家兔近视眼后巩膜加固术后不同时期成纤维细胞力学特性及CTGF表达的研究
发布时间：2018-07-25 16:37
【摘要】： 目的:明确后巩膜加固术后不同时期结缔组织生长因子(connective tissue growth factor,CTGF)表达的变化,并结合成纤维细胞力学特性的研究,从组织-细胞-生物力学的角度进一步探讨后巩膜加固术的作用机制。 方法:3周龄新西兰家兔45只随机选取一眼为实验眼(experimental eye),用眼睑缝合方法制备近视动物模型,对侧为自身对照眼(阴性对照组)。造模60天后,实验眼随机分为两组,A组(实验组)行后巩膜加固术(posterior scleral Reinforcement,PSR),B组(阳性对照组)行相似手术,仅不放置加固条带。实验前后均检测每只家兔双眼屈光状态、眼轴长度,分别于术后3月、6月取材。部分组织用组织块培养法培养巩膜加固区及过渡组织的成纤维细胞,并传2代。用免疫细胞化学法对培养的细胞进行鉴定,A组细胞分为A1组(3月巩膜组)、A2组(3月过渡区组)、A3组(6月巩膜组)、A4组(6月过渡区组),B组分为B1组(3月巩膜组)、B3组(6月巩膜组),利用免疫细胞化学法对各组细胞CTGF进行半定量分析;部分组织行组织学检测,对各组的CTGF的表达行半定量分析;利用细胞微管吸吮的方法结合半无限体细胞力学模型(Half-space model)测定各组成纤维细胞的力学特性(包括成纤维细胞的杨氏弹性模量和粘弹性参数)。结果用SPSS13.0统计软件进行统计分析。 结果: 1家兔实验性近视的诱导结果 眼睑缝合前家兔双眼屈光度无明显差异,经过60天形觉剥夺诱导后,实验眼与对照眼屈光度比较,诱导出(1.80±1.37)D的相对近视,眼轴相对延长了(0.45±0.29)mm,差异有统计学意义(P0.05)。 2后巩膜加固术后的屈光度与眼轴长度 后巩膜加固术后3个月,A组与B组比较,两者屈光度与眼轴长度之间均无统计学意义(P0.05)。后巩膜加固术后6个月,A组与B组比较,眼轴相对变化了(0.59±0.35)mm,两者之间有统计学意义(P0.05)。 3细胞培养及鉴定结果 家兔巩膜组织块培养4-6天,过渡区组织培养3-5天,周围开始有细胞长出,2-3周融合,免疫细胞化学鉴定为成纤维细胞。 4组织学研究结果 光镜下实验眼巩膜胶原纤维排列紊乱,粗细不均,纤维间隙变大,纤维走向不一,板层结构不清,胶原纤维部分或完全溶解,有纤维断裂现象,而对照眼未见上述改变。 5不同时期巩膜组织CTGF的表达 CTGF的表达以细胞核及细胞浆为主,正常对照组的表达多呈淡黄色,为+,实验组着色较深,多呈棕黄色,为+++。后巩膜加固术后3个月、6个月,A组与B组比较,其着色程度均较高,差异均有统计学意义(P0.05)。 6不同组织中成纤维细胞CTGF的表达 后巩膜加固术后3个月、6个月,A1组与B1组、A3组与B3组比较,前者着色程度均较高,差异有统计学意义(P0.05)。A2组与A1组、A4组与A3比较,前者着色程度均较高,差异有统计学意义(P0.05)。A1组与A3组、A2组与A4组比较,两者之间均无统计学意义(P0.05)。 7成纤维细胞力学特性及统计学分析 7.1后巩膜加固术后3个月、6个月, A组中巩膜成纤维细胞平衡杨氏模量分别为E∝= 0.35 21±0.0853kPa、0.3612±0.1211kPa,B组的巩膜成纤维细胞平衡杨氏模量分别为E∝=0.3418±0.0911kPa、0.3476±0.0821kPa,经统计学分析,两组巩膜成纤维细胞在同一时间的平衡杨氏模量均无统计学意义(P0.05)。 对成纤维细胞进行微管吸吮测试发现,在某一恒定负压作用下,成纤维细胞表现出典型的粘弹性固体蠕变特征。A组巩膜成纤维细胞表观黏性分别为μ=2.8733±0.6154kPa.s、2.9282±0.6694 kPa.s,B组的巩膜成纤维细胞表观黏性μ=2.8183±0.590 kPa.s、2.8206±0.5935kPa.s,统计学分析表明在同一时间两组巩膜成纤维细胞的表观黏性差异均无统计学意义(P 0.05)。 7.2不同时期的加固区巩膜成纤维细胞力学特性及统计学分析 后巩膜加固术后3月组、6月组的巩膜成纤维细胞,无论是平衡杨氏模量还是细胞表观黏性,其差异均无统计学意义(P0.05)。 7.3不同时期过渡区成纤维细胞力学特性及统计学分析 后巩膜加固术后3个月、6个月的过渡区的成纤维细胞的力学特性,无论是平衡杨氏模量还是细胞表观黏性,其差异均无统计学意义(P 0.05)。 结论: 1通过眼睑缝合的方法,可以成功诱导幼兔实验性近视眼的形成。 2后巩膜加固术后实验组巩膜无论在组织还是细胞水平,CTGF的表达均高于同期阳性对照组,说明手术及加固条带本身的存在均刺激过渡区的形成。 3后巩膜加固术后不同时期过渡区的组织及其培养的细胞的CTGF表达均高于同期的加固区巩膜组,说明CTGF参与了手术本身及加固条带引起过渡区的形成。 4后巩膜加固术后不同时期巩膜成纤维细胞生物力学特性(平衡杨氏模量、表观黏性)未发生明显变化,说明后巩膜加固术的增强作用并不是原位巩膜成纤维细胞本身生物力学的加强,而是由于加固条带及引起过渡区的存在。 5后巩膜加固术后不同时期过渡区的成纤维细胞生物力学特性无明显变化,说明后巩膜加固术是通过细胞间联合的整体效应及加固条带的机械作用,而非通过单个细胞的力学变化起作用。
[Abstract]:Objective: to identify the changes in the expression of connective tissue growth factor (CTGF) in different periods after posterior scleral reinforcement and to study the mechanical properties of fibroblasts, and to further explore the mechanism of posterior scleral reinforcement from the point of tissue cell biomechanics.
Methods: 45 rabbits at 3 weeks of age were randomly selected as experimental eyes (experimental eye). The myopic animal model was prepared by the eyelid suture method. The contralateral was self opposite to the eye (negative control group). After 60 days, the experimental eyes were randomly divided into two groups, and the A group (experimental group) underwent posterior scleral reinforcement (posterior scleral Reinforcement, PSR), B group (positive). In the control group, the similar operation was performed without the reinforcement strip. The diopter of each rabbit was detected before and after the experiment. The length of the eye axis was measured in March and June after the operation. Some tissue culture method was used to cultivate the fibroblasts of the scleral reinforcement area and the transitional tissue by tissue mass culture, and the 2 generations were passed through the immunocytochemical method. The cells of group A were divided into group A1 (group of sclera in March), group A2 (group of transition in March), group A3 (June sclera group), group A4 (June transition zone), group B, B1 group (March sclera group), B3 group (June sclera group), semi quantitative analysis of CTGF in each group by immunocytochemical method; the expression of CTGF in each group was half quantified by histological examination. The mechanical properties of each constituent fiber cells (including young's modulus of elasticity and viscoelastic parameters of fibroblasts) were measured by the method of cell micropipette sucking combined with the semi infinite cell mechanical model (Half-space model). The results were statistically analyzed by the SPSS13.0 statistical software.
Result:
The induction of experimental myopia in 1 rabbits
There was no significant difference in the diopter of the rabbits before the eyelid suture. After 60 days of shape deprivation, the experimental eye was compared with the control eye, and the relative myopia was induced (1.80 + 1.37) D, and the ocular axis was prolonged (0.45 + 0.29) mm, and the difference was statistically significant (P0.05).
The diopter and axial length of 2 posterior scleral reinforcement
3 months after the posterior scleral reinforcement, there was no significant difference between the A group and the B group (P0.05). 6 months after the posterior scleral reinforcement, the A group was compared with the B group (0.59 + 0.35) mm, and there was a statistically significant difference between the two groups (P0.05).
3 cell culture and identification results
The sclera tissue of rabbits was cultured for 4-6 days, and the transitional zone was cultured for 3-5 days. The cells began to grow and began to merge for 2-3 weeks. The immunocytochemistry was identified as fibroblasts.
4 results of histology
In the light microscope, the collagenous fibers in the experimental eye were disorganized, the thickness of the fiber was uneven, the fiber gap became larger, the fiber trend was uneven, the lamellar structure was not clear, the collagen fibers were partially or completely dissolved, and the fibers were broken, but the above changes were not seen in the eyes.
5 expression of CTGF in scleral tissue at different stages
The expression of CTGF was mainly the nucleus and cytoplasm. The expression of the normal control group was mostly light yellow, and was +. The experimental group was stained darker and more brown and yellow, plus + + + + + +. After 3 months and 6 months after the sclera reinforcement, the degree of coloring was higher in group A and in B group, the difference was statistically significant (P0.05).
6 expression of fibroblast CTGF in different tissues
After posterior scleral reinforcement, 3 months, 6 months, group A1 and group B1, group A3 and group B3, the former coloring degree was higher, the difference was statistically significant (P0.05).A2 group and A1 group, A4 group and A3, the former coloring degree was higher, the difference was statistically significant (P0.05).A1 group and A3 group, there was no statistical significance between the two groups.
Mechanical properties and statistical analysis of 7 fibroblasts
7.1 3 months and 6 months after posterior scleral reinforcement, the balance Young's modulus of scleral fibroblasts in group A was E = 0.3521 + 0.0853kPa and 0.3612 + 0.1211kPa. The equilibrium Young's modulus of scleral fibroblasts in group B was E =0.3418 + 0.0911kPa and 0.3476 + 0.0821kPa respectively. Two groups of scleral fibroblasts were at the same time. The balance Young's modulus was not statistically significant (P0.05).
The micropipette test of fibroblasts showed that the fibroblasts showed typical viscoelastic solid creep characteristics under a constant negative pressure. The apparent viscosity of scleral fibroblasts in group.A was =2.8733 + 0.6154kPa.s, 2.9282 + 0.6694 kPa.s respectively, and the apparent viscosity of scleral fibroblasts in group B was + 0.590 kPa.s, 2.820 6 + 0.5935kPa.s, statistical analysis showed that at the same time, the apparent viscosity of scleral fibroblasts in the two groups was not statistically significant (P 0.05).
7.2 mechanical characteristics and statistical analysis of scleral fibroblasts in different areas of reinforcement
The scleral fibroblasts in the group of March after the posterior scleral reinforcement and in the June group had no significant difference in both the balance Young's modulus and the apparent viscosity of the cells (P0.05).
7.3 the mechanical properties and statistical analysis of fibroblasts in transitional areas at different times
The mechanical properties of fibroblasts in the 3 months and 6 months after the posterior scleral reinforcement were not statistically significant (P 0.05), regardless of the balance Young's modulus or the apparent viscosity of the cells.
Conclusion:
1 the method of eyelid suture can successfully induce the formation of experimental myopia in young rabbits.
2 after the posterior scleral reinforcement, the expression of CTGF in the experimental group was higher than that of the positive control group at the level of tissue and cell, indicating that the existence of the operation and the reinforcement strip itself stimulated the formation of the transitional zone.
3 after the posterior scleral reinforcement, the CTGF expression of the tissue and the cultured cells in different periods were higher than that in the same period, indicating that CTGF was involved in the formation of the transition zone in the operation itself and the reinforcement strip.
4 the biomechanical properties of scleral fibroblasts (balance Young's modulus and apparent viscosity) after the posterior scleral reinforcement were not significantly changed, indicating that the strengthening effect of the posterior scleral reinforcement is not the strengthening of the biomechanics of the scleral fibroblast itself, but the reinforcement strip and the existence of the transition zone.
5 after the posterior scleral reinforcement, the biomechanical properties of the fibroblasts in the transitional zone were not significantly changed, indicating that the posterior scleral reinforcement was through the overall effect of the intercellular Union and the mechanical action of reinforcing the strip, but not by the mechanical changes of the single cell.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R779.6
[Abstract]:Objective: to identify the changes in the expression of connective tissue growth factor (CTGF) in different periods after posterior scleral reinforcement and to study the mechanical properties of fibroblasts, and to further explore the mechanism of posterior scleral reinforcement from the point of tissue cell biomechanics.
Methods: 45 rabbits at 3 weeks of age were randomly selected as experimental eyes (experimental eye). The myopic animal model was prepared by the eyelid suture method. The contralateral was self opposite to the eye (negative control group). After 60 days, the experimental eyes were randomly divided into two groups, and the A group (experimental group) underwent posterior scleral reinforcement (posterior scleral Reinforcement, PSR), B group (positive). In the control group, the similar operation was performed without the reinforcement strip. The diopter of each rabbit was detected before and after the experiment. The length of the eye axis was measured in March and June after the operation. Some tissue culture method was used to cultivate the fibroblasts of the scleral reinforcement area and the transitional tissue by tissue mass culture, and the 2 generations were passed through the immunocytochemical method. The cells of group A were divided into group A1 (group of sclera in March), group A2 (group of transition in March), group A3 (June sclera group), group A4 (June transition zone), group B, B1 group (March sclera group), B3 group (June sclera group), semi quantitative analysis of CTGF in each group by immunocytochemical method; the expression of CTGF in each group was half quantified by histological examination. The mechanical properties of each constituent fiber cells (including young's modulus of elasticity and viscoelastic parameters of fibroblasts) were measured by the method of cell micropipette sucking combined with the semi infinite cell mechanical model (Half-space model). The results were statistically analyzed by the SPSS13.0 statistical software.
Result:
The induction of experimental myopia in 1 rabbits
There was no significant difference in the diopter of the rabbits before the eyelid suture. After 60 days of shape deprivation, the experimental eye was compared with the control eye, and the relative myopia was induced (1.80 + 1.37) D, and the ocular axis was prolonged (0.45 + 0.29) mm, and the difference was statistically significant (P0.05).
The diopter and axial length of 2 posterior scleral reinforcement
3 months after the posterior scleral reinforcement, there was no significant difference between the A group and the B group (P0.05). 6 months after the posterior scleral reinforcement, the A group was compared with the B group (0.59 + 0.35) mm, and there was a statistically significant difference between the two groups (P0.05).
3 cell culture and identification results
The sclera tissue of rabbits was cultured for 4-6 days, and the transitional zone was cultured for 3-5 days. The cells began to grow and began to merge for 2-3 weeks. The immunocytochemistry was identified as fibroblasts.
4 results of histology
In the light microscope, the collagenous fibers in the experimental eye were disorganized, the thickness of the fiber was uneven, the fiber gap became larger, the fiber trend was uneven, the lamellar structure was not clear, the collagen fibers were partially or completely dissolved, and the fibers were broken, but the above changes were not seen in the eyes.
5 expression of CTGF in scleral tissue at different stages
The expression of CTGF was mainly the nucleus and cytoplasm. The expression of the normal control group was mostly light yellow, and was +. The experimental group was stained darker and more brown and yellow, plus + + + + + +. After 3 months and 6 months after the sclera reinforcement, the degree of coloring was higher in group A and in B group, the difference was statistically significant (P0.05).
6 expression of fibroblast CTGF in different tissues
After posterior scleral reinforcement, 3 months, 6 months, group A1 and group B1, group A3 and group B3, the former coloring degree was higher, the difference was statistically significant (P0.05).A2 group and A1 group, A4 group and A3, the former coloring degree was higher, the difference was statistically significant (P0.05).A1 group and A3 group, there was no statistical significance between the two groups.
Mechanical properties and statistical analysis of 7 fibroblasts
7.1 3 months and 6 months after posterior scleral reinforcement, the balance Young's modulus of scleral fibroblasts in group A was E = 0.3521 + 0.0853kPa and 0.3612 + 0.1211kPa. The equilibrium Young's modulus of scleral fibroblasts in group B was E =0.3418 + 0.0911kPa and 0.3476 + 0.0821kPa respectively. Two groups of scleral fibroblasts were at the same time. The balance Young's modulus was not statistically significant (P0.05).
The micropipette test of fibroblasts showed that the fibroblasts showed typical viscoelastic solid creep characteristics under a constant negative pressure. The apparent viscosity of scleral fibroblasts in group.A was =2.8733 + 0.6154kPa.s, 2.9282 + 0.6694 kPa.s respectively, and the apparent viscosity of scleral fibroblasts in group B was + 0.590 kPa.s, 2.820 6 + 0.5935kPa.s, statistical analysis showed that at the same time, the apparent viscosity of scleral fibroblasts in the two groups was not statistically significant (P 0.05).
7.2 mechanical characteristics and statistical analysis of scleral fibroblasts in different areas of reinforcement
The scleral fibroblasts in the group of March after the posterior scleral reinforcement and in the June group had no significant difference in both the balance Young's modulus and the apparent viscosity of the cells (P0.05).
7.3 the mechanical properties and statistical analysis of fibroblasts in transitional areas at different times
The mechanical properties of fibroblasts in the 3 months and 6 months after the posterior scleral reinforcement were not statistically significant (P 0.05), regardless of the balance Young's modulus or the apparent viscosity of the cells.
Conclusion:
1 the method of eyelid suture can successfully induce the formation of experimental myopia in young rabbits.
2 after the posterior scleral reinforcement, the expression of CTGF in the experimental group was higher than that of the positive control group at the level of tissue and cell, indicating that the existence of the operation and the reinforcement strip itself stimulated the formation of the transitional zone.
3 after the posterior scleral reinforcement, the CTGF expression of the tissue and the cultured cells in different periods were higher than that in the same period, indicating that CTGF was involved in the formation of the transition zone in the operation itself and the reinforcement strip.
4 the biomechanical properties of scleral fibroblasts (balance Young's modulus and apparent viscosity) after the posterior scleral reinforcement were not significantly changed, indicating that the strengthening effect of the posterior scleral reinforcement is not the strengthening of the biomechanics of the scleral fibroblast itself, but the reinforcement strip and the existence of the transition zone.
5 after the posterior scleral reinforcement, the biomechanical properties of the fibroblasts in the transitional zone were not significantly changed, indicating that the posterior scleral reinforcement was through the overall effect of the intercellular Union and the mechanical action of reinforcing the strip, but not by the mechanical changes of the single cell.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R779.6
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