OPTC-shRNA干扰对牛玻璃体细胞与视网膜色素上皮细胞共培养胶原凝胶收缩系统的影响

The effect of OPTC-shRNA on the bovine retinal pigment epithelial cells and hyalocytes co-culture collagen gel contraction system

目的探讨抑制opticin蛋白表达对牛玻璃体细胞与视网膜色素上皮（RPE）细胞共培养胶原凝胶收缩系统的调控作用。方法实验研究。设计并转染OPTC-shRNA质粒至体外培养的牛RPE细胞，通过免疫印迹法检测转染后3、5、7dopticin蛋白的相对表达量和抑制率。在牛I型胶原凝胶内加入牛玻璃体细胞和RPE细胞，构建细胞共培养的胶原凝胶体外收缩模型。实验共分为6组：OPTC-shRNA质粒转染RPE及玻璃体细胞组（A组）、空质粒转染RPE及玻璃体细胞组（B组）、未转染RPE及玻璃体细胞组（c组）、仅含未转染RPE细胞组（D组）、仅含玻璃体细胞组（E组）以及不含任何细胞空白对照组（F组）。应用One—wayANOVA、SNK-q检验和单因素回归分析比较各组凝胶收缩差异，并分析A、B、C组玻璃体细胞数的差异对胶原凝胶收缩性状的影响。结果成功构建并转染了有显著抑制作用的OPTC—shRNA质粒至牛RPE细胞。免疫印迹法检测结果显示，opticin蛋白表达抑制率在转染后3、5、7d分别为（83．91±2．88）、（84．71±4．27）、（82．85±2．72）％，差异无统计学意义（F=1．15，P〉0．05）。胶原模型构建后3d，A、B、C组中不同密度（2×10^7、1×10^8、5×10^8个／L）亚组玻璃体细胞密度凝胶收缩率分别为A组：（23．52±2．08）、（56．00±1．02）、（61．62±1．73）％；B组：（16．56±2．01）、（36．41±1．33）、（49．56±1．75）％；C组：（15．75±1．37）、（37．45±1．14）、（48．45±1．97）％，D、E组凝胶收缩率分别为（12．18±0．95）、（10．95±0．93）％，F组未观察到凝胶收缩。在相同玻璃体细胞密度下A、B、C组间凝胶收缩性两两比较，2×10^7个／L细胞密度：A组与B组、A组与C组间差异有统计学意义（q=11．38、12．72，P值均〈0．05）；B组与C组间差异无统计学意义（q=1．34，P〉0．05）；1×10^8个／L细胞密度：A组与B组、A组与c组差异有统计学意义（q=48．83、46．22，P值均〈0．05）；B组与C组差异无统计学意义（q=-2．61，P〉0．05）；5×10^8个／L细胞密度：A组与B组、A组与C组间差异有统计学意义（q=48．83、46．22，P值均〈0．05）；B组与C组间差异无统计学意义（q=1．74，P〉0．05）。A、B、c各组内不同玻璃体细胞密度之间凝胶收缩性两两比较（2×10^7个／L与1×10^8个／L、2×10^7个／L与5×10^8个／L、2×10^7个／L与2×10^7个／L）：A、B、C3组内各细胞密度之间差异均有统计学意义（A组：q=-55．97、-65．66、-9．69；B组：q=-34．53、-57．41、-22．88；C组：q=-41．94、-63．19、-21．25；P值均〈0．05）。进一步对组内不同玻璃体细胞密度与对应的凝胶收缩率进行回归分析，结果显示，共培养模型的凝胶收缩率与其中的玻璃体细胞密度呈一定的正相关性（A、B、C组r=0．919、0．981、0．937，P值均〈0．05）。D、E、F组凝胶收缩率两两比较，D组与E组、D组与F组、E组与F组差异均有统计学意义（q=54．87、49．33、5．54，P值均〈0．05）。结论opticin蛋白可调控牛玻璃体细胞与RPE细胞共培养胶原凝胶收缩性状。

Objective To investigate the OPTC-shRNA inhibiting effect on the opticin expression by the bovine hyalocytes and retina pigment epithelial （RPE） cells co-culture collagen gel contraction system. Methods Experimental study. The OPTC-shRNA expression vector was designed and transfected into bovine RPE cells cultured in vitro. The relative expression and the inhibition rate of the opticin protein were measured by Western blot on days 3,5 and 7. An in vitro cells co-culture bovine type I collagen gel contraction assay was constructed consisting of the hyalocytes and RPE cells. Six groups were established in this experiments： OPTC-shRNA plasmid transfected RPE cells and hyalocytes （group A）, empty plasmid transfected RPE cells and hyalocytes（ group B）, non-transfected RPE cells and hyalocytes （group C）, non- transfected RPE cells （ group D ）, only hyalocytes （ group E ）, and no cells （ group F ） . The collagen gel contractile activities of these groups were compared by One-way ANOVA, SNK-q tests and regression analysis ;and the influence of the hyalocytes density variance on the collagen gel contraction in groups A, B and C were also analyzed. Results The OPTC-shRNA expression vector with significant inhibition effect was constructed and transfected into bovine RPE cells successfully. The results of Western blot analysis showed that the inhibitory rates on the opticin expression on days 3,5 and 7 were （ 83.91 ± 2. 88 ）, （ 84. 71 ± 4. 27 ） and （ 82. 85 ± 2. 72） % , respectively. Furthermore, the differences among days 3,5 and 7 were insignificant（ F = 1.15 ,P 〉 0. 05 ）. On day 3, the gel contraction rates for the sub-groups with various hyalocytes densities （2 × 10^7 ,1× 10^8 and 5 ×10^8/L） in groups A,B and C were：group A： （23.52 ±2. 08）, （56.00 ± 1.02）, （61.62 ± 1.73）% ;group B： （ 16. 56 ±2.01）, （36.41 ± 1.33）, （49. 56 ± 1.75）% ;group C： （ 15.75 ± 1.37）, （37.45 ± 1.14）, （48. 45 ± 1.97）%. The gel contraction rates for groups D and E were （ 12. 18 ± 0. 95 ） % and （ 10. 95 ± 0. 93 ） %, respectively ; no gel contraction was observed in group F. Pairwise comparisons of the gel contraction rates were performed by SNK-q test among groups A, B and C for various hyalocyte densities. In the 2 × 10^7/L cell density group,the differences between groups A and B or C were significant（q = 11.38,2. 72, respectively, P both 〈 0. 05 ）, the differences between B and C were insignificant（q = 1.34,P 〉0. 05）. In the 1 × 10^8/L cell density group, the differences between groups A and B or C were significant（q = 8.83,46. 22, respectively,P both 〈 0. 05 ）, the differences between B and C were insignificant（q = 1.34,P 〉 0. 05）. In the 5× 10^8/L cell density group, the differences between groups A and B or C were significant（q = 48.83,46.22, respectively, P both 〈 0. 05 ） , the differences between groups B and C were insignificant （ q = 1.74, P 〉 0. 05 ）. Pairwise comparisons of the sub-groups with different hyalocyte densities in groups A,B and C （comparisons of 2 × 10^7/L and 1 × 10^8/L,2 × 10^7/L and 5× 10^8/L, 2 × 10^7/L and 2 ×10^7/L, respectively）, the differences were all significant（ group A：q = -55.97, -65.66, - 9. 69, respectively; group B ： q = - 34. 53, - 57.41, - 22. 88, respectively ; group C ： q = - 41.94, -63. 19, -21.25, P all 〈 0. 05 ）. Furthermore, the regression analysis was performed between the hyalocyte density and the collage gel contraction rates in each group. The results showed that there was a positive correlation between the gel contraction rates of the co-culture collagen gel contraction system and its hyalocyte density （ groups A, B, C ： r = 0. 919,0. 981,0. 937, respectively, P all 〈 0.05 ）. Pairwise comparison of groups D and E, D and F, E and F by SNK-q test revealed q = 54. 87,49.33,5.54, respectively, P all 〈 0.05. Conclusion Opticin is capable of regulating the contraction of bovine hyalocytes and RPE cells co-culture collagen gel.