阻断Notch信号对兔耳增生性瘢痕形成的影响及作用机制

Blocking of Notch signaling decreased scar formation in rabbit ear hypertrophic scar model

目的探讨Notch信号相关分子对增生性瘢痕形成的影响。方法用16只新西兰大耳白兔建立耳增生性瘢痕模型，分别在伤后1、3、5、7和14d向左耳瘢痕灶内注射含有100μmol／LNotch信号途径抑制剂N-[N-（3，5-二氟苯乙酰基-L-丙氨酰基）]-（S）-苯基甘氨酸叔丁酯（DAPT）的稀释液（二甲基亚砜和生理盐水稀释）0．1ml（DAPT组），右耳在相同时点瘢痕灶内注射生理盐水稀释的二甲基亚砜（对照组）。术后14、21、28和35d各处死4只兔，切取瘢痕标本，HE染色观察并计算瘢痕增生指数和成纤维细胞密度，免疫组织化学染色观察表皮分化标志物角蛋白19（K19）、K14、外皮蛋白和Notch下游分子P21、P63的表达。结果DAPT组术后21、28、35d瘢痕增生指数（分别为1．93±0．32、1．82±0．36、1．79±0．25）和成纤维细胞密度[分别为（4．08±0．88）、（3．30±0．53）、（3．19±0．73）×10^3／mm^2]均明显低于对照组[2．56±0．29、2．61±0．30、2．58±0．39和（5．45±0．99）、（4．80±1．13）、（4．43±1．17）×10^3／mm^2，均P〈0．01]。DAPT组术后14d瘢痕表皮K19、K14和P63表达阳性率（28．6％±5．7％、53．1％±4．5％、57．0％±5．8％）均明显高于对照组（10．1％±2．8％、30．8％±4．9％、16．5％±2．2％，均P〈0．01），而外皮蛋白和P21表达阳性率（12．3％±1．9％、11．0％±1．7％）均明显低于对照组（29．3％±4．6％、44．3％±3．5％，均P〈0．01）。结论阻断Notch信号可以抑制表皮细胞的过度分化，从而抑制兔耳增生性瘢痕的形成。

Objective To investigate the effects of Notch signaling on scars in a rabbit ear model of hypertrophic scarring. Methods The hypertrophic scar of rabbit ears was reproduced. The left rabbit ear wounds as the N- [ N- （3, 5-difluorophenacetyl-L-alanyl） ] - （S） -phenylglycine t-butyl ester （DAPT） treated group were treated intradermally with the γ-secretase inhibitor DAPT to inhibit the activation of Notch on day 1, 3, 7 and 14. The right ears as the control group were treated with normal saline at the same time points. Experimental and control wounds were harvested on day 14, 21, 28 and 35 post-wounding, and then examined histologically to quantify hypertrophic index and fibroblasts. The expression of epidermal differentiation markers -keratin 19 （K19）, keratin 19 （K14）, involucrin and Notch downstream molecules - P21, P63 were examined and analyzed with immunohistochemical staining. Results On day 21,28 and 35 post-wounding, both hypertrophic index （ 1.93 ± 0. 32, 1.82 ± 0. 36, and 1.79 ± 0. 25, respectively） and number of fibroblasts [ （4.08 ± 0. 88）, （3.30 ± 0. 53 ）, and （3.19 ± 0. 73 ） × 10^3/mm^2, respectively] in the DAPT treated group were significantly lower than those of the control group [ 2. 56 ± 0.29, 2. 61 ± 0. 30, 2.58＋0.39, and （5.45±0.99）, （4.80±1.13）, （4.43 ±1.17） ×10^3/mm^2, allP〈0.01）]. The expression of K19, K14 and P63 increased in the DAPT treated group （28.6% ±5.7% , 53.1% ±4. 5%, and 57.0% ± 5.8 %, respectively） as compared with the control group （ 10. 1% ± 2. 8%, 30. 8% ± 4. 9%, and 16. 5% ±2. 2%, all P 〈0. 01） on day 14 post-wounding, while the expression of involucrin and P21 decreased in the DAPT-treated group （12. 3% ± 1.9% and 1l. 0% ± 1.7% respectively） as compared with the control group （29. 3% ± 4. 6% and 44. 3% ± 3.5%, both P 〈 0. 01 ）. Conclusion Blocking of Notch signaling will inhibit the over-differentiation of scar epidermis, and thereby suppress the proliferation of hypertrophic scars in rabbit ears.