基因轉(zhuǎn)染的虹膜色素上皮細胞治療RCS大鼠視網(wǎng)膜色素變性的實驗研究
發(fā)布時間：2018-08-31 20:06
【摘要】：遺傳性視網(wǎng)膜變性是一組以進行性光感受器細胞及色素上皮細胞功能喪失為共同表現(xiàn)的疾病。目前為止,這些疾病尚無有效治療方法[1, 2]。全世界有數(shù)百萬人因此永久致盲,危害性極大。臨床上迫切需要有效措施以改善和維護RPE功能,防止感光細胞變性及凋亡,這是多年來全世界范圍內(nèi)一大熱點和難點。目前發(fā)現(xiàn)腦源性神經(jīng)營養(yǎng)因子(brain derived neurotrophic factor, BDNF)能促進培養(yǎng)的人和牛的視網(wǎng)膜色素上皮(retinal pigment epithelium, RPE)細胞分化,通過自分泌或通過膠質(zhì)細胞調(diào)節(jié)視網(wǎng)膜變性中感光細胞的生存,使視網(wǎng)膜細胞存活[3, 4, 5]。 本研究擬在視網(wǎng)膜變性的RCS(Royal College of Surgeons)鼠早期進行干預,將BDNF基因轉(zhuǎn)染的虹膜色素上皮細胞(iris pigment epitheium,IPE)(AAV-BDNF-IPE)移植入早期RCS大鼠視網(wǎng)膜下腔后,觀察不同時期視網(wǎng)膜電圖(electroretinogram, ERG)、視網(wǎng)膜組織BDNF表達及視網(wǎng)膜形態(tài)變化,分析移植后RCS鼠視網(wǎng)膜改善情況,為臨床對由于RPE功能障礙以及感光細胞變性引起的疾病患者提供更多的思路。 方法本研究通過以下四部分進行: 第一部分:出生后18-22天(3周齡)RCS大鼠48只,雌雄不拘。通過改進的鞏膜外路移植法將AAV-BDNF-IPE細胞移植到RCS大鼠雙眼視網(wǎng)膜下腔,術(shù)后觀察眼底情況,并行HE染色,觀察移植細胞的存活情況。 第二部分:SD正常對照大鼠,按照出生后3周齡、6、8、10、12、14周齡分組,每周齡組3只,共18只,RCS大鼠及AAV-BDNF-IPE細胞移植RCS大鼠(已在第一部分實驗中完成AAV-BDNF-IPE細胞移植)各18只,按出生后周齡分組同SD大鼠,分別行雙眼暗適應ERG記錄,電生理檢查參照國際臨床視覺電生理標準。每次每眼至少檢查3次,疊加后取波形穩(wěn)定、干擾小的ERG圖形,計算ERG-a、b波振幅值并進行統(tǒng)計學處理。 第三部分:對照組RCS大鼠按照出生后3周齡、6、8、10、12、14周齡分組,每周齡組4只,共24只;AAV-BDNF-IPE細胞移植RCS大鼠(已在第一部分實驗中完成AAV-BDNF-IPE細胞移植)24只,按出生后周齡分組同對照組RCS大鼠。分別于相應時間點即手術(shù)當天、術(shù)后3、5、7、9、11周取雙眼球,用酶聯(lián)免疫吸附法(Elisa)檢測視網(wǎng)膜組織中BDNF的表達水平,比較分析這些數(shù)據(jù)并進行統(tǒng)計學處理。 第四部分:實驗動物及分組同第二部分,行電生理檢測后處死大鼠,取出雙眼球,取視網(wǎng)膜組織進行HE染色、TUNEL染色和免疫組化染色檢測。 結(jié)果 1.共計43只RCS大鼠實施了雙眼視網(wǎng)膜下腔移植,發(fā)生大量玻璃體積血、廣泛視網(wǎng)膜脫離、眼內(nèi)炎及誤注入玻璃體腔18眼。AAV-BDNF-IPE細胞成功移植入視網(wǎng)膜下腔68眼,手術(shù)成功率79%。 RCS大鼠視網(wǎng)膜及睫狀體部位HE染色發(fā)現(xiàn),睫狀體及視網(wǎng)膜均缺乏色素細胞層;AAV-BDNF-IPE細胞移植到RCS大鼠視網(wǎng)膜下腔5周后,HE染色可見大鼠視網(wǎng)膜下腔有散在存活的褐色上皮細胞,推測為移植后存活的AAV-BDNF-IPE細胞;視網(wǎng)膜或脈絡膜內(nèi)未見明顯炎性細胞浸潤反應及結(jié)構(gòu)破壞(Fig.6)。 2.三組大鼠成功完成暗適應FERG記錄,從出生后第3周到第14周,共記錄正常SD大鼠15只30次,未干預對照RCS大鼠15只30次,細胞移植組RCS大鼠17只34次。 三組大鼠生后8周內(nèi)FERG的暗適應最大反應a、b波振幅無明顯差別,出生10周以后RCS大鼠FERG的暗適應最大反應a、b波振幅均明顯降低,與同周齡正常SD大鼠相比顯著下降(P㩳0.05),出生12周以后RCS大鼠ERG波形呈低平型,未見明顯波峰波谷,a、b波模糊。AAV-BDNF-IPE移植手術(shù)組RCS大鼠出生后10周齡時b波仍保持較高水平,盡管細胞移植組RCS大鼠b波呈現(xiàn)緩慢下降趨勢,但振幅仍顯著高于同周齡未干預對照組RCS大鼠(12w和14w組P0.05)。 3.對照組RCS大鼠出生后3周齡時視網(wǎng)膜組織中BDNF仍保持較高水平,其后迅速降低,其中3周齡組與其它周齡組比較,P0.01;手術(shù)組RCS大鼠術(shù)時、術(shù)后3、5、7、9、11周各組間兩兩比較,BDNF表達無顯著差異(P㧐0.05);出生后6周齡直到14周齡的不同時期,AAV-BDNF-IPE移植手術(shù)組RCS大鼠視網(wǎng)膜BDNF表達水平均明顯高于對照組(其中6周齡組P0.05,其它各周齡組P0.01)。 4. HE染色結(jié)果示:AAV-BDNF-IPE移植手術(shù)組和對照組相比視桿層靠近外核層的一側(cè)厚度略有改善;TUNEL結(jié)果:實驗組凋亡指數(shù)減少,凋亡峰值后延,凋亡高峰發(fā)生于移植術(shù)后9wk;免疫組化染色結(jié)果示:實驗組大鼠原外層視網(wǎng)膜部位GFAP呈弱陽性表達,Muller細胞著色較淺;SYN除內(nèi)叢狀層著色外,外叢狀層呈弱陽性反應。 小結(jié) 1、本實驗采用自制的注射器,經(jīng)單通道鞏膜外路法成功將AAV-BDNF-IPE移植至幼年RCS大鼠視網(wǎng)膜下腔,有效縮小了鞏膜切口,減少了視網(wǎng)膜解剖結(jié)構(gòu)的破壞,手術(shù)成功率達到79%。 RCS大鼠視網(wǎng)膜及睫狀體部位HE染色發(fā)現(xiàn),睫狀體及視網(wǎng)膜均缺乏色素細胞層;AAV-BDNF-IPE細胞移植到RCS大鼠視網(wǎng)膜下腔5周后,HE染色可見大鼠視網(wǎng)膜下腔有散在存活的褐色上皮細胞,推測為移植后存活的AAV-BDNF-IPE細胞;視網(wǎng)膜或脈絡膜內(nèi)未見明顯炎性細胞浸潤反應及結(jié)構(gòu)破壞。 雖然不能判定移植細胞與其宿主視網(wǎng)膜組織間建立了正確的解剖聯(lián)系,但我們的實驗結(jié)果證實,通過改進的鞏膜外路移植法將AAV-BDNF-IPE細胞移植至視網(wǎng)膜下腔,移植細胞不但能夠較長期存活,而且不發(fā)生免疫排斥反應,是安全可靠的。 2、本實驗發(fā)現(xiàn)SD正常對照大鼠、RCS大鼠及AAV-BDNF-IPE細胞移植RCS大鼠三組大鼠生后8周內(nèi)FERG的暗適應最大反應a、b波振幅無明顯差別,出生10周以后RCS大鼠FERG的暗適應最大反應a、b波振幅均明顯降低,與同周齡正常SD大鼠相比顯著下降(P㩳0.05),出生12周以后RCS大鼠ERG波形呈低平型,未見明顯波峰波谷,a、b波模糊,這些結(jié)果證實RCS大鼠視網(wǎng)膜色素變性進展過程中,視網(wǎng)膜功能逐步喪失。RCS大鼠視網(wǎng)膜電圖的改變滯后于視網(wǎng)膜形態(tài)改變。 將AAV-BDNF-IPE細胞移植入RCS大鼠視網(wǎng)膜下腔后,可以明顯改善FERG b波振幅,盡管隨著術(shù)后時間延長,細胞移植組RCS大鼠b波呈現(xiàn)緩慢下降趨勢,但出生后10周齡后b波仍保持相對較高水平,且振幅顯著高于同周齡未干預對照組RCS大鼠(12w和14w組P0.05),提示AAV-BDNF-IPE細胞移植可以在一定程度上延緩RCS大鼠視網(wǎng)膜色素變性的進展,改善部分視網(wǎng)膜功能。 3.對照組RCS大鼠出生后3周齡時視網(wǎng)膜組織中BDNF仍保持較高水平,其后迅速降低;出生后6周齡直到14周齡的不同時期,AAV-BDNF-IPE移植手術(shù)組RCS大鼠視網(wǎng)膜BDNF表達水平均明顯高于對照組。這些結(jié)果表明,BDNF基因轉(zhuǎn)染的虹膜色素上皮細胞在RCS大鼠視網(wǎng)膜下腔移植后,視網(wǎng)膜組織中BDNF可以持續(xù)穩(wěn)定高水平表達,這是AAV-BDNF-IPE移植改善宿主視網(wǎng)膜功能的一個重要原因。 4. AAV-BDNF-IPE細胞移植效果比未干預組略好:視桿層靠近外核層的一側(cè)厚度也略有改善,這與以往的研究一致;TUNEL檢測證實視細胞的喪失是一種凋亡現(xiàn)象,也提示AAV-BDNF-IPE的治療可能是抑制細胞凋亡而發(fā)揮作用的;免疫組化結(jié)果提示可能減輕了代償反應性膠質(zhì)纖維增生,也可能表現(xiàn)了以突觸恢復為特征的超微結(jié)構(gòu)的變化。 綜上所述,該實驗延緩了視網(wǎng)膜變性的進一步發(fā)展,表明這一方法可能拯救變性的光感受器,使之重新恢復功能。
[Abstract]:Hereditary retinal degeneration is a group of diseases characterized by progressive photoreceptor cell and pigment epithelial cell dysfunction. Up to now, there is no effective treatment for these diseases [1,2]. Preventing degeneration and apoptosis of photoreceptor cells has been a hot and difficult issue worldwide for many years. Brain derived neurotrophic factor (BDNF) has been found to promote the differentiation of cultured human and bovine retinal pigment epithelium (RPE) cells through autocrine or glial fineness. The cells regulate the survival of photoreceptors in retinal degeneration, so that retinal cells survive [3, 4, 5].
This study was designed to observe the expression of BDNF and electroretinogram (ERG) in the retina of early RCS rats after transplantation of iris pigment epithelium (IPE) transfected with BDNF gene into the subretinal space of early RCS rats. The morphological changes of the retina and the improvement of the retina in RCS mice after transplantation were analyzed to provide more ideas for the clinical treatment of patients with RPE dysfunction and photoreceptor degeneration.
Methods the study was carried out in the following four parts:
The first part: 48 RCS rats aged from 18 to 22 days after birth, male and female. AAV-BDNF-IPE cells were transplanted into the subretinal space of both eyes of RCS rats by improved scleral transplantation. The fundus of the eyes was observed after operation, and the survival of the transplanted cells was observed by HE staining.
The second part: SD normal control rats were divided into 3 weeks old, 6,8,10,12,14 weeks old, 3 rats in each age group, 18 rats in each age group. RCS rats and 18 RCS rats transplanted with AAV-BDNF-IPE cells (which had been transplanted with AAV-BDNF-IPE cells in the first experiment) were divided into two groups according to the age of birth and SD rats were recorded by binocular dark adaptation ERG. Physical examination refers to the international clinical visual electrophysiology standard. Each eye is examined at least three times. After stacking, ERG patterns with stable waveform and little interference are obtained. ERG-a and b wave amplitudes are calculated and statistically processed.
The third part: The control group RCS rats were divided into 3 weeks old, 6,8,10,12,14 weeks old, 4 rats in each week group, 24 rats in total; 24 RCS rats transplanted with AAV-BDNF-IPE cells (which had been transplanted into AAV-BDNF-IPE cells in the first part of the experiment), and the control group RCS rats were divided into two groups according to the age after birth. The expression of BDNF in retinal tissue was detected by enzyme linked immunosorbent assay (Elisa) at 7,9 and 11 weeks after operation.
The fourth part: The experimental animals and groups were the same as the second part. The rats were sacrificed after electrophysiological examination. The eyeballs were taken out and the retinal tissues were stained with HE, TUNEL and immunohistochemical staining.
Result
1. A total of 43 RCS rats underwent bilateral subretinal cavity transplantation, which resulted in massive vitreous hemorrhage, extensive retinal detachment, endophthalmitis and misinjection into the vitreous cavity in 18 eyes. AAV-BDNF-IPE cells were successfully transplanted into the subretinal cavity in 68 eyes, with a success rate of 79%.
HE staining in the retina and ciliary body of RCS rats showed that there was no pigment cell layer in both ciliary body and retina. After transplantation of AAV-BDNF-IPE cells into the subretinal space of RCS rats for 5 weeks, HE staining showed that there were scattered surviving Brown epithelial cells in the subretinal space of the rats, presumed to be surviving AV-BDNF-IPE cells in the retina or choroid. No obvious inflammatory cell infiltration and structural damage were observed (Fig.6).
2. Three groups of rats successfully completed dark adaptation FERG recording. From the 3rd week to the 14th week after birth, 15 normal SD rats were recorded 30 times, 15 non-intervention RCS rats 30 times, and 17 RCS rats 34 times in cell transplantation group.
There was no significant difference in the amplitudes of a and B waves of the maximum dark adaptation response of FERG among the three groups within 8 weeks after birth. The amplitudes of a and B waves of the maximum dark adaptation response of FERG in RCS rats decreased significantly after 10 weeks of birth compared with normal SD rats of the same age (P?0.05). After 12 weeks of birth, the ERG waveforms of RCS rats showed a low flat pattern, and no obvious peaks, troughs, A and B modes. The B wave of RCS rats in AAV-BDNF-IPE transplantation group remained high at 10 weeks after birth. Although the B wave of RCS rats in cell transplantation group showed a slowly decreasing trend, the amplitude of B wave was still significantly higher than that of RCS rats in non-intervention control group (P 0.05 in 12W and 14W groups).
3. The levels of BDNF in the retina of RCS rats in the control group remained high at 3 weeks after birth, and then decreased rapidly. Compared with other age groups, the levels of BDNF in the retina of RCS rats in the 3-week-old group were P 0.01; there was no significant difference in the expression of BDNF between the two groups at 3, 5, 7, 9 and 11 weeks after operation (P? The expression of BDNF in the retina of RCS rats after BDNF-IPE transplantation was significantly higher than that in the control group (P 0.05 in the 6-week-old group and P 0.01 in the other groups).
4. HE staining showed that the thickness of the optic rod layer near the outer nuclear layer was slightly improved in AAV-BDNF-IPE transplantation group compared with the control group; TUNEL results: the apoptosis index of the experimental group was reduced, the apoptosis peak was delayed, and the apoptosis peak occurred 9 wk after transplantation; immunohistochemical staining showed that GFAP was weakly positive in the original outer retina of the experimental group rats. The expression of Muller cells was relatively pale, while the outer plexiform layer of SYN was weakly positive except for the inner plexiform layer.
Summary
1. AAV-BDNF-IPE was successfully transplanted into the subretinal space of young RCS rats by single-channel scleral approach with a self-made injector. The scleral incision was effectively reduced and the damage of retinal anatomy was reduced. The success rate of the operation was 79%.
HE staining in the retina and ciliary body of RCS rats showed that there was no pigment cell layer in both ciliary body and retina. After transplantation of AAV-BDNF-IPE cells into the subretinal space of RCS rats for 5 weeks, HE staining showed that there were scattered surviving Brown epithelial cells in the subretinal space of the rats, presumed to be surviving AV-BDNF-IPE cells in the retina or choroid. No obvious inflammatory cell infiltration and structural damage were observed.
Although it is not possible to determine the correct anatomical relationship between the transplanted cells and their host retinal tissues, our experimental results confirm that the transplantation of AAV-BDNF-IPE cells into the subretinal space by improved scleral transplantation is safe and reliable, not only for long-term survival, but also for the absence of immune rejection.
2. In this study, we found that there was no significant difference in the amplitude of a and B waves of the maximum dark adaptation response of FERG between the three groups of SD normal control rats, RCS rats and AAV-BDNF-IPE cells transplanted RCS rats within 8 weeks after birth. After 10 weeks of birth, the amplitude of a and B waves of the maximum dark adaptation response of FERG in RCS rats decreased significantly, compared with normal SD rats of the same age (P?0.05). After 12 weeks of birth, the ERG waveforms of RCS rats were low and flat, and there were no obvious peaks and troughs, and a and B waves were blurred. These results confirmed that the retinal function was gradually lost during the development of RPD in RCS rats.
After transplantation of AAV-BDNF-IPE cells into the subretinal space of RCS rats, the amplitude of FERG-b wave could be significantly improved. Although the amplitude of B wave in RCS rats decreased slowly with the prolonged time after transplantation, the amplitude of B wave remained relatively high after 10 weeks of birth, and was significantly higher than that in RCS rats without intervention at the same age (12 weeks and 14 weeks). Group P 0.05), suggesting that AAV-BDNF-IPE cell transplantation can delay the progress of retinitis pigmentosa in RCS rats to some extent and improve some retinal function.
3. BDNF levels in the retina of RCS rats in the control group remained high at 3 weeks after birth, and then decreased rapidly; BDNF expression levels in the retina of RCS rats in the AAV-BDNF-IPE transplantation group were significantly higher than those in the control group from 6 weeks to 14 weeks after birth. After transplantation of RCS rats into the subretinal space, the expression of BDNF in the retina can be maintained at a high level, which is an important reason why AAV-BDNF-IPE transplantation can improve the function of the host retina.
4. The transplantation effect of AAV-BDNF-IPE cells was slightly better than that of non-intervention group: the thickness of rod layer near the outer nuclear layer was also slightly improved, which was consistent with previous studies; TUNEL detection confirmed that loss of optic cells was an apoptotic phenomenon, and suggested that AAV-BDNF-IPE treatment might play a role in inhibiting apoptosis; immunohistochemical results suggested that It may alleviate the compensatory reactive glial fibrillary hyperplasia and may also show the ultrastructural changes characterized by synaptic recovery.
In summary, this experiment delayed the further development of retinal degeneration, suggesting that this method may save degenerated photoreceptors and restore their function.
【學位授予單位】：福建醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R774.1
本文編號：2216023
[Abstract]:Hereditary retinal degeneration is a group of diseases characterized by progressive photoreceptor cell and pigment epithelial cell dysfunction. Up to now, there is no effective treatment for these diseases [1,2]. Preventing degeneration and apoptosis of photoreceptor cells has been a hot and difficult issue worldwide for many years. Brain derived neurotrophic factor (BDNF) has been found to promote the differentiation of cultured human and bovine retinal pigment epithelium (RPE) cells through autocrine or glial fineness. The cells regulate the survival of photoreceptors in retinal degeneration, so that retinal cells survive [3, 4, 5].
This study was designed to observe the expression of BDNF and electroretinogram (ERG) in the retina of early RCS rats after transplantation of iris pigment epithelium (IPE) transfected with BDNF gene into the subretinal space of early RCS rats. The morphological changes of the retina and the improvement of the retina in RCS mice after transplantation were analyzed to provide more ideas for the clinical treatment of patients with RPE dysfunction and photoreceptor degeneration.
Methods the study was carried out in the following four parts:
The first part: 48 RCS rats aged from 18 to 22 days after birth, male and female. AAV-BDNF-IPE cells were transplanted into the subretinal space of both eyes of RCS rats by improved scleral transplantation. The fundus of the eyes was observed after operation, and the survival of the transplanted cells was observed by HE staining.
The second part: SD normal control rats were divided into 3 weeks old, 6,8,10,12,14 weeks old, 3 rats in each age group, 18 rats in each age group. RCS rats and 18 RCS rats transplanted with AAV-BDNF-IPE cells (which had been transplanted with AAV-BDNF-IPE cells in the first experiment) were divided into two groups according to the age of birth and SD rats were recorded by binocular dark adaptation ERG. Physical examination refers to the international clinical visual electrophysiology standard. Each eye is examined at least three times. After stacking, ERG patterns with stable waveform and little interference are obtained. ERG-a and b wave amplitudes are calculated and statistically processed.
The third part: The control group RCS rats were divided into 3 weeks old, 6,8,10,12,14 weeks old, 4 rats in each week group, 24 rats in total; 24 RCS rats transplanted with AAV-BDNF-IPE cells (which had been transplanted into AAV-BDNF-IPE cells in the first part of the experiment), and the control group RCS rats were divided into two groups according to the age after birth. The expression of BDNF in retinal tissue was detected by enzyme linked immunosorbent assay (Elisa) at 7,9 and 11 weeks after operation.
The fourth part: The experimental animals and groups were the same as the second part. The rats were sacrificed after electrophysiological examination. The eyeballs were taken out and the retinal tissues were stained with HE, TUNEL and immunohistochemical staining.
Result
1. A total of 43 RCS rats underwent bilateral subretinal cavity transplantation, which resulted in massive vitreous hemorrhage, extensive retinal detachment, endophthalmitis and misinjection into the vitreous cavity in 18 eyes. AAV-BDNF-IPE cells were successfully transplanted into the subretinal cavity in 68 eyes, with a success rate of 79%.
HE staining in the retina and ciliary body of RCS rats showed that there was no pigment cell layer in both ciliary body and retina. After transplantation of AAV-BDNF-IPE cells into the subretinal space of RCS rats for 5 weeks, HE staining showed that there were scattered surviving Brown epithelial cells in the subretinal space of the rats, presumed to be surviving AV-BDNF-IPE cells in the retina or choroid. No obvious inflammatory cell infiltration and structural damage were observed (Fig.6).
2. Three groups of rats successfully completed dark adaptation FERG recording. From the 3rd week to the 14th week after birth, 15 normal SD rats were recorded 30 times, 15 non-intervention RCS rats 30 times, and 17 RCS rats 34 times in cell transplantation group.
There was no significant difference in the amplitudes of a and B waves of the maximum dark adaptation response of FERG among the three groups within 8 weeks after birth. The amplitudes of a and B waves of the maximum dark adaptation response of FERG in RCS rats decreased significantly after 10 weeks of birth compared with normal SD rats of the same age (P?0.05). After 12 weeks of birth, the ERG waveforms of RCS rats showed a low flat pattern, and no obvious peaks, troughs, A and B modes. The B wave of RCS rats in AAV-BDNF-IPE transplantation group remained high at 10 weeks after birth. Although the B wave of RCS rats in cell transplantation group showed a slowly decreasing trend, the amplitude of B wave was still significantly higher than that of RCS rats in non-intervention control group (P 0.05 in 12W and 14W groups).
3. The levels of BDNF in the retina of RCS rats in the control group remained high at 3 weeks after birth, and then decreased rapidly. Compared with other age groups, the levels of BDNF in the retina of RCS rats in the 3-week-old group were P 0.01; there was no significant difference in the expression of BDNF between the two groups at 3, 5, 7, 9 and 11 weeks after operation (P? The expression of BDNF in the retina of RCS rats after BDNF-IPE transplantation was significantly higher than that in the control group (P 0.05 in the 6-week-old group and P 0.01 in the other groups).
4. HE staining showed that the thickness of the optic rod layer near the outer nuclear layer was slightly improved in AAV-BDNF-IPE transplantation group compared with the control group; TUNEL results: the apoptosis index of the experimental group was reduced, the apoptosis peak was delayed, and the apoptosis peak occurred 9 wk after transplantation; immunohistochemical staining showed that GFAP was weakly positive in the original outer retina of the experimental group rats. The expression of Muller cells was relatively pale, while the outer plexiform layer of SYN was weakly positive except for the inner plexiform layer.
Summary
1. AAV-BDNF-IPE was successfully transplanted into the subretinal space of young RCS rats by single-channel scleral approach with a self-made injector. The scleral incision was effectively reduced and the damage of retinal anatomy was reduced. The success rate of the operation was 79%.
HE staining in the retina and ciliary body of RCS rats showed that there was no pigment cell layer in both ciliary body and retina. After transplantation of AAV-BDNF-IPE cells into the subretinal space of RCS rats for 5 weeks, HE staining showed that there were scattered surviving Brown epithelial cells in the subretinal space of the rats, presumed to be surviving AV-BDNF-IPE cells in the retina or choroid. No obvious inflammatory cell infiltration and structural damage were observed.
Although it is not possible to determine the correct anatomical relationship between the transplanted cells and their host retinal tissues, our experimental results confirm that the transplantation of AAV-BDNF-IPE cells into the subretinal space by improved scleral transplantation is safe and reliable, not only for long-term survival, but also for the absence of immune rejection.
2. In this study, we found that there was no significant difference in the amplitude of a and B waves of the maximum dark adaptation response of FERG between the three groups of SD normal control rats, RCS rats and AAV-BDNF-IPE cells transplanted RCS rats within 8 weeks after birth. After 10 weeks of birth, the amplitude of a and B waves of the maximum dark adaptation response of FERG in RCS rats decreased significantly, compared with normal SD rats of the same age (P?0.05). After 12 weeks of birth, the ERG waveforms of RCS rats were low and flat, and there were no obvious peaks and troughs, and a and B waves were blurred. These results confirmed that the retinal function was gradually lost during the development of RPD in RCS rats.
After transplantation of AAV-BDNF-IPE cells into the subretinal space of RCS rats, the amplitude of FERG-b wave could be significantly improved. Although the amplitude of B wave in RCS rats decreased slowly with the prolonged time after transplantation, the amplitude of B wave remained relatively high after 10 weeks of birth, and was significantly higher than that in RCS rats without intervention at the same age (12 weeks and 14 weeks). Group P 0.05), suggesting that AAV-BDNF-IPE cell transplantation can delay the progress of retinitis pigmentosa in RCS rats to some extent and improve some retinal function.
3. BDNF levels in the retina of RCS rats in the control group remained high at 3 weeks after birth, and then decreased rapidly; BDNF expression levels in the retina of RCS rats in the AAV-BDNF-IPE transplantation group were significantly higher than those in the control group from 6 weeks to 14 weeks after birth. After transplantation of RCS rats into the subretinal space, the expression of BDNF in the retina can be maintained at a high level, which is an important reason why AAV-BDNF-IPE transplantation can improve the function of the host retina.
4. The transplantation effect of AAV-BDNF-IPE cells was slightly better than that of non-intervention group: the thickness of rod layer near the outer nuclear layer was also slightly improved, which was consistent with previous studies; TUNEL detection confirmed that loss of optic cells was an apoptotic phenomenon, and suggested that AAV-BDNF-IPE treatment might play a role in inhibiting apoptosis; immunohistochemical results suggested that It may alleviate the compensatory reactive glial fibrillary hyperplasia and may also show the ultrastructural changes characterized by synaptic recovery.
In summary, this experiment delayed the further development of retinal degeneration, suggesting that this method may save degenerated photoreceptors and restore their function.
【學位授予單位】：福建醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R774.1
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