【摘要】： 目的:明確后鞏膜加固術后不同時期結締組織生長因子(connective tissue growth factor,CTGF)表達的變化,并結合成纖維細胞力學特性的研究,從組織-細胞-生物力學的角度進一步探討后鞏膜加固術的作用機制。 方法:3周齡新西蘭家兔45只隨機選取一眼為實驗眼(experimental eye),用眼瞼縫合方法制備近視動物模型,對側為自身對照眼(陰性對照組)。造模60天后,實驗眼隨機分為兩組,A組(實驗組)行后鞏膜加固術(posterior scleral Reinforcement,PSR),B組(陽性對照組)行相似手術,僅不放置加固條帶。實驗前后均檢測每只家兔雙眼屈光狀態(tài)、眼軸長度,分別于術后3月、6月取材。部分組織用組織塊培養(yǎng)法培養(yǎng)鞏膜加固區(qū)及過渡組織的成纖維細胞,并傳2代。用免疫細胞化學法對培養(yǎng)的細胞進行鑒定,A組細胞分為A1組(3月鞏膜組)、A2組(3月過渡區(qū)組)、A3組(6月鞏膜組)、A4組(6月過渡區(qū)組),B組分為B1組(3月鞏膜組)、B3組(6月鞏膜組),利用免疫細胞化學法對各組細胞CTGF進行半定量分析;部分組織行組織學檢測,對各組的CTGF的表達行半定量分析;利用細胞微管吸吮的方法結合半無限體細胞力學模型(Half-space model)測定各組成纖維細胞的力學特性(包括成纖維細胞的楊氏彈性模量和粘彈性參數(shù))。結果用SPSS13.0統(tǒng)計軟件進行統(tǒng)計分析。 結果: 1家兔實驗性近視的誘導結果 眼瞼縫合前家兔雙眼屈光度無明顯差異,經(jīng)過60天形覺剝奪誘導后,實驗眼與對照眼屈光度比較,誘導出(1.80±1.37)D的相對近視,眼軸相對延長了(0.45±0.29)mm,差異有統(tǒng)計學意義(P0.05)。 2后鞏膜加固術后的屈光度與眼軸長度 后鞏膜加固術后3個月,A組與B組比較,兩者屈光度與眼軸長度之間均無統(tǒng)計學意義(P0.05)。后鞏膜加固術后6個月,A組與B組比較,眼軸相對變化了(0.59±0.35)mm,兩者之間有統(tǒng)計學意義(P0.05)。 3細胞培養(yǎng)及鑒定結果 家兔鞏膜組織塊培養(yǎng)4-6天,過渡區(qū)組織培養(yǎng)3-5天,周圍開始有細胞長出,2-3周融合,免疫細胞化學鑒定為成纖維細胞。 4組織學研究結果 光鏡下實驗眼鞏膜膠原纖維排列紊亂,粗細不均,纖維間隙變大,纖維走向不一,板層結構不清,膠原纖維部分或完全溶解,有纖維斷裂現(xiàn)象,而對照眼未見上述改變。 5不同時期鞏膜組織CTGF的表達 CTGF的表達以細胞核及細胞漿為主,正常對照組的表達多呈淡黃色,為+,實驗組著色較深,多呈棕黃色,為+++。后鞏膜加固術后3個月、6個月,A組與B組比較,其著色程度均較高,差異均有統(tǒng)計學意義(P0.05)。 6不同組織中成纖維細胞CTGF的表達 后鞏膜加固術后3個月、6個月,A1組與B1組、A3組與B3組比較,前者著色程度均較高,差異有統(tǒng)計學意義(P0.05)。A2組與A1組、A4組與A3比較,前者著色程度均較高,差異有統(tǒng)計學意義(P0.05)。A1組與A3組、A2組與A4組比較,兩者之間均無統(tǒng)計學意義(P0.05)。 7成纖維細胞力學特性及統(tǒng)計學分析 7.1后鞏膜加固術后3個月、6個月, A組中鞏膜成纖維細胞平衡楊氏模量分別為E∝= 0.35 21±0.0853kPa、0.3612±0.1211kPa,B組的鞏膜成纖維細胞平衡楊氏模量分別為E∝=0.3418±0.0911kPa、0.3476±0.0821kPa,經(jīng)統(tǒng)計學分析,兩組鞏膜成纖維細胞在同一時間的平衡楊氏模量均無統(tǒng)計學意義(P0.05)。 對成纖維細胞進行微管吸吮測試發(fā)現(xiàn),在某一恒定負壓作用下,成纖維細胞表現(xiàn)出典型的粘彈性固體蠕變特征。A組鞏膜成纖維細胞表觀黏性分別為μ=2.8733±0.6154kPa.s、2.9282±0.6694 kPa.s,B組的鞏膜成纖維細胞表觀黏性μ=2.8183±0.590 kPa.s、2.8206±0.5935kPa.s,統(tǒng)計學分析表明在同一時間兩組鞏膜成纖維細胞的表觀黏性差異均無統(tǒng)計學意義(P 0.05)。 7.2不同時期的加固區(qū)鞏膜成纖維細胞力學特性及統(tǒng)計學分析 后鞏膜加固術后3月組、6月組的鞏膜成纖維細胞,無論是平衡楊氏模量還是細胞表觀黏性,其差異均無統(tǒng)計學意義(P0.05)。 7.3不同時期過渡區(qū)成纖維細胞力學特性及統(tǒng)計學分析 后鞏膜加固術后3個月、6個月的過渡區(qū)的成纖維細胞的力學特性,無論是平衡楊氏模量還是細胞表觀黏性,其差異均無統(tǒng)計學意義(P 0.05)。 結論: 1通過眼瞼縫合的方法,可以成功誘導幼兔實驗性近視眼的形成。 2后鞏膜加固術后實驗組鞏膜無論在組織還是細胞水平,CTGF的表達均高于同期陽性對照組,說明手術及加固條帶本身的存在均刺激過渡區(qū)的形成。 3后鞏膜加固術后不同時期過渡區(qū)的組織及其培養(yǎng)的細胞的CTGF表達均高于同期的加固區(qū)鞏膜組,說明CTGF參與了手術本身及加固條帶引起過渡區(qū)的形成。 4后鞏膜加固術后不同時期鞏膜成纖維細胞生物力學特性(平衡楊氏模量、表觀黏性)未發(fā)生明顯變化,說明后鞏膜加固術的增強作用并不是原位鞏膜成纖維細胞本身生物力學的加強,而是由于加固條帶及引起過渡區(qū)的存在。 5后鞏膜加固術后不同時期過渡區(qū)的成纖維細胞生物力學特性無明顯變化,說明后鞏膜加固術是通過細胞間聯(lián)合的整體效應及加固條帶的機械作用,而非通過單個細胞的力學變化起作用。
[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.
【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R779.6

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