糖尿病大鼠玻璃体腔注射缺氧诱导因子-1α siRNA对血管内皮生长因子蛋白表达的影响

目的：观察缺氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)小干扰RNA(siRNA)对糖尿病大鼠视网膜组织中血管内皮生长因子(vascular endothelial growth factor,VEGF)蛋白表达的抑制作用,及其用于糖尿病新生血管性疾病治疗的可行性。

方法：以pSilencer2.1-U6neo为质粒载体,构建HIF-1α.siRNA 重组质粒。选取雄性Sprague-Dawley(SD)大鼠54只,随机分为正常对照组(15只)和实验组(39只)。实验组采用尾静脉注射链尿佐菌素(STZ)的方法建立糖尿病大鼠模型,造模成功后将实验组随机分为糖尿病组(15只)、基因治疗组(12只)和空载体组(12只)。正常对照组及糖尿病组大鼠均不做转染; 基因治疗组和空载体组分别转染HIF-1α.siRNA 重组质粒和pSilencer空载体质粒。行苏木精-伊红染色(hematoxylin-eosin staining,HE染色)观察视网膜组织形态,并采用免疫组织化学法检测VEGF蛋白的表达。分别于干扰后24、48、72h,1wk时计算VEGF蛋白的抑制效率。采用单因素方差分析和LSD-t检验进行统计学分析。

结果：HIF-1α.siRNA 重组质粒经酶切、测序鉴定,确定为目的序列。HE染色显示：正常对照组大鼠视网膜各层细胞排列整齐,细胞形态基本正常。糖尿病大鼠视网膜各层细胞排列紊乱,内界膜不完整,新生血管芽、新生血管簇呈垂直状突破内界膜生长。免疫组织化学染色显示：VEGF阳性表达为细胞浆出现棕黄色颗粒,主要位于神经节细胞层。正常对照组VEGF蛋白呈弱阳性表达,而DR对照组和空载体组表达明显增强,基因治疗组较DR组和空载体组表达明显减少,差异有统计学意义(P<0.05)。VEGF蛋白抑制率24、48、72h和1wk时分别为：27.4%、40.6%、47.5%、64.5%。

结论：HIF-1α.siRNA重组质粒能够抑制糖尿病大鼠视网膜中VEGF蛋白的表达,可能成为一种治疗糖尿病性新生血管疾病的新方法。     

糖尿病视网膜病变激光面积与疗效的分析

目的 通过自行设计的计算机软件量化激光斑面积,计算糖尿病视网膜病变(diabetic retinopathy,DR)患者全视网膜光凝(panretinal photocoagulation,PRP)后激光斑所占眼底图总面积百分比,探讨激光斑面积与视力及黄斑厚度、黄斑容积之间的关系.方法 选择重度非增生性DR患者30例60眼行PRP治疗.将不伴有黄斑水肿的患者分为A、B两组,每组24眼;将伴有黄斑水肿的患者分为C、D两组,每组6眼.其中A、B两组行PRP,C、D两组先行黄斑格栅样光凝,1周后再行PRP;A组、C组PRP间隔1个激光斑;B组、D组PRP间隔2个激光斑.记录PRP前、PRP后3个月各组视力及黄斑厚度、黄斑容积,并测量4组眼底激光癍面积占眼底图总面积的百分比,比较激光斑面积比值与激光治疗效果的关系.结果 PRP间隔1个激光斑时,激光斑面积(像素)为20 000 ～ 50 000(34 398.27±8808.51),激光斑面积与眼底图总面积的百分比为(32.64±4.43)％(25％ ～40％).PRP间隔2个激光斑时,激光斑面积为10 000 ～20 000(15 442.33 ±2737.77),激光斑面积与眼底图总面积的百分比为(18.35±3.43)％(10％～25％).A组与B组比较,PRP后视力、黄斑厚度及黄斑容积差异均无统计学意义(均为P＞0.05)[C组与D组比较,PRP后视力恢复更好,黄斑厚度及黄斑容积均有显著降低(均为P＜0.05).结论 有效的激光斑面积是PRP治疗成功的关键,对于伴有黄斑水肿的重度非增生性DR患者,激光斑所占视网膜总面积为25％ ～ 40％时,术后视力及黄斑厚度、黄斑容积恢复较好.     

Inhibition effect of interfering RNA targeting hypoxia inducible factor-1α on vascular endothelial growth factor mRNA expression in the retina of diabetic rats

Objective To observe the inhibition effect of the hypoxia inducible factor-1α(HIF-1α)specific siRNA on the expression of vascular endothelial growth factor(VEGF)mRNA in retinal tissues in diabetic rat.Methods This is a randomized controlled study.HIF-1α specific siRNA recombinant plasmid was built in pSilencer2.1-U6neo vector.Fifty-four healthy Sprague Dawley(SD)rats were divided into control group(15 rats)and experimental group(39 rats).The experimental rats were induced with streptozotocin injection for diabetic retinopathy model,and then randomly divided into diabetic retinopathy (DR)group(15 rats),vector group(12 rats)and gene therapy group(12 rats).LipofectamineTM2000mixed with pSilencer2.1-U6neo plasmid or HiF-1α siRNA plasmid were injected into the vitreous in the vector group and gene therapy group respectively.Nothing was transfected into DR and control group.The expression of VEGF mRNA in retinas was measured by real-time RT-PCR.The inhibition efficiency of VEGFmRNA was calculated at 24,48,72 hours and 1 week after injection respectively.Significant differences between groups were evaluated by one-way analysis and LSD-t analysis.Results HIF-1 α siRNA recombinant plasmid was confirmed by enzyme digestion and sequence analysis.Real-time RT-PCR revealed that the expression of VEGFmRNA was faint in the control group.increased obviously in the DR and vector group,decreased in the gene therapy group.There was no statistically significant between DR and vector group(t=0.669,0.142,0.151,0.025;P=0.514,0.889,0.882,0.980).The expression of VEGFmRNA in the gene therapy group were obviously decreased compared with DR and vector group(t=8.768,13.695,11.285,8.253;P=0.000).The inhibition efficiency of VEGFmRNA was 32.76% ,43.60% ,47.70% ,50.86% at 24,48,72 hours and 1 week after injection.Conclusions The expression of VEGFmRNA can be efficiently inhibited by HIF-1α siRNA recombinant plasmid.     

PDR患者玻璃体视网膜术后高眼压的危险因素及疗效分析

目的 探讨增生性糖尿病视网膜病变(proliferative diabetic retinopathy,PDR)玻璃体视网膜手术(vitreorefinal surgery,VRS)后高眼压发生的危险因素及治疗效果.方法 回顾性分析136例(161眼)行VRS的PDR患者的临床资料,观察治疗前后及随访结束时患者的眼压、视网膜解剖复位情况及最佳矫正视力,术后随访12-56个月,平均23个月.对手术后发生高眼压的危险因素进行多因素Logistic回归分析.结果 VRS术后43例(43眼)患者发生高眼压,发生于术后1 d-15个月,发生率为26.7%(43/161);其中48.8%的惠者(21眼)为术后暂时性高眼压,51.2%的患者(22眼)需手术控制眼压.患者治疗前平均眼压(34.26±8.76)mmHg(1 kPa=7.5 mmHg);治疗后6-8周时平均眼压为(17.44 ±3.79)mmHg;随访结束时平均眼压为(15.58±3.80)mmHg,三者之间两两比较差异均有统计学意义(均为P<0.05).多因素Logistic回归分析显示,术后高眼压发生的独立危险因素为:患者血糖水平及前房炎性反应(OR=2.866、3.027;P=0.013、0.028).与患者年龄、术中填充物的类型、是否一期行白内障手术、术后再次玻璃体出血及是否联合扣带术等无关.本组患者视网膜一次解剖复住成功率90.70%.发生高眼压前最佳矫正视力为无光感～0.08,随访结束时最佳矫正视力为无光感～0.7.结论 PDR患者血糖水平及前房炎性反应为VRS术后高眼压发生的独立危险因素.早期发现、个体化治疗可以最大程度地挽救患者的视力.     

玻璃体视网膜手术治疗增生性糖尿病视网膜病变合并牵拉性视网膜脱离视力预后影响因素

目的　观察玻璃体视网膜手术治疗增生性糖尿病视网膜病变合并牵拉性视网膜脱离（DTRD）患者视力预后的影响因素。方法　回顾分析接受玻璃体视网膜手术治疗的86例DTRD患者102只眼的临床资料。所有患者均在散瞳后经间接检眼镜及B型超声检查确诊。手术后随访12~56个月,平均随访23个月。随访观察手术前、后视力及手术后视网膜解剖复位情况。采用t检验、χ2检验、多因素Logistic回归分析等统计学方法,分析视力提高组及未提高组间疾病自然因素及手术后并发症与视力预后的关系。结果　87只眼视网膜首次解剖复位成功,占85.29%;15只眼视网膜首次未完全解剖复位,占14.71%。49只眼手术后视力提高且最佳矫正视力（BCVA）大于0.05,占48.04%;53只眼手术后视力降低或BCVA较手术前有所提高但仍低于0.05,占51.96%。两组间疾病自然因素比较,是否一期行白内障手术及是否合并视神经萎缩差异有统计学意义(χ²=5.266,9.274;P=0.022,0.002)。两组手术后并发症比较,视网膜脱离(RD)复发差异有统计学意义(χ²=12.059,P=0.000)。Logistic回归分析显示RD复发、视神经萎缩是影响DTRD患者玻璃体视网膜手术后视力的独立危险因素(OR=33.518,4.079;P=0.003,0.041);手术前完成全视网膜激光光凝是手术后视力的保护性因素(OR=0.270,P=0.034)。结论　RD复发、视神经萎缩是影响DTRD患者玻璃体视网膜手术后视力预后的独立危险因素。      

Inhibition effect of interfering RNA targeting hypoxia inducible factor-1α on vascular endothelial growth factor mRNA expression in the retina of diabetic rats

Objective To observe the inhibition effect of the hypoxia inducible factor-1α(HIF-1α)specific siRNA on the expression of vascular endothelial growth factor(VEGF)mRNA in retinal tissues in diabetic rat.Methods This is a randomized controlled study.HIF-1α specific siRNA recombinant plasmid was built in pSilencer2.1-U6neo vector.Fifty-four healthy Sprague Dawley(SD)rats were divided into control group(15 rats)and experimental group(39 rats).The experimental rats were induced with streptozotocin injection for diabetic retinopathy model,and then randomly divided into diabetic retinopathy (DR)group(15 rats),vector group(12 rats)and gene therapy group(12 rats).LipofectamineTM2000mixed with pSilencer2.1-U6neo plasmid or HiF-1α siRNA plasmid were injected into the vitreous in the vector group and gene therapy group respectively.Nothing was transfected into DR and control group.The expression of VEGF mRNA in retinas was measured by real-time RT-PCR.The inhibition efficiency of VEGFmRNA was calculated at 24,48,72 hours and 1 week after injection respectively.Significant differences between groups were evaluated by one-way analysis and LSD-t analysis.Results HIF-1 α siRNA recombinant plasmid was confirmed by enzyme digestion and sequence analysis.Real-time RT-PCR revealed that the expression of VEGFmRNA was faint in the control group.increased obviously in the DR and vector group,decreased in the gene therapy group.There was no statistically significant between DR and vector group(t=0.669,0.142,0.151,0.025;P=0.514,0.889,0.882,0.980).The expression of VEGFmRNA in the gene therapy group were obviously decreased compared with DR and vector group(t=8.768,13.695,11.285,8.253;P=0.000).The inhibition efficiency of VEGFmRNA was 32.76% ,43.60% ,47.70% ,50.86% at 24,48,72 hours and 1 week after injection.Conclusions The expression of VEGFmRNA can be efficiently inhibited by HIF-1α siRNA recombinant plasmid.     

云南瑞丽47例输入性登革热的临床特点

2006年～2009年瑞丽市发生过2次输入性登革热病例,本文采用回顾性方法对瑞丽2次输入性登革热（DF）期间收治的47例患者的临床特点、实验实检查及诊疗结果分析报道如下。     

缺氧诱导因子-1α小干扰RNA对糖尿病大鼠视网膜血管内皮生长因子mRNA抑制作用的动态观察

目的 观察缺氧诱导因子-1α(HIF-1α)特异性小干扰RNA(siRNA)对糖尿病大鼠视网膜组织中血管内皮生长因子(VEGF)mRNA表达的抑制作用.方法 随机对照研究.以pSilencer 2.1-U6neo为质粒载体,构建HIF-1α siRNA重组质粒.雄性Sprague-Dawley大鼠54只,随机分为正常对照组和实验组,分别为15、39只大鼠.实验组大鼠尾静脉注射链尿佐菌素(STZ)诱导建立糖尿病动物模型,再分为糖尿病模型组、空载体组和基因治疗组,分别为15、12、12只大鼠.脂质体Lipofectamine TM 2000介导,空载体组和基因治疗组分别转染pSilencer空载体质粒和HIF-1αsiRNA重组质粒,糖尿病模型组和正常组对照组不做转染.采用实时逆转录(RT)-聚合酶链式反应(PCR)检测各组大鼠VEGF mRNA的表达.分别于干扰后24、48、72 h,1周时计算VEGF mRNA的抑制效率.采用单因素方差分析和LSD-t检验进行统计学分析.结果 HIF-1α siRNA重组质粒经酶切、测序鉴定,确定为目的 序列.实时RT-PCR检测结果显示,正常对照组仅见弱的VEGF mRNA表达,糖尿病模型组及空载体组表达明显上调,基因治疗组表达下调;各时间点空载体组与糖尿病模型组比较,差异无统计学意义(t=0.669,0.142,0.151,0.025;P=0.514,0.889,0.882,0.980),基因治疗组较糖尿病模型组及空载体组表达下调,差异有统计学意义(t=8.768、13.695、11.285、8.253,t=9.437、13.554、11.436、8.228;P值均=0.000);VEGF mRNA抑制率24、48、72 h和1周时分别为32.76%、43.60%、47.70%、50.86%.结论 HIF-1α siRNA重组质粒能有效抑制糖尿病大鼠视网膜中VEGFmRNA的表达.

Abstract：

Objective To observe the inhibition effect of the hypoxia inducible factor-1α(HIF-1α)specific siRNA on the expression of vascular endothelial growth factor(VEGF)mRNA in retinal tissues in diabetic rat.Methods This is a randomized controlled study.HIF-1α specific siRNA recombinant plasmid was built in pSilencer2.1-U6neo vector.Fifty-four healthy Sprague Dawley(SD)rats were divided into control group(15 rats)and experimental group(39 rats).The experimental rats were induced with streptozotocin injection for diabetic retinopathy model,and then randomly divided into diabetic retinopathy (DR)group(15 rats),vector group(12 rats)and gene therapy group(12 rats).LipofectamineTM2000mixed with pSilencer2.1-U6neo plasmid or HiF-1α siRNA plasmid were injected into the vitreous in the vector group and gene therapy group respectively.Nothing was transfected into DR and control group.The expression of VEGF mRNA in retinas was measured by real-time RT-PCR.The inhibition efficiency of VEGFmRNA was calculated at 24,48,72 hours and 1 week after injection respectively.Significant differences between groups were evaluated by one-way analysis and LSD-t analysis.Results HIF-1 α siRNA recombinant plasmid was confirmed by enzyme digestion and sequence analysis.Real-time RT-PCR revealed that the expression of VEGFmRNA was faint in the control group.increased obviously in the DR and vector group,decreased in the gene therapy group.There was no statistically significant between DR and vector group(t=0.669,0.142,0.151,0.025;P=0.514,0.889,0.882,0.980).The expression of VEGFmRNA in the gene therapy group were obviously decreased compared with DR and vector group(t=8.768,13.695,11.285,8.253;P=0.000).The inhibition efficiency of VEGFmRNA was 32.76% ,43.60% ,47.70% ,50.86% at 24,48,72 hours and 1 week after injection.Conclusions The expression of VEGFmRNA can be efficiently inhibited by HIF-1α siRNA recombinant plasmid.     

玻璃体内注射血管内皮生长因子抑制剂并发症的研究进展

玻璃体内注射血管内皮生长因子（VEGF）抑制剂广泛用于年龄相关性黄斑变性（AMD）、中心性渗出性脉络膜视网膜病变及病理性近视引起的脉络膜新生血管（CNV）的治疗,也用于增生型糖尿病视网膜病变（PDR）、视网膜血管阻塞、早产儿视网膜病变及新生血管性青光眼等的治疗,已取得一定的疗效,但玻璃体内注射VEGF抑制剂也出现了很多并发症。就玻璃体内注射VEGF抑制剂对眼正常结构和生理功能的影响以及引起的眼压升高、视网膜色素上皮撕裂（RRPE）、视网膜脱离（RD）、眼内炎、注射相关损伤、全身不良反应等并发症进行综述。