nephrin、血管内皮生长因子及其受体表达与先兆子痫大鼠蛋白尿的关系

Objective To investigate the association of the expressions of glomerular nephrin, vascular endothelial growth factor (VEGF) and its receptor (VEGFR) with proteinuria in preeclampsia rats. Methods A rat model of preeclampsia was developed by inhibitor of nitric oxide synthase (L-NAME). The systolic blood pressure (SBP) and 24 h urine protein were compared among the normal female group (n=6), the normal pregnant group (n=8), nonpregnant control group (n=6) and preeclampsia group(n=8). The kidney biopsies of each group were observed by light and electron microscopy. The glomerular nephrin was detected by Western blotting and real-time PCR. Immunofluorescence was used to detect the expression of WT1. The level of glomerular VEGF and VEGFR (Flt-1 and Flk-1) were evaluated by Western blotting. Results The level of glomerular nephrin protein in the rats with preeclampsia (0.0726±0.0074) was significantly lower compared with normal female group (0.3795±0.0509), normal pregnant group (0.2361±0.0437) and nonpregnant control group (0.7265±0.0503) (P＜0.01, respectively), while the levels of nephrin mRNA were not significantly different among 4 groups. The expression of WT1 was not significantly different among 4 groups as well. The level of glomerular VEGF in preeclampsia group (1.5429±0.0898) was significantly higher compared with normal female group (1.1870±0.1160), normal pregnant group (1.3741 ±0.1165) and nonpregnant control group (1.0155±0.0742)(P＜0.01,respectively). VEGFR (Flt-1 and Flk-1) was also significantly higher in preeclampsia rats compared with other control groups (P＜0.05, respectively). Conclusions In preeclampsia rats, nephrin is decreased significantly and the glomerular VEGF-VEGFR is increased significantly compared with the other control groups. The abnormal expression of nephrin and VEGF-VEGFR may be involved in the preeclampsia proteinuria. The underlying mechanism of this phenomenon needs further research.     

nephrin、血管内皮生长因子及其受体表达与先兆子痫大鼠蛋白尿的关系

Objective To investigate the association of the expressions of glomerular nephrin, vascular endothelial growth factor (VEGF) and its receptor (VEGFR) with proteinuria in preeclampsia rats. Methods A rat model of preeclampsia was developed by inhibitor of nitric oxide synthase (L-NAME). The systolic blood pressure (SBP) and 24 h urine protein were compared among the normal female group (n=6), the normal pregnant group (n=8), nonpregnant control group (n=6) and preeclampsia group(n=8). The kidney biopsies of each group were observed by light and electron microscopy. The glomerular nephrin was detected by Western blotting and real-time PCR. Immunofluorescence was used to detect the expression of WT1. The level of glomerular VEGF and VEGFR (Flt-1 and Flk-1) were evaluated by Western blotting. Results The level of glomerular nephrin protein in the rats with preeclampsia (0.0726±0.0074) was significantly lower compared with normal female group (0.3795±0.0509), normal pregnant group (0.2361±0.0437) and nonpregnant control group (0.7265±0.0503) (P＜0.01, respectively), while the levels of nephrin mRNA were not significantly different among 4 groups. The expression of WT1 was not significantly different among 4 groups as well. The level of glomerular VEGF in preeclampsia group (1.5429±0.0898) was significantly higher compared with normal female group (1.1870±0.1160), normal pregnant group (1.3741 ±0.1165) and nonpregnant control group (1.0155±0.0742)(P＜0.01,respectively). VEGFR (Flt-1 and Flk-1) was also significantly higher in preeclampsia rats compared with other control groups (P＜0.05, respectively). Conclusions In preeclampsia rats, nephrin is decreased significantly and the glomerular VEGF-VEGFR is increased significantly compared with the other control groups. The abnormal expression of nephrin and VEGF-VEGFR may be involved in the preeclampsia proteinuria. The underlying mechanism of this phenomenon needs further research.     

人血管内皮生长因子165基因转染人骨髓间充质干细胞的实验研究

Objective To establish an effective method of transfecting human marrow mesenchymal stem cells (MSC) with human vascular endothelial growth factor 165 ( VEGF 165) gene. Methods MSCs isolated and cultured in vitro were divided into transfection group (pShuttle-CMV/VEGF 165 plasmid was transfected into MSCs through liposome-mediating method), empty plasmid group (pShuttle-CMV vehi-cle was transfected into MSCs as control), liposome group (liposome was transfected into MSCs as control) and control group(normal culture). Expressions of Mrna and protein of MSCs were determined by RT-PCR, enzyme-linked immunosorbent assay and Western Blot. Sensitivity to MSCs on VEGF plasmid transfec-tion was detected by MTT test. Results Expression level of VEGF 165 gene Mrna in transfection group, empty plasmid group, liposome group, and control group was respectively 0.89 ± 0.03, 0. 34 ± 0.04, 0.40 ± 0.03, and 0.30 ± 0.03, and the difference between transfection group and the other three groups was statistically significant ( P <0. 01 ). Content of VEGF protein in transfection group, empty plasmid group, liposome group, and control group was respectively (778 ± 35 ), (543 ± 24), (561 ± 28), (571 ± 23) pg/Ml, and the difference between transfection group and the other three groups was statistically significant ( P <0.01 ). In the transfection group, expression level of VEGF protein peaked on 7th day after transfec-tion, which was decreased gradually later. In transfection group, expression level of V EGF 165 protein was obviously higher than that of the other three groups ( P <0. 01 ), and no inhibitory effect of VEGF plasmid transfection on MSCs proliferation was found. Conclusions The method for transfecting human VEGF 165 gene into MSCs is established in this research, through which target gene and protein can express effectively.     

Effect of rhVEGF gene transfection on survival of grafts after autologous free granular fat transplantation in rats

Objective： To investigate the effect of recombinant human vascular endothelial growth factor （rhVEGF） on autologous free granular fat grafts in rats.
Methods： Forty-eight Sprague Dawley （ SD ） rats, weighing 190-280 g and regardless sex, were randomly divided into three groups, sixteen in each. After fat transplantation, the rats were treated with plasmid DNA encoding rhVEGF protein （the experimental group ）, plasmid DNA （ the negative group） and normal saline （ the blank control group ）, respectively. At 3, 7, 15 and 30 days after transplantation, the rats were killed and the grafts were weighed, respectively. Histopathological changes were evaluated. Microvessel density and the expression of VEGF were examined by immunohistochemical staining and Western blotting.
Results： The weights of the negative and blank control groups were significantly reduced on the 7th, 15th and 30th days compared with those of the experimental group. The expression of VEGF and the microvessel density in the experimental group were significantly higher than the other two groups during the latter periods.
Conclusion： The plasmid encoding VEGF can induce expression of VEGF and angiogenesis in fat grafts and reduce the absorption of free fat grafts.     

急性胰腺炎时骨髓和循环内皮祖细胞的变化研究

Objective To investigate changes in number of endothelial progenitor cells(EPCs)from bone marrow and circulation in mice with acute pancreatitis.Methods BALB/c mice were assigned randomly to saline group and cerulein group.Animals were sacrificed at 12, 24 and 48 hours after injection.Bone marrow and circulating EPCs were detected by flow cyzometric analysis.Plasma VEGF, TNF-α and ET-1 were determined by enzyme-linked immunosorbent assay.The expression of VEGF in the pancreas was assessed by Western blotting.Apoptosis in situ was detected by TUNEL.Results The amounts of EPCs in bone marrow and circulation increased remarkably after cerulein injection(P < 0.05), also the levels of plasma VEGF TNF-α and ET-1(P < 0.05), the EPCs levels in bone marrow and circulation seen in the study closely mirrors the levels of VEGF detected in the circulation(r = 0.77, 0.67 individually).VEGF expression in pancreas was up-regulated after 12 h of cerulein injection compared with that of control group.Apoptosis of endothelial cells also increased in the cerulein group.Conclusion EPCs were mobilized by acute pancreatitis, which may be due to the mobilizing effect of increased levels of VEGF, EPCs may participate in the repair process of injured endothelium induced by acute pancreatitis.     

不同剂量己烯雌酚对新生小鼠睾丸表皮生长因子及受体的影响

目的 观察不同剂量己烯雌酚(DES)对新生小鼠睾丸组织中表皮生长因子(EGF)及其受体(EGFR)的影响,探讨DES对睾丸发育影响的机制.方法 建立小鼠DES模型.将72只怀孕的雌性昆明小鼠随机分成3组:正常组、对照组及实验组1～4(DES 10、25、50、100 μg/kg).采用免疫组织化学方法检测各组新生小鼠睾丸组织中EGF、EGFR的表达.结果 EGF和EGFR主要表达在新生小鼠睾丸的间质细胞.实验组1～4中EGF阳性细胞的累积吸光度值(IA)分别为75.43±1. 42、52.22±5.67、13.75±3.14、6.38±3.20,显著低于正常组及对照组中阳性细胞的IA值433.88±11.64、23.44±4.70;实验组EGFR阳性细胞的IA值分别为198.16±34.35、138.00±12.04、46.03±6.74、27.22±5.52,显著低于正常组及对照组中阳性细胞的IA值804.74±22.52、800.03±21.96.两者在正常、不同剂量DES实验组的两两比较差异有统计学意义(P＜0.05).随DES剂量增加,EGF和EGFR表达减弱,各组间差异有统计学意义(P＜0.05).EGF与EGFR呈强正线性相关(r=0.750,P＜0.01).结论 不同剂量DES对新生小鼠睾丸组织中EGF和EGFR表达强度均有影响,可能是影响睾丸发育机制之一.

Abstract：

Objective To investigate the effects of prenatal exposure to diethylstilbestrol (DES)with different dosages on epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) in offspring mice testis, and the possible mechanism. Methods DES model was induced in mice by DES.Female Kungmiag mice were randomly divided into normal group, control group and experimental groups 1-4 ( DES 10, 25, 50, 100 μg/kg). Immunohistochemistry was applied to detect the expression of EGF and EGFR in the testicular tissue in each group. Results EGF and EGFR were expressed mainly in sffspring mice testis Leydig cells. The cumulative absorbance ( IA ) values of EGF positive cells in experimental groups 1-4 were 75. 43 ± 1.42, 52. 22 ± 5.67, 13.75 ± 3. 14, and 6. 38 ± 3.20 respectively, which were significantly lower than in normal and control groups (433.88 ± 11.64,423.44 ±4. 70 respectively).The IA values of EGFR positive cells in experimental groups 1-4 were 198. 16 ± 34. 35, 138.00 ± 12.04,46.03 ± 6. 74, 27.22 ± 5.52 respectively, which were significantly lower than in normal and control groups (804. 74 ± 22. 52,800. 03 ± 21.96 respectively). The expression levels of EGF and EGFR could be detected in each subgroup and statistically significant differences existed in the expression of EGF and EGFR between any two groups ( P ＜ 0. 05 ). With the increase of DES dosage, the expression of EGF and EGFR was decreased, with the difference being significant amond the groups ( P ＜ 0. 05 ). Conclusion DES can influence the expression of EGF and EGFR in mice testis, which might be one of possible mechanisms effecting the development of testis.     

Possible mechanisms inducing glomerulations in interstitial cystitis: relationship between endoscopic findings and expression of angiogenic growth factors

PURPOSE: Glomerulation has been one of the requisite criteria for the diagnosis of interstitial cystitis (IC) but the mechanisms for glomerulation remain unclear. Therefore, we investigated the relationship between the cystoscopic findings of vascular events and the expression of angiogenic growth factors in IC bladders to identify the possible mechanisms inducing glomerulations in IC. MATERIALS AND METHODS: In 45 patients suspected of having IC continuous, fixed point cystoscopic observation was performed during hydrodistention using spinal anesthesia. Bladder biopsies were performed in these cases and in an additional 5 asymptomatic cases. Thereafter, the expression of platelet derived endothelial cell growth factor/thymidine phosphorylase (PDECGF/TP) was measured by enzyme-linked immunosorbent assay. Immunohistochemical staining for PDECGF/TP and vascular endothelial growth factor was also performed. RESULTS: Of 45 symptomatic patients 38 had glomerulations during cystoscopic examination. In these patients during hydrodistention the blood flow in bladder wall vessels was interrupted by whitish fibrous bundles. Thereafter petechial bleeding began from capillaries distal to obstructed vessels during bladder emptying. PDECGF/TP expression in patients with glomerulation was significantly higher than in symptomatic patients without glomerulation or asymptomatic patients (p < 0.001). In patients with glomerulations a high positive rate for PDECGF/TP (97.4%) and vascular endothelial growth factor (68.4%) staining was observed, while no positive staining was found in asymptomatic patients. CONCLUSIONS: Glomerulations during hydrodistention are highly associated with the over expression of angiogenic growth factors in the bladder. Thus, it seems likely that neovascularization promoted by angiogenic growth factors has an important role in the pathogenesis of IC, inducing glomerulations during hydrodistension.     

Subtypes of bladder mast cells in interstitial cystitis

BACKGROUND: Because the types of mast cells present in the bladder of patients with interstitial cystitis (IC) have not been elucidated, we have used immunohistochemical techniques to determine which of the mast cell types is present in biopsy specimens. METHODS: For all patients diagnosed with IC (n = 10; female) their symptoms satisfied the criteria proposed by the National Institutes of Health criteria of IC and six patients suffering from bladder tumors were selected as control patients. Adjacent sections of paraffin-embedded tissues that had been fixed in Carnoy's solution were reacted with either antitryptase or antichymase antibodies. RESULTS: In detrusor and in mucosa, the number of tryptase-positive and chymase-negative mast cells (MC(T)) was 146+/-25 and 81+/-31 cells/mm2, respectively, and the number of tryptase-positive and chymase-positive mast cells (MC(TC)) was 124+/-50 and 54+/-20 cells/mm2, respectively. These numbers were significantly greater than those of the control group. A significant negative correlation (P<0.005; R = 0.943) was observed between the number of MC(TC) and the bladder capacity. The number of mast cells obtained by toluidine blue staining in detrusor and in mucosa was 95+/-68 and 71+/-39 cells/mm2, respectively, suggesting that staining with toluidine blue underestimated the number of mast cells. CONCLUSIONS: Mast cells were significantly increased in number in both the mucosa and detrusor of bladder specimens from IC patients compared with those from control. The MC(TC) may be the type of mast cell dominantly present in the bladder of IC patients. The MC(TC) in detrusor increased with the progression of contracted bladder.     

血管内皮生长因子转染胚胎成骨细胞的实验研究

目的 观察血管内皮生长因子(vascular endothelial growth factor,VEGF)转染胚胎成骨细胞后的成骨细胞增殖情况.方法 取SD大鼠的胎鼠颅骨成骨细胞作原代培养后再进行体外培养扩增传代,同时构建含有VEGF基因片段(ad5-h-VEGF)的人5型腺病毒载体,以进入对数生长期的成骨细胞为靶细胞进行转染,免疫组化、免疫荧光定性检测实验组即转染(+)组与对照组即转染(-)组细胞,绘制转染前后的生长曲线,并进行统计分析.结果 VEGF在成骨细胞的表达可通过免疫组化、免疫荧光定性检测;实验组细胞增殖能力较对照组增强,统计分析转染组的细胞计数(＞48 h)显著高于空白对照组(P＜0.05).结论 VEGF能够在成骨细胞中成功表达,VEGF转染后能够促进成骨细胞生长及成骨.     

肥大细胞特征检测与盆腔症状评分联合应用——间质性膀胱炎诊断标准研究

目的 探讨间质性膀胱炎(IC)黏膜组织中肥大细胞特征与盆腔症状评分(PUF)联合应用诊断IC的应用价值.方法 IC患者膀胱黏膜活检组织标本18例,12例正常膀胱组织标本作为对照组.标本0.5%甲苯胺蓝染色,光镜下计数肥大细胞密度,连续10个视野取其均数;锇酸液固定,透射电镜下观察肥大细胞与脱颗粒肥大细胞的超微结构;比较2组标本肥大细胞浸润密度、脱颗粒肥大细胞构成比.统计学分析肥大细胞浸润密度、脱颗粒比率与PUF评分的相关性.结果 IC组织标本中肥大细胞密度(28～76个/mm2)明显高于对照组(7～15个/mm2),差异有统计学意义(Z=3.927,P＜0.01);IC组织中75.3%的肥大细胞呈不同程度脱颗粒状态;IC患者PUF评分(17～35分)明显高于对照组(0～8分),差异有统计学意义(t=14.736,P＜0.01);PUF评分与肥大细胞浸润数量无线性关系(rs=-0.618,P=0.601).结果 肥大细胞浸润是IC组织特征性的病理表现;IC组织中肥大细胞浸润数量与PUF评分无线性相关关系.联合检测IC组织中肥大细胞浸润密度、肥大细胞激活脱颗粒状态与PUF评分可作为IC诊断的有效指标.     

肌动蛋白交联蛋白与血管内皮生长因子在肾细胞癌中的表达及其相关性

目的 探讨肌动蛋白交联蛋白(Fascin)与血管内皮生长因子(VEGF)在肾细胞癌组织的表达及与其生物学行为的关系.方法 采用免疫组织化学SP法检测Fascin与VEGF在92例肾细胞癌组织及20例正常肾脏组织中的表达.结果 Fascin及VEGF在肾细胞癌组织中的表达阳性率明显高于正常肾组织(P＜0.05);肾细胞癌组织中两种蛋白的表达与肿瘤组织学分级和临床分期均呈正相关(P＜0.05);与年龄、性别及肿瘤病理类型无明显相关(P＞0.05).且Fascin和VEGF的表达之间呈正相关(P＜0.05).结论 Fascin与VEGF可作为反映肾细胞癌生物学行为的参考指标.

Abstract：

Objective To investigate the expression of Fascin and vascular endothelial growth factor (VEGF) in renal cell carcinoma (RCC) and the correlation with the biological behaviors. Methods The immunohistochemistry staining method was used to detect the expression of Fascin and VEGF in 92 cases of RCC and 20 cases of normal renal tissues as controls. Results The expression of Fascin in carcinoma tissue was significantly higher than that in normal tissues ( P ＜ 0. 05 ). Positive expression of Fascin and VEGF in renal cell carcinoma tissue was correlated with tumor grade ( P ＜ 0. 05 ) and clinical stage (P ＜0. 05 ) , but not with age, gender and different histological categories (P ＞ 0. 05 ). There was also a positive correlation between Fascin and VEGF (P ＜ 0. 05 ). Conclusion Fascin and VEGF are objective markers to estimate the behaviors of renal cell carcinoma.     

过表达核转录因子Kr(u)ppel样因子4下调胃癌AGS细胞血管内皮生长因子的表达

目的 观察过表达核转录因子Kr(u)ppel样因子4(KLF4)是否下调胃癌AGS细胞血管内皮生长因子(VEGF)的表达.方法 体外培养人胃癌AGS细胞,分为4组:转染空质粒24 h对照组、转染空质粒48 h对照组、转染KLF4表达质粒24 h组、转染KLF4表达质粒48 h组.构建KLF4表达质粒,脂质体LipofectamineTM2000分别转染空质粒、KLF4表达质粒到AGS细胞,24、48 h后提取总RNA和蛋白,实时荧光定量逆转录-聚合酶链反应(RT-PCR)法、Western blot法分别检测KLF4和VEGF mRNA和蛋白水平的表达.结果 AGS细胞在24 h转染率为65%-75%,48 h转染率为40%～45%.实验组与对照组转染24、48 h后,KLF4 mRNA的表达量分别为:563.584±250.744比4.997±5.729(P＜0.05);351.852±212.439比2.4420±1.3770(P＜0.05).VEGF mRNA表达量分别为:0.008 929±0.003 810比0.002 294±0.000720(P＜0.05);0.018 375±0.008 263比0.002 193±0.001 698(P＜0.05);胃癌AGS细胞KLF4蛋白表达量显著性增加,相对应地VEGF蛋白表达水平显著性降低(P＜0.05).结论 体外胃癌AGS细胞过表达KLF4导致VEGF mRNA和蛋白表达下调,KLF4可能参与抑制调节胃癌AGS细胞VEGF的表达.     

间质性膀胱炎大鼠动物模型膀胱黏膜保护前后组胺受体变化及意义

目的 探讨透明质酸膀胱灌注治疗间质性膀胱炎(IC)后膀胱组织中组胺受体变化的意义. 方法 IC模型大鼠20只,随机分为2组,每组10只.实验组尿道灌注0.8 g/L透明质酸.对照组即时处死.HE染色计数膀胱固有层单核炎症细胞,特殊染色计数肥大细胞.免疫组化染色2组大鼠膀胱组织,IPP4.5图像分析软件计算吸光度(A)值,比较2组间的差异. 结果 实验组单核炎症细胞数和肥大细胞数分别为(12.20±2.48)、(2.90±0.87)个/视野;组胺H1～H4受体A值分别为0.015±0.007、0.006±0.001、0.007±0.004、0.061±0.026;对照组单核炎症细胞数和肥大细胞数分别为(23.90±3.07)、(7.08±1.23)个/视野;组胺H1～H4受体A值分别为0.055±0.033、0.031±0.023、0.033±0.017、0.091±0.059.2组单核炎症细胞和肥大细胞数比较差异均有统计学意义(P＜0.01);H1、H2、H3受体A值明显减小(P＜0.05);组胺H4受体A值差异无统计学意义(P＞0.05). 结论 组胺H1、H2、H3受体参与IC发生,其受体拮抗剂可能用于临床治疗IC.     

血管内皮生长因子对关节软骨细胞诱导型一氧化氮合酶的影响

目的 观察血管内皮生长因子(VEGF)对体外培养的关节软骨细胞诱导型一氧化氮合酶(iNOS)表达的影响。方法 体外培养SD乳鼠关节软骨细胞,用白细胞介素(IL)-1β诱导的方法建立骨关节炎(OA)体外模型,实验分为4组,每组加入不同处理因素进行干预,A组:(正常对照组)不加任何处理因素;B组:10 μg/L VEGF;C组:10 μg/L IL-1β;D组:10 μg/L VEGF+ 10 μg/LIL-1β。采用实时荧光定量PCR( Real Time PCR)检测iNOS mRNA的表达,采用蛋白免疫印迹法( Western blot)检测iNOS蛋白的表达。结果 iNOS mRNA的表达:A组iNOS mRNA无表达,B组(9.64±1.64)、C组(17.27±2.01)及D组(28.93±6.63),3组的iNOS mRNA表达量显著升高,进一步组间比较,D组软骨细胞iNOS的mRNA表达水平明显高于B组(P＜0.01)及C组(P＜0.05),C组软骨细胞iNOS的mRNA表达水平高于B组(P＜0.05)。iNOS蛋白的表达:A组iNOS蛋白无表达,B组(0.44±0.12)、C组(0.74±0.07)及D组(1.38±0.38),3组的iNOS蛋白表达量显著升高,进一步组间比较,D组软骨细胞iNOS的蛋白表达水平明显高于B组(P＜0.01)及C组(P＜0.05),C组软骨细胞iNOS的mRNA表达水平高于B组(P＜0.01)。结论 在OA的发病过程中,VEGF可能通过上调软骨细胞iNOS的表达发挥重要作用。