【摘要】： 目的:明確后鞏膜加固術(shù)后不同時(shí)期結(jié)締組織生長因子(connective tissue growth factor,CTGF)表達(dá)的變化,并結(jié)合成纖維細(xì)胞力學(xué)特性的研究,從組織-細(xì)胞-生物力學(xué)的角度進(jìn)一步探討后鞏膜加固術(shù)的作用機(jī)制。 方法:3周齡新西蘭家兔45只隨機(jī)選取一眼為實(shí)驗(yàn)眼(experimental eye),用眼瞼縫合方法制備近視動(dòng)物模型,對(duì)側(cè)為自身對(duì)照眼(陰性對(duì)照組)。造模60天后,實(shí)驗(yàn)眼隨機(jī)分為兩組,A組(實(shí)驗(yàn)組)行后鞏膜加固術(shù)(posterior scleral Reinforcement,PSR),B組(陽性對(duì)照組)行相似手術(shù),僅不放置加固條帶。實(shí)驗(yàn)前后均檢測每只家兔雙眼屈光狀態(tài)、眼軸長度,分別于術(shù)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(xì)胞,并傳2代。用免疫細(xì)胞化學(xué)法對(duì)培養(yǎng)的細(xì)胞進(jìn)行鑒定,A組細(xì)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(xì)胞化學(xué)法對(duì)各組細(xì)胞CTGF進(jìn)行半定量分析;部分組織行組織學(xué)檢測,對(duì)各組的CTGF的表達(dá)行半定量分析;利用細(xì)胞微管吸吮的方法結(jié)合半無限體細(xì)胞力學(xué)模型(Half-space model)測定各組成纖維細(xì)胞的力學(xué)特性(包括成纖維細(xì)胞的楊氏彈性模量和粘彈性參數(shù))。結(jié)果用SPSS13.0統(tǒng)計(jì)軟件進(jìn)行統(tǒng)計(jì)分析。 結(jié)果: 1家兔實(shí)驗(yàn)性近視的誘導(dǎo)結(jié)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(dǎo)后,實(shí)驗(yàn)眼與對(duì)照眼屈光度比較,誘導(dǎo)出(1.80±1.37)D的相對(duì)近視,眼軸相對(duì)延長了(0.45±0.29)mm,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 2后鞏膜加固術(shù)后的屈光度與眼軸長度 后鞏膜加固術(shù)后3個(gè)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(jì)學(xué)意義(P0.05)。后鞏膜加固術(shù)后6個(gè)月,A組與B組比較,眼軸相對(duì)變化了(0.59±0.35)mm,兩者之間有統(tǒng)計(jì)學(xué)意義(P0.05)。 3細(xì)胞培養(yǎng)及鑒定結(jié)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(xì)胞長出,2-3周融合,免疫細(xì)胞化學(xué)鑒定為成纖維細(xì)胞。 4組織學(xué)研究結(jié)果 光鏡下實(shí)驗(yàn)眼鞏膜膠原纖維排列紊亂,粗細(xì)不均,纖維間隙變大,纖維走向不一,板層結(jié)構(gòu)不清,膠原纖維部分或完全溶解,有纖維斷裂現(xiàn)象,而對(duì)照眼未見上述改變。 5不同時(shí)期鞏膜組織CTGF的表達(dá) CTGF的表達(dá)以細(xì)胞核及細(xì)胞漿為主,正常對(duì)照組的表達(dá)多呈淡黃色,為+,實(shí)驗(yàn)組著色較深,多呈棕黃色,為+++。后鞏膜加固術(shù)后3個(gè)月、6個(gè)月,A組與B組比較,其著色程度均較高,差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。 6不同組織中成纖維細(xì)胞CTGF的表達(dá) 后鞏膜加固術(shù)后3個(gè)月、6個(gè)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(jì)學(xué)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(jì)學(xué)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(jì)學(xué)意義(P0.05)。 7成纖維細(xì)胞力學(xué)特性及統(tǒng)計(jì)學(xué)分析 7.1后鞏膜加固術(shù)后3個(gè)月、6個(gè)月, A組中鞏膜成纖維細(xì)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(xì)胞平衡楊氏模量分別為E∝=0.3418±0.0911kPa、0.3476±0.0821kPa,經(jīng)統(tǒng)計(jì)學(xué)分析,兩組鞏膜成纖維細(xì)胞在同一時(shí)間的平衡楊氏模量均無統(tǒng)計(jì)學(xué)意義(P0.05)。 對(duì)成纖維細(xì)胞進(jìn)行微管吸吮測試發(fā)現(xiàn),在某一恒定負(fù)壓作用下,成纖維細(xì)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(xì)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(xì)胞表觀黏性μ=2.8183±0.590 kPa.s、2.8206±0.5935kPa.s,統(tǒng)計(jì)學(xué)分析表明在同一時(shí)間兩組鞏膜成纖維細(xì)胞的表觀黏性差異均無統(tǒng)計(jì)學(xué)意義(P 0.05)。 7.2不同時(shí)期的加固區(qū)鞏膜成纖維細(xì)胞力學(xué)特性及統(tǒng)計(jì)學(xué)分析 后鞏膜加固術(shù)后3月組、6月組的鞏膜成纖維細(xì)胞,無論是平衡楊氏模量還是細(xì)胞表觀黏性,其差異均無統(tǒng)計(jì)學(xué)意義(P0.05)。 7.3不同時(shí)期過渡區(qū)成纖維細(xì)胞力學(xué)特性及統(tǒng)計(jì)學(xué)分析 后鞏膜加固術(shù)后3個(gè)月、6個(gè)月的過渡區(qū)的成纖維細(xì)胞的力學(xué)特性,無論是平衡楊氏模量還是細(xì)胞表觀黏性,其差異均無統(tǒng)計(jì)學(xué)意義(P 0.05)。 結(jié)論: 1通過眼瞼縫合的方法,可以成功誘導(dǎo)幼兔實(shí)驗(yàn)性近視眼的形成。 2后鞏膜加固術(shù)后實(shí)驗(yàn)組鞏膜無論在組織還是細(xì)胞水平,CTGF的表達(dá)均高于同期陽性對(duì)照組,說明手術(shù)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(shù)后不同時(shí)期過渡區(qū)的組織及其培養(yǎng)的細(xì)胞的CTGF表達(dá)均高于同期的加固區(qū)鞏膜組,說明CTGF參與了手術(shù)本身及加固條帶引起過渡區(qū)的形成。 4后鞏膜加固術(shù)后不同時(shí)期鞏膜成纖維細(xì)胞生物力學(xué)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(shù)的增強(qiáng)作用并不是原位鞏膜成纖維細(xì)胞本身生物力學(xué)的加強(qiáng),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(shù)后不同時(shí)期過渡區(qū)的成纖維細(xì)胞生物力學(xué)特性無明顯變化,說明后鞏膜加固術(shù)是通過細(xì)胞間聯(lián)合的整體效應(yīng)及加固條帶的機(jī)械作用,而非通過單個(gè)細(xì)胞的力學(xué)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(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R779.6

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