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
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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(P<0.05)。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(P<0.05)；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(P<0.05)；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(P>0.05)。
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(P<0.05)。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(P<0.05), A、 B、 C三组自身对照眼的阳性率不变,差异无统计学意义(P>0.05)；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(P>0.05)。
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(P<0.05)。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(P<0.05)；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(P<0.05)；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(P>0.05)。
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(P<0.05)。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(P<0.05)。
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(P<0.05)。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。(P>0.05)。
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(P<0.05)。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(P>0.05)。
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(P<0.05)。
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(P<0.05),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(P<0.05)。
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(P<0.05)。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(P>0.05),30天组与6天组、15天组比较差异均有统计学意义(P<0.05)。
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(P>0.05),6天组与15天组、30天组比较差异均有统计学意义(P<0.05)。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(P>0.05)；两两比较差异均无统计学意义(P>0.05)。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(P<0.05)；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(P<0.05)。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(P<0.05)。
(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(P<0.05)。
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(P<0.05)。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(P>0.05)。
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(P<0.05)：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(P>0.05)。
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。
Objective:To determine the cell proliferation and the growth period of anterior and posterior retinal pigment epithelial (RPE) cells and scleral fibroblast (SF) cells in cultured guinea pig model of lens-induced myopia (LIM), and to determine the expression changes of MMP-2, TGF-β2and bFGF in RPE and SF cells.
To determine the impact of TGF-β2on the cell proliferation of anterior and posterior scleral fibroblast cells in cultured guinea pig and the impact of TGF-P2on the expression of MMP-2, TβRII and bFGF in scleral fibroblast cells; and also to study the biomechanical characteristics changes. Some conclusions were valuable for revealing the pathological mechanism and the treatment of myopia.
Methods:Divide30two-week-old guinea pigs into three groups,10of each group. The guinea pigs were randomly lens-induced monocularly. The unilateral eye was treated by defocus method as the experimental eye, and induced by a spherical lens of-10.00D to build the myopia model. The other eye in each animal was untreated as self-control (SC) eye.5two-week-old guinea pigs were randomly closed as normal-control (NC). Each group was raised for6,15and30days separately. Before and after the experiment, the refraction was measured using retinoscopy and the axis oculi lengths of binoculus were determined by A-scan ultrasonography.
The retinal pigment epithelial cells of each group were cultured and passed continually for2generations. The cultured cells were verified to be retinal pigment epithelial cells using immunocytochemical method. Immunocytochemistry, Real-Time PCR Analysis and Western Blot method were used to detect the expressions of MMP-2, TGF-β2and bFGF in anterior and posterior retinal pigment epithelial cells of the experimental eyes and self-control eyes. MTT was used to detect the proliferation of each group of scleral fibroblast cells. The cultured cells of each group were studied by Flow Cytometry (FCM). SPSS13.0system was used to do statistical analysis of the observed data.
The scleral fibroblasts of each group were cultured and passed continually for2generations. The cultured cells were verified to be scleral fibroblasts using immunocytochemistry method. Immunocytochemistry, Real-Time PCR Analysis and Western Blot method were used to detect the expressions of MMP-2, TGF-β2and bFGF in anterior and posterior scleral fibroblast cells of the experimental eyes and self-control eyes. MTT was used to detect the proliferation of each group of scleral fibroblast cells. The cultured cells of each group were studied by Flow Cytometry (FCM). SPSS13.0system was used to do statistical analysis of the observed data.
The scleral fibroblasts of each group were purified with the tissue explant method and passed continually for2generations. The cultured cells were verified to be scleral fibroblasts using immunocytochemistry method. Add TGF-β2of different mass concentrations (0,1,10, and100ng/ml) to serum-free DMEM to react for24hours. Immunocytochemistry, Real-Time PCR Analysis and Western Blot method were used to detect the expressions of MMP-2, TGF-β2and bFGF in anterior and posterior scleral fibroblast cells of the experimental eyes and self-control eyes. MTT was used to detect the proliferation of each group of scleral fibroblast cells. The cultured cells of each group were studied by Flow Cytometry (FCM). SPSS13.0system was used to do statistical analysis of the observed data.
Results:1The expressions of TGF-β2, bFGF, MMP-2in cultured guinea pig anterior and posterior retinal pigment epithelial cells and scleral fibroblasts in lens-induced myopia shown by the results of Immunocytochemistry, Real-Time PCR Analysis and Western blot method:1.1The expression of TGF-β2was higher in experimental eye than in control eye. There was a significant difference between them (P<0.05). The expression of TGF-β2in posterior cells was higher than the in anterior cells (P>0.05) on the6th,15th and30th day. There was a significant difference of the expression of TGF-β2between model eyes and control eyes (P<0.05).1.2The expression of bFGF was lower in experimental eye than in control eye. There was a significant difference between them (P<0.05). The expression of bFGF in anterior cells was almost the same with the expression in posterior cells on the6th,15th and30th day (P>0.05).
1.3The expression of MMP-2was higher in experimental eye than in control eye. There was a significant difference between them (P<0.05). The expression of TGF-β2in posterior cells was higher than the expression in anterior cells (P>0.05) on the6th,15th and30th day. There was a significant difference of the expression of HGF between model eyes and control eyes (P<0.05).
2The effect of TGF-β2on the expressions of MMP-2, TβR II and bFGF (mRNA, protein)
2.1The expression of MMP-2was located in cytoplasm, and it was higher in experimental eye than in control eye. There was a significant difference between them (P<0.05). The expression of TGF-β2becomes gradually higher as the concentration of TGF-β2increases.
2.2The expression of TPR II was located in cytoplasm and nuclear membrane, and it was higher in experimental eye than in control eye. There was a significant difference between them (P<0.05). The expression of TβRⅡ becomes gradually higher as the concentration of TGF-β2increases.2.3The expression of bFGF was located in cytoplasm and nuclear membrane, and it was lower in experimental eye than in control eye. There was a significant difference between them (P<0.05). The expression of bFGF does not have a significant change as the concentration of TGF-β2increases.
3The observation of growth period
The ratio of G1/G0phrase of anterior and posterior retinal pigment epithelial cells increased after6-day and15-day inductions separately (P<0.05), while the ratio in S period of anterior and posterior retinal pigment epithelial cells decreased significantly (P<0.05) after6-day and15-day inductions separately.
4The observation of ultrastructure
After the anterior and posterior retinal pigment epithelial cells and scleral fibroblast cells were induced for6days, it was observed that the endoplasmic reticulum was dilated and some organelles decreased, such as endoplasmic reticulum and ribosome. The proliferation of the anterior retinal pigment epithelial cells is reduced after a15-day induction, same results with those after a6-day induction.
5The mechanical behaviors of the anterior part of LIM group and the statistical analysis
5.1In comparison of the mechanical behaviors of sclerotic desmocytes between LIM group and SC group, it was found that, whether on equilibrium Young's modulus or on cellular apparent viscosity, all the anterior parts of LIM group were longer than those of SC group on the30th day, which was significant statistically (P<0.05); and all the extreme posterior parts of LIM group were larger than SC group on the15th and the30th day, which was significant statistically too (P<0.05).
5.2The effect of TGF-β on the mechanical behaviors of the posterior parts of LIM and SC group and the statistical analysis Compared with the fellow eyes and normal control eyes, the equilibrium modulus and apparent viscosity in model eyes were significantly higher (P<0.05). After treatment of TGF-β2, the equilibrium modulus and apparent viscosity in the model group and fellow eyes were positively correlated to the concentrations of TGF-β2(r=0.743, r=0.533, r=0.654, r=0.576, P<0.05). Following the addition of1ng/ml TGF-β2and10ng/ml TGF-β2, the equilibrium modulus and apparent viscosity of scleral fibroblasts were significantly reduced in model eyes compared with fellow eyes (P<0.05). No significant difference was found in the equilibrium modulus and apparent viscosity of scleral fibroblasts between model eyes and fellow eyes after treatment with100ng/ml TGF-β2(P>0.05).
Conclusions:
1LIM could up-regulate the expressions of TGF-p2and MMP2in both RPE and scleral fibroblasts cells, while the bFGF expression was down-regulated.
2LIM and TGF-β2could inhibit the proliferation of RPE and scleral fibroblasts cells.
3TGF-β2could up-regulated the expression of MMP-2and TβR Ⅱ and remodel the ultrastructure of RPE and scleral fibroblasts cells, but has no effects on bFGF expression.
4LIM could increase the equilibrium modulus and apparent viscosity of scleral fibroblasts cells, and there was a difference between the anterior and the posterior part of sclera cells.
5Light concentration of TGF-β2could decrease the equilibrium modulus and apparent viscosity of scleral fibroblasts cells.
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(P<0.05)。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(P<0.05)；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(P<0.05)；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(P>0.05)。
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(P<0.05)。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(P<0.05), A、 B、 C三组自身对照眼的阳性率不变,差异无统计学意义(P>0.05)；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(P>0.05)。
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(P<0.05)。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(P<0.05)；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(P<0.05)；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(P>0.05)。
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(P<0.05)。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(P<0.05)。
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(P<0.05)。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。(P>0.05)。
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(P<0.05)。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(P>0.05)。
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(P<0.05)。
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(P<0.05),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(P<0.05)。
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(P<0.05)。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(P>0.05),30天组与6天组、15天组比较差异均有统计学意义(P<0.05)。
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(P>0.05),6天组与15天组、30天组比较差异均有统计学意义(P<0.05)。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(P>0.05)；两两比较差异均无统计学意义(P>0.05)。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(P<0.05)；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(P<0.05)。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(P<0.05)。
(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(P<0.05)。
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(P<0.05)。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(P>0.05)。
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(P<0.05)：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(P>0.05)。
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。
Objective:To determine the cell proliferation and the growth period of anterior and posterior retinal pigment epithelial (RPE) cells and scleral fibroblast (SF) cells in cultured guinea pig model of lens-induced myopia (LIM), and to determine the expression changes of MMP-2, TGF-β2and bFGF in RPE and SF cells.
To determine the impact of TGF-β2on the cell proliferation of anterior and posterior scleral fibroblast cells in cultured guinea pig and the impact of TGF-P2on the expression of MMP-2, TβRII and bFGF in scleral fibroblast cells; and also to study the biomechanical characteristics changes. Some conclusions were valuable for revealing the pathological mechanism and the treatment of myopia.
Methods:Divide30two-week-old guinea pigs into three groups,10of each group. The guinea pigs were randomly lens-induced monocularly. The unilateral eye was treated by defocus method as the experimental eye, and induced by a spherical lens of-10.00D to build the myopia model. The other eye in each animal was untreated as self-control (SC) eye.5two-week-old guinea pigs were randomly closed as normal-control (NC). Each group was raised for6,15and30days separately. Before and after the experiment, the refraction was measured using retinoscopy and the axis oculi lengths of binoculus were determined by A-scan ultrasonography.
The retinal pigment epithelial cells of each group were cultured and passed continually for2generations. The cultured cells were verified to be retinal pigment epithelial cells using immunocytochemical method. Immunocytochemistry, Real-Time PCR Analysis and Western Blot method were used to detect the expressions of MMP-2, TGF-β2and bFGF in anterior and posterior retinal pigment epithelial cells of the experimental eyes and self-control eyes. MTT was used to detect the proliferation of each group of scleral fibroblast cells. The cultured cells of each group were studied by Flow Cytometry (FCM). SPSS13.0system was used to do statistical analysis of the observed data.
The scleral fibroblasts of each group were cultured and passed continually for2generations. The cultured cells were verified to be scleral fibroblasts using immunocytochemistry method. Immunocytochemistry, Real-Time PCR Analysis and Western Blot method were used to detect the expressions of MMP-2, TGF-β2and bFGF in anterior and posterior scleral fibroblast cells of the experimental eyes and self-control eyes. MTT was used to detect the proliferation of each group of scleral fibroblast cells. The cultured cells of each group were studied by Flow Cytometry (FCM). SPSS13.0system was used to do statistical analysis of the observed data.
The scleral fibroblasts of each group were purified with the tissue explant method and passed continually for2generations. The cultured cells were verified to be scleral fibroblasts using immunocytochemistry method. Add TGF-β2of different mass concentrations (0,1,10, and100ng/ml) to serum-free DMEM to react for24hours. Immunocytochemistry, Real-Time PCR Analysis and Western Blot method were used to detect the expressions of MMP-2, TGF-β2and bFGF in anterior and posterior scleral fibroblast cells of the experimental eyes and self-control eyes. MTT was used to detect the proliferation of each group of scleral fibroblast cells. The cultured cells of each group were studied by Flow Cytometry (FCM). SPSS13.0system was used to do statistical analysis of the observed data.
Results:1The expressions of TGF-β2, bFGF, MMP-2in cultured guinea pig anterior and posterior retinal pigment epithelial cells and scleral fibroblasts in lens-induced myopia shown by the results of Immunocytochemistry, Real-Time PCR Analysis and Western blot method:1.1The expression of TGF-β2was higher in experimental eye than in control eye. There was a significant difference between them (P<0.05). The expression of TGF-β2in posterior cells was higher than the in anterior cells (P>0.05) on the6th,15th and30th day. There was a significant difference of the expression of TGF-β2between model eyes and control eyes (P<0.05).1.2The expression of bFGF was lower in experimental eye than in control eye. There was a significant difference between them (P<0.05). The expression of bFGF in anterior cells was almost the same with the expression in posterior cells on the6th,15th and30th day (P>0.05).
1.3The expression of MMP-2was higher in experimental eye than in control eye. There was a significant difference between them (P<0.05). The expression of TGF-β2in posterior cells was higher than the expression in anterior cells (P>0.05) on the6th,15th and30th day. There was a significant difference of the expression of HGF between model eyes and control eyes (P<0.05).
2The effect of TGF-β2on the expressions of MMP-2, TβR II and bFGF (mRNA, protein)
2.1The expression of MMP-2was located in cytoplasm, and it was higher in experimental eye than in control eye. There was a significant difference between them (P<0.05). The expression of TGF-β2becomes gradually higher as the concentration of TGF-β2increases.
2.2The expression of TPR II was located in cytoplasm and nuclear membrane, and it was higher in experimental eye than in control eye. There was a significant difference between them (P<0.05). The expression of TβRⅡ becomes gradually higher as the concentration of TGF-β2increases.2.3The expression of bFGF was located in cytoplasm and nuclear membrane, and it was lower in experimental eye than in control eye. There was a significant difference between them (P<0.05). The expression of bFGF does not have a significant change as the concentration of TGF-β2increases.
3The observation of growth period
The ratio of G1/G0phrase of anterior and posterior retinal pigment epithelial cells increased after6-day and15-day inductions separately (P<0.05), while the ratio in S period of anterior and posterior retinal pigment epithelial cells decreased significantly (P<0.05) after6-day and15-day inductions separately.
4The observation of ultrastructure
After the anterior and posterior retinal pigment epithelial cells and scleral fibroblast cells were induced for6days, it was observed that the endoplasmic reticulum was dilated and some organelles decreased, such as endoplasmic reticulum and ribosome. The proliferation of the anterior retinal pigment epithelial cells is reduced after a15-day induction, same results with those after a6-day induction.
5The mechanical behaviors of the anterior part of LIM group and the statistical analysis
5.1In comparison of the mechanical behaviors of sclerotic desmocytes between LIM group and SC group, it was found that, whether on equilibrium Young's modulus or on cellular apparent viscosity, all the anterior parts of LIM group were longer than those of SC group on the30th day, which was significant statistically (P<0.05); and all the extreme posterior parts of LIM group were larger than SC group on the15th and the30th day, which was significant statistically too (P<0.05).
5.2The effect of TGF-β on the mechanical behaviors of the posterior parts of LIM and SC group and the statistical analysis Compared with the fellow eyes and normal control eyes, the equilibrium modulus and apparent viscosity in model eyes were significantly higher (P<0.05). After treatment of TGF-β2, the equilibrium modulus and apparent viscosity in the model group and fellow eyes were positively correlated to the concentrations of TGF-β2(r=0.743, r=0.533, r=0.654, r=0.576, P<0.05). Following the addition of1ng/ml TGF-β2and10ng/ml TGF-β2, the equilibrium modulus and apparent viscosity of scleral fibroblasts were significantly reduced in model eyes compared with fellow eyes (P<0.05). No significant difference was found in the equilibrium modulus and apparent viscosity of scleral fibroblasts between model eyes and fellow eyes after treatment with100ng/ml TGF-β2(P>0.05).
Conclusions:
1LIM could up-regulate the expressions of TGF-p2and MMP2in both RPE and scleral fibroblasts cells, while the bFGF expression was down-regulated.
2LIM and TGF-β2could inhibit the proliferation of RPE and scleral fibroblasts cells.
3TGF-β2could up-regulated the expression of MMP-2and TβR Ⅱ and remodel the ultrastructure of RPE and scleral fibroblasts cells, but has no effects on bFGF expression.
4LIM could increase the equilibrium modulus and apparent viscosity of scleral fibroblasts cells, and there was a difference between the anterior and the posterior part of sclera cells.
5Light concentration of TGF-β2could decrease the equilibrium modulus and apparent viscosity of scleral fibroblasts cells.
引文
1 胡诞宁.近视的病因与发病机制研究进展.眼视光学杂志.2004,6(1)：1-5
2 Rada JA, Nickla DL, Troilo D. Decreased proteoglycan synthesis associated with form deprivation myopia in mature primate eyes [J]. Invest Ophthalmol Vis Sci.2000,41(1):2050-2058
3 Zhu X, Park TW, Winawer J, Walman J. In a matter of minutes, the eye can know which way to grow [J]. Invest Ophthalmol Vis Sci.2005,46(7): 2238-2241
4 欧阳朝祜,胡文政,褚仁远.凹透镜对豚鼠眼生长及屈光发展的影响.眼科研究.2002,20(5)：391-393
5 Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C (T)) Method[J]. Methods.2001,25(4):402-408
6 Burger H, Foekens JA, Look MP, et al. RNA expression of breast cancer resistance protein, lung resistance-related protein, multidrug resistance-associated proteins 1 and 2, and multidrug resistance gene 1 in breast cancer:Correlation with chemotherapeutic response [J]. Clin Cancer Res.2003,9(2):827-836
7 McBrien NA, Moghaddam HO, Cottriall CL, Leech EM, Cornell LM.The effects of blockade of retinal cell action potentials cm ocular growth, emmetropization and form dep rivation myopia in young chick [J]. Vision Res.1995,35(9):1141-1152
8 Norton TT, Essinger JA, MC Brien NA. Lid-suture myopia in tree shrews with retinal ganglion cell blockade[J]. Vis Neurosci.1994,11(1):143-153
9 Wildsoet C. Neural pathways subserving negative lens-induced emmetropization in chicks-in sights from selective lesions of the optic nerve and ciliary nerve[J]. Curr Eye Res.2003,27(6):371-385
10 Kee CS, Ramamirtliam R, Qiao-Grider Y, Hung LF, Word M, Smith EL.The role of peripheral vision in the refractive-en-or development of infant monkeys Macaca mulatta [J].Invest Ophthalmol VisSci.2004,45: E-1157
11 Shih YF, Fitzgerald ME, Reiner A. The effects of choroidal of nerve transection on myopic eye growth induced by googles [J]. Invest Ophthalmol Vis Sci.1994,35:3691-3701
12胡诞宁,M cCormick SA.视网膜色素上皮-脉络膜在近视发病中的作用[J].眼视光学杂志.2000,2(4)：197-200
13 Laihu MO, Saksela O, Andresen PA, et al. Enhanced production and extracellular deposition of die endothelial-type plasminogen activator inhibitor in cultured human lung fibroblasts by transfoming growth factor-beta[J]. J Cell B ion.1986,103:2403-2410
14 Seko Y, Shimokawa H, Tokoro T. Expression of bFGF and TGF-β2 in experimental myopia in chickens[J]. Invest ophthalmol Vis Sci.1995, 36(6):1183-1187
15曾爱萍,曾水清.转化生长因子β-1对培养的人RPE细胞MMP和TIMP-1 mRNA表达的影响.眼科新进展.2006,26(2)：81-84
16 Zheng Xiaofen, Chu Renyuan.Effects of TGF-β on human embryonic retinal pigment epithelium cell. Chin Ophthal Res. January 2007,125(1): 14-17
17陈有信,何世坤.细胞外基质蛋白和转化生长因子β2共同作用诱导人视网膜色素上皮细胞向肌纤维母细胞转化.中华眼底病杂志.2006,22(5)：328-332
18 Hayashi T, Inoko H. Exclusion of transforming growth factor-beta 1 as a candidate gene for myopia in the Japanese. Jpn J Ophthalmol.2007,51 (2):96-99
19魏海英,崔浩.鸡形觉剥夺性近视眼形态学及bFGF免疫组织化学研究.眼科研究.2007,25(1)：22-24
20 Rohrer B, Iuvone PM, Stell WK. Stimulation of dopaminergic amacrine cells by stroboscopic illumination or fibroblast growth factor (bFGF, FGF-2)injections:possible roles in prevention of form-deprivation myopia in the chick. Brain Res.1995,686:169
1 曾衍钧,李秀云,任庆华等.猪眼角膜的生物力学特性[J].生物物理学报.1993,9(2)：323-327
2 Kee CS, Ramamirtliam R, Qiao-Grider Y, Hung LF, Word M, Smith EL, The role of peripheral vision in the refractive-error development of infant monkeys(Macaca mulatta)[J]. Invest Ophthalmol Vis Sci.2004,45: E-1157
3 Kusakari T, Sato T, Tokoro T. Regional scleral changes in form-deprivation myopia in chicks [J]. Exp Eye Res.1997,64 (3):465-476
4 Fullwood NJ, Troilo D, Wallman J, et al. Synchrotron x-ray diffraction and histo-cheanical studies of normal and myopic chick eyes[J].Tissue Cell.1993,25 (1):73-85
5 McBrien NA, Cornell LM, Gentle A. Structural and ultrastructural changes to the sclera in a mammalian model of high myopia[J].Invest Ophthalmol Vis Sci.2001,42 (10):2179-2187
6 Gental A, McBrien NA. Modulation of seleral DNA synthesis in development of and recovery from axial myopia in the tree shrew [J]. Exp Eye Res.1999,68 (2):155-163
7 Curtin BJ, Iwamoto T, Benalclo DP. Normal and staphylomatous sclera of high myopia. An electron microscopic study [J]. Arch Ophthalmol.1979, 97 (5):912-915
8 McBrien SA, Lawlor P, Gentle A. Scleral remodeling during the development of and recovery from axial myopia in the tree shrew [J]. InVeSt Ophthalmol Vis Sci.2000,41 (12):3713-3719
9 Le MD, Besson A, Fogg DK, et al. Explotation of astroeytes by glioma cells to facilitate invasiveness:a mechanism involving matrix metalloproteinase-2 and the arokinase-type plasminogen activator-plasmin cascade[J]. J Neurosci.2003,23 (10):4034-4043
10 Jones BE, Thompson EW, Hodos W, et al.Scleral matrix metalloproteinases, serine proteinase activity and hydrational capcity are inceased in myopia induced by retinal image degradation[J]. ExpEyeRes. 1996,63:369-381.
11 Rada JA, Perry CA, Slover ML, et al.Gelatinase A and MMP2 expresson in the fibrous sclera of myoia and recovering chicks eyes[J].Invest Ophthalmol Vis Sci.1999,40:3091-3099
12 GuggenheimJA, Mcbrien NA. Form-deprivation myopia induces activation of scleral matrix metalloproteinase-2 in tree shrew[J].Invest Ophthalmol Vis Sci.1996,37:1380-1395
13吴文灿,刘双珍,王剑锋等.小鸡行觉剥夺性近视眼后极部巩膜MMP-2与TIMP-2 mRNA表达的动态变化[J].眼科新进展.2004,24(3) ：73-1770
14 Siegwart JT Jr, Norton TT. Steady state mRNA levels in tree shew sclera with form-deprivation myopia and during recovery [J]. Invest Ophthalmol Vis Sci.2001,42:1153-1159
15 Kenning MS, Gentle A, Mcbrien NA. Expression and cDNA Sequence of Matrix metalloproteinase-2 in a Mammalian Model of Human Disesase Processes:Tupaia belangeri. DNA Seq.2004,15:332-337
16 Rohrer B, Stell WK. Basic fibroblast growth factor and transforming growth factor act as stop and go signals to modulate postnatal ocular growth in the chick. Exp Eye Res.1994,58:553-561
17 seko Y, Tanaka Y, Tokoro T. Influence of bFGF as a potent growth stimulator and TGF-β as a growth regulator on scleral chondrocytes and scleral fibroblasts in vitro [J]. Ophthalmic Res.1995,27 (3):144-152
18 Hu DN, McCormick SA. Effect of TGF-β and cAMP-elevating agents on the growth of human scleral fibroblasts in vitro (A)//Lin LK edi. Myopia updates Ⅱ (Proceedings, Ⅶ international conference on myopia [M]. Tokyo:Springer.2000:131-132
19 Kee CS, Marazani D, Wallman J. Differences in time course and visual requirements of ocular responses to lenses and diffusers[J]. Invest Ophthalmol Vis Sci.2001,42:575-583
20 Ejedor J, de la Villa P. Refractive changes induced by form deprivation in the mouse eye [J]. Invest Ophthalmol Vis Sci.2003,44:32-36
21 Kenning MS, Gentle A, Mcbrien NA. Expression and cDNA Sequence of Matrix metalloproteinase-2 in a Mammalian Model of Human Disesase Processes-.Tupaia belangeri. DNA Seq.2004,15:332-337
22 Rohrer B, Tao J, Stell WK. Basic fibroblast growth factor,its high and low affinity receptors and their relationship to form deprivation myopia in the chick[J].NeuroSci.1997,79 (3):775
23 Seko Y, Shimokawa H, Tokoro T. Expression of bFGF and TGF-β2 in experimental myopia in chickens[J]. Invest ophthalmol Vis Sci.1995, 36(6):1183-1187
1 白灵,樊瑜波,张明.离体培养细胞的力学实验方法.生物医学工程学杂志.2002,19(2)：324-328
2 王超英,陈维毅,郝岚,等.高度近视眼巩膜生物力学特性初步研究.眼科研究.2003,21(2)：113-115
3 王晓君,李涛,陈维毅.巩膜胶原含量与其生物力学性能的关系研究[J].太原理工大学学报.2007,38(4)：371-373
4 孙朝晖,王超英,靳胜利等.实验性近视眼巩膜生物力学特征研究[J].眼视光学杂志.2006,8(4)：209-213
5 Gentle A, McBrien NA. Modulation of scleral DNA synthesis in development of and. recovery from induced axial myopia in the tree shrew. Exp Eye Res.1999,68:155-163
6 Cui W, Bryant MR, Sweet PM. McDonnell PJ. Changes in gene expression in response to mechanicalstrain in human scleral fibroblasts[J]. Experimental Eye Research.2004,78:275-284
7 Stamenovic D. Effects of cytoskeletal prestress on cell rheological behavior[J]. Acta Biomaterialia.2005:255-262
8 NortonTT, Rada JA. Reduced extracellular matrix in mammalian sclera with induced myopia. Vision Res.1995,35 (9):1271-1281
9 PhillipsJR, McBrien NA. Form deprivation myopia:elastic properties of sclera[J].Ophthalmic Physiol Opt.1995,15 (5):357-362
10 Shelton Lilian, Jody Summers Rada. Effects of cyclic mechanical stretch on extracellular matrix synthesis by human scleral fibroblasts[J]. Experimental Eye Research.2006,10(004):1-9
1 McBrien NA, Gentle A. Role of the sclera in the development and pathological complications of myopia. Prog Retin Eye Res.2003,22: 307-338
2 Rohrer B, Stell WK. Basic fibroblast growth factor and transforming growth factor act as stop and go signals to modulate postnatal ocular growth in the chick. Exp Eye Res.1994,58:553-561
3 seko Y, Tanaka Y, Tokoro T. Influence of bFGF as a potent growth stimulator and TGF-β as a growth regulator on scleral chondrocytes and scleral fibroblasts in vitro [J]. Ophthalmic Res.1995,27 (3):144-152
4 吴文灿,刘双珍,王剑锋等.小鸡形觉剥夺性近视眼后极部巩膜MMP-2与TIMP-2 mRNA表达的动态变化[J].眼科新进展.2004,24(3)：73-1770
5 Hu DN, McCormick SA. Effect of TGF-β and cAMP-elevating agents on the growth of human scleral fibroblasts in vitro (A)//Lin LK edi. Myopia updates II (Proceedings, VII international conference on myopia) [M]. Tokyo:Springer.2000:131-132
6 Overall CM, Wrana JL, Sodex JL, Sodek J. Post-transcriptional regulation of 72-kDa gelatinase/type transforming growth factor-betalin human fibroblasts:Transcriptional and IV collagenase by comparisons with collagenase and tissue inhibitor of matrix metalloproteinase gene expression. J Biol Chem.1991,266:14064
7 Kim HS, Shang T, Chen Z et al. TGF-betal stimulates production of gelatinase (MMP-9), collagenases (MMP-1,-13) and stromelysins (MMP-3,-10,-11)by human corneal epithelial cells. Exp Eye Res.2004, 79:263
8 Seomun Y, Kim J, Lee EH et al. Overexpression of metalloproteinase-2 mediates phenotypic transformation of lens epithelial cells. Biochem J. 2001,15:41
9 Behzadian MA, Wang XL, Windsor LJ et al. TGF-pincreases retinal endothelial cell permeability by increasing MMP-9:possible role of glial cells in endothelial cells in endothelial barrier function. Invest Ophthalmo Vis Sci.2001,42:853
10 Kee CS, Marazani D, Wallman J. Differences in time course and visual requirements of ocular responses to lenses and diffusers[J]. Invest Ophthalmol VisSci 2001.42:575-583
11 Ejedor J, de la Villa P. Refractive changes induced by form deprivation in the mouse eye [J]. Invest Ophthalmol Vis Sci.2003,44:32-36
12 Rada JA, Nickla DL. Troilo D. Decreased proteoglycan synthesis associated with form deprivation myopia in mature primate eyes [J]. Invest Ophthalmol VisSci.2000; 41 (1):2050-2058
13郑瑾,佘振珏,周国民.EGF与bFGF对体外大鼠SF细胞增殖的影响.Fudan Univ Jmed Sci.2006May; 33 (3):301-304
14 Rohrer B, Stell WK. Basic fibroblast growth factor and transforming growth factor act as stop and go signals to modulate postnatal ocular growth in the chick. Exp Eye Res.1994,58:553-561
15胡诞宁,M cCormick SA视网膜色素上皮-脉络膜在近视发病中的作用[J].眼视光学杂志.2000,2(4)：197-200
16 Massague J. Rcecptors for the TGF-beta family.Cell.1992,69 (70): 1067-70
17 Warana JL, Attisano L, Wieser R,et al. Mechanism of activation of the TGF-beta receptor. Nature.1994,370 (6488):341-7
18 Savage C, Das P, Firelli AL, et al. Caenorhabditis elegans genes sma-2, sma-3, and sma-4 define a conserved family of transforming growth factor beta pathway components. Proc Natl Acad Sci USA.1996,93 (2):790-4
19 Shipley GD, Tucker RF, Mouses HL. Type beta transforming growth factor/growth inhibitor stimulates entry of monolayer cultures of AKR-2B cells into S phase after a prolonged prereplicative interval. Proc Natl Sci USA.1985,82 (12):4147-51
20 Anzano MA, Roberts AB, Sporn MB, et al. Anchorage-independent growth of primary rat embryo cells is induced by platelet-derived growth factor and inhibited by type-beta transforming growth factor. Cell Physiol. 1986,126 (2):312-8
21 Heimark RL, Twardzik DR, Schwartz SM,et al. Inhibition of endothelial regeneration by type-beta transforming growth factor from platelets. Science.1986,233 (4768):1078-80
22 Kehrl JH, Robert AB, Wakefield LM, te al.Transforming growth factor beta is an important immanomodulatory protein for human B lymphocytes. J Immunol.1986,137 (12):3855-60
23 Kehrl JH, Wakefield LM, Robert AB,te al. Production of transforming growth factor beta by human T lymphocytes and its potential role in the regulation of T cell growth. J Exp Med.1986,163 (5):1037-50
24 Carr BI, Hayashi T, Branum EL, et al. Inhibition of DNA synthesis in rate hepatocytes by platelet-derived type beta transforming growth factor. Cancer Res.1986,46 (5):2330-4
25 Sporn MB, Robert AB, Wakefield LM, et al. Some recent advances in the chemistry and biology of transforming growth factor-beta. J Cell Biol. 1987,105 (3):1039-45
26王红,曲毅,郑水军.豚鼠透镜诱导性近视眼巩膜细胞凋亡的研究[J].山东大学学报(医学版).2006,44(7)：671-675
27潘卫红,曾俊文.TGF-β1对小鼠SF细胞增殖和细胞周期影响[J].眼科研究.2006,23 (3)：232-234
28傅亚娜,周翔天,付小莹等.碱性成纤维细胞生长因子和转化生长因子-β对人SF细胞的生长调控研究.眼视光学杂志.2008,10(3)186-189
1 王超英,陈维毅,郝岚等.高度近视眼巩膜生物力学特性初步研究[J].眼科研究.2003,21(2)：113-115
2王晓君,李涛,陈维毅,巩膜胶原含量与其生物力学性能的关系研究[J].太原理工大学学报.2007,38(4)：371-373
3 孙朝晖,王超英,靳胜利等.实验性近视眼巩膜生物力学特征研究[J].眼视光学杂志.2006,8(4)：209-213
4 Gentle A,McBrien NA. Modulation of scleral DNA synthesis in development of and. recovery from induced axial myopia in the tree shrew. Exp Eye Res.1999,68:155-163
5 陈维毅,王超英,张全有,李阳阳等.实验性近视眼SF细胞黏弹性研究[J].医用生物力学.2007,22(1)：22-29
6 Cui W, Bryant MR, Sweet PM, et al. Changes in gene expression in response to mechanicalstrain in human scleral fibroblasts[J]. Experimental Eye Research.2004,78:275-284
7 Shelton Lilian, Jody Summers Rada. Effects of cyclic mechanical stretch on extracellular matrix synthesis by human scleral fibroblasts[J]. Experimental Eye Research.2006,10 (004):1-9
1 许银娥,吴小影,刘双珍,夏晓波,王育科.近视眼视网膜神经纤维层厚度分析[J].国际眼科杂志.2006,6(1)：116-118
2 Harm an AM, HoskinsR, Beazley LD. Experimental eye en-largement in mature animals changes the retinal pigment epithelium [J]. Vis Neurosci. 1999,16 (4):619-628
3 任力,李永平,易玉珍等.近视眼模型的形态学及视网膜超微结构观察[J].中华眼底病杂志.1999,15(1)：20-23
4席晓勃,褚仁远.实验性近视眼组织病理及超微结构观察.眼科新进展.2001,21(5)： 329-331
5 Zhong xingwu, Ge jian, Chen xiaolian. Comparision of retinal morphology and ultrastructure in defocus-iuduced myopia and form-deprivation myopia in rhesus monkeys. ChinJ Ophthalmol.2005,41 (7):625-630
6 王红,高蕾,曹加会,陈红梅.碱性成纤维细胞生长因子对豚鼠透镜诱导性近视眼巩膜细胞凋亡的影响.环境与健康杂志.2008,25(8)：676-679
7汪芳润编著.近视眼[M].上海：上海医科人学出版社.1996：212-229
8 Janet R, Bolin C. Blue light-induced apoptosis of A2E-containing RPE: involvement of caspase-3 and protection by Bcl-2. Invest Ophthalmol Vis Sci.2001,42:1356
9 Hu DN, Woodward DF, McCormick ST. Influence of autonomic. neuro-transmitters on human uveal melanocytes in vitro. Exp Eye Res. 2000,71:217-224
10 Roberts JE, Wiechmann AF, Hu DN. Melatonin receptors in human u-veal melanocytes and melanoma cells. J Pineal Res.2000,28:16-171
11 Hu DN. Role of RPE-Choroid axis on the occurrence of myopia. Chin J Optom Ophthalmol.2000,2 (4):197-200
12 Haque R, Maltseva O, Ivanova T, et al. Dopamine D1 recpptor ex-pression in cultured human andmonkey retinal pigment epithelial cells. Invest Ophthalmol Vis Sci.2000,41:S843
13 Rymer J, Wildsoet CF. The role of the pigment epithelium in eye growth regulation and myopia. Vis Neurosci.2005,22 (3):251-261
14 Laihu MO, Saksela O, Andresen PA,et al. Enhanced production and extracellular deposition of die endothelial-type plasminogen activator inhibitor in cultured human lung fibroblasts by transfoming growth factor-beta[J]. J Cell Bion.1986,103:2403-2410
15 Kvanta A. Expression and secretion of transforming growth factor-beta in transformed and nontransformed retinal pigment epithelial cells.Ophthahmic Res.1994,26:361-367
16 Sakamoto T, Soriano D, Nassaralla J, et al. Effect of intravitreal administration of indomethacin onexperimental subretinal neovascularization in the subhuman primate.Arch Ophthalmol.1995,113: 222-226
17 Seko Y, Shimokawa H, Tokoro T. Expression of bFGF and TGF-β 2 in experimental myopia in chickens[J]. Invest ophthalmol Vis Sci.1995,36 (6):1183-1187
18曾爱萍,曾水清.转化生长因子β-1对培养的人RPE细胞MMP和TIMP-1 mRNA表达的影响.眼科新进展.2006,26(2)： 81-84
19 Zheng Xiaofen, Chu Renyuan. Effects of TGF-β on human embryonic retinal pigment epithelium cell. Chin Ophthal Res. January 2007,125 (1): 14-17
20陈有信,何世坤.细胞外基质蛋白和转化生长因子β 2共同作用诱导人视网膜色素上皮细胞向肌纤维母细胞转化.中华眼底病杂志.2006,22(5)：328-332
21 Hayashi T, Inoko H. Exclusion of transforming growth factor-betal as a candidate gene for myopia in the Japanese. Jpn J Ophthalmol.2007,51 (2):96-99
22 Kimizuka Y, Yamada T, Tamai M.Quantitative study on regenerated retinal pigment epithelium and the effects of growth factor[J]. Curr Eye Res 1997; 16 (11):1081-1087
23周浩,褚仁远,周行涛,戴锦晖.体内实验观察bFGF对视网膜色素上皮细胞再生的促进作用[J].眼科新进展.2003；23(2)：97-99
24魏海英,崔浩.鸡形觉剥夺性近视眼形态学及bFGF免疫组织化学研究.眼科研究.2007,25(1)：22-24
25 Rohrer B, Iuvone PM, Stell WK. Stimulation of dopaminergic amacrine cells by stroboscopic illumination or fibroblast growth factor (bFGF, FGF-2) injections:possible roles in prevention of form-deprivation myopia in the chick. Brain Res.1995,686:169
26毛俊峰,刘双珍.bFGF对鸡形觉剥夺性近视眼的抑制作用.国际眼科杂志.2005,5(4)：652-654
27 Rohrer B, Stell WK. Basic fibroblast growth factor and transforming growth factor act as stop and go signals to modulate postnatal ocular growth in the chick. Exp Eye Res.1994,58:553-561
28 seko Y, Tanaka Y, Tokoro T. Influence of bFGF as a potent growth stimulator and TGF-β as a growth regulator on scleral chondrocytes and scleral fibroblasts in vitro [J]. Ophthalmic Res.1995,27 (3):144-152
29 Hu DN, McCormick SA. Effect of TGF-β and cAMP-elevating agents on the growth of human scleral fibroblasts in vitro (A)//Lin LK edi. Myopia updates II (Proceedings, VII international conference on myopia) [M]. Tokyo:Springer.2000:131-132
30 Kee CS, Marazani D, Wallman J. Differences in time course and visual requirements of ocular responses to lenses and diffusers[J]. Invest Ophthalmol Vis Sci.2001,42:575-583
31 Ejedor J, de la Villa P, Refractive changes induced by form deprivation in the mouse eye [J]. Invest Ophthalmol VisSci.2003,44:32-36
32 Derynck R Feng XH.TGF-beta receptor signaling. Biochim BiophysActa. 1997(24); 1333 (2):105
33 Simmons ML, Murphy S. Induction of nitric oxide synthase in glialcells[J]. Neurochem.1992,59 (3):897
34 Le MD, Besson A, Fogg DK,et al. Explotation of astroeytes by glioma cells to facilitate invasiveness:a mechanism involving matrix metalloproteinase-2and the arokinase-type plasminogen activator-plasmin cascade[J]. J Neuroscib.2003,23 (10):4034-4043
35 Jones BE, Thompson EW, Hodos W, et al.Scleral matrix metalloproteinases, serine proteinase activity and hydrational capcity are inceased in myopia induced by retinal image degradation[J]. ExpEyeRes. 1996,63:369-381
36 Rada JA, Perry CA, Slover ML,et al. Gelatinase A and MMP2 expresson in the fibrous sclera of myoia and recovering chicks eyes[J].Invest Ophthalmol Vis Sci.1999,40:3091-3099
37 Guggenheim JA, IcBrien NA. Form-deprivation myopia induces activation of scleral matrix metalloproteinase-2 in tree shrew[J]. Invest Ophthalmol Vis Sci.1996,37:1380-1395
38吴文灿,刘双珍,王剑锋等.小鸡行觉剥夺性近视眼后极部巩膜MMP-2与TIMP-2 mRNA表达的动态变化[J].眼科新进展.2004,24(3)：73-1770
39 Siegwart JT Jr, Norton TT. Steady state mRNA levels in tree shew sclera with form-deprivation myopia and during recovery[J]. Invest Ophthalmol Vis Sci.2001,42:1153-1159
40 Kenning MS, Gentle A, Mcbrien NA. Expression and cDNA Sequence of Matrix metalloproteinase-2 in a Mammalian Model of Human Disesase Processes:Tupaia belangeri. DNA Seq.2004,15:332-337
41 Rohrer B, Tao J, Stell WK. Basic fibroblast growth factor,its high and low affinity receptors and their relationship to form deprivation myopia in the chick[J]. NeuroSci.1997,79 (3):775
42 Seko Y, Shimokawa H, Tokoro T. Expression of bFGF and TGF-β2 in experimental myopia in chickens[J]. Invest ophthalmol Vis Sci.1995,36 (6):1183-1187
43 Cowan DB, Lye SJ, Langille BL. Regulation of vascular connexin43 gene expression by mechanical loads. Circ Res.1998,82 (7):786-793
44 Matsumoto T, Kawakami M, Kuribayashi K, Takenaka T, Tamaki T. Cyclic mechanical stretch stress increases the growth rate and collagen synthesis of nucleus pulposus cells in vitro. Spine.1999,24 (4):315-319
45王建洲,惠延年,侯旭等.磁珠包裹的人视网膜色素上皮细胞受磁场 牵拉后ET-1的表达.国际眼科杂志.2006,6(4)：775-777
46张晓光,惠延年等.培养人视网膜色素上皮细胞牵拉模型中细胞骨架的改变.中华眼科杂志.2006,42(2)：121-126
47候旭,惠延年,韩泉洪等.机械牵拉对培养人视网膜色素上皮细胞内钙离子的影响.国际眼科杂志.2005,5(1)：50-54
48王超英,陈维毅,郝岚,等.高度近视眼巩膜生物力学特性初步研究.眼科研究.2003,21(2)：113-115
49王晓君,李涛,陈维毅.巩膜胶原含量与其生物力学性能的关系研究[J].太原理工大学学报.2007,38(4)：371-373
50孙朝晖,王超英,靳胜利等.实验性近视眼巩膜生物力学特征研究[J].眼视光学杂志.2006,8(4)：209-213
51 Gentle A, McBrien NA. Modulation of scleral DNA synthesis in development of and. recovery from induced axial myopia in the tree shrew. Exp Eye Res.1999,68155-163
52胡萍,李镜海.透镜诱导豚鼠近视眼巩膜基质中基质金属蛋白酶2及其组织抑制剂和生长因子的变化[J].眼视光学杂志.2008,10(1)：17-20
53 Cui W, Bryant MR, Sweet PM, McDonnell PJ. Changes in gene expression in response to mechanical strain in human scleral fibroblasts[J]. Experimental Eye Research,2004,78:275-284
54 Shelton Lilian，Jody Summers Rada. Effects of cyclic mechanical stretch on extracellular matrix synthesis by human scleral fibroblasts[J]. Experimental Eye Research (2006).10.004:1-9
55 Stamenovic D. Effects of cytoskeletal prestress on cell rheological behavior[J]. Acta Biomaterialia,2005,255-262
56胡诞宁,Steven A, McComick视网膜色素上皮-脉络膜在近视发病中的作用[J].眼视光学杂志.2000,2(4)：197-200
57 Hu DN, MC-Cormick SA. Role of RPE-choroid axis on production, binding and response to various growth factors modulate the occurrence of myopia[C]. In Thorn F, etal, (edi):Myopia 2000 (Proceed-ings,,International Conference on Myopia) [M]USA:Conference on Myopia, Inc.2000.213-216
2 Rada JA, Nickla DL, Troilo D. Decreased proteoglycan synthesis associated with form deprivation myopia in mature primate eyes [J]. Invest Ophthalmol Vis Sci.2000,41(1):2050-2058
3 Zhu X, Park TW, Winawer J, Walman J. In a matter of minutes, the eye can know which way to grow [J]. Invest Ophthalmol Vis Sci.2005,46(7): 2238-2241
4 欧阳朝祜,胡文政,褚仁远.凹透镜对豚鼠眼生长及屈光发展的影响.眼科研究.2002,20(5)：391-393
5 Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C (T)) Method[J]. Methods.2001,25(4):402-408
6 Burger H, Foekens JA, Look MP, et al. RNA expression of breast cancer resistance protein, lung resistance-related protein, multidrug resistance-associated proteins 1 and 2, and multidrug resistance gene 1 in breast cancer:Correlation with chemotherapeutic response [J]. Clin Cancer Res.2003,9(2):827-836
7 McBrien NA, Moghaddam HO, Cottriall CL, Leech EM, Cornell LM.The effects of blockade of retinal cell action potentials cm ocular growth, emmetropization and form dep rivation myopia in young chick [J]. Vision Res.1995,35(9):1141-1152
8 Norton TT, Essinger JA, MC Brien NA. Lid-suture myopia in tree shrews with retinal ganglion cell blockade[J]. Vis Neurosci.1994,11(1):143-153
9 Wildsoet C. Neural pathways subserving negative lens-induced emmetropization in chicks-in sights from selective lesions of the optic nerve and ciliary nerve[J]. Curr Eye Res.2003,27(6):371-385
10 Kee CS, Ramamirtliam R, Qiao-Grider Y, Hung LF, Word M, Smith EL.The role of peripheral vision in the refractive-en-or development of infant monkeys Macaca mulatta [J].Invest Ophthalmol VisSci.2004,45: E-1157
11 Shih YF, Fitzgerald ME, Reiner A. The effects of choroidal of nerve transection on myopic eye growth induced by googles [J]. Invest Ophthalmol Vis Sci.1994,35:3691-3701
12胡诞宁,M cCormick SA.视网膜色素上皮-脉络膜在近视发病中的作用[J].眼视光学杂志.2000,2(4)：197-200
13 Laihu MO, Saksela O, Andresen PA, et al. Enhanced production and extracellular deposition of die endothelial-type plasminogen activator inhibitor in cultured human lung fibroblasts by transfoming growth factor-beta[J]. J Cell B ion.1986,103:2403-2410
14 Seko Y, Shimokawa H, Tokoro T. Expression of bFGF and TGF-β2 in experimental myopia in chickens[J]. Invest ophthalmol Vis Sci.1995, 36(6):1183-1187
15曾爱萍,曾水清.转化生长因子β-1对培养的人RPE细胞MMP和TIMP-1 mRNA表达的影响.眼科新进展.2006,26(2)：81-84
16 Zheng Xiaofen, Chu Renyuan.Effects of TGF-β on human embryonic retinal pigment epithelium cell. Chin Ophthal Res. January 2007,125(1): 14-17
17陈有信,何世坤.细胞外基质蛋白和转化生长因子β2共同作用诱导人视网膜色素上皮细胞向肌纤维母细胞转化.中华眼底病杂志.2006,22(5)：328-332
18 Hayashi T, Inoko H. Exclusion of transforming growth factor-beta 1 as a candidate gene for myopia in the Japanese. Jpn J Ophthalmol.2007,51 (2):96-99
19魏海英,崔浩.鸡形觉剥夺性近视眼形态学及bFGF免疫组织化学研究.眼科研究.2007,25(1)：22-24
20 Rohrer B, Iuvone PM, Stell WK. Stimulation of dopaminergic amacrine cells by stroboscopic illumination or fibroblast growth factor (bFGF, FGF-2)injections:possible roles in prevention of form-deprivation myopia in the chick. Brain Res.1995,686:169
1 曾衍钧,李秀云,任庆华等.猪眼角膜的生物力学特性[J].生物物理学报.1993,9(2)：323-327
2 Kee CS, Ramamirtliam R, Qiao-Grider Y, Hung LF, Word M, Smith EL, The role of peripheral vision in the refractive-error development of infant monkeys(Macaca mulatta)[J]. Invest Ophthalmol Vis Sci.2004,45: E-1157
3 Kusakari T, Sato T, Tokoro T. Regional scleral changes in form-deprivation myopia in chicks [J]. Exp Eye Res.1997,64 (3):465-476
4 Fullwood NJ, Troilo D, Wallman J, et al. Synchrotron x-ray diffraction and histo-cheanical studies of normal and myopic chick eyes[J].Tissue Cell.1993,25 (1):73-85
5 McBrien NA, Cornell LM, Gentle A. Structural and ultrastructural changes to the sclera in a mammalian model of high myopia[J].Invest Ophthalmol Vis Sci.2001,42 (10):2179-2187
6 Gental A, McBrien NA. Modulation of seleral DNA synthesis in development of and recovery from axial myopia in the tree shrew [J]. Exp Eye Res.1999,68 (2):155-163
7 Curtin BJ, Iwamoto T, Benalclo DP. Normal and staphylomatous sclera of high myopia. An electron microscopic study [J]. Arch Ophthalmol.1979, 97 (5):912-915
8 McBrien SA, Lawlor P, Gentle A. Scleral remodeling during the development of and recovery from axial myopia in the tree shrew [J]. InVeSt Ophthalmol Vis Sci.2000,41 (12):3713-3719
9 Le MD, Besson A, Fogg DK, et al. Explotation of astroeytes by glioma cells to facilitate invasiveness:a mechanism involving matrix metalloproteinase-2 and the arokinase-type plasminogen activator-plasmin cascade[J]. J Neurosci.2003,23 (10):4034-4043
10 Jones BE, Thompson EW, Hodos W, et al.Scleral matrix metalloproteinases, serine proteinase activity and hydrational capcity are inceased in myopia induced by retinal image degradation[J]. ExpEyeRes. 1996,63:369-381.
11 Rada JA, Perry CA, Slover ML, et al.Gelatinase A and MMP2 expresson in the fibrous sclera of myoia and recovering chicks eyes[J].Invest Ophthalmol Vis Sci.1999,40:3091-3099
12 GuggenheimJA, Mcbrien NA. Form-deprivation myopia induces activation of scleral matrix metalloproteinase-2 in tree shrew[J].Invest Ophthalmol Vis Sci.1996,37:1380-1395
13吴文灿,刘双珍,王剑锋等.小鸡行觉剥夺性近视眼后极部巩膜MMP-2与TIMP-2 mRNA表达的动态变化[J].眼科新进展.2004,24(3) ：73-1770
14 Siegwart JT Jr, Norton TT. Steady state mRNA levels in tree shew sclera with form-deprivation myopia and during recovery [J]. Invest Ophthalmol Vis Sci.2001,42:1153-1159
15 Kenning MS, Gentle A, Mcbrien NA. Expression and cDNA Sequence of Matrix metalloproteinase-2 in a Mammalian Model of Human Disesase Processes:Tupaia belangeri. DNA Seq.2004,15:332-337
16 Rohrer B, Stell WK. Basic fibroblast growth factor and transforming growth factor act as stop and go signals to modulate postnatal ocular growth in the chick. Exp Eye Res.1994,58:553-561
17 seko Y, Tanaka Y, Tokoro T. Influence of bFGF as a potent growth stimulator and TGF-β as a growth regulator on scleral chondrocytes and scleral fibroblasts in vitro [J]. Ophthalmic Res.1995,27 (3):144-152
18 Hu DN, McCormick SA. Effect of TGF-β and cAMP-elevating agents on the growth of human scleral fibroblasts in vitro (A)//Lin LK edi. Myopia updates Ⅱ (Proceedings, Ⅶ international conference on myopia [M]. Tokyo:Springer.2000:131-132
19 Kee CS, Marazani D, Wallman J. Differences in time course and visual requirements of ocular responses to lenses and diffusers[J]. Invest Ophthalmol Vis Sci.2001,42:575-583
20 Ejedor J, de la Villa P. Refractive changes induced by form deprivation in the mouse eye [J]. Invest Ophthalmol Vis Sci.2003,44:32-36
21 Kenning MS, Gentle A, Mcbrien NA. Expression and cDNA Sequence of Matrix metalloproteinase-2 in a Mammalian Model of Human Disesase Processes-.Tupaia belangeri. DNA Seq.2004,15:332-337
22 Rohrer B, Tao J, Stell WK. Basic fibroblast growth factor,its high and low affinity receptors and their relationship to form deprivation myopia in the chick[J].NeuroSci.1997,79 (3):775
23 Seko Y, Shimokawa H, Tokoro T. Expression of bFGF and TGF-β2 in experimental myopia in chickens[J]. Invest ophthalmol Vis Sci.1995, 36(6):1183-1187
1 白灵,樊瑜波,张明.离体培养细胞的力学实验方法.生物医学工程学杂志.2002,19(2)：324-328
2 王超英,陈维毅,郝岚,等.高度近视眼巩膜生物力学特性初步研究.眼科研究.2003,21(2)：113-115
3 王晓君,李涛,陈维毅.巩膜胶原含量与其生物力学性能的关系研究[J].太原理工大学学报.2007,38(4)：371-373
4 孙朝晖,王超英,靳胜利等.实验性近视眼巩膜生物力学特征研究[J].眼视光学杂志.2006,8(4)：209-213
5 Gentle A, McBrien NA. Modulation of scleral DNA synthesis in development of and. recovery from induced axial myopia in the tree shrew. Exp Eye Res.1999,68:155-163
6 Cui W, Bryant MR, Sweet PM. McDonnell PJ. Changes in gene expression in response to mechanicalstrain in human scleral fibroblasts[J]. Experimental Eye Research.2004,78:275-284
7 Stamenovic D. Effects of cytoskeletal prestress on cell rheological behavior[J]. Acta Biomaterialia.2005:255-262
8 NortonTT, Rada JA. Reduced extracellular matrix in mammalian sclera with induced myopia. Vision Res.1995,35 (9):1271-1281
9 PhillipsJR, McBrien NA. Form deprivation myopia:elastic properties of sclera[J].Ophthalmic Physiol Opt.1995,15 (5):357-362
10 Shelton Lilian, Jody Summers Rada. Effects of cyclic mechanical stretch on extracellular matrix synthesis by human scleral fibroblasts[J]. Experimental Eye Research.2006,10(004):1-9
1 McBrien NA, Gentle A. Role of the sclera in the development and pathological complications of myopia. Prog Retin Eye Res.2003,22: 307-338
2 Rohrer B, Stell WK. Basic fibroblast growth factor and transforming growth factor act as stop and go signals to modulate postnatal ocular growth in the chick. Exp Eye Res.1994,58:553-561
3 seko Y, Tanaka Y, Tokoro T. Influence of bFGF as a potent growth stimulator and TGF-β as a growth regulator on scleral chondrocytes and scleral fibroblasts in vitro [J]. Ophthalmic Res.1995,27 (3):144-152
4 吴文灿,刘双珍,王剑锋等.小鸡形觉剥夺性近视眼后极部巩膜MMP-2与TIMP-2 mRNA表达的动态变化[J].眼科新进展.2004,24(3)：73-1770
5 Hu DN, McCormick SA. Effect of TGF-β and cAMP-elevating agents on the growth of human scleral fibroblasts in vitro (A)//Lin LK edi. Myopia updates II (Proceedings, VII international conference on myopia) [M]. Tokyo:Springer.2000:131-132
6 Overall CM, Wrana JL, Sodex JL, Sodek J. Post-transcriptional regulation of 72-kDa gelatinase/type transforming growth factor-betalin human fibroblasts:Transcriptional and IV collagenase by comparisons with collagenase and tissue inhibitor of matrix metalloproteinase gene expression. J Biol Chem.1991,266:14064
7 Kim HS, Shang T, Chen Z et al. TGF-betal stimulates production of gelatinase (MMP-9), collagenases (MMP-1,-13) and stromelysins (MMP-3,-10,-11)by human corneal epithelial cells. Exp Eye Res.2004, 79:263
8 Seomun Y, Kim J, Lee EH et al. Overexpression of metalloproteinase-2 mediates phenotypic transformation of lens epithelial cells. Biochem J. 2001,15:41
9 Behzadian MA, Wang XL, Windsor LJ et al. TGF-pincreases retinal endothelial cell permeability by increasing MMP-9:possible role of glial cells in endothelial cells in endothelial barrier function. Invest Ophthalmo Vis Sci.2001,42:853
10 Kee CS, Marazani D, Wallman J. Differences in time course and visual requirements of ocular responses to lenses and diffusers[J]. Invest Ophthalmol VisSci 2001.42:575-583
11 Ejedor J, de la Villa P. Refractive changes induced by form deprivation in the mouse eye [J]. Invest Ophthalmol Vis Sci.2003,44:32-36
12 Rada JA, Nickla DL. Troilo D. Decreased proteoglycan synthesis associated with form deprivation myopia in mature primate eyes [J]. Invest Ophthalmol VisSci.2000; 41 (1):2050-2058
13郑瑾,佘振珏,周国民.EGF与bFGF对体外大鼠SF细胞增殖的影响.Fudan Univ Jmed Sci.2006May; 33 (3):301-304
14 Rohrer B, Stell WK. Basic fibroblast growth factor and transforming growth factor act as stop and go signals to modulate postnatal ocular growth in the chick. Exp Eye Res.1994,58:553-561
15胡诞宁,M cCormick SA视网膜色素上皮-脉络膜在近视发病中的作用[J].眼视光学杂志.2000,2(4)：197-200
16 Massague J. Rcecptors for the TGF-beta family.Cell.1992,69 (70): 1067-70
17 Warana JL, Attisano L, Wieser R,et al. Mechanism of activation of the TGF-beta receptor. Nature.1994,370 (6488):341-7
18 Savage C, Das P, Firelli AL, et al. Caenorhabditis elegans genes sma-2, sma-3, and sma-4 define a conserved family of transforming growth factor beta pathway components. Proc Natl Acad Sci USA.1996,93 (2):790-4
19 Shipley GD, Tucker RF, Mouses HL. Type beta transforming growth factor/growth inhibitor stimulates entry of monolayer cultures of AKR-2B cells into S phase after a prolonged prereplicative interval. Proc Natl Sci USA.1985,82 (12):4147-51
20 Anzano MA, Roberts AB, Sporn MB, et al. Anchorage-independent growth of primary rat embryo cells is induced by platelet-derived growth factor and inhibited by type-beta transforming growth factor. Cell Physiol. 1986,126 (2):312-8
21 Heimark RL, Twardzik DR, Schwartz SM,et al. Inhibition of endothelial regeneration by type-beta transforming growth factor from platelets. Science.1986,233 (4768):1078-80
22 Kehrl JH, Robert AB, Wakefield LM, te al.Transforming growth factor beta is an important immanomodulatory protein for human B lymphocytes. J Immunol.1986,137 (12):3855-60
23 Kehrl JH, Wakefield LM, Robert AB,te al. Production of transforming growth factor beta by human T lymphocytes and its potential role in the regulation of T cell growth. J Exp Med.1986,163 (5):1037-50
24 Carr BI, Hayashi T, Branum EL, et al. Inhibition of DNA synthesis in rate hepatocytes by platelet-derived type beta transforming growth factor. Cancer Res.1986,46 (5):2330-4
25 Sporn MB, Robert AB, Wakefield LM, et al. Some recent advances in the chemistry and biology of transforming growth factor-beta. J Cell Biol. 1987,105 (3):1039-45
26王红,曲毅,郑水军.豚鼠透镜诱导性近视眼巩膜细胞凋亡的研究[J].山东大学学报(医学版).2006,44(7)：671-675
27潘卫红,曾俊文.TGF-β1对小鼠SF细胞增殖和细胞周期影响[J].眼科研究.2006,23 (3)：232-234
28傅亚娜,周翔天,付小莹等.碱性成纤维细胞生长因子和转化生长因子-β对人SF细胞的生长调控研究.眼视光学杂志.2008,10(3)186-189
1 王超英,陈维毅,郝岚等.高度近视眼巩膜生物力学特性初步研究[J].眼科研究.2003,21(2)：113-115
2王晓君,李涛,陈维毅,巩膜胶原含量与其生物力学性能的关系研究[J].太原理工大学学报.2007,38(4)：371-373
3 孙朝晖,王超英,靳胜利等.实验性近视眼巩膜生物力学特征研究[J].眼视光学杂志.2006,8(4)：209-213
4 Gentle A,McBrien NA. Modulation of scleral DNA synthesis in development of and. recovery from induced axial myopia in the tree shrew. Exp Eye Res.1999,68:155-163
5 陈维毅,王超英,张全有,李阳阳等.实验性近视眼SF细胞黏弹性研究[J].医用生物力学.2007,22(1)：22-29
6 Cui W, Bryant MR, Sweet PM, et al. Changes in gene expression in response to mechanicalstrain in human scleral fibroblasts[J]. Experimental Eye Research.2004,78:275-284
7 Shelton Lilian, Jody Summers Rada. Effects of cyclic mechanical stretch on extracellular matrix synthesis by human scleral fibroblasts[J]. Experimental Eye Research.2006,10 (004):1-9
1 许银娥,吴小影,刘双珍,夏晓波,王育科.近视眼视网膜神经纤维层厚度分析[J].国际眼科杂志.2006,6(1)：116-118
2 Harm an AM, HoskinsR, Beazley LD. Experimental eye en-largement in mature animals changes the retinal pigment epithelium [J]. Vis Neurosci. 1999,16 (4):619-628
3 任力,李永平,易玉珍等.近视眼模型的形态学及视网膜超微结构观察[J].中华眼底病杂志.1999,15(1)：20-23
4席晓勃,褚仁远.实验性近视眼组织病理及超微结构观察.眼科新进展.2001,21(5)： 329-331
5 Zhong xingwu, Ge jian, Chen xiaolian. Comparision of retinal morphology and ultrastructure in defocus-iuduced myopia and form-deprivation myopia in rhesus monkeys. ChinJ Ophthalmol.2005,41 (7):625-630
6 王红,高蕾,曹加会,陈红梅.碱性成纤维细胞生长因子对豚鼠透镜诱导性近视眼巩膜细胞凋亡的影响.环境与健康杂志.2008,25(8)：676-679
7汪芳润编著.近视眼[M].上海：上海医科人学出版社.1996：212-229
8 Janet R, Bolin C. Blue light-induced apoptosis of A2E-containing RPE: involvement of caspase-3 and protection by Bcl-2. Invest Ophthalmol Vis Sci.2001,42:1356
9 Hu DN, Woodward DF, McCormick ST. Influence of autonomic. neuro-transmitters on human uveal melanocytes in vitro. Exp Eye Res. 2000,71:217-224
10 Roberts JE, Wiechmann AF, Hu DN. Melatonin receptors in human u-veal melanocytes and melanoma cells. J Pineal Res.2000,28:16-171
11 Hu DN. Role of RPE-Choroid axis on the occurrence of myopia. Chin J Optom Ophthalmol.2000,2 (4):197-200
12 Haque R, Maltseva O, Ivanova T, et al. Dopamine D1 recpptor ex-pression in cultured human andmonkey retinal pigment epithelial cells. Invest Ophthalmol Vis Sci.2000,41:S843
13 Rymer J, Wildsoet CF. The role of the pigment epithelium in eye growth regulation and myopia. Vis Neurosci.2005,22 (3):251-261
14 Laihu MO, Saksela O, Andresen PA,et al. Enhanced production and extracellular deposition of die endothelial-type plasminogen activator inhibitor in cultured human lung fibroblasts by transfoming growth factor-beta[J]. J Cell Bion.1986,103:2403-2410
15 Kvanta A. Expression and secretion of transforming growth factor-beta in transformed and nontransformed retinal pigment epithelial cells.Ophthahmic Res.1994,26:361-367
16 Sakamoto T, Soriano D, Nassaralla J, et al. Effect of intravitreal administration of indomethacin onexperimental subretinal neovascularization in the subhuman primate.Arch Ophthalmol.1995,113: 222-226
17 Seko Y, Shimokawa H, Tokoro T. Expression of bFGF and TGF-β 2 in experimental myopia in chickens[J]. Invest ophthalmol Vis Sci.1995,36 (6):1183-1187
18曾爱萍,曾水清.转化生长因子β-1对培养的人RPE细胞MMP和TIMP-1 mRNA表达的影响.眼科新进展.2006,26(2)： 81-84
19 Zheng Xiaofen, Chu Renyuan. Effects of TGF-β on human embryonic retinal pigment epithelium cell. Chin Ophthal Res. January 2007,125 (1): 14-17
20陈有信,何世坤.细胞外基质蛋白和转化生长因子β 2共同作用诱导人视网膜色素上皮细胞向肌纤维母细胞转化.中华眼底病杂志.2006,22(5)：328-332
21 Hayashi T, Inoko H. Exclusion of transforming growth factor-betal as a candidate gene for myopia in the Japanese. Jpn J Ophthalmol.2007,51 (2):96-99
22 Kimizuka Y, Yamada T, Tamai M.Quantitative study on regenerated retinal pigment epithelium and the effects of growth factor[J]. Curr Eye Res 1997; 16 (11):1081-1087
23周浩,褚仁远,周行涛,戴锦晖.体内实验观察bFGF对视网膜色素上皮细胞再生的促进作用[J].眼科新进展.2003；23(2)：97-99
24魏海英,崔浩.鸡形觉剥夺性近视眼形态学及bFGF免疫组织化学研究.眼科研究.2007,25(1)：22-24
25 Rohrer B, Iuvone PM, Stell WK. Stimulation of dopaminergic amacrine cells by stroboscopic illumination or fibroblast growth factor (bFGF, FGF-2) injections:possible roles in prevention of form-deprivation myopia in the chick. Brain Res.1995,686:169
26毛俊峰,刘双珍.bFGF对鸡形觉剥夺性近视眼的抑制作用.国际眼科杂志.2005,5(4)：652-654
27 Rohrer B, Stell WK. Basic fibroblast growth factor and transforming growth factor act as stop and go signals to modulate postnatal ocular growth in the chick. Exp Eye Res.1994,58:553-561
28 seko Y, Tanaka Y, Tokoro T. Influence of bFGF as a potent growth stimulator and TGF-β as a growth regulator on scleral chondrocytes and scleral fibroblasts in vitro [J]. Ophthalmic Res.1995,27 (3):144-152
29 Hu DN, McCormick SA. Effect of TGF-β and cAMP-elevating agents on the growth of human scleral fibroblasts in vitro (A)//Lin LK edi. Myopia updates II (Proceedings, VII international conference on myopia) [M]. Tokyo:Springer.2000:131-132
30 Kee CS, Marazani D, Wallman J. Differences in time course and visual requirements of ocular responses to lenses and diffusers[J]. Invest Ophthalmol Vis Sci.2001,42:575-583
31 Ejedor J, de la Villa P, Refractive changes induced by form deprivation in the mouse eye [J]. Invest Ophthalmol VisSci.2003,44:32-36
32 Derynck R Feng XH.TGF-beta receptor signaling. Biochim BiophysActa. 1997(24); 1333 (2):105
33 Simmons ML, Murphy S. Induction of nitric oxide synthase in glialcells[J]. Neurochem.1992,59 (3):897
34 Le MD, Besson A, Fogg DK,et al. Explotation of astroeytes by glioma cells to facilitate invasiveness:a mechanism involving matrix metalloproteinase-2and the arokinase-type plasminogen activator-plasmin cascade[J]. J Neuroscib.2003,23 (10):4034-4043
35 Jones BE, Thompson EW, Hodos W, et al.Scleral matrix metalloproteinases, serine proteinase activity and hydrational capcity are inceased in myopia induced by retinal image degradation[J]. ExpEyeRes. 1996,63:369-381
36 Rada JA, Perry CA, Slover ML,et al. Gelatinase A and MMP2 expresson in the fibrous sclera of myoia and recovering chicks eyes[J].Invest Ophthalmol Vis Sci.1999,40:3091-3099
37 Guggenheim JA, IcBrien NA. Form-deprivation myopia induces activation of scleral matrix metalloproteinase-2 in tree shrew[J]. Invest Ophthalmol Vis Sci.1996,37:1380-1395
38吴文灿,刘双珍,王剑锋等.小鸡行觉剥夺性近视眼后极部巩膜MMP-2与TIMP-2 mRNA表达的动态变化[J].眼科新进展.2004,24(3)：73-1770
39 Siegwart JT Jr, Norton TT. Steady state mRNA levels in tree shew sclera with form-deprivation myopia and during recovery[J]. Invest Ophthalmol Vis Sci.2001,42:1153-1159
40 Kenning MS, Gentle A, Mcbrien NA. Expression and cDNA Sequence of Matrix metalloproteinase-2 in a Mammalian Model of Human Disesase Processes:Tupaia belangeri. DNA Seq.2004,15:332-337
41 Rohrer B, Tao J, Stell WK. Basic fibroblast growth factor,its high and low affinity receptors and their relationship to form deprivation myopia in the chick[J]. NeuroSci.1997,79 (3):775
42 Seko Y, Shimokawa H, Tokoro T. Expression of bFGF and TGF-β2 in experimental myopia in chickens[J]. Invest ophthalmol Vis Sci.1995,36 (6):1183-1187
43 Cowan DB, Lye SJ, Langille BL. Regulation of vascular connexin43 gene expression by mechanical loads. Circ Res.1998,82 (7):786-793
44 Matsumoto T, Kawakami M, Kuribayashi K, Takenaka T, Tamaki T. Cyclic mechanical stretch stress increases the growth rate and collagen synthesis of nucleus pulposus cells in vitro. Spine.1999,24 (4):315-319
45王建洲,惠延年,侯旭等.磁珠包裹的人视网膜色素上皮细胞受磁场 牵拉后ET-1的表达.国际眼科杂志.2006,6(4)：775-777
46张晓光,惠延年等.培养人视网膜色素上皮细胞牵拉模型中细胞骨架的改变.中华眼科杂志.2006,42(2)：121-126
47候旭,惠延年,韩泉洪等.机械牵拉对培养人视网膜色素上皮细胞内钙离子的影响.国际眼科杂志.2005,5(1)：50-54
48王超英,陈维毅,郝岚,等.高度近视眼巩膜生物力学特性初步研究.眼科研究.2003,21(2)：113-115
49王晓君,李涛,陈维毅.巩膜胶原含量与其生物力学性能的关系研究[J].太原理工大学学报.2007,38(4)：371-373
50孙朝晖,王超英,靳胜利等.实验性近视眼巩膜生物力学特征研究[J].眼视光学杂志.2006,8(4)：209-213
51 Gentle A, McBrien NA. Modulation of scleral DNA synthesis in development of and. recovery from induced axial myopia in the tree shrew. Exp Eye Res.1999,68155-163
52胡萍,李镜海.透镜诱导豚鼠近视眼巩膜基质中基质金属蛋白酶2及其组织抑制剂和生长因子的变化[J].眼视光学杂志.2008,10(1)：17-20
53 Cui W, Bryant MR, Sweet PM, McDonnell PJ. Changes in gene expression in response to mechanical strain in human scleral fibroblasts[J]. Experimental Eye Research,2004,78:275-284
54 Shelton Lilian，Jody Summers Rada. Effects of cyclic mechanical stretch on extracellular matrix synthesis by human scleral fibroblasts[J]. Experimental Eye Research (2006).10.004:1-9
55 Stamenovic D. Effects of cytoskeletal prestress on cell rheological behavior[J]. Acta Biomaterialia,2005,255-262
56胡诞宁,Steven A, McComick视网膜色素上皮-脉络膜在近视发病中的作用[J].眼视光学杂志.2000,2(4)：197-200
57 Hu DN, MC-Cormick SA. Role of RPE-choroid axis on production, binding and response to various growth factors modulate the occurrence of myopia[C]. In Thorn F, etal, (edi):Myopia 2000 (Proceed-ings,,International Conference on Myopia) [M]USA:Conference on Myopia, Inc.2000.213-216