HIF-1α特异性小干扰RNA抑制糖尿病大鼠视网膜HIF-1α表达的研究

发布时间：2018-02-06 07:29

本文关键词： 糖尿病视网膜病变缺氧诱导因子-1α 小干扰RNA 视网膜新生血管基因治疗　出处：《天津医科大学》2010年硕士论文　论文类型：学位论文

【摘要】：目的以pSilencer2.1-U6neo为质粒载体,构建缺氧诱导因子-1α (HIF-1α)特异性小干扰RNA (siRNA),研究其对糖尿病大鼠视网膜HIF-1α表达的影响。观察玻璃体腔注射HIF-1α siRNA后不同时间糖尿病大鼠视网膜表达HIF-1α的变化情况。方法 1.构建HIF-1α特异性siRNA重组质粒。SD大鼠共100只,随机分成正常组(A)组(21只)和糖尿病组(79只)。链脲佐菌素(STZ)尾静脉注射制作糖尿病大鼠模型。饲养18周后,FITC-Dextran荧光造影视网膜铺片证实DR模型成功后,糖尿病大鼠又随机分成对照(B)组、空载体(C)组和基因治疗(D)组。向空载体组玻璃体腔注射pSilencer空载体质粒,基因治疗组玻璃体腔注射HIF-1α特异性siRNA重组质粒。根据玻璃体腔注射后24小时、48小时和72小时,A、 B、C、D各组随机分成三个组。 2.分别于注药后24小时、48小时、72小时后处死相应组动物,摘除眼球,应用组织病理学观察不同时间点各组大鼠视网膜的病理改变。采用实时荧光RT-PCR法检测各组视网膜HIF-1α mRNA水平的差异；免疫组化SP法观察视网膜各层HIF-1蛋白的染色情况。Image-Pro Plus6.0图像处理分析软件分析各组染色阳性区的平均累积光密度(integrated optic density,IOD)。 3.运用SPSS11.5统计软件,经正态性检验及方差齐性检验,对同一组间不同时间,同一时间不同组间数据进行分析,采用单因素方差分析,并用SNK检验进行组间比较,P0.05差异具有统计学意义。结果 1.STZ诱导的糖尿病大鼠高血糖状态持久稳定,表现为明显的“三多一少”典型症状。在各时间点,正常组与糖尿病组大鼠体重、血糖均有统计学差异(P0.05)。 2. FITC-Dextran荧光造影视网膜铺片显示糖尿病组视网膜血管形态和分布发生明显改变,可见微血管瘤及异常吻合的新生血管团、高荧光素渗漏和无灌注区,证实DR模型制作成功。正常大鼠视网膜血管走行良好,视网膜浅层大血管及深层血管网均清晰可见,未见视网膜微血管瘤形成和视网膜新生血管形成。 3.实时荧光RT-PCR结果显示,同一时间点C组和B组视网膜HIF-1α mRNA表达较A组明显上调,D组视网膜组织中HIF-1α mRNA较B组、C组下调,差异具有统计学意义(P0.05),B组和C组表达差异无统计学意义(P0.05)。D组不同时间点视网膜HIF-1α mRNA的表达差异具有统计学差异(F=9.437,P=0.002)。进一步进行组间比较,玻璃体腔注射HIF-1α siRNA48小时及72小时后视网膜HIF-1α mRNA表达较注射24小时后明显减少(P1=0.003,P2=0.001),注射72小时后较注射48小时后视网膜HIF-1αamRNA表达差异不具有统计学意义(P=0.749)。HIF-1α siRNA干扰24小时、48小时和72小时的抑制效率分别为32.59%、46.92%和52.28%。 4. HIF-1α蛋白在正常大鼠视网膜神经节细胞层和外丛状层有少量阳性染色细胞,糖尿病组染色阳性细胞数表达增多,主要位于视网膜神经节细胞层、内丛状层、内核层、外丛状层。同一时间点C组和B组视网膜HIF-1α染色阳性区的平均IOD较正常组明显上调(P0.05),B组和C组表达差异无统计学意义(P0.05),D组视网膜组织中HIF-1α蛋白染色阳性区的平均IOD较B、C组下调,差异具有统计学意义(P0.05)。D组不同时间点视网膜HIF-1α蛋白的表达差异具有统计学差异(F=26.373,P=0.000)。进一步进行组间比较,玻璃体腔注射HIF-1α siRNA48小时及72小时后视网膜HIF-1a蛋白表达较24小时明显减少(P1=0.000,P2=0.000),注射72小时后较注射48小时后视网膜HIF-1α蛋白表达差异不具有统计学意义(P=0.215)。结论本实验构建HIF-1α特异性siRNA重组质粒,将HIF-1α siRNA成功的转染至大鼠视网膜,通过实时荧光RT-PCR和免疫组织化学染色方法观察糖尿病大鼠视网膜HIF-1α的表达,发现HIF-1α特异性siRNA降低了糖尿病大鼠视网膜HIF-1α的表达,从而有望抑制其靶基因VEGF等促进血管新生因子的表达,抑制视网膜新生血管的形成,成为治疗DR的一种新的途径。
[Abstract]:objective
With pSilencer2.1-U6neo plasmid to construct hypoxia inducible factor -1 alpha (HIF-1 alpha) specific small interfering RNA (siRNA), to study its effect on retinal HIF-1 alpha in diabetic rats. To observe the expression of intravitreal injection of HIF-1 alpha siRNA at different time after the retina of diabetic rats of HIF-1 alpha changes.
Method
1. construction of HIF-1 alpha specific recombinant plasmid siRNA 100.SD rats were randomly divided into normal control group (A) group (21 rats) and diabetic group (79 rats). Streptozotocin (STZ) diabetic rat model was established by tail vein injection. After 18 weeks of feeding, FITC-Dextran fluorescence angiography was the DR model proved successful, and diabetic rats were randomly divided into control group (B) (C), empty vector group and gene therapy group (D). To empty vector group pSilencer intravitreal injection of empty plasmid, gene therapy group HIF-1 intravitreal injection of alpha specific siRNA recombinant plasmid. According to the 24 hours after the intravitreal injection, 48 hours and 72 hours, A, B, C, D were randomly divided into three groups.
2. to 24 hours after injection, 48 hours, 72 hours after the corresponding group of animal, removal of the eye, histopathological observation on pathological changes of retina in rats at different time points. The difference by using real-time fluorescence RT-PCR method to detect the retinal HIF-1 alpha mRNA levels; immunohistochemical SP method was used to observe the retinal layers HIF-1 protein staining of.Image-Pro Plus6.0 image processing and analysis software were analyzed with positive staining area cumulative average optical density (integrated optic, density, IOD).
3., using SPSS11.5 statistical software, by normality test and homogeneity test of variance, the data of different groups at the same time and at the same time between different groups were analyzed. One-way ANOVA and SNK test were used to compare the data between groups. The difference of P0.05 was statistically significant.
Result
High blood glucose in diabetic rats induced by 1.STZ lasting stability, is obviously a little more than three typical symptoms. At each time point, the normal weight group and diabetic rats, there were differences in blood glucose (P0.05).
2. FITC-Dextran fluorescein angiography retinal flatmount showed retinal vascular morphology and distribution of diabetes group changed obviously, visible micro hemangioma and abnormal neovascularization anastomosis group, high fluorescein leakage and non perfusion area, confirmed that the DR model was established successfully. The retinal vessels in normal rats running well, the superficial layer of the retina vessels and deep vascular network visible, and retinal neovascularization no retinal microaneurysm.
3. real time RT-PCR results showed that at the same time C group and B group of retinal HIF-1 alpha mRNA expression compared with A group was significantly increased in the D group, the retinal tissue HIF-1 alpha mRNA compared with B group, C group decreased, the difference was statistically significant (P0.05), B group and C group was no statistically significant difference (P0.05) the differential expression of.D group at different time points of retinal HIF-1 alpha mRNA with statistical difference (F=9.437, P=0.002). Further comparison between groups, the expression of mRNA in retinal HIF-1 alpha was significantly less than 24 hours after the injection of intravitreal injection of HIF-1 alpha siRNA48 and 72 hours (P1=0.003, P2=0.001), 72 h after injection with injection of 48 hours after the retinal expression of HIF-1 alpha amRNA difference was not statistically significant (P=0.749).HIF-1 alpha siRNA interference for 24 hours, the inhibition efficiency of 48 hours and 72 hours were 32.59%, 46.92% and 52.28%.
There is a small amount of 4. HIF-1 protein positive staining cells in the retinal ganglion cell layer and outer plexiform layer of normal rats, diabetic group staining positive cells expression increased, mainly located in the retinal ganglion cell layer, inner plexiform layer, inner nuclear layer, outer plexiform layer. At the same time C group and B group of retinal HIF-1 alpha the average IOD positive staining area was significantly increased compared with normal group (P0.05), B group and C group was no statistically significant difference (P0.05), the average IOD positive area than B staining of HIF-1 protein in retinal tissue in the D group, C group decreased, the difference was statistically significant (P0.05) expression of.D group at different time points retinal HIF-1 alpha protein has a statistically significant difference (F=26.373, P=0.000). Further comparison between groups, the retinal expression of HIF-1a protein was significantly reduced by 24 hours compared with intravitreal injection of HIF-1 alpha siRNA48 and 72 hours (P1=0.000, P2=0.000), injection of 72 hours There was no significant difference in the expression of HIF-1 alpha protein in the retina after 48 hours after injection (P=0.215).
conclusion
The constructed HIF-1 alpha specific siRNA recombinant plasmid, HIF-1 alpha siRNA successfully transfected into rat retina, observe the expression of retinal HIF-1 alpha in diabetic rats by real-time RT-PCR and immunohistochemistry method, HIF-1 alpha specific siRNA decreased the expression of retinal HIF-1 alpha in diabetic rats, which is expected to inhibit the the target gene VEGF promotes the expression of angiogenic factors, inhibit retinal neovascularization, become a new approach to the treatment of DR.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R587.2;R774.1

【参考文献】