【摘要】：目的:(1)通過熒光素眼底血管造影和視網(wǎng)膜眼底血管內(nèi)皮細(xì)胞CD105免疫組化來檢測3組試驗(yàn)犬增殖性糖尿病視網(wǎng)膜病變的成模率,探究VEGF對犬PDR形成的作用。(2)通過相對熒光定量PCR方法測定3組試驗(yàn)犬玻璃體內(nèi)TGF-β1 m RNA相對表達(dá)量,探究VEGF對玻璃體內(nèi)TGF-β1表達(dá)的影響。方法:(1)選取身體健康狀況大致相同的中華田園犬,建造糖尿病模型犬,隨機(jī)分成S1和S2兩組,每組各5只。其中S1組雙眼玻璃體腔內(nèi)注射VEGF(0.2 ug/kg);S2組雙眼玻璃體腔內(nèi)注射等量生理鹽水;另外隨機(jī)挑選5只健康犬雙眼玻璃體腔內(nèi)注射等量生理鹽水,作為空白對照,設(shè)為S3組。(2)玻璃體腔內(nèi)注射后,連續(xù)8 w,每周對3組試驗(yàn)犬進(jìn)行熒光素眼底血管造影雙眼檢查,觀察并記錄各組試驗(yàn)犬眼底血管滲漏情況,在玻璃體腔內(nèi)注射后第8 w,采集各組試驗(yàn)犬左眼相同部位的視網(wǎng)膜組織,制成病理切片,進(jìn)行CD105免疫組織化學(xué)染色,顯微鏡下觀察記錄各組免疫組織化學(xué)染色情況。(3)玻璃體腔內(nèi)注射后第8 w,采集各組犬左眼玻璃體2 m L,移入離心管中液氮保存。通過相對熒光定量PCR的方法測定記錄各組試驗(yàn)犬玻璃體內(nèi)TGF-β1m RNA相對表達(dá)量。(4)最后通過Spass分析軟件進(jìn)行數(shù)據(jù)分析。試驗(yàn)結(jié)果:(1)熒光素眼底血管造影檢查顯示,S1組在第5 w時(shí),1只試驗(yàn)犬眼底血管開始出現(xiàn)輕微滲漏,到第8 w時(shí),S1組試驗(yàn)犬眼底血管均出現(xiàn)熒光滲漏。而S2與S3組,在8 w時(shí)間內(nèi),眼底血管均未出現(xiàn)熒光滲漏。(2)通過光學(xué)顯微鏡鏡觀察各組試驗(yàn)犬視網(wǎng)膜石蠟切片,S1組血管內(nèi)皮細(xì)胞CD105免疫組化染色均為陽性,CD105陽性表達(dá)率為100%;S2與S3組血管內(nèi)皮細(xì)胞CD105免疫組化染色均為陰性,CD105陽性表達(dá)率為0%。(3)通過相對熒光定量PCR的方法測定出:S1組玻璃體內(nèi)TGF-β1m RNA的表達(dá)量極顯著高于S2和S3組,是S2組的3.180倍(p0.01),S3組的10.657倍(p0.01)。S2組玻璃體內(nèi)TGF-β1m RNA的表達(dá)量顯著高于S3組,是S3組的3.351倍(p0.05)。說明S1組玻璃體內(nèi)TGF-β1 m RNA表達(dá)量最高,S2組次之,S3組最低。結(jié)論:(1)在糖尿病模型犬的基礎(chǔ)上,外源性的玻璃體注射VEGF能夠增大眼底視網(wǎng)膜血管壁的通透性,加速血管的滲漏,可以促進(jìn)犬增殖性糖尿病視網(wǎng)膜病變的形成。(2)VEGF可以顯著提高玻璃體內(nèi)TGF-β1 m RNA的表達(dá)量,有正向調(diào)控作用,加快了視網(wǎng)膜新生血管形成。
[Abstract]:Objective: (1) to detect the model forming rate of proliferative diabetic retinopathy in three groups by fluorescein fundus angiography and CD105 immunohistochemistry. To investigate the effect of VEGF on the formation of PDR in dogs. (2) to determine the relative expression of TGF- 尾 1 m RNA in vitreous of three groups of dogs by relative fluorescence quantitative PCR, and to explore the effect of VEGF on the expression of TGF- 尾 1 in vitreous. Methods: (1) Chinese idyllic dogs with similar health status were selected to construct diabetic model dogs. The dogs were randomly divided into S1 and S2 groups with 5 dogs in each group. In S1 group, VEGF (0.2 ug/kg) and S2 group were injected intravitreous with the same amount of saline, and 5 healthy dogs were randomly selected to inject the same amount of saline into the binocular vitreous cavity as the blank control. (2) after intravitreal injection, three groups of dogs were examined with fluorescein fundus angiography every week for 8 weeks, and the fundus vascular leakage was observed and recorded in each group. At the 8th week after intravitreal injection, the retinal tissues in the same part of the left eye of the dogs in each group were collected and made into pathological sections. CD105 immunohistochemical staining was performed. The immunohistochemical staining of each group was observed under microscope. (3) at the 8th week after intravitreous injection, the vitreous body of the left eye of each group was collected and transplanted into the centrifuge tube to preserve liquid nitrogen. The relative expression of TGF- 尾 1m RNA in vitreous of experimental dogs was measured by relative fluorescence quantitative PCR. (4) finally, the data were analyzed by Spass software. Results: (1) fundus fluorescein angiography showed that the fundus vessels of 1 dog in the S _ 1 group began to leak slightly at the 5th week, and the fundus vessels of all the dogs in the S _ 1 group showed fluorescence leakage at the 8th week. In S2 and S3 groups, within 8 weeks, No fluorescein leakage was found in the fundus vessels. (2) the positive rate of CD105 expression of vascular endothelial cells was 100% in S _ 2 and S _ 3 groups by optical microscopy in S _ 1 group and S _ 1 group. The positive expression rate of CD105 was 0. (3) the expression of TGF- 尾 1m RNA in vitreous of group 1 was significantly higher than that in group S2 and S3 by relative fluorescence quantitative PCR. The expression of TGF- 尾 1m RNA in vitreous of S2 group was 10.657 times as much as that of S3 group (p0.01). The expression of TGF- 尾 1m RNA in S3 group was significantly higher than that in S3 group and 3.351 times higher than that in S3 group (p0.05). The results showed that the expression of TGF- 尾 1 m RNA was the highest in S1 group and the lowest in S2 group, followed by S3 group. Conclusion: (1) on the basis of diabetic model dogs, exogenous vitreous injection of VEGF can increase the permeability of retinal vascular wall and accelerate the leakage of blood vessels. (2) VEGF can significantly increase the expression of TGF- 尾 1 m RNA in vitreous body, which has positive regulation effect and accelerate the formation of retinal neovascularization.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S858.292

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