豚鼠实验性近视眼巩膜成纤维细胞的力学特性变化及其与色素上皮细胞和部分细胞因子关系的研究

发布时间：2018-05-22 17:44

本文选题：近视 + 凹透镜诱导　；参考：《河北医科大学》2012年博士论文

【摘要】：目的：建立豚鼠镜片诱导型近视眼模型(Lens induced myopia, LIM),观察前部及后极部视网膜色素上皮(Retinal pigment epithelial cells, RPE)细胞及巩膜成纤维(Scleral fibroblasts, SF)细胞的生长周期变化,观察RPE细胞和SF细胞内基质金属蛋白酶(matrix metallopeptidase2, MMP-2)、转化生长因子-β2(transforming growth factor-beta2, TGF-(32)和碱性成纤维细胞生长因子(basic fibroblast growth factor, bFGF)的表达变化；观察外源性转化生长因子(TGF-β2)对豚鼠眼球前部及后极部SF细胞生长周期的影响,观察TGF-β2对豚鼠眼球前部及后极部SF细胞基质金属蛋白酶(MMP-2)、转化生长因子-β2受体(Transforming Growth Factor-β receptor type2, TβR II)和碱性成纤维细胞生长因子(bFGF)衣达的影响；结合TGF-β2对细胞力学特性的影响,观察SF细胞的力学特性变化。方法：取出生后2周龄的豚鼠30只(60只眼)雌雄不限,分为A、B、C三组,每组10只,随机选取一只眼为实验眼(LIM眼),对侧眼为自身对照眼(Self-control, SC眼)。实验眼采用离焦点方法,用-10.00D凹透镜诱导成近视眼模型。 A、B、C三组试验眼-10.00D凹透镜诱导时间分别为6天、15天、30天。实验前后检测每组豚鼠双眼屈光状态,用A超测双眼眼轴长度。另取5只(10眼)豚鼠不作任何干预,作为正常对照眼(Norma-contro, NC组)。细胞酶消化法原代培养豚鼠眼球前部及后极部RPE细胞,并传2代。免疫细胞化学法鉴定培养的细胞,利用免疫细胞化学法、荧光定量PCR (Q-PCR)法和Western Blot法对每组实验眼和对照眼前部及后极部RPE细胞的TGF-β2、bFGF的表达进行半定量及定量检测；用流式细胞仪检测每组实验眼和对照眼前部及后极部RPE细胞的生长周期。结果用SPSS13.0统计软件进行统计分析。组织块培养法培养豚鼠眼球前部及后极部SF细胞,并传2代。免疫细胞化学法鉴定培养的细胞,免疫细胞化学法、荧光定量PCR (Q-PCR)法和Western Blot法对每组实验眼和对照眼前部及后极部SF细胞MMP-2、 TGF-β2、 bFGF的表达进行半定量及定量检测；用流式细胞仪检测每组实验眼和对照眼前部及后极部SF细胞的生长周期；利用细胞微管吸吮的方法测定每组实验眼和对照眼前部及后极部SF细胞的力学特性。结果用SPSS13.0统计软件进行统计分析。用组织块培养法培养豚鼠后极部SF细胞并传2代,免疫细胞化学法鉴定培养的细胞。将不同质量浓度的TGF-β2(分别为0、1、10、100ng/ml)加入无血清DMEM中作用24h,利用免疫细胞化学法、荧光定量PCR (Q-PCR)法和Western Blot法对每组实验眼和对照眼后极部SF细胞MMP-2、 TβRⅡ、bFGF的表达进行半定量及定量检测；用流式细胞仪检测每组后极部SF细胞的生长周期；用细胞微管吸吮的方法测定各组实验眼和对照眼SF细胞的力学特性。结果用SPSS13.0统计软件进行统计分析。结果：1RPE细胞和SF细胞中TGF-β2、 bFGF、 MMP-2的表达：免疫细胞化学、荧光定量PCR (Q-PCR)及Western Blot去结果显示： ①A、B、C三组实验眼和对照眼前部及后极部RPE、 SF细胞均有TGF-β2的表达,三组实验眼前部及后极部与自身对照眼相应前部及后极部比较,实验眼中TGF-β2的阳性率高于自身对照眼(P0.05)。实验眼中前部RPE细胞、前部SF细胞于15天阳性率增高,30天阳性率最高；后极部RPE和SF细胞于6天阳性率开始增高,至15天阳性率最高,以后保持较高的阳性率(P0.05)；30天时后极部RPE细胞、SF细胞TGF-β2的阳性率明显低于同期前部RPE细胞、SF细胞阳性率(P0.05)；三组自身对照眼自身前部及后极部RPE、 SF细胞TGF-β2阳性率比较,差异无统计学意义(P0.05)。 ②A、B、C三组实验眼和对照眼前部及后极部RPE细胞、SF细胞均有bFGF的表达,实验眼前部及后极部与自身对照眼相应前部及后极部比较,实验眼中bFGF的阳性率低于自身对照眼,差异有显著统计学意义(P0.05)。而且,随着诱导时间的延长,A、B、C三组实验眼的阳性率逐渐降低,差异有显著统计学意义(P0.05), A、 B、 C三组自身对照眼的阳性率不变,差异无统计学意义(P0.05)；实验眼和自身对照眼各组自身前部及后极部比较,差异无统计学意义(P0.05)。 ③A、B、C三组实验眼和对照眼前部及后极部SF细胞均有MMP-2的表达,实验眼前部及后极部与自身对照眼相应前部及后极部比较,(LIM组)实验眼中MMP-2的阳性率高于自身对照眼(P0.05)。实验眼中前部SF细胞于15天阳性率增高,30天阳性率最高；后极部SF细胞于6天阳性率开始增高,至15天阳性率最高,以后保持较高的阳性率(P0.05)；30天时后极部SF细胞MMP-2阳性率仍明显低于同期前部SF细胞阳性率(P0.05)；各组自身对照眼自身前部及后极部SF细胞MMP-2阳性率比较,差异无统计学意义(P0.05)。 2TGF-β2对SF细胞MMP-2.TβRⅡ、bFGF表达的影响免疫细胞化学、荧光定量PCR(Q-PCR)及Western Blot法结果显示：实验眼0ng/ml TGF-β2组与对照眼0ng/ml TGF-β2组比较,实验眼MMP-2的阳性率明显增高,差异有统计学意义(P0.05)。实验眼和对照眼SF细胞在TGF-β2浓度为10ng/ml时MMP-2的阳性率最高(P0.05)。实验眼0ng/ml TGF-β2组与对照眼0ng/ml TGF-β2组比较,实验眼TβR Ⅱ的阳性率明显增高,差异有统计学意义(P0.05)。实验眼和对照眼SF细胞随着TGF-β2浓度的增高TβRⅡ的阳性率越来越高。经统计学分析SF细胞阳性率和TGF-β2浓度有明显的相关性。(P0.05)。实验眼0ng/ml TGF-β2组与对照眼0ng/ml TGF-β2组比较,实验眼bFGF的阳性率明显降低,差异有统计学意义(P0.05)。实验眼和对照眼SF细胞随着TGF-β2浓度的增高bFGF的阳性率变化无明显统计学意义(P0.05)。 3流式细胞仪检测细胞生长周期结果： A、B、C三组实验眼后极部RPE细胞、SF细胞于诱导6天后,G0／G1期细胞百分比明显升高,S期百分比明显下降,15天时G0／G1期细胞百分比继续升高,S期百分比继续下降。但到30天时G0／G1期细胞百分比又有所降低,S期百分比又有所升高,与对照组相比差异仍有统计学意义(P0.05)。 A、B、C三组实验眼前部RPE细胞、SF细胞于诱导15天后,G0／G1期细胞百分比才开始明显升高,S期百分比明显降低,与对照眼相比差异有统计学意义(P0.05),但到30天时G0／G1期细胞百分比又有所降低,S期百分比又有所升高,与对照眼相比差异仍有统计学意义(P0.05)。 A、B、C三组实验眼相同诱导时间前部RPE细胞、SF细胞与后极部RPE细胞、SF细胞比较,两者差异有统计学意义(P0.05)。4细胞超微结构变化 A、B、C三组实验眼后极部RPE细胞、SF细胞于诱导6天后,胞核外形不规则,胞质水中,胞浆内线粒体较多而小、部分线粒体水肿,粗面内质网扩张、内质网及核糖体等细胞器均减少,细胞界膜不清,部分界膜消失。 A、B、C三组实验眼前部RPE细胞、SF细胞于诱导15天后,始见细胞核外形不规则,胞质水肿,胞浆内线粒体较多而小、部分线粒体水肿,粗面内质网扩张、内质网及核糖体等细胞器均减少,细胞界膜不清,部分界膜消失。 5细胞力学研究结果 (1)透镜诱导后巩膜成纤维细胞自身力学特性改变 ①不同诱导时间实验眼前部SF细胞力学特性比较及统计学分析实验眼前部SF细胞平衡杨氏模量、表观黏性6天组与15天组比较差异无统计学意义(P0.05),30天组与6天组、15天组比较差异均有统计学意义(P0.05)。 ②不同诱导时间实验组后极部SF细胞力学特性比较及统计学分析实验眼后极部SF细胞平衡杨氏模量、表观黏性15天组与30天组比较差异无统计学意义(P0.05),6天组与15天组、30天组比较差异均有统计学意义(P0.05)。③不同诱导时间自身对照眼前部及后极部SF细胞力学特性比较及统计学分析在诱导6天、15天、30天时,自身对照眼前部与后极部SF细胞平衡杨氏模量、表观黏性结果比较,差异无统计学意义(P0.05)；两两比较差异均无统计学意义(P0.05)。④各组实验眼的SF细胞力学特性与各组对照眼比较,论是平衡杨氏模量还是细胞表观黏性,实验眼前部于30天时与自身对照眼前部比较均增高,其差异有统计学意义(P0.05)；实验眼后极部于15天、30天时与自身对照眼后极部比较均增高,其差异有统计学意义(P0.05)。⑤经统计学分析：各诱导时间段自身对照眼巩膜细胞的杨氏模量、细胞表观黏性均与实验眼巩膜细胞的杨氏模量、细胞表观黏性有显著统计学差异(P0.05)。 (2)TGF-β2对SF细胞的力学特性变化的影响实验眼0ng/ml TGF-β2组与对照眼O ng/ml TGF-β2组比较,实验眼SF细胞的平衡杨氏模量、黏弹性参数明显升高,二者之间差异有统计学意义(P0.05)。实验眼与对照眼在TGF-β2作用下,0ng/ml组与1ng/ml组、10ng/ml组比较,细胞的平衡杨氏模量、黏弹性参数差异均有统计学意义(P0.05)。实验眼和对照眼培养细胞的平衡杨氏模量、黏弹性参数与TGF-β2的质量浓度均呈正相关(r=0.743、r=0.533;r=0.654、r=0.576),实验眼和对照眼中的0ng/ml组与100ng/ml组比较,SF细胞的平衡杨氏模量、黏弹性参数差异均无统计学意义(P0.05)。实验眼1ng/ml组、10ng/ml组与对照眼1ng/ml组、10ng/ml组比较,SF细胞的平衡杨氏模量、黏弹性参数之间差异有统计学意义(P0.05)：实验眼100ng/ml组与对照眼100ng/ml组比较,SF细胞的平衡杨氏模量、黏弹性参数差异无统计学意义(P0.05)。结论： 1LIM模型可使RPE及SF细胞TGF-β2及MMP-2表达上调,并且表达水平存在时间和部位上的差异；同时使bFGF表达下调,但不存在明显的时间和部位上的差异。 2LIM模型及TGF-β2刺激均可诱导RPE及SF细胞增殖抑制。 3TGF-β2刺激可诱导RPE及SF细胞超微结构变化,同时上调MMP-2及TβRⅡ的表达,但对bFGF表达无明显作用。 4LIM模型可使SF细胞平衡杨氏模量及粘弹性参数明显升高,产生“硬化”现象,且前部与后极部细胞发生变化的时间存在差异。 5低浓度TGF-β2刺激可降低SF细胞的平衡杨氏模量及粘弹性参数。
[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.
【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R778.11

【参考文献】