本文選題：近視 + 凹透鏡誘導(dǎo)　； 參考：《河北醫(yī)科大學(xué)》2012年博士論文

【摘要】：目的：建立豚鼠鏡片誘導(dǎo)型近視眼模型(Lens induced myopia, LIM),觀察前部及后極部視網(wǎng)膜色素上皮(Retinal pigment epithelial cells, RPE)細(xì)胞及鞏膜成纖維(Scleral fibroblasts, SF)細(xì)胞的生長周期變化,觀察RPE細(xì)胞和SF細(xì)胞內(nèi)基質(zhì)金屬蛋白酶(matrix metallopeptidase2, MMP-2)、轉(zhuǎn)化生長因子-β2(transforming growth factor-beta2, TGF-(32)和堿性成纖維細(xì)胞生長因子(basic fibroblast growth factor, bFGF)的表達(dá)變化；觀察外源性轉(zhuǎn)化生長因子(TGF-β2)對(duì)豚鼠眼球前部及后極部SF細(xì)胞生長周期的影響,觀察TGF-β2對(duì)豚鼠眼球前部及后極部SF細(xì)胞基質(zhì)金屬蛋白酶(MMP-2)、轉(zhuǎn)化生長因子-β2受體(Transforming Growth Factor-β receptor type2, TβR II)和堿性成纖維細(xì)胞生長因子(bFGF)衣達(dá)的影響；結(jié)合TGF-β2對(duì)細(xì)胞力學(xué)特性的影響,觀察SF細(xì)胞的力學(xué)特性變化。 方法：取出生后2周齡的豚鼠30只(60只眼)雌雄不限,分為A、B、C三組,每組10只,隨機(jī)選取一只眼為實(shí)驗(yàn)眼(LIM眼),對(duì)側(cè)眼為自身對(duì)照眼(Self-control, SC眼)。實(shí)驗(yàn)眼采用離焦點(diǎn)方法,用-10.00D凹透鏡誘導(dǎo)成近視眼模型。 A、B、C三組試驗(yàn)眼-10.00D凹透鏡誘導(dǎo)時(shí)間分別為6天、15天、30天。實(shí)驗(yàn)前后檢測(cè)每組豚鼠雙眼屈光狀態(tài),用A超測(cè)雙眼眼軸長度。另取5只(10眼)豚鼠不作任何干預(yù),作為正常對(duì)照眼(Norma-contro, NC組)。 細(xì)胞酶消化法原代培養(yǎng)豚鼠眼球前部及后極部RPE細(xì)胞,并傳2代。免疫細(xì)胞化學(xué)法鑒定培養(yǎng)的細(xì)胞,利用免疫細(xì)胞化學(xué)法、熒光定量PCR (Q-PCR)法和Western Blot法對(duì)每組實(shí)驗(yàn)眼和對(duì)照眼前部及后極部RPE細(xì)胞的TGF-β2、bFGF的表達(dá)進(jìn)行半定量及定量檢測(cè)；用流式細(xì)胞儀檢測(cè)每組實(shí)驗(yàn)眼和對(duì)照眼前部及后極部RPE細(xì)胞的生長周期。結(jié)果用SPSS13.0統(tǒng)計(jì)軟件進(jìn)行統(tǒng)計(jì)分析。 組織塊培養(yǎng)法培養(yǎng)豚鼠眼球前部及后極部SF細(xì)胞,并傳2代。免疫細(xì)胞化學(xué)法鑒定培養(yǎng)的細(xì)胞,免疫細(xì)胞化學(xué)法、熒光定量PCR (Q-PCR)法和Western Blot法對(duì)每組實(shí)驗(yàn)眼和對(duì)照眼前部及后極部SF細(xì)胞MMP-2、 TGF-β2、 bFGF的表達(dá)進(jìn)行半定量及定量檢測(cè)；用流式細(xì)胞儀檢測(cè)每組實(shí)驗(yàn)眼和對(duì)照眼前部及后極部SF細(xì)胞的生長周期；利用細(xì)胞微管吸吮的方法測(cè)定每組實(shí)驗(yàn)眼和對(duì)照眼前部及后極部SF細(xì)胞的力學(xué)特性。結(jié)果用SPSS13.0統(tǒng)計(jì)軟件進(jìn)行統(tǒng)計(jì)分析。 用組織塊培養(yǎng)法培養(yǎng)豚鼠后極部SF細(xì)胞并傳2代,免疫細(xì)胞化學(xué)法鑒定培養(yǎng)的細(xì)胞。將不同質(zhì)量濃度的TGF-β2(分別為0、1、10、100ng/ml)加入無血清DMEM中作用24h,利用免疫細(xì)胞化學(xué)法、熒光定量PCR (Q-PCR)法和Western Blot法對(duì)每組實(shí)驗(yàn)眼和對(duì)照眼后極部SF細(xì)胞MMP-2、 TβRⅡ、bFGF的表達(dá)進(jìn)行半定量及定量檢測(cè)；用流式細(xì)胞儀檢測(cè)每組后極部SF細(xì)胞的生長周期；用細(xì)胞微管吸吮的方法測(cè)定各組實(shí)驗(yàn)眼和對(duì)照眼SF細(xì)胞的力學(xué)特性。結(jié)果用SPSS13.0統(tǒng)計(jì)軟件進(jìn)行統(tǒng)計(jì)分析。 結(jié)果：1RPE細(xì)胞和SF細(xì)胞中TGF-β2、 bFGF、 MMP-2的表達(dá)： 免疫細(xì)胞化學(xué)、熒光定量PCR (Q-PCR)及Western Blot去結(jié)果顯示： ①A、B、C三組實(shí)驗(yàn)眼和對(duì)照眼前部及后極部RPE、 SF細(xì)胞均有TGF-β2的表達(dá),三組實(shí)驗(yàn)眼前部及后極部與自身對(duì)照眼相應(yīng)前部及后極部比較,實(shí)驗(yàn)眼中TGF-β2的陽性率高于自身對(duì)照眼(P0.05)。實(shí)驗(yàn)眼中前部RPE細(xì)胞、前部SF細(xì)胞于15天陽性率增高,30天陽性率最高；后極部RPE和SF細(xì)胞于6天陽性率開始增高,至15天陽性率最高,以后保持較高的陽性率(P0.05)；30天時(shí)后極部RPE細(xì)胞、SF細(xì)胞TGF-β2的陽性率明顯低于同期前部RPE細(xì)胞、SF細(xì)胞陽性率(P0.05)；三組自身對(duì)照眼自身前部及后極部RPE、 SF細(xì)胞TGF-β2陽性率比較,差異無統(tǒng)計(jì)學(xué)意義(P0.05)。 ②A、B、C三組實(shí)驗(yàn)眼和對(duì)照眼前部及后極部RPE細(xì)胞、SF細(xì)胞均有bFGF的表達(dá),實(shí)驗(yàn)眼前部及后極部與自身對(duì)照眼相應(yīng)前部及后極部比較,實(shí)驗(yàn)眼中bFGF的陽性率低于自身對(duì)照眼,差異有顯著統(tǒng)計(jì)學(xué)意義(P0.05)。而且,隨著誘導(dǎo)時(shí)間的延長,A、B、C三組實(shí)驗(yàn)眼的陽性率逐漸降低,差異有顯著統(tǒng)計(jì)學(xué)意義(P0.05), A、 B、 C三組自身對(duì)照眼的陽性率不變,差異無統(tǒng)計(jì)學(xué)意義(P0.05)；實(shí)驗(yàn)眼和自身對(duì)照眼各組自身前部及后極部比較,差異無統(tǒng)計(jì)學(xué)意義(P0.05)。 ③A、B、C三組實(shí)驗(yàn)眼和對(duì)照眼前部及后極部SF細(xì)胞均有MMP-2的表達(dá),實(shí)驗(yàn)眼前部及后極部與自身對(duì)照眼相應(yīng)前部及后極部比較,(LIM組)實(shí)驗(yàn)眼中MMP-2的陽性率高于自身對(duì)照眼(P0.05)。實(shí)驗(yàn)眼中前部SF細(xì)胞于15天陽性率增高,30天陽性率最高；后極部SF細(xì)胞于6天陽性率開始增高,至15天陽性率最高,以后保持較高的陽性率(P0.05)；30天時(shí)后極部SF細(xì)胞MMP-2陽性率仍明顯低于同期前部SF細(xì)胞陽性率(P0.05)；各組自身對(duì)照眼自身前部及后極部SF細(xì)胞MMP-2陽性率比較,差異無統(tǒng)計(jì)學(xué)意義(P0.05)。 2TGF-β2對(duì)SF細(xì)胞MMP-2.TβRⅡ、bFGF表達(dá)的影響 免疫細(xì)胞化學(xué)、熒光定量PCR(Q-PCR)及Western Blot法結(jié)果顯示： 實(shí)驗(yàn)眼0ng/ml TGF-β2組與對(duì)照眼0ng/ml TGF-β2組比較,實(shí)驗(yàn)眼MMP-2的陽性率明顯增高,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。實(shí)驗(yàn)眼和對(duì)照眼SF細(xì)胞在TGF-β2濃度為10ng/ml時(shí)MMP-2的陽性率最高(P0.05)。 實(shí)驗(yàn)眼0ng/ml TGF-β2組與對(duì)照眼0ng/ml TGF-β2組比較,實(shí)驗(yàn)眼TβR Ⅱ的陽性率明顯增高,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。實(shí)驗(yàn)眼和對(duì)照眼SF細(xì)胞隨著TGF-β2濃度的增高TβRⅡ的陽性率越來越高。經(jīng)統(tǒng)計(jì)學(xué)分析SF細(xì)胞陽性率和TGF-β2濃度有明顯的相關(guān)性。(P0.05)。 實(shí)驗(yàn)眼0ng/ml TGF-β2組與對(duì)照眼0ng/ml TGF-β2組比較,實(shí)驗(yàn)眼bFGF的陽性率明顯降低,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。實(shí)驗(yàn)眼和對(duì)照眼SF細(xì)胞隨著TGF-β2濃度的增高bFGF的陽性率變化無明顯統(tǒng)計(jì)學(xué)意義(P0.05)。 3流式細(xì)胞儀檢測(cè)細(xì)胞生長周期結(jié)果： A、B、C三組實(shí)驗(yàn)眼后極部RPE細(xì)胞、SF細(xì)胞于誘導(dǎo)6天后,G0／G1期細(xì)胞百分比明顯升高,S期百分比明顯下降,15天時(shí)G0／G1期細(xì)胞百分比繼續(xù)升高,S期百分比繼續(xù)下降。但到30天時(shí)G0／G1期細(xì)胞百分比又有所降低,S期百分比又有所升高,與對(duì)照組相比差異仍有統(tǒng)計(jì)學(xué)意義(P0.05)。 A、B、C三組實(shí)驗(yàn)眼前部RPE細(xì)胞、SF細(xì)胞于誘導(dǎo)15天后,G0／G1期細(xì)胞百分比才開始明顯升高,S期百分比明顯降低,與對(duì)照眼相比差異有統(tǒng)計(jì)學(xué)意義(P0.05),但到30天時(shí)G0／G1期細(xì)胞百分比又有所降低,S期百分比又有所升高,與對(duì)照眼相比差異仍有統(tǒng)計(jì)學(xué)意義(P0.05)。 A、B、C三組實(shí)驗(yàn)眼相同誘導(dǎo)時(shí)間前部RPE細(xì)胞、SF細(xì)胞與后極部RPE細(xì)胞、SF細(xì)胞比較,兩者差異有統(tǒng)計(jì)學(xué)意義(P0.05)。4細(xì)胞超微結(jié)構(gòu)變化 A、B、C三組實(shí)驗(yàn)眼后極部RPE細(xì)胞、SF細(xì)胞于誘導(dǎo)6天后,胞核外形不規(guī)則,胞質(zhì)水中,胞漿內(nèi)線粒體較多而小、部分線粒體水腫,粗面內(nèi)質(zhì)網(wǎng)擴(kuò)張、內(nèi)質(zhì)網(wǎng)及核糖體等細(xì)胞器均減少,細(xì)胞界膜不清,部分界膜消失。 A、B、C三組實(shí)驗(yàn)眼前部RPE細(xì)胞、SF細(xì)胞于誘導(dǎo)15天后,始見細(xì)胞核外形不規(guī)則,胞質(zhì)水腫,胞漿內(nèi)線粒體較多而小、部分線粒體水腫,粗面內(nèi)質(zhì)網(wǎng)擴(kuò)張、內(nèi)質(zhì)網(wǎng)及核糖體等細(xì)胞器均減少,細(xì)胞界膜不清,部分界膜消失。 5細(xì)胞力學(xué)研究結(jié)果 (1)透鏡誘導(dǎo)后鞏膜成纖維細(xì)胞自身力學(xué)特性改變 ①不同誘導(dǎo)時(shí)間實(shí)驗(yàn)眼前部SF細(xì)胞力學(xué)特性比較及統(tǒng)計(jì)學(xué)分析 實(shí)驗(yàn)眼前部SF細(xì)胞平衡楊氏模量、表觀黏性6天組與15天組比較差異無統(tǒng)計(jì)學(xué)意義(P0.05),30天組與6天組、15天組比較差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。 ②不同誘導(dǎo)時(shí)間實(shí)驗(yàn)組后極部SF細(xì)胞力學(xué)特性比較及統(tǒng)計(jì)學(xué)分析 實(shí)驗(yàn)眼后極部SF細(xì)胞平衡楊氏模量、表觀黏性15天組與30天組比較差異無統(tǒng)計(jì)學(xué)意義(P0.05),6天組與15天組、30天組比較差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。③不同誘導(dǎo)時(shí)間自身對(duì)照眼前部及后極部SF細(xì)胞力學(xué)特性比較及統(tǒng)計(jì)學(xué)分析 在誘導(dǎo)6天、15天、30天時(shí),自身對(duì)照眼前部與后極部SF細(xì)胞平衡楊氏模量、表觀黏性結(jié)果比較,差異無統(tǒng)計(jì)學(xué)意義(P0.05)；兩兩比較差異均無統(tǒng)計(jì)學(xué)意義(P0.05)。④各組實(shí)驗(yàn)眼的SF細(xì)胞力學(xué)特性與各組對(duì)照眼比較,論是平衡楊氏模量還是細(xì)胞表觀黏性,實(shí)驗(yàn)眼前部于30天時(shí)與自身對(duì)照眼前部比較均增高,其差異有統(tǒng)計(jì)學(xué)意義(P0.05)；實(shí)驗(yàn)眼后極部于15天、30天時(shí)與自身對(duì)照眼后極部比較均增高,其差異有統(tǒng)計(jì)學(xué)意義(P0.05)。⑤經(jīng)統(tǒng)計(jì)學(xué)分析：各誘導(dǎo)時(shí)間段自身對(duì)照眼鞏膜細(xì)胞的楊氏模量、細(xì)胞表觀黏性均與實(shí)驗(yàn)眼鞏膜細(xì)胞的楊氏模量、細(xì)胞表觀黏性有顯著統(tǒng)計(jì)學(xué)差異(P0.05)。 (2)TGF-β2對(duì)SF細(xì)胞的力學(xué)特性變化的影響 實(shí)驗(yàn)眼0ng/ml TGF-β2組與對(duì)照眼O ng/ml TGF-β2組比較,實(shí)驗(yàn)眼SF細(xì)胞的平衡楊氏模量、黏彈性參數(shù)明顯升高,二者之間差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 實(shí)驗(yàn)眼與對(duì)照眼在TGF-β2作用下,0ng/ml組與1ng/ml組、10ng/ml組比較,細(xì)胞的平衡楊氏模量、黏彈性參數(shù)差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。實(shí)驗(yàn)眼和對(duì)照眼培養(yǎng)細(xì)胞的平衡楊氏模量、黏彈性參數(shù)與TGF-β2的質(zhì)量濃度均呈正相關(guān)(r=0.743、r=0.533;r=0.654、r=0.576),實(shí)驗(yàn)眼和對(duì)照眼中的0ng/ml組與100ng/ml組比較,SF細(xì)胞的平衡楊氏模量、黏彈性參數(shù)差異均無統(tǒng)計(jì)學(xué)意義(P0.05)。 實(shí)驗(yàn)眼1ng/ml組、10ng/ml組與對(duì)照眼1ng/ml組、10ng/ml組比較,SF細(xì)胞的平衡楊氏模量、黏彈性參數(shù)之間差異有統(tǒng)計(jì)學(xué)意義(P0.05)：實(shí)驗(yàn)眼100ng/ml組與對(duì)照眼100ng/ml組比較,SF細(xì)胞的平衡楊氏模量、黏彈性參數(shù)差異無統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論： 1LIM模型可使RPE及SF細(xì)胞TGF-β2及MMP-2表達(dá)上調(diào),并且表達(dá)水平存在時(shí)間和部位上的差異；同時(shí)使bFGF表達(dá)下調(diào),但不存在明顯的時(shí)間和部位上的差異。 2LIM模型及TGF-β2刺激均可誘導(dǎo)RPE及SF細(xì)胞增殖抑制。 3TGF-β2刺激可誘導(dǎo)RPE及SF細(xì)胞超微結(jié)構(gòu)變化,同時(shí)上調(diào)MMP-2及TβRⅡ的表達(dá),但對(duì)bFGF表達(dá)無明顯作用。 4LIM模型可使SF細(xì)胞平衡楊氏模量及粘彈性參數(shù)明顯升高,產(chǎn)生“硬化”現(xiàn)象,且前部與后極部細(xì)胞發(fā)生變化的時(shí)間存在差異。 5低濃度TGF-β2刺激可降低SF細(xì)胞的平衡楊氏模量及粘彈性參數(shù)。
[Abstract]:Objective: to establish the Lens induced myopia (LIM) model of guinea pig lens induced myopia (LIM), and to observe the growth cycle of the retinal pigment epithelium (Retinal pigment epithelial cells, RPE) cells and scleral fibroblasts (Scleral fibroblasts, SF) cells in the anterior and posterior parts. Tallopeptidase2, MMP-2), the expression of transforming growth factor - beta 2 (transforming growth factor-beta2, TGF- (32) and basic fibroblast growth factor (basic fibroblast growth factor, bFGF)), and the effect of exogenous TGF (TGF- beta 2) on the growth cycle of the anterior and posterior polar part of the eyeball of the porpoise The effects of the matrix metalloproteinase (MMP-2), transforming growth factor - beta 2 receptor (Transforming Growth Factor- beta receptor type2, T beta R II) and basic fibroblast growth factor (bFGF) clothing on the anterior and posterior part of the eyeball of the eyeball of guinea pigs were affected, and the changes of the mechanical properties of SF cells were observed in combination with the effect of TGF- beta 2 on the cell force.
Methods: 30 (60 eyes) of the guinea pigs, 2 weeks of age after birth, were divided into A, B, and C three, group 10. One eye was selected as experimental eye (LIM eye), and the eye was self to eye (Self-control, SC eye). The experimental eye was induced by the method of focal point and induced into myopia model with -10.00D concave lens. A, B, C three test eyes -10.00D concave lens The induction time was 6 days, 15 days and 30 days, respectively. Before and after the experiment, the binocular refraction was detected in each group of guinea pigs, and the binocular axis length was measured by A. 5 (10 eyes) guinea pigs were taken without any intervention as normal eyes (group Norma-contro, NC).
RPE cells in the anterior and posterior part of the eyeball of the guinea pig were cultured by cell enzyme digestion method and passed through 2 generations. Immunocytochemical method was used to identify the cultured cells. The expression of TGF- beta 2 and bFGF in the eyes of each group and the RPE cells in the anterior and posterior part of each group were semi quantified and determined by immunocytochemical method, fluorescence quantitative PCR (Q-PCR) method and Western Blot method. A flow cytometry was used to detect the growth cycle of RPE cells in the eyes of each group and the anterior and posterior parts of the control group. The results were statistically analyzed with the SPSS13.0 software.
SF cells in the anterior and posterior part of the eyeball of the guinea pig were cultured by tissue mass culture and passed on for 2 generations. The expression of MMP-2, TGF- beta 2 and bFGF in the anterior and posterior SF cells of each group was semi quantified and quantitatively measured by immunocytochemical method, immunocytochemical method, fluorescence quantitative PCR (Q-PCR) method and Western Blot method. The growth cycle of SF cells in the eyes of each group and the control of the anterior and posterior part of the eyes was detected by flow cytometry. The mechanical properties of the SF cells in the experimental eyes and the control eyes and the posterior pole were measured by the method of cell micropipette sucking. The results were statistically analyzed by the SPSS13.0 statistical software.
SF cells in the posterior polar part of guinea pig were cultured with tissue mass culture and passed through 2 generations. Immunocytochemical method was used to identify the cultured cells. The TGF- beta 2 (0,1,10100ng/ml) with different mass concentration was added to the serum free DMEM, and 24h was added to the serum-free DMEM. The immunocytochemical method, the fluorescence quantitative PCR (Q-PCR) method and Western Blot method were applied to the experimental and control eyes of each group. The expression of MMP-2, T beta, R II, bFGF was detected by semi quantitative and quantitative detection, and the growth cycle of SF cells in each group was detected by flow cytometry, and the mechanical properties of SF cells in the experimental and control eyes of each group were measured by cell micropipette sucking. The results were statistically analyzed with SPSS13.0 statistics software.
Results: the expressions of TGF- beta 2, bFGF and MMP-2 in 1RPE cells and SF cells were as follows:
The results of immunocytochemistry, fluorescence quantitative PCR (Q-PCR) and Western Blot showed that:
(1) the A, B, C three groups of experimental eyes and the control eyes and the posterior pole RPE, SF cells all had TGF- beta 2 expression. The three groups of experimental eyes and the posterior pole were compared with the corresponding anterior and posterior part of the self control eye. The positive rate of TGF- beta 2 in the experimental eyes was higher than that of the eye (P0.05). The positive rate of the anterior SF cells in the anterior part of the experimental eye was higher than the 15 day positive rate, 30 The positive rate of the day was the highest, the positive rate of the RPE and SF cells in the posterior pole began to increase at 6 days, and the positive rate of the 15 days was the highest, and the positive rate was higher (P0.05). The positive rate of TGF- beta 2 in the SF cells at the 30 day was lower than that of the RPE cells in the same period and the positive rate of SF cells (P0.05), and the three groups of self control eyes themselves and the posterior polar part RP. There was no significant difference in the positive rate of TGF- beta 2 between E and SF cells (P0.05).
(2) A, B, C three groups of experimental eyes and the control of the anterior and posterior RPE cells, SF cells all had bFGF expression. The experimental eyes and the posterior pole were compared with the corresponding anterior and posterior part of the self control eye. The positive rate of bFGF in the experimental eyes was lower than that of the eye, the difference was statistically significant (P0.05). Moreover, A, B, C three with the prolongation of the induction time. The positive rate of experimental eyes decreased gradually, and the difference was statistically significant (P0.05). The positive rate of self control eyes in A, B, C three groups remained unchanged, the difference was not statistically significant (P0.05), and there was no statistical significance (P0.05) in the comparison between the anterior and the posterior parts of the experimental and self control eyes.
(3) the expression of MMP-2 in the A, B and C three groups of experimental eyes and the SF cells in the anterior and posterior part of the control was MMP-2. The positive rate of MMP-2 in the experimental eyes was higher than that of the eye (P0.05). The positive rate of SF cells in the anterior part of the experimental eyes was higher than that in the experimental eyes (group LIM). The positive rate of the 30 days was the highest in the anterior part of the experimental eyes. The positive rate of the SF cells in the posterior pole began to increase at 6 days, and the positive rate was the highest to 15 days, and the positive rate was higher (P0.05). The positive rate of SF cells in the posterior polar part of the posterior pole was still lower than that of the SF cell positive rate (P0.05) at the same time in the same period (P0.05), and the positive rate of MMP-2 in the anterior and posterior SF cells of the control eyes of each group was not statistically significant. Meaning (P0.05).
Effect of 2TGF- beta 2 on expression of MMP-2.T beta R II and bFGF in SF cells
The results of immunocytochemistry, fluorescence quantitative PCR (Q-PCR) and Western Blot showed that:
In experimental eye 0ng/ml TGF- beta 2 and control eyes 0ng/ml TGF- beta 2, the positive rate of MMP-2 in experimental eyes was significantly higher, the difference was statistically significant (P0.05). The positive rate of SF cells in experimental and control eye SF was the highest when the concentration of TGF- beta 2 was 10ng/ml (P0.05).
Compared with the control eye 0ng/ml TGF- beta 2 group, the positive rate of T beta R II in the experimental eyes was significantly higher than that of the control eye 0ng/ml TGF- beta group (P0.05). The positive rate of T beta R II in experimental and control eye SF cells increased with the increase of TGF- beta 2 concentration, and there was a significant correlation between the positive rate of SF fine cell and the concentration of TGF- beta 2. P0.05).
In experimental eye 0ng/ml TGF- beta 2 and control eyes 0ng/ml TGF- beta 2, the positive rate of bFGF in experimental eyes was significantly lower, the difference was statistically significant (P0.05). There was no significant difference in the positive rate of bFGF in experimental and control eye SF cells with the increase of the concentration of TGF- beta 2 (P0.05).
The results of cell growth cycle were detected by 3 flow cytometry.
A, B, C three groups of RPE cells in the posterior polar part, the percentage of G0 / G1 cells increased significantly after 6 days of induction, the percentage of S phase decreased significantly, and the percentage of G0 / G1 phase cells continued to rise at 15 days, and the percentage of S phase continued to decline. The difference was statistically significant (P0.05).
A, B, C three groups of three groups of experimental RPE cells, SF cells in 15 days after induction, the percentage of G0 / G1 cells began to increase obviously, the percentage of S phase decreased significantly, compared with the control eyes, the difference was statistically significant (P0.05), but the percentage of G0 / G1 in the 30 days decreased and the percentage of S phase increased, and there was still a difference compared with the control eye. Statistical significance (P0.05).
A, B, C three groups of experimental eyes with the same induction time of the anterior RPE cells, SF cells and the posterior polar RPE cells, SF cells, the difference is statistically significant (P0.05).4 cell ultrastructural changes
A, B, C three groups of experimental RPE cells in the posterior polar region. After 6 days of induction, SF cells were irregular in shape, cytoplasmic water, cytoplasmic mitochondria were more and smaller, some mitochondria were edema, rough endoplasmic reticulum dilated, endoplasmic reticulum and ribosome and other organelles decreased, the cell boundary membrane was not clear, and partial boundary membranes disappeared.
A, B, and C three groups experiment with anterior RPE cells. After 15 days of induction, SF cells began to see irregular nuclei, cytosolic edema, more mitochondria in the cytoplasm, partial mitochondria edema, rough endoplasmic reticulum dilation, endoplasmic reticulum and ribosome and other organelles decreased, the cell boundary membrane was not clear, and partial boundary membranes disappeared.
The results of 5 cell mechanics
(1) changes in mechanical properties of scleral fibroblasts after lens induction
Statistical analysis of the mechanical properties of SF cells in the anterior chamber at different induction times
There was no significant difference between the 6 days of apparent viscosity and the 15 day group (P0.05), and there was a significant difference between the 30 day group and the 6 day group in the 30 day group and the 15 day group, and the difference was statistically significant (P0.05).
2. Comparison of mechanical properties of SF cells in the posterior pole of the experimental group at different induction times and statistical analysis
The balance of SF cells in the posterior polar part of the experimental eye had no significant difference between the 15 day group and the 30 day group (P0.05), the 6 day group and the 15 day group, and the 30 day group had statistical significance (P0.05). (3) the comparison and statistical analysis of the mechanical properties of the SF cell in the anterior and posterior part of the different induction time self control
In the induction of 6 days, 15 days, and 30 days, the balance of the young's modulus of the SF cells in the anterior and the posterior poles of the eyes was not statistically significant (P0.05), and there was no significant difference in the apparent viscosity (P0.05). (P0.05). (4) the mechanical properties of the SF cells in the experimental eyes were compared with the control eyes of each group, or the balance of Young's modulus or cells. Apparent stickiness increased in the anterior part of the experiment at 30 days, and the difference was statistically significant (P0.05). The posterior polar part of the experimental eye was increased at the 15 day and 30 days, and the difference was statistically significant (P0.05). 5. The young's modulus and cell apparent viscosity were significantly different from the young's modulus and apparent viscosity of scleral cells in experimental eyes (P0.05).
(2) the effect of TGF- beta 2 on the mechanical properties of SF cells.
The experimental eye 0ng/ml TGF- beta 2 group and the control eye O ng/ml TGF- beta 2 group, the experimental eye SF cell's balance Young's modulus, the viscoelastic parameter obviously rises, the difference between the two is statistically significant (P0.05).
Under the action of TGF- beta 2 in the experimental and control eyes, there were significant differences in the balance Young's modulus and the viscoelastic parameters of the 0ng/ml group with the 1ng/ml group and the 10ng/ml group (P0.05). The viscoelastic parameters were positively correlated with the mass concentration of TGF- beta 2 in both experimental and control eyes (r=0.743, r=0.533; r=0.654, r=0.57). 6) compared with group 0ng/ml, there was no significant difference in the balance of Yang's modulus and viscoelastic parameters between the experimental group and the control group (100ng/ml) in group 0ng/ml (P0.05).
The experimental eye group 1ng/ml, 10ng/ml group and control eye 1ng/ml group, 10ng/ml group, SF cell balance Young's modulus, the difference between the viscoelastic parameters is statistically significant (P0.05): experimental eye 100ng/ml group and the control eye 100ng/ml group, SF cell balance Young's modulus, viscoelastic parameter difference is not statistically significant (P0.05).
Conclusion:
1LIM model could increase the expression of TGF- beta 2 and MMP-2 in RPE and SF cells, and the expression level was different in time and location, and the expression of bFGF was downregulated at the same time, but there was no significant difference in time and location.
2LIM and TGF- beta 2 stimulated the proliferation of RPE and SF cells.
3TGF- beta 2 stimulates the ultrastructural changes of RPE and SF cells, and upregulates the expression of MMP-2 and T beta R II, but has no significant effect on bFGF expression.
The 4LIM model can significantly increase the balance of Young's modulus and viscoelastic parameters of SF cells, resulting in "hardening" phenomenon.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R778.11

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