本文選題：小分子RNA + 基因表達　； 參考：《第二軍醫(yī)大學》2012年博士論文

【摘要】：視網(wǎng)膜新生血管性疾病，如糖尿病性視網(wǎng)膜病變、視網(wǎng)膜靜脈阻塞和早產(chǎn)兒視網(wǎng)膜病變等均會導致視網(wǎng)膜新生血管化。目前視網(wǎng)膜新生血管性疾病，是世界范圍內(nèi)最嚴重的致盲性眼病之一，在發(fā)達國家已上升為致盲性眼病的首要原因。隨著我國人民生活水平提高，眼底新生血管性疾病也日漸成為威脅我國人民視力健康的重要原因。視網(wǎng)膜新生血管的形成可引起玻璃體出血、牽拉性視網(wǎng)膜脫離和新生血管性青光眼，其發(fā)病機制并未完全清楚。但近年來生長因子在視網(wǎng)膜新生血管形成中的作用已形成共識。局部組織新生血管刺激因子和抑制因子動態(tài)平衡的失調(diào)是產(chǎn)生新生血管的關(guān)鍵。已發(fā)現(xiàn)的刺激血管生長的因子包括：血管內(nèi)皮生長因子(vascular endothelial growthfactor，VEGF)、成纖維細胞生長因子(fibroblast growth factor，F(xiàn)GF)、白細胞介素8(interlukin-8)等。已發(fā)現(xiàn)的抑制血管生長的因子有轉(zhuǎn)化生長因子(transforming growthfactor beta，TGF-β)和血栓素(thrombospondin)等。VEGF是視網(wǎng)膜新生血管形成最主要的生長因子。雖然視網(wǎng)膜新生血管形成的機制尚未完全明了，許多調(diào)節(jié)因素也仍然不清，但是許多研究表明，抑制VEGF的表達，可以減少視網(wǎng)膜新生血管的形成。 microRNA (miRNA)是一種長度約22nt的內(nèi)源性，非編碼單鏈小分子RNA，與靶基因mRNA的3′非翻譯區(qū)進行堿基互補配對后，發(fā)揮對mRNA的降解作用或抑制mRNA的翻譯，從而對基因進行轉(zhuǎn)錄后表達的調(diào)控。大約30%的蛋白質(zhì)編碼基因是由miRNA調(diào)控的。miRNA在細胞的增殖，分化和凋亡以及所報道的所有生物學進程中都發(fā)揮重要的作用。microRNA已經(jīng)在腫瘤、心血管等方面研究取得了一定的進展，其在眼科的應用也引起了學者們的興趣，有研究發(fā)現(xiàn)在視網(wǎng)膜和眼部其他組織中，microRNA的表達具有組織特異性和發(fā)育階段特異性，提示其在視網(wǎng)膜和眼部其他組織中具有潛在的組織和細胞的功能特異性。 本實驗通過構(gòu)建小鼠microRNA-410質(zhì)粒載體，以高氧誘導的小鼠視網(wǎng)膜新生血管模型為實驗對象，將構(gòu)建好的濃縮質(zhì)粒通過以下兩種不同的方式：1.玻璃體腔注射濃縮質(zhì)粒入小鼠眼球，5天后取材觀察療效。2.將濃縮質(zhì)粒制成眼藥水，每天對實驗動物局部點眼藥水觀察治療效果，持續(xù)5天。最后研究結(jié)果表明microRNA-410可通過下調(diào)VEGF的表達抑制血管內(nèi)皮細胞的增殖，減少視網(wǎng)膜新生血管數(shù)量，從而阻礙新生血管的形成。 第一部分miRNA-410質(zhì)粒載體的構(gòu)建及驗證 目的構(gòu)建miRNA-410質(zhì)粒載體，并在細胞水平驗證其對VEGF的抑制作用 方法構(gòu)建miRNA-410重組表達質(zhì)粒（pLKO.1-miRNA-410）；采用脂質(zhì)體法分別將表達質(zhì)粒和空質(zhì)粒轉(zhuǎn)入人臍靜脈內(nèi)皮細胞(HUVEC)。轉(zhuǎn)染后采用RT-PCR法檢測轉(zhuǎn)染后重組質(zhì)粒的表達情況；實時定量PCR及western blot檢測VEGF表達的改變。 結(jié)果成功構(gòu)建了miRNA-410重組質(zhì)粒。轉(zhuǎn)染了pLKO.1-miRNA-410的HUVEC中VEGF表達較轉(zhuǎn)染pLKO.1-mock中的表達明顯下調(diào)(p0.05),轉(zhuǎn)染了pLKO.1-miRNA-410-antisense的HUVEC中VEGF表達較轉(zhuǎn)染pLKO.1-mock中的表達明顯上調(diào)(p0.05)。 結(jié)論成功構(gòu)建了miRNA-410重組質(zhì)粒，并在細胞水平驗證其對VEGF的抑制作用。 第二部分可定量的氧誘導小鼠視網(wǎng)膜新生血管模型的建立及VEGFmRNA的動態(tài)表達 目的建立可對視網(wǎng)膜新生血管進行定量研究的動物模型，為下一步研究視網(wǎng)膜新生血管發(fā)生機制及治療提供實驗基礎。 方法選擇健康7日齡(P7)C57BL/6J小鼠，雌雄不限。共16窩100只，隨機分為高氧組(OIR組)和對照組(C組)。高氧組：將50只7日齡的C57BL／6J小鼠（P7）連同母鼠放在濃度為(75±2)％的氧濃度環(huán)境中，5天后回到正常空氣中，作為氧誘導模型組；對照組：另50只同日齡新生小鼠置于正�？諝猸h(huán)境中生活，作為正常對照組。兩組小鼠分別在P13、P15、P17、P19、P23分批處死，摘除眼球，通過FITC-Dextran熒光造影視網(wǎng)膜鋪片觀察視網(wǎng)膜新生血管形態(tài)變化；組織切片觀察并計數(shù)矢狀面5mm視網(wǎng)膜切片中突破內(nèi)界膜的血管內(nèi)皮細胞核數(shù)，，反映視網(wǎng)膜血管的增生情況；real-time RT-PCR檢測視網(wǎng)膜中VEGFmRNA的動態(tài)表達。 結(jié)果熒光素血管灌注法視網(wǎng)膜鋪片結(jié)果：正常組小鼠視網(wǎng)膜血管分布呈均勻的網(wǎng)狀，未見新生血管和無灌注區(qū)。高氧誘導組小鼠回到正常空氣環(huán)境1天（P13）后，視網(wǎng)膜開始出現(xiàn)新生血管；到空氣中3天（P15）后，視網(wǎng)膜新生血管增多明顯，到空氣中5天（P17）后，視網(wǎng)膜新生血管形成達到高峰，到空氣中7天（P19）后，視網(wǎng)膜新生血管逐漸減少，到空氣中12天(P23)后，視網(wǎng)膜新生血管消退。視網(wǎng)膜切片結(jié)果：突破視網(wǎng)膜內(nèi)界膜的血管內(nèi)皮細胞數(shù)，在P13.P15.P17.P19.P23氧誘導的模型組分別為(1.39±1.12個)、（16.67±3.51個）、（42.31±4.69個）、（39.23±4.5個）、（0.93±0.85個），正常對照組分別為（0.96±0.91個）、（0.94±0.92個）、(0.89±0.91個)、（0.9±0.89個）、（0.89±0.97個），二者相比差異有顯著意義(p0.05)。高氧誘導模型組各時間點視網(wǎng)膜組織中的VEGFmRNA表達水平較同時間點對照組明顯上調(diào)，二者相比差異有顯著意義(p0.01)。 結(jié)論該動物模型成模率高，可重復性好，并可進行定量研究，是進行小鼠視網(wǎng)膜新生血管發(fā)生機制和藥物治療研究的合適模型。RT-PCR結(jié)果顯示在氧誘導增殖性視網(wǎng)膜病變小鼠視網(wǎng)膜組織中VEGFmRNA表達明顯上調(diào)，其變化趨勢與視網(wǎng)膜新生血管形成相對應。 第三部分miRNA-410抑制小鼠視網(wǎng)膜新生血管形成的研究 目的觀察miRNA-410對視網(wǎng)膜新生血管形成的抑制作用，進一步闡明miRNA-410抑制視網(wǎng)膜新生血管形成中的調(diào)控作用。 方法選擇健康7日齡(P7)C57BL/6J小鼠，雌雄不限。共10窩共60只C57BL／6J小鼠。隨機取10只在正常氧環(huán)境下飼養(yǎng)的C57BL／6J小鼠為正常對照組。剩余50只鼠齡為7天的C57BL／6J小鼠置于濃度為(75±2)％高氧環(huán)境中生活5天，在P12返回正常氧環(huán)境中。在高氧處理過的50只小鼠中，隨機取其中10只，不做任何藥物處理，作為高氧誘導模型組。剩余40只從高氧環(huán)境中取出的小鼠，隨機取10只小鼠于出氧艙后每日右眼局部點pLKO.1-miRNA-410眼藥水，2/日,作為pLKO.1-miRNA-410眼藥水組。隨機取10只小鼠于出氧艙后每日右眼局部點pLKO.1-mock眼藥水，2/日，作為pLKO.1-mock眼藥水組。10只小鼠于出氧艙后當日右眼玻璃體腔內(nèi)注射0.4μlpLKO.1-miRNA-410，作為眼內(nèi)注射pLKO.1-miRNA-410組，10只小鼠于出氧艙后當日右眼玻璃體腔內(nèi)注射0.4μl pLKO.1-mock，作為眼內(nèi)注射pLKO.1-mock組。以上5組小鼠的左眼均不做外源性處理。所有小鼠在P17時，在麻醉狀態(tài)下取材，組織學切片觀察突破視網(wǎng)膜內(nèi)界膜的血管內(nèi)皮細胞核數(shù)量；FITC-Dextran熒光造影視網(wǎng)膜鋪片了解視網(wǎng)膜血管形態(tài)的改變；RT-PCR檢測視網(wǎng)膜組織中VEGFmRNA表達水平。 結(jié)果組織學切片結(jié)果表明：高氧誘導組突破視網(wǎng)膜內(nèi)界膜的血管內(nèi)皮細胞核數(shù)與正常組比較差異有顯著性(p0.05)；局部使用pLKO.1-miRNA-410眼藥水組和玻璃體腔注射pLKO.1-miRNA-410組與高氧誘導組比較差異有顯著性(p0.05)，與正常組比較差異無顯著性(p＞0.05)；局部使用pLKO.1-miRNA-410眼藥水組與局部使用pLKO.1-miRNA-mock眼藥水組比較差異有顯著性(p0.05)；玻璃體腔注射pLKO.1-miRNA-410組與玻璃體腔注射pLKO.1-miRNA-mock組比較差異有顯著性(p0.05)；局部使用pLKO.1-miRNA-410眼藥水組和玻璃體腔注射pLKO.1-miRNA-410組與高氧誘導組比較差異無顯著性(p＞0.05) 視網(wǎng)膜鋪片結(jié)果顯示：玻璃體腔注射pLKO.1-miRNA-410組較高氧誘導模型組、玻璃體腔內(nèi)注射pLKO.1-mock組新生血管叢明顯減少，滲漏明顯減輕；局部使用pLKO.1-miRNA-410眼藥水組較高氧誘導模型組、玻璃體腔注射pLKO.1-miRNA-410組、玻璃體腔內(nèi)注射pLKO.1-mock組和局部pLKO.1-mock眼藥水組新生血管叢明顯減少，熒光滲漏最輕。 視網(wǎng)膜組織VEGFmRNA水平檢測：1.玻璃體腔注射pLKO.1-miRNA-410組與玻璃體腔注射pLKO.1-mock相比，VEGFmRNA表達降低。差異有顯著性（p0.05）2.局部使用pLKO.1-miRNA-410眼藥水組與局部使用pLKO.1-mock眼藥水組相比，VEGFmRNA表達降低。差異有顯著性（p0.05）3.局部使用pLKO.1-miRNA-410眼藥水組與玻璃體腔注射pLKO.1-miRNA-410組相比，VEGFmRNA表達降低。差異無顯著性（p＞0.05） 結(jié)論miRNA-410可有效抑制視網(wǎng)膜新生血管的形成，進一步證明了miRNA-410在視網(wǎng)膜新生血管形成中所起的調(diào)控作用,同時為血管增生性視網(wǎng)膜病變的治療提供了新的途徑。
[Abstract]:Retinal neovascularization, such as diabetic retinopathy, retinal vein occlusion and retinopathy of prematurity, can all lead to neovascularization of the retina. At present, retinal neovascularization is one of the most serious blindness diseases in the world, and is the primary cause of blindness in the country of hair. With the improvement of the living standard of the people in our country, the new vascular diseases in the fundus have become an important cause of the people's vision health. The formation of retinal neovascularization can cause vitreous hemorrhage, traction retinal detachment and neovascular glaucoma, its pathogenesis is not completely clear. However, the growth factor has been in the optic network in recent years. A consensus has been made on the role of neovascularization in the membrane. The maladjustment of the dynamic balance of neovascularization and inhibitory factors in local tissue is the key to the production of neovascularization. The factors that have been found to stimulate vascular growth include vascular endothelial growth factor (vascular endothelial growthfactor, VEGF), fibroblast growth factor (fibrob Last growth factor, FGF), interleukin 8 (interlukin-8), and so on. The factors that have been found to inhibit vascular growth are transforming growth factors (transforming growthfactor beta, TGF- beta) and thromboxane (thrombospondin), etc..VEGF is the most important growth factor in retinal neovascularization. Although the mechanism of retinal neovascularization has not been completed yet Although many regulatory factors are still unclear, many studies have shown that inhibiting the expression of VEGF can reduce the formation of retinal neovascularization.
MicroRNA (miRNA) is an endogenous, non coded single strand small molecule RNA, which is paired with the 3 'non translation region of the target gene mRNA to play a role in the degradation of mRNA or to inhibit the translation of mRNA, thus regulating the post transcriptional expression of the gene. A large about 30% of the protein encoding genes are.MiRNA regulated by.MiRNA. The important role of.MicroRNA in cell proliferation, differentiation and apoptosis, as well as all the biological processes reported, has made some progress in the research of tumor, cardiovascular and other aspects. Its application in the ophthalmology has also aroused the interest of scholars. Studies have found that the expression of microRNA in the retina and other tissues of the eye has been found. Tissue-specific and developmental stage specificity suggests that it has potential tissue and cell function specificity in the retina and other tissues of the eye.
In this experiment, the mouse microRNA-410 plasmid vector was constructed and the mouse retinal neovascularization model induced by hyperoxia was used as the experimental object. The condensed plasmids were constructed in two different ways: 1. vitreous cavity was injected into the eye of the mice by injection of plasmids, and the concentrated plasmid was made into eye drops after 5 days. The effect of local eye drops for 5 days was observed. The results showed that microRNA-410 could inhibit the proliferation of vascular endothelial cells by down regulating the expression of VEGF and reduce the number of retinal neovascularization, thus hindering the formation of neovascularization.
Part 1 construction and verification of miRNA-410 plasmid vector
Objective to construct miRNA-410 plasmid vector and to verify its inhibitory effect on VEGF at cellular level.
Methods the recombinant expression plasmid (pLKO.1-miRNA-410) of miRNA-410 was constructed, and the expression plasmid and empty plasmid were transferred into human umbilical vein endothelial cells (HUVEC) by liposome method. The expression of the recombinant plasmid was detected by RT-PCR method, and the changes of VEGF expression were detected by real-time quantitative PCR and Western blot.
Results the recombinant plasmid of miRNA-410 was successfully constructed. The expression of VEGF in the transfected HUVEC was significantly lower than that in the transfected pLKO.1-mock (P0.05). The expression of VEGF in pLKO.1-miRNA-410-antisense's HUVEC was obviously up regulated than that in the transfected pLKO.1-mock (P0.05).
Conclusion miRNA-410 recombinant plasmid was successfully constructed, and its inhibitory effect on VEGF was verified at cell level.
The second part is the establishment of quantitative oxygen induced retinal neovascularization in mice and the dynamic expression of VEGFmRNA.
Objective to establish an animal model for quantitative study of retinal neovascularization, and to provide an experimental basis for the next study of the mechanism and treatment of retinal neovascularization.
Methods the healthy 7 days old (P7) C57BL/6J mice were selected and 16 litters and 100 rats were randomly divided into hyperoxia group (group OIR) and control group (group C). 50 7 day old C57BL / 6J mice (P7) were placed in the oxygen concentration environment with a concentration of (75 + 2)% in the concentration of (75 + 2)%), and returned to the normal air after 5 days as the oxygen induced model group; the control group: the control group: 50 newborn mice of the same age were placed in the normal air environment as the normal control group. The two groups were killed in P13, P15, P17, P19, P23, respectively. The eyeball was removed and the retinal neovascularization was observed by the FITC-Dextran fluorescein retina sheet. The tissue sections were observed and counted in the sagittal 5mm retina slices. The number of endothelial cells in the inner limiting membrane reflects the proliferation of retinal vessels, and the dynamic expression of VEGFmRNA in the retina is detected by real-time RT-PCR.
Results the retinal vasculature of fluorescein perfusion method showed that the retinal vessels in the normal group showed a uniform network, no neovascularization and no perfusion area. After 1 days (P13) of the high oxygen induction group, the retina began to appear in the retina. After 3 days (P15) in the air, the neovascularization of the retina was increased obviously. After 5 days (P17) in the air, the formation of retinal neovascularization reached its peak. After 7 days (P19) in the air, the neovascularization of the retina decreased gradually. After 12 days (P23) in the air, the neovascularization of the retina subsided. The retinal section was broken through the number of vascular endothelial cells in the inner retina membrane, and the model group induced by P13.P15.P17.P19.P23 oxygen was respectively (1.39 + 1.12), (16.67 + 3.51), (42.31 + 4.69), (39.23 + 4.5), (0.93 + 0.85), normal control group (0.96 + 0.91), (0.94 + 4.69), (4.69), compared with the difference (P0.05). The expression of the retinal tissue at each time point in the hyperoxic induced model group The level of the control group was significantly higher than that of the control group at the same time point, and the difference between the two groups was significant (P0.01).
Conclusion the model rate is high, the reproducibility is good, and the quantitative study can be carried out. It is a suitable model for the study of the mechanism of retinal neovascularization and the study of drug therapy in mice retina. The result of.RT-PCR shows that VEGFmRNA surface Daming in retinal tissue of mice with oxygen induced proliferative retinopathy is up to be up-regulated. The formation of blood vessels should be corresponding.
The third part of miRNA-410 inhibits retinal neovascularization in mice.
Objective To observe the inhibitory effect of miRNA-410 on retinal neovascularization, and further clarify the regulatory role of miRNA-410 in inhibiting retinal neovascularization.
Methods the healthy 7 days old (P7) C57BL/6J mice were selected and male and female were not limited. A total of 10 nests and 60 C57BL / 6J mice were selected. 10 C57BL / 6J mice fed in normal oxygen environment were randomly selected as normal control group. The remaining 50 mice aged 7 days of C57BL / 6J mice were placed in the concentration of (75 + 2)% hyperoxic environment for 5 days, and returned to normal oxygen environment in P12. Of the 50 mice treated with hyperoxia, 10 were taken randomly, without any treatment, as a hyperoxic induction model group. The remaining 40 mice removed from the hyperoxic environment were randomly selected from 10 mice in the oxygen chamber and at the local point of pLKO.1-miRNA-410 eye daily in the right eye, and 2/ days as the pLKO.1-miRNA-410 eye drops. 10 mice were randomly selected. After the oxygen chamber, pLKO.1-mock eye drops in the right eye, 2/ day, as pLKO.1-mock eye drops,.10 mice were injected with 0.4 mu lpLKO.1-miRNA-410 in the right eye glass cavity of the right eye on the day after the oxygen capsule, as the intraocular injection pLKO.1-miRNA-410 group, and the 10 mice were injected with 0.4 Mu L pLKO.1-mock in the right eye glass cavity on the same day after the oxygen chamber. The left eye of the 5 groups of mice were not treated with exogenous treatment. All the mice in the above 5 groups were not treated with exogenous treatment. All the mice were harvested in the state of anesthesia at P17. The number of vascular endothelial nuclei in the inner boundary membrane of the retina was observed by histological section, and the changes of the retinal vascular morphology of the retina were detected by FITC-Dextran fluorescein angiography; RT-PCR was used to detect the retina. The expression level of VEGFmRNA in the tissue.
Results the results of histological section showed that the number of vascular endothelial nuclei in the hyperoxia induced group was significantly different from that of the normal group (P0.05), and the ratio of the local use of pLKO.1-miRNA-410 eye drug group and the intravitreal injection pLKO.1-miRNA-410 group with the hyperoxia induction group was significant (P0.05), and the difference was worse than that of the normal group. There was no significant difference (P > 0.05); there was a significant difference between the local use of pLKO.1-miRNA-410 eye drops and the local use of pLKO.1-miRNA-mock eye drops (P0.05), and there was a significant difference between the pLKO.1-miRNA-410 group and the pLKO.1-miRNA-mock group injected with the vitreous cavity (P0.05), and the local use of pLKO.1-miRNA-410 eye medicine group (P0.05). There was no significant difference between pLKO.1-miRNA-410 group and intravitreal injection group (P > 0.05).
The results of retina spread showed that the hyperoxia induced model group was injected into the pLKO.1-miRNA-410 group by injection of the vitreous cavity, and the new vascular plexus of the pLKO.1-mock group was reduced and the leakage was lightened. The high oxygen induction group in the pLKO.1-miRNA-410 eye medicine group, the pLKO.1-miRNA-410 group injected with the glass cavity, the intravitreal injection in the vitreous cavity. The neovascularization group of pLKO.1-mock group and local pLKO.1-mock eye drops group decreased significantly and fluorescence leakage was lightest.
VEGFmRNA level detection in retinal tissue: 1. the expression of VEGFmRNA in group pLKO.1-miRNA-410 injected with vitreous cavity was lower than that of intravitreal injection of pLKO.1-mock. The difference was significant (P0.05) 2. local use of pLKO.1-miRNA-410 eye medicine group and local use of pLKO.1-mock ophthalmic water group, the expression of VEGFmRNA decreased. The difference was significant (P0.05) 3. Compared with intravitreal injection of pLKO.1-miRNA-410, the expression of VEGFmRNA in local pLKO.1-miRNA-410 eye drops was not significantly different (P > 0.05).
Conclusion miRNA-410 can effectively inhibit the formation of retinal neovascularization, and further demonstrate the regulatory role of miRNA-410 in the formation of retinal neovascularization, and provide a new way for the treatment of vascular proliferative retinopathy.

【學位授予單位】：第二軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R774.1

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相關(guān)期刊論文 前2條

1 孔祥梅;孫興懷;俞道義;郭文毅;姜春暉;;眼科常用實驗動物視網(wǎng)膜血管的比較[J];中國實驗動物學報;2007年06期

2 孔怡淳;韓梅;趙堪興;李寧東;林錦鏞;;小鼠視網(wǎng)膜新生血管模型熒光素灌注造影[J];中華眼科雜志;2006年07期

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