Blood

BackgroundDiabetes is an autoimmunologic disease that may have a different background. The aim of our study was to show that type 1 diabetes is accompanied by changes in gene expression in peripheral blood mononuclear cells. We analyzed the genes characteristic of pancreatic islet cells and genes playing a big part in autoimmune diseases and cancer.DesignThe study included 21 patients and was performed to examine the expression of 9 genes. The patients were divided into 3 research groups: people with type 1 diabetes, people with diabetes after pancreas transplant, and a control group of healthy patients. To assess the level of expression, RNA material was obtained from peripheral blood collected from individuals qualified for the study.ResultsThe results of the study showed many interesting changes in the expression level of the analyzed genes. It was demonstrated that CASR gene expression was significantly higher in transplant patients than in diabetic patients. Differences in the level of activity are also noted in genes that take part in autoimmune diseases.ProposalProfiling gene expression in peripheral blood samples may be a useful and noninvasive diagnostic tool that allows early detection of changes leading to the onset or resumption of diabetes.     

人源Dickkopf1原核表达产物对体外培养的黑素细胞的抑制作用

目的:观察人源Dickkopf1原核表达产物(DKK1蛋白)对体外培养的黑素细胞的抑制作用。方法:采用MTT法测定细胞增殖情况;光学显微镜观察细胞形态学变化;氢氧化钠裂解法测定黑素合成;多巴氧化法测定酪氨酸酶活性。结果:DKK1蛋白对黑素细胞的增殖有抑制作用,能使细胞数明显减少(P<0.05);光学显微镜观察到DKK1蛋白作用的黑素细胞胞体较大,形态略呈多角形,树突较粗短;并能使黑素合成显著下降(P<0.05);使酪氨酸酶活性显著减弱(P<0.05)。结论:DKK1蛋白能抑制黑素细胞增殖、黑素合成、酪氨酸酶活性,为DKK1蛋白应用于治疗色素增多性疾病奠定实验基础。     

Changes on distribution of CD4+/CD45RA- and CD8+/CD11- cells in tumor-infiltrating lymphocytes of renal cell carcinoma associated with tumor progression.

To study the distribution of subsets of T cells in renal cell carcinoma, peripheral blood lymphocytes (PBL) and tumor-infiltrating lymphocytes (TIL) were analyzed in 43 untreated patients using two-color flow cytometry. An increase in the relative number of CD4+/CD45RA-, CD8+/CD11- and HLA-DR+/CD3+ cells was shown in TIL when compared with PBL. When the influence of various tumor factors on subsets of TIL was examined, a decrease in CD4, CD4+/CD45RA- and CD16+/CD57- cells and an increase in CD8+ and CD8+/CD11- cells was observed along with the aggravation of tumor stage and grade. In TIL of stage III/IV and grade III/IV disease, most patients showed an increase in CD8+/CD11- associated with a decrease in CD4+/CD45RA- cells, or the reverse, resulting in changes of the CD4+/CD45RA- to CD8+/CD11- ratio. The prognosis for these patients was poor, suggesting that changes in the ratio were a sign of the impairment of local immune status associated with disease progression.     

腺病毒介导LRIG1过表达联合顺铂对膀胱癌细胞株EJ的抑制作用

目的 观察腺病毒介导的LRIG1过表达是否能增强顺铂对膀胱癌细胞株EJ的损伤作用.方法 构建重组腺病毒载体包装pLRIG1-GFP质粒,采用逆转录-聚合酶链反应(RT-PCR)及Western blot法鉴定其是否能在膀胱癌细胞株EJ中上调LRIG1表达,并下调表皮生长因子(EGFR)的表达水平.通过采用单细胞凝胶电泳实验、流式细胞技术、免疫细胞化学染色及Matrigel侵袭实验,对比对照组、顺铂单药组、顺铂联合腺病毒载体组和顺铂联合腺病毒介导的LRIG1过表达组间细胞DNA损伤,细胞凋亡、增殖及侵袭能力.结果与对照组比较,顺铂在联合腺病毒介导的LRIG1过表达后,能下调细胞EGFR表达水平,同时显著增强肿瘤细胞DNA损伤程度(OTM值,对照组:2.54±0.54;CDDP组:4.57±0.79;CDDP/Ad-GFP组:5.38±1.16;CDDP/Ad-LRIG1组:9.45±2.64);引起细胞周期抑制[S期抑制,对照组:(40.82±2.11)%;CDDP组:(37.31±1.12)%,CDDP/Ad-GFP组:(37.57±2.52)%,CDDP/Ad-LRIG1组:(55.04±4.28)%];诱导细胞凋亡[细胞凋亡率,对照组:(2.63±2.49)%,CDDP组(3.49±1.94)%,CDDP/Ad-GFP组:(3.96±4.68)%,CDDP/Ad-LRIG1组:(12.56±0.77)%];抑制细胞增殖[细胞计数,对照组:(371.33±16.17)个;CDDP组:(224.67±88.06)个;CDDP/Ad-GFP组:(176.33±69.79)个;CDDP/Ad-LRIG1组:(138.33±8.39)个]并逆转细胞侵袭性[细胞侵袭数量,对照组:(259.40±9.21)个;CDDP组:(175.00±25.78)个;CDDP/Ad-GFP组:(157.20±22.79)个;CDDP/Ad-LRIG1组:(114.20±25.11)个].结论 腺病毒介导LRIG1过表达能增强顺铂对膀胱肿瘤细胞株EJ的损伤作用.     

前列腺素E1抑制血吸虫病家兔肝脏星状细胞活化研究

目的　探讨血吸虫病家兔肝纤维化形成过程中 ,静脉应用前列腺素 (PG)E1对肝脏星状细胞 (HSCs)活化和胶原含量的影响。方法　血吸虫尾蚴皮肤敷贴法感染家兔 ,构建肝脏纤维化模型 ,其中 7只家兔在感染后 60d开始给予PGE1(2 .5 μg/kg .d-1)。至 12 0d透射电镜比较HSCs超微结构变化 ,免疫组织化学检测肝窦周围平滑肌肌动蛋白 (α SMA)表达 ,苦味酸天狼星红染色测定Ⅰ、Ⅲ型胶原纤维含量。结果　血吸虫病家兔肝脏纤维化形成过程中HSCs活化增加 ,收缩特性相关的α SMA表达增强 ,胶原纤维含量增高。外源性PGE1可以抑制HSCs活化 ;显著降低肝窦周围α SMA表达 (P <0 .0 1) ;肝窦周围胶原纤维含量显著降低 ,模型组和干预组Ⅰ、Ⅲ型胶原显色面积与单个偏振光显微镜视野面积比例 (% )分别为 3 7.2 5± 9.71和 13 .3 8± 4.2 4(P <0 .0 1) ;9.66±3 .5 2和 6.2 3± 1.81(P <0 .0 5 )。结论　HSCs活化是血吸虫病家兔肝纤维化形成的重要环节。PGE1可以有效地抑制血吸虫家兔肝纤维化形成 ,这种作用至少部分地是通过抑制HSCs活化实现的。     

胰岛素泵治疗小儿1型糖尿病的临床研究

目的探讨胰岛素泵治疗小儿1型糖尿病的疗效。方法将1型糖尿病患儿随机分成胰岛素泵治疗组和每日多次皮下注射胰岛素组,比较两组的疗效。结果胰岛素泵治疗组餐后血糖、胰岛素用量、治疗时间、低血糖发生次数与每日多次皮下注射胰岛素组相比差异有统计学意义（P〈0.05）。结论胰岛素泵治疗可更快更好地控制高血糖,减少低血糖的发生。     

Influence of Bushen Huoxue Recipe on Senescence-associated Factors HIRA and ASF1a Expression in Articular Cartilage of Rats with Knee Osteoarthritis

[目的]探讨补肾活血方对膝骨关节炎大鼠关节软骨衰老信号分子HIRA、ASF1a表达的影响.[方法]选用雌性SD大鼠,随机分为假手术组、模型组和中药组;采用切除双侧卵巢并切断右侧膝交叉韧带法复制骨关节炎动物模型,术后第4周开始灌服补肾活血中药,模型组、假手术组灌服等量的生理盐水,分别于术后8、12、16周取材,采用骨关节炎软骨病理变化评价系统(OARSI)评价关节软骨的病变,运用western blot法检测H1RA、ASF1a表达水平.[结果]术后8、12、16周,模型组OARSI评分较假手术组显著升高,差异有显著性意义(P＜0.01),中药组OARSI评分较模型组显著降低,差异有显著意义(P＜0.05);HIRA、ASF1a在模型组中表达明显增强,补肾活血中药能下调HIRA、ASF1a的表达.[结论]补肾活血方可通过下调HIRA、ASF1a表达保护关节软骨.     

前列腺素E1对血吸虫病家兔门静脉平滑肌细胞增殖的影响

目的 观察血吸虫病家兔门静脉高压症形成过程中,静脉应用前列腺素E1对门静脉血管平滑肌细胞(VSMC)增殖的影响及可能机制.方法 血吸虫尾蚴皮肤敷贴法感染家兔,构建门静脉高压模型,其中7只在感染尾蚴后60 d开始耳缘静脉给予PGEI(2.5μg/kg体重).至150 d透射电镜比较门静脉VSMC超微结构变化,Western blot和免疫组织化学分别检测门静脉血小板衍生生长因子B(PDGF-B)表达水平和表达部位.结果 血吸虫病家兔门静脉中膜增厚,VSMC活化增殖,PDGF-B表达增强并集中在中膜平滑肌层.外源性PGE1可以抑制VSMC增殖;显著降低门静脉中膜厚度[(76.35±11.41)μm比(118.29±36.52)μm,P《0.01]和PDGF-B[(3.63±1.39)比(6.64±1.95),P《0.05]表达.结论 VSMC增殖是血吸虫病家兔门静脉高压血管病变的重要环节.PGE1可以有效地抑制血吸虫家兔门静脉VSMC增殖,抑制PDGF-B表达可能是其中机制之一.     

HIV一1感染者外周血I型干扰素产生细胞水平变化的研究

目的探讨我国HIV感染者外周血Ⅰ型干扰素产生细胞（IPC）水平及其与疾病进展的关系。方法应用流式细胞术以全血染色、全白细胞设门对92例HIV-1感染者和59例健康对照进行IPC水平的测定,并分析外周血IPC变化与CD4＋T细胞计数和HIV血浆病毒载量的关系。结果 HIV感染者外周血CD4＋T细胞及IPC绝对计数的均数分别为342.000个/μl和3.431个/μl,显著低于正常对照（965.000个/μl和5.995个/μl,P均〈0.001）;HIV感染者IPC细胞数量与CD4＋T细胞计数成正比（r=0.430,P〈0.001）,与HIV血浆病毒载量成反比（r=-0.483,P〈0.001）;CD4＋T淋巴细胞计数〈200个/μl的感染者IPC水平明显低于CD4＋T淋巴细胞计数〉200个/μl者（P〈0.005）。HIV慢性感染者的IPC水平显著高于艾滋病患者（P〈0.001）,新发感染者的IPC水平（5.080个/μl）明显低于正常对照（P=0.038）,但新发感染与慢性进展者的IPC水平间的差异无统计学意义。结论 HIV感染可显著降低机体的IPC水平,IPC水平变化与HIV疾病进展密切相关。     

WTp53基因联合TK/GCV、CD/5-FC系统抑制结肠癌肝转移的实验研究

目的研究WTp53基因联合TK／GCV、CD／5-FC系统对结肠癌肝转移的抑制作用。方法32只裸鼠随机分为4组，每组8只。脾内注射法建立结肠癌肝转移动物模型。第1组：脾内注射SW480细胞（对照组）；第2组：脾内注射SW480／p53细胞；第3组：脾内注射SW480／TK-CD细胞，腹腔注射GCV、5-FC；第4组：脾内注射SW480／p53与SW480／TK-CD等比例混合细胞，腹腔注射GCV、5-FC。观察各组裸小鼠的肝转移率、肝转移瘤数目、肿瘤细胞凋亡指数（AI）、生存期等指标。结果各治疗组裸鼠肝转移的发生率下降，平均肝转移瘤数减少，其动物的生存期延长，肝转移瘤内的癌细胞凋亡率增高。联合基因治疗组效果最好，且WTp53与TK／GCV、CD／5-FC有交互协同作用。结论WTp53基因与TK／GCV、CD／5-FC系统联合应用具有协同效应，可有效地抑制结肠癌肝转移的形成。     

慢性肾小球肾炎患者红细胞补体受体Ⅰ型分子及其基因多态性变化

目的：探讨慢性肾小球肾炎（CGN）患者红细胞补体受体Ⅰ型分子（CR1）数量表达和黏附活性及其基因多态性的变化及临床意义。方法：采用聚合酶链反应（PCR）和HindⅢ酶切技术测定红细胞CR1分子基因型，采用酶联法定量测定红细胞CR1分子的数量，以红细胞天然免疫黏附试验测定红细胞CRl分子黏附活性。结果：50例CGN患者红细胞CR1基因多态性分布（HH：56．0％，HL：32．0％，LL：12．0％）与50名健康人（HH：68．0％，HL：26．0％，LL：6．0％）比较无统计学差异；CGN患者红细胞CR1数量表达和黏附活性明显低于健康人（P〈0．01）。并且CGN患者红细胞CR1高、中、低表达基因型的数量表达和黏附活性均低于健康人高、中、低表达基因型（P〈0．05）。结论：CGN患者红细胞CR1数量表达和黏附活性的变化主要是后天因素引起，红细胞CR1数量表达和黏附活性与CGN的发生和发展有着非常密切的关系。