羊膜上皮细胞损伤后在纤维蛋白支架及细胞生长因子作用下修复状况的研究

Experimental study of repairing damaged human amniotic epithelial cells with formulated fibrin clot and cell growth factor

目的探讨人羊膜上皮细胞（HAEC）损伤后能否在体外构建的纤维蛋白支架上修复，以及表皮生长因子（EGF）、碱性成纤维细胞生长因子（bFGF）和转化生长因子β1（TGF-β1）对HAEC增殖的影响。方法采用环钻钻切培养板上的HAEC建立定量损伤模型，用制备的纤维蛋白块覆盖环钻钻切范围，分别加入不同浓度的培养液进行培养。其中加入EGF为EGF组、加入bFGF为bFGF组、加入TGF-β1为TGF-β组，不加任何细胞生长因子作为对照组。显微镜下观察各组HAEC生长移行情况，5-溴脱氧尿嘧啶掺入法检测HAEC的增殖效应。结果（1）各组HAEC均能向环钻缺损处移行并向内生长，EGF组、bFGF组的HAEC移行速度快，细胞数量多，对照组次之，TGF-β1组最少。（2）EGF组不同浓度的EGF（1．0、5．0、10．0、20．0、40．0、80．0及160．0ng／ml）培养下，HAEC细胞增殖率分别为17．8％、28．0％、35．3％、51．6％、34．1％、34．2％及26．0％，EGF组10～80ng／ml的HAEC增殖效应均明显大于对照组（17．1％，P〈0．05）。（3）bFGF组不同浓度的bFGF（1．0、5．0、10．0、20．0．40．0、80．0及160．0ng／ml）培养下，HAEC细胞增殖率分别为18．0％、35．7％、43．0％、52．7％、67．4％、43．6％及30．5％。bFGF组5～80ng／ml的HAEC增殖效应均明显大于对照组（P〈0．05）。其中40ng／ml时的细胞增殖率最高（P〈0．05）。（4）TGF-β1组不同浓度的TGF-β1（0．1、0．2、0．4、0．8、1．6、3．2、6．4及12．8ng／ml）培养下，HAEC细胞增殖率分别为17．1％、15．1％、9．3％、6．2％、4．8％、3．6％、2．0％、1．2％。TGF-β1组0．8～12．8ng／ml的HAEC增殖效应明显小于对照组（P〈0．05）。结论通过纤维蛋白支架，HAEC能移行修复缺损部位。EGF、bFGF能促进HAEC的增殖，而TGF-β1则抑制HAEC的增殖。

Objective To investigate whether damaged human amniotic epithelial cells （HAEC） could be repaired on the matrix of formulated fibrin clot in vitro and the effects of epidermal growth factor （EGF） ,basic fibroblast growth factor（bFGF） and transforming growth factor β1 （TGF-β1）on the proliferation of HAEC. Methods Ring drill was used to drill the HAEC layer on culture sheets to make quantified models of damaged HAEC, on which the lacks were then covered with fibrin clot. Subsequently, EGF （EGF group）, bFGF（ bFGF group） and TGF-β1 （TGF-β1 group） of different concentration were added into the sheets respectively. After the predesigned culturing time, the growing and transiting conditions of HAEC were observed under inverted microscope after Giemsa stain. Also, the proliferating conditions of HAEC were detected by using 5-bromodeoxyuridine （BrdU）. Results In all groups, HAEC could transit toward damaged area on fibrin clot and grow there. Higher transiting speed and larger cell numbers were observed in the EGF and bFGF groups followed by the control group, while the TGF-β1 group showed the relatively poorer results. Proliferating rates of HAEC were 17. 8% , 28.0%, 35.3%, 51.6% , 34. 1% , 34. 2% and 26. 0% respectively by EGF of different cultured concentration （ 1.0ng/ml, 5. 0ng/ml, 10. 0ng/ml, 20. 0ng/ml, 40. 0ng/ml, 80. 0ng/ml and 160. 0ng/ml） . Proliferating rates of HAEC were 18. 0%, 35. 7%, 43. 0% , 52. 7%, 67.4% , 43.6% and 30. 5% respectively by bFGF of different cultured concentration （ 1.0ng/ml, 5.0ng/ml, 10. 0ng/ml, 20. 0 ng/ml, 40. 0 ng/ml, 80. 0 ng/ml and 160. 0ng/ ml）. Compared with the control group, EGF groups （EGF concentration ranging from 10ng/ml to 80ng/ml）and bFGF groups （bFGF concentration ranging from 5ng/ml to 80ng/ml ） showed better proliferating effects of HAEC （P 〈 0. 05 ）, especially the 20ng/ml EGF group and 40ng/ml bFGF group had the best proliferating results among their own respective groups（P 〈0. 05）. Proliferating rates of HAEC were 17. 1%, 15. 1%, 9. 3%, 6. 2%, 4. 8%, 3. 6%, 2. 0% and 1.2% respectively by TGF-β1 of different cultured concentration （0. 1 ng/ml, 0.2 ng/ml, 0.4 ng/ml, 0.8 ng/ml, 1.6 ng/ml, 3.2 ng/ml, 6.4 ng/ml and 12.8 ng/ml）. Proliferating rates of HAEC in TGF-β1 groups （TGF-β1 concentration ranging from 0. 8ng/ml to 12. 8ng/ml） were significantly lower than that in the control group （ P 〈 0.05 ）. Conclusions HAEC could transit and grow on the matrix of fibrin clot and repair the damaged area. EGF and bFGF could obviously stimulate HAEC proliferation ,while TGF-β1 might have the inhibitive effects.